abstract tot - 5° NPCF Nuove Prospettive in Chimica Farmaceutica

Transcript

5° Meeting
NUOVE PROSPETTIVE IN
CHIMICA FARMACEUTICA
ABSTRACTS
Trieste
28-30 marzo 2011
Plenary Lectures
2
Wandering around New Targets. Opportunities and Risks for
Medicinal Chemistry Research
G. Costantino
Dipartimento Farmaceutico, Università di Parma Via G.P. Usberti 27/A 43100
Parma
[email protected]
The rapidly changing scenario of industrial drug discovery is also impacting
academic medicinal chemistry research. Cost- and risk-reducing strategies and
outsourcing are expected to bring about an increasing importance to exploratory
preclinical research carried out in Universities. It is expected that most of the risky
and mainly exploratory projects of target selection will be taken up by academic
groups, which must be prepared to tackle the challenge. Besides scientific excellence,
keys to success will be flexibility and the ability to select targets and pathways
endowed with high potential for development.
In this presentation a survey of opportunities and risks associated with
identification and druggability of new targets will be presented, with a special
emphasis on CNS targets.
3
Biomaterials and Nanomedicine
M. Prato
Dipartimento di Scienze Chimiche e Farmaceutiche, Università degli Studi di Trieste,
Piazzale Europa 1, 34127 Trieste, Italy
[email protected]
Nanometer-scale structures represent a novel and intriguing field, where scientists
and engineers manipulate objects at the atomic and molecular levels to produce
innovative materials for composites, electronic, sensing, and biomedical applications.
Carbon nanomaterials such as carbon nanotubes constitute a relatively new class of
materials exhibiting exceptional mechanical and electronic properties and are also
promising candidates for gas storage and drug delivery.
However, processing carbon nanotubes is
severely limited by a number of inherent
problems: purification from a variety of
byproducts, difficult manipulation and low
solubility in organic solvents and in water are
only some of these problems. For these reasons,
several strategies have been devised to make
nanotubes “easier” materials. The chemical
modification of carbon nanotubes is a very young
field, but carbon nanotubes represent today a
class of materials expected to play a significant
role in innovative discoveries.
Covalent
and
supramolecular
functionalizations are the basic synthetic
techniques for solubilization and use in different
fields. Their use as drug delivery scaffolds and
substrates for vaccines has already been
demonstrated.
When functionalized, they become less toxic, and exhibit a high propensity to
cross cell membranes. The use of carbon nanotubes as active substrates for neuronal
growth has also given so far very exciting results. Not only nanotubes are compatible
with neurons, but they play a very interesting role in interneuron communication.
Future studies will determine the opportunities as well as the limitations of such
materials.
4
Main Lectures 5
Receptor insight: recent advances in the development of novel
therapeutic agents
C. Martini, S. Daniele, M. L. Trincavelli
Dep. of Psychiatry, Neurobiology, Pharmacology and Biotechnology. Pisa University
[email protected]
G protein-coupled receptors (GPCRs) are the largest family of cell surface
receptors, and mediate effects of neurotransmitters, hormones, ions, odorants and
light. Their signals are essentially mediated via the activation of heterotrimeric G
proteins and their effectors (e.g. adenylyl cyclase, phospholipase C, potassium and
calcium channels). Because of their involvement in a large number of physiological
and pathological processes, GPCRs have been subject to intensive investigation and
represent major targets for current pharmacological intervention.1 However, recent
findings necessitate revison of the traditional view of receptor-receptor interactions
and GPCR signalling.
Receptor-receptor interactions. Traditionally, mechanisms of ligand binding and
signal transduction by GPCRs have been modelled on the assumption that monomeric
receptors participate in the processes. Really, several receptor subtypes form heterooligomers, and these ‘heteromeric’ receptors have functional characteristics that differ
from homogeneous populations.2,3 Given that dimeric ligands seem to have altered
properties, such as increased efficacy and potency,4 there seems to be great potential
in developing novel therapies based on GPCR oligomerization. First of all, by
identifying receptors that directly interact with each other, it might be possible to
develop new therapies simply by using available drugs in new ways. Based on the
direct interactions among receptors, it is possible to develop novel therapies simply by
employing already available drugs. Moreover, it is possible to develop dimeric
ligands, selective for heteromeric GPCRs, in order to stimulate or selectively block
the novel signalling of heteromers, or to have drugs that can enhance or disrupt GPCR
oligomer formation in order to regulate oligomerization-dependent functions.
Receptor regulations. Similar to other classes of receptors, after binding of the
activating ligands, all GPCRs undergo a complex series of reactions to turn off the
signal transduction.1 Prolonged stimulation of GPCRs often leads to their
desensitisation and internalization into endosomes.1,5 Although originally considered
as a major mechanism of signal desensitization, internalization has other pivotal
functions, such as: i) controlling the recovery of receptor responsiveness following
agonist-mediated activation; ii) signalling to intracellular protein kinases (i.e. the
mitogen activated protein kinase (MAPK) cascade), which in turn regulate several
cellular processes, such as cell differentiation and cell proliferation/death. The
complex machinery that follows GPCR activation not only confer signal specificity
and diversity, but may partecipate in a range of other cellular processes, such as
cellular differentiation and maturation. On this basis, it’s clear that the functional state
of receptors and the regulation of the intracellular pathways, involved in receptormediated responses have to be taken in account in the development of therapeutic
agents, the choose of the doses and time of somministration.
References
[1]Pierce KL, Premont RT, Lefkowitz RJ. Nat Rev Mol Cell Biol 2002; 3:639–650. [2] Trincavelli ML, Cuboni S,
Catena Dell’Osso M, Maggio R, Klotz K-N, Novi F, Panighini A, Daniele S, Martini C. Purinergic Signalling
2010; 6:373-381. [3]Maggio R, Novi F, Scarselli M, Corsini GU. FEBS J 2005; 272:2939-2946. [4]Martini C,
Daniele S, Picchetti M, Panighini A, Carlini M, Trincavelli ML, Cesari D, Da Pozzo E, Golia F, Dell'osso L.
Neuropsychobiology 2011; 63:154-159. [5]Trincavelli ML, Tonazzini I, Montali M, Abbracchio MP, Martini C. J
Cell Biochem. 2008; 104:150-161.
6
The role of chirality in drug discovery and development
C. Bertucci
Dipartimento di Scienze Farmaceutiche, Università di Bologna, Bologna, Italy
[email protected]
1. Introduction
Chirality has a fundamental role in determining the pharmacodynamic and
pharmacokinetics properties of drugs.1 Furthermore chirality contributes significantly
to the understanding of the mechanisms lying behind biorecognition phenomena.
Circular dichroism (CD) is the technique of choice to obtain information on chirality,
and to monitor and characterize biomolecular recognition phenomena in solution.2,3
2. Results and Discussion
Several cases will be presented to underline the role of chirality in the
understanding of the recognition phenomena at molecular level, and giving
information on the structural features that modulate these processes. The examples
underline the usefulness of CD in drug discovery and development, and its potential
in the emerging studies of the interactome. In particular the use of CD to assess the
stereochemistry of chiral leads and drugs will be discussed. This aspect is
fundamental for assessing a reliable relationship between stereochemistry and
pharmacological activity. Furthermore, the use of induced CD analysis will be
presented for determining the stereochemistry of a drug when bound to the target
protein, and to determine the binding parameters. Finally, it will be shown how the
CD monitoring of the conformational transitions of peptides and proteins yields
information on the oligomerization process and molecular recognition phenomena.
Both these aspects are often key steps in physiological and pathological events, and
also relevant to stability and quality control studies of recombinant protein
therapeutics.
3. References
[1] Aboul-Enein HY, Wainer IW (eds.) (1997), The impact of stereochemistry on drug
development and use, Chemical Analysis Series. John Wiley and Sons, New York.
[2] Ascoli GA, Domenici E, Bertucci C (2006) Chirality 18(9):667-79.
[3] Bertucci C., Pistolozzi M., De Simone A. Anal Bioanal Chem 2010, 398:155-166.
7
Development of novel drugs for the treatment of colorectal cancer:
focus on recently identified and potential molecular targets
E. Mini
Department of Pharmacology, University of Florence, Firenze, Italy
[email protected]
The prognosis of advanced colorectal cancer remains poor in spite of the advances
obtained in recent years with new approaches in surgical procedures, new diagnostic
methods and new therapeutic agents. Our understanding of existing drugs, such as
fluoropyrimidines, oxaliplatin and irinotecan, has become more refined. The
incorporation of newer drugs, such as monoclonal antibodies directed against the
vascular endothelial growth factor (VEGF) (bevacizumab) and the epidermal growth
factor receptor (EGFR) (cetuximab, panitumumab) has expanded treatment options as
well. An emerging understanding of the molecular pathways that characterise cell
growth, cell cycle, apoptosis, angiogenesis and invasion has led to the identification
of other novel targets for cancer therapy. Improvements in our understanding of
numerous signaling proteins being implicated in colorectal cancer progression have
opened the door toward potentially efficient, selective, but also complex therapeutic
approaches. Beyond EGFR, other growth factor membrane receptors such as vascular
endothelial growth factor receptor (VEGFR), insulin-like growth factor receptor
(IGFR), platelet-derived growth factor receptor (PDGFR), tumour necrosis factorrelated apoptosis-inducing ligand receptor (TRAIL-R), and hepatocyte growth factor
receptor (c-Met) may represent promising targets of novel drugs. Also, intracellular
signalling targets (the Ras/Raf/MAPK pathway, the phosphatidylinositol-3-kinase
(PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, src kinase) and other
protein kinases that regulate cell division, including aurora kinases, have given rise to
the discovery of selective compounds that are currently under clinical development in
the setting of colorectal cancer. Emerging data from these clinical investigations are
providing novel opportunities for the treatment of colorectal cancer patients that will
probably translate into efficacy benefits in the coming years.
8
Protein-ligand binding free energy and kinetics
F. Colizzi,a J. Patel,b D. Branduardi,b M. Recanatini,a A. Cavallia,b
a
Dipartimento di Scienze Farmaceutiche, Università di Bologna; bDipartimento di
Scoperta e Sviluppo Farmaci, Istituto Italiano di Tecnologia, Genova
[email protected]
1. Introduction
In structure-based drug discovery, an accurate prediction of the protein-ligand
binding free energy is extremely challenging.1 A possible alternative is represented by
the estimation of the protein-ligand binding kinetics, which is a direct measure of the
stability of the binary complex.2 Some experimental methods are nowadays available
for measuring the rate of binary complex dissociation (i.e. the protein-ligand binding
koff). Conversely, from a computational standpoint, a few examples have appeared in
the literature aimed at predicting the koff of binary complexes.
Recently, we used steered molecular dynamics (SMD) to pull a series of inhibitors
out of their complexes with a target enzyme.3 In particular, we investigated a series of
flavonoid derivatives, and we computed the force that was required to extract
inhibitors from complexes with the FabZ enzyme, a promising anti-malarial target.
Although we were not able to determine the flavonoids-FabZ binding free energy, we
could clearly distinguish active from inactive inhibitors, by estimating the kinetics
stability of these complexes. This was accomplished by calculating the forces
required for pulling each ligand out of the protein embrace.4 In a subsequent study,
we further explore the importance of the protein-ligand binding kinetics by studying
the local mechanical response experienced by a ligand during SMD-based undocking
simulations. These studies have clearly shown the pivotal role of protein-ligand
binding kinetics and SMD-based predictions in structure-based drug design.
3. References
[1] Gilson, M.K. and Zhou, H.X. Annu. Rev. Biophys. Biomol. Struct. 2007, 36, 21-42
[2] Copeland, R.A., Pompliano, D.R. and Meek, T.D. Nat. Rev. Drug Discov. 2006, 5, 21-42.
[3] Colizzi, F., Perozzo, R., Scapozza, L., Recanatini, M. and Cavalli, A. J. Am. Chem. Soc. 2010, 132,
7361-7371.
[4] Jorgensen, W.L. Nature 2010, 466, 42-43.
9
Targeting CNS: opportunities and challenges
for medicinal chemistry
F. Micheli
Aptuit (VR) srl, via Fleming 4, 37135 Verona (Italy)
[email protected]
Targeting the CNS “chemical space” with potential new drugs represents both a
great opportunity and a formidable challenge for medicinal chemists.
Beyond the usual parameters to be addressed (e.g. potency, selectivity, appropriate
PK parameters), the CNS drugs need to cross the blood brain barrier and to remain in
the brain to exert their action.
A case study on selective Dopamine D3 antagonist (Figure 1) recently published by
GSK [1-2] will be presented.
Figure 1
N
N
N
N
H3C
N
O
N
S
N N
1
H3C
2
N
N
N
S
N N
N
N
N
S
3
CF3
O
O
N N
N
N
NC
N N
4
H
F
F F
N N
S
N
N
5
References
[1] Micheli, F., Bonanomi, G., Blaney, F. E., Braggio, S., Capelli, A. et al. 2007, J. Med. Chem. 50
(21), 5076-5089.
[2] Micheli, F., Arista, L., Bonanomi, G., Blaney, F. E., Braggio, S., Capelli, A. M et al.. 2010, J. Med.
Chem. 53 (1), 374-391.
10
Protein-Protein interactions modulation for therapeutic intervention
A. Padova
Molecular Informatics and Medicinal Chemistry Department
[email protected]
1. Introduction
Protein-protein interactions play a key role in biological processes. In the past few
years, there has been a wealth of research activities aimed at identifying proteinprotein interactions in the context of cellular function and studying the interactions at
molecular and atomic level. Modulation of protein-protein interaction may represent
the paradigm for the discovery of novel, selective therapeutic agents. However, it has
proved challenging to develop small molecule capable of inhibiting protein-protein
interactions,1 in general characterized by large, rather flat contact interactions.
After a general introduction on the interactome,2
the seminar will give an overview of on-going
drug discovery efforts to identify, optimize and
develop protein-protein interaction modulators.
Hence, the role of in silico and biophysical
approaches to aid understanding of dynamic bimolecular interaction will be discussed and
exemplified.3 The seminar will conclude with a
perspective on PPI inhibitors for cell signaling
pathways modulation.4
3. References
[1] Wells et al, doi:10.1038/nature06526.
[2] De Las Rivas et al, doi:10.1371/journal.pcbi.1000807.
[2] Agamennone et al, DOI: 10.1002/cmdc.200900393.
[3] Guarracino et al, DOI: 10.1002/bip.21546.
11
Short Communications 12
A novel series of potent calcium-sensing receptor antagonist for the
treatment of osteoporosis
L. Arista
Novartis Institutes for BioMedical Research, Basel Switzerland.
[email protected]
Parathyroid hormone (PTH) is an effective bone anabolic agent. However, only when
administered by daily subcutaneous injections exposure of short duration is achieved,
a prerequisite for an anabolic response.
Instead of applying exogenous PTH, mobilization of endogenous stores of the
hormone can be envisaged. The secretion of PTH stored in the parathyroid glands is
mediated by a calcium sensing receptor (CaSR) a GPCR localized at the cell surface.
Antagonists of CaSR (calcilytics) mimic a state of hypocalcaemia and stimulate PTH
release to the bloodstream. Screening of the internal compound collection for
inhibition of CaSR signaling function afforded compound 1.
N
O
O
N
Compound 1
During the Lead Optimization phase in vitro potency could be improved >1000 fold
by derivatization of its chemical structure. While the compounds readily induced PTH
release after iv application a special formulation was needed for oral activity. The
required profile was achieved by using microemulsions. Excellent PK/PD correlation
was found in rats and dogs.
13
D2 dopamine receptor agonists as allosteric modulators of A2A
adenosine receptor signalling
S. Daniele,a M. L. Trincavelli,a E. Orlandini,b G. Navarro,c V. Casadó,c S. Nencetti,b
E. Nuti,b M. Macchia,b H. Huebner,d P. Gmeiner,d A. Rossello,b C. Martinia
a
Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology,
University of Pisa, Via Bonanno 6, 56126 Pisa, Italy; bDepartment of Pharmaceutical
Sciences, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy; cCentro de
Investigacón Biomédica en Red sobre Enfermedades Neurodegenerativas
(CIBERNED), and Department of Biochemistry and Molecular Biology, Faculty of
Biology, University of Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain;
d
Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich Alexander
University, 91052, Erlangen, Germany.
[email protected]
1. Introduction
The structural and functional interaction between D2 dopamine receptor (DR) and
A2A adenosine receptor (AR) has suggested these two receptors as a pharmacological
target in pathologies associated with dopamine dysfunction, such as Parkinson's
disease [1-4]. In transfected cell lines it has been demonstrated the activation of D2DR
induces a significant negative regulation of A2AAR-mediated responses [4-5], whereas
few data are at now available about the regulation of A2AAR by D2DR agonists at
receptor recognition site.
In this work we confirmed that in A2A-D2 co-transfected cells, these receptors
exist as homo and heterodimers. The classical D2DR agonists were able to negatively
modulate both A2AAR affinity and functionality. These effects occurred even if any
significant changes in A2AAR/D2DR energy transfer interaction could be detected in
BRET experiments.
Since the development of new molecules able to target A2A-D2 dimers may represent
an attractive tool for innovative pharmacological therapy, we also identified a new
small
molecule,
3-(3,4-dimethylphenyl)-1-(2-piperidin-1-yl)ethyl)piperidine
(compound 1), full agonist of D2DR and modulator of A2A-D2 dimer. This compound,
although showed a low affinity for D2DR, was able to negatively modulate A2AAR
binding properties and functional responsiveness in a manner comparable to classical
D2DR agonists. In contrast to classical agonists, compound 1 led to conformational
changes in the quaternary structure in D2DR homomers or heteromers. These results
suggest that compound 1, even if shows low D2DR affinity, exerts a high control of
the function of heteromers and could represent a starting point for the development of
new drugs targeting A2A/D2 heteromers.
3. References
[1] L.F. Agnati, S. Ferrè, C. Lluı´s, R. Franco, K. Fuxe, Pharmacol. Rev. 55 (2003) 509-550. [2] M.
Canals, J. Burgueño, D. Marcellino, N. Cabello, E.I. Canela, J. Mallol, L. Agnati, S. Ferré, M. Bouvier,
K. Fuxe, F. Ciruela, C. Lluis, R. Franco, J. Neurochem. 88 (2004) 726–734. [3] K. Fuxe, D.
Marcellino, S. Genedani, L. Agnati, Movement Disord. 22 (2007) 1990-2017. [4] S. Ferré, G. Von
Euler, B. Johansson, B.B. Fredholm, K. Fuxe, Proc. Natl Acad. Sci. USA 88 (1991) 7238-7241. [5] S.
Ferré, K. Fuxe, E.G. von, B. Johansson, B. B. Fredholm, Neuroscience 51 (1992) 501-512.
14
1,2,4-triazolo[1,5-a]-1,3,5-triazine nucleus:
a possible template for adenosine receptor subtypes
S. Federico,a S. Paoletta,b S. L. Cheong,c G. Pastorin,c B. Cacciari,d S. Stragliotto,b K.N. Klotz,e J. Siegel,f Z.-G. Gao,f K. A. Jacobson,f S. Moro,b and G. Spallutoa
a
Università di Trieste, Piazzale Europa 1, I-34127 Trieste, Italy; bUniversità di
Padova, via Marzolo 5, I-35131 Padova, Italy; cNational University of Singapore, 3
Science Drive 2, Singapore 117543; dUniversità degli Studi di Ferrara, via Fossato di
Mortara 17-19, I-44100 Ferrara, Italy; eUniversität of Würzburg, Versbacher Str. 9,
D-97078 Würzburg, Germany; fNational Institute of Diabetes and Digestive and
Kidney Diseases, National Institutes of Health, MD 20892-0810, Bethesda, Maryland,
United States
[email protected]
A new series of triazolo-triazines variously substituted at the C5 and N7 (5–25)
positions was synthesized and fully characterized at the four adenosine receptor (AR)
subtypes. In general, compounds with a free amino group at the 7 position showed
good affinity at the rA2AAR, while the introduction of a phenylcarbamoyl moiety at
the N7 position slightly increased the affinity at the hA3AR with respect to the
unsubstituted derivatives. At the hA2BAR, the most potent derivative contains a free
amino group at the 7 position and could represent a starting point for searching new
non-xanthine hA2BAR antagonists. Molecular models of the rA2A and hA3 ARs were
constructed by homology to the recently reported crystallographic structure of the
hA2AAR. Then, the hA2AAR X-ray structure was used to propose a structural basis for
the activity and selectivity of the analogues and to direct the synthetic design strategy
to provide access to solvent-exposed regions. Thus, we have identified a point of
substitution for the attachment of solubilizing groups to enhance both aqueous
solubility and physicochemical properties, maintaining potent interactions with the
A2AAR and, in some cases, receptor subtype selectivity. Also the new analogues were
docked in the crystallographic structure of the hA2AAR and in a homology model of
the hA3AR, and the per residue electrostatic and hydrophobic contributions to the
binding were assessed and stabilizing factors were proposed.
References
[1]Pastorin, G., et al. Bioorg. Med. Chem. 2010, 18, 2524-2536.
[2]Federico, S. et al. J. Med.Chem. 2011, 54, 877-889.
15
Pyrrole-based selective COX-2 inhibiting nitric oxide donors:
synthesis, in vitro and in vivo evaluation
C. Battilocchio,a G. C. Porretta,a G. Poce,a S. Alfonso,a V. Calderone,b L. Sautebin,c
C. Ghelardini,d A. Giordani,e M. Anzini,f M. Biava a
a
Dipartimento di Chimica e Tecnologie del Farmaco, Università di Roma
“Sapienza”, P.le A. Moro, 5, 00185 Roma; bDipartimento di Psichiatria,
Neurobiologia, Farmacologia e Biotecnologie, Università di Pisa, Via Bonanno 6, I56126 Pisa; cDipartimento di Farmacologia Sperimentale, Università di Napoli
“Federico II”, via D. Montesano 49, I-80131 Napoli; dDipartimento di
Farmacologia, Università di Firenze, viale G. Pieraccini 6, I-50139 Firenze;
e
Rottapharm S.p.A., via Valosa di Sopra 7, I-20052 Monza; fDipartimento Farmaco
Chimico Tecnologico, Università degli Studi di Siena, Via Alcide de Gasperi 2, I53100 Siena
[email protected]
1. Introduction
In the past we synthesized several classes of diarylpyrrole-based selective
Cyclooxigenase-2 (COX-2) inhibitors, endowed with unique analgesic and antiinflammatory profiles.1 Considering that the development of COX-inhibiting nitric
oxide donors (CINODs) has been driven by the pursuit for novel analgesic/antiinflammatory agents devoid of adverse side effects,2 we planned the synthesis of
novel COX-2 selective CINODs, in which the nitric oxide (NO) release aims at
cutting down the side effects displayed by COX-2-like structures. All compounds
were evaluated to assess their inhibition towards both COX-1 and COX-2 through a
cell-based assay, the NO-dependent vasorelaxing effect and their in vivo analgesic and
anti-inflammatory profiles.3
All compounds were characterized by activities ranging from micromolar to
nanomolar values against COX-2 isoenzyme. A slow-kinetic NO-dependent
vasorelaxing effect was shown. The in vivo tests displayed an antinociceptic effect of
compounds comparable to that shown by the reference drug (celecoxib). The antiinflammatory effect of these molecules was less outstanding than the analgesic one,
probably because the two profiles are prompted by different driving forces. Further
structural developments of these scaffolds are ongoing.3
3. References
[1] Biava, M. et al J. Med. Chem. 2010, 53, 723–33.
[2] Fiorucci S. Science direct, Digestive and Liver Disease, 2007 , 39, 1043-1051
[3] Biava, M. et al J. Med. Chem. submitted for publication.
16
Exploration of multivalency in 5-HT3 receptor
M. Paolino
Dipartimento Farmaco-Chimico-Tecnologico, Università di Siena
[email protected]
1. Introduction
The serotonin 5-HT3 receptor (5-HT3R) belongs to the family of the ligand-gated
ion channels (LGICs) and shows a pentameric structure similar to that of nicotinic
acetylcholine (nACh), glycine, and type A γ-aminobutyric acid (GABAA) receptors.
Our long-lasting interest in the development of 5-HT3R ligands started from the study
of arylpiperazine derivatives related to quipazine and has produced a considerable
amount of structure-activity relationship data for the interaction of this class of
ligands with their receptor.1 Recently, the exploration of multivalency in this receptor
represents an interesting development with significant results in our research.
We show the results of a study focused
on the characterization of the interaction
of the 5-HT3 receptors with bivalent
ligands assembled by (a) an optimized
arylpiperazine 5-HT3 receptor ligand
(playing the role of an anchor in
interacting with the main binding site of the 5-HT3 receptor) (b) a spacer with
different lengths and (c) a probe showing different stereo electronic and functional
features. In particular, two types of probes have been used in the exploration of
multivalency: the ureido acetic acid moiety as a chemofunctional probe of
heterobivalent ligands and the same arylpiperazine anchor as a biofunctional probe
present in the corresponding homobivalent ligands. The conjugation with an ureido
acetic acid moiety provides binding capabilities toward a synthetic receptor localized
on the surface of urea adamantyl-functionalized poly(propylene imine) dendrimers to
assemble supramolecular complexes based on the dendrimeric host and a bioactive
guest by specific non-covalent interactions.2 Moreover, the high affinity shown by
homobivalent ligands suggests that bivalency is a promising approach in 5-HT3
receptor modulation. Since bivalency can be considered the first step of multivalency,
the results obtained provide the rational basis for applying the concepts of
multivalency to the study of 5-HT3 receptor function.
3. References
[1] Cappelli, A.; Butini, S.; Brizzi, A.; Gemma, S.; Valenti, S.; Giuliani, G.; Anzini, M.; Mennuni, L.;
Campiani, G.; Brizzi, V.; Vomero, S. Curr. Top. Med. Chem., 2010, 10, 504-526
[2] Galeazzi, S.; Hermans, T. M.; Paolino, M.; Anzini, M.; Mennuni, L.; Giordani, A.; Caselli, G.;
Makovec, F.; Meijer, E. W.; Vomero, S.; Cappelli, A. Biomacromolecules, 2010, 11, 182-186
17
Anthranilic acid based CCK1 receptor antagonists
M. V. Pavan, L. Lassiani, A. Varnavas
Dipartimento di Scienze Chimiche e Farmaceutiche, Università degli Studi di Trieste,
P.le Europa 1, 34127 Trieste
[email protected]
Cholecystokinin (CCK) is a brain-gut peptide discovered in the small intestine that,
together with secretin and gastrin, constitutes the classical gut hormone triad. CCK is
involved in a bunch of physiological processes, both peripheral (gallbladder
contraction, intestinal motility, pancreatic enzyme secretion and growth, satiety
signalling, inhibition of gastric acid secretion) and central (nociception, anxiety
emotional and cognitive processes).
The biological effects of CCK are mediated by two specific G protein coupled
receptors subtypes, termed CCK1 and CCK2 [1]. Available data suggest that CCK1
receptor antagonists might be therapeutically useful in GERD, IBS, functional
dyspepsia and acute pancreatitis.
Recently, we have reported a new class of CCK1 receptor antagonists based on the
anthranilic scaffold [2]. The II generation lead compound is characterized by the
presence of the 2-indolyl moiety, reflecting thus the Trp residue of CCK4, and the
HPhe residue mimicking the C-terminal amino acid of the endogenous ligand.
HN
O
NH
O
N
H
OH
O
VL- 0797
Starting from this lead compound we have investigated the role of the anthranilic
and aminoacidic moiety and, in order to verify whether the C-terminal free carboxylic
acid is essential for the activity and selectivity or it plays a pharmacokinetic role only,
have studied the effects of its substitution on VL-0797 affinity.
The results of these studies, together with those of site directed mutagenesis of human
CCK-1R and VL-0797 docking in pharmacophore CCK-1R model [3], will be
discussed during the congress.
References
[1] Herranz, R. Medicinal research Review, 2003, 23, 559-605.
[2] Varnavas, A.; Lassiani, L.; Valenta, V.; Berti, F.; Tontini, A.; Mennuni, L.; Makovec, F.
European Journal of Medicinal Chemistry 2004, 39, 85-97.
[3] Archer-Lahlou E.; Tikhonova I.; Escrieut C.; Dufresne M.; Seva C.; Pradayorl L.; Moroder L.;
Maigret B.; Fourmy D. Journal of Medicinal Chemistry 2005, 48, 180-191.
18
Overview on σ 1 receptors: a 3D homology model
to solve a part of the enigma
E. Laurini,a V. Dal Col,b D. Zampieri,a M. G. Mamolo,a P. Posocco,b S. Pricl,b L. Vioa
a
Dipartimento di Scienze Chimiche e Farmaceutiche, Università degli Studi di
Trieste, piazzale Europa 1, 34127, Trieste; bDipartimento di Ingegneria Industriale e
dell'Informazione, Università degli Studi di Trieste, Via A. Valerio, 10 34127 Trieste
[email protected]
1. Introduction
Sigma binding site were first defined and classified as being an opioid receptor
subtype. Later on, further investigations demonstrated that σ receptors were distinct
from opioid and phencyclidine receptors and at least two distinct σ receptor subtypes,
designated σ1 and σ2 have been pharmacologically characterized.1 Their large
distribution in the CNS seems to explain the modulatory role in cellular, biochemical
and neurotransmission systems.2 For this reason σ1 ligands may be involved in the
treatment of diseases such as depression, anxiety, cognitive impairments, psychosis,
analgesia and against the effects of drugs of abuse.3 To date, the information available
from the 3D pharmacophore models were the only way to design a new σ1 ligands.4
The lack of σ1 receptor’s crystallographic structure has
led to create a 3D model using homology modeling to find
out more details about his physiology. The resulting
structure has been optimized by molecular simulation
(Fig.1) and used for generation of protein binding pocket.
Subsequently the amino acids, probably involved in
binding pocket region, have been analyzed with alaninescanning technique in order to understand their
fundamental role in the binding with the σ ligands.
Fig.1
3. References
[1] Bowen, W.D.; Pharm. Acta Helv. 2000, 74, 211-218
[2] Bermack JE, Debonnel G; J Pharmacol Sci, 2005, 97, 317-336
[3] Ishikawa M, Hashimoto K J Receptor Ligand Channel Res 2010, 3,25-36
[4] Zampieri D, Mamolo MG, Laurini E, Florio C, Zanette C, Fermeglia M, Posocco P, Pricl S, Vio L J
Med Chem 2009, 52, 5380-5393
19
Selective GluK1 receptor ligands structurally based upon (S)CPW399: synthesis, pharmacological, and
biostructural characterization
S. Butini,a,b S. Gemma,a,b S. Maramai,a,b S. Valenti,a,b R. Venskutonytė,c,d J.S.
Kastrup,d E. Novellino,b,e D.S. Pickering,c G. Campiania,b
a
Dip. Farmaco Chimico Tecnologico and bEuropean Research Centre for Drug
Discovery and Development, Università di Siena, Italy; cDep. of Pharmacology and
Pharmacotherapy and dDep. of Medicinal Chemistry, University of Copenhagen,
Denmark; eDip. di Chimica Farmaceutica e Tossicologica, Università di Napoli
Federico II, Italy.
[email protected]
1. Introduction
Glutamate (Glu) is the main excitatory neurotransmitter in the central nervous
system (CNS) and plays an important role in neuronal plasticity, neurotoxicity, and in
degenerative disorders1. Glu actions are mediated by metabotropic and ionotropic
receptors (iGluRs). Kainate receptors (KARs) belong to the family of iGluR which
also consists of the AMPA and NMDA receptor subfamilies. The GluK1 subunit is
predominantly associated to pain pathways, as shown in different areas of the CNS
and its involvement in pain signaling was demonstrated through the use of GluK1
competitive antagonists2. One of the aims of this study was the investigation of
bicyclic scaffold of 1 and 23 in order to identify potent and selective GluK1 receptors
agonists/antagonists.
Pharmacological and functional characterization of the
analogues of 3a,b4 (3c-e and 4a-d) highlighted that all
compounds were selective for Kainate-GluK1 receptors
(GluK3/GluK1). Depending upon the nature of the R
substituent, they behaved as functional agonist or
antagonists. The crystal structure of the GluK1 ligand
binding domain in complex with the novel selective
antagonist 4a was also resolved and revealed flexibility in
the binding mode of 4a.
3. References
[1]
[2]
[3]
[4]
Bleakman, D. et al, Semin Cell Dev Biol, 2006, 17, 592-604.
Dolman, N.P. et al, J Med Chem, 2007, 50, 1558-1570.
Campiani, G. et al, J Med Chem, 2001, 44, 4501-4504.
Butini, S. et al, J Med Chem, 2008, 51, 6614-6618.
20
CB2 Selective Agonists as Neuroprotective Agents
S. Pasquini,a C. Mugnaini,a M. Frosini,b F. Corellia
a
Dipartimento Farmaco Chimico Tecnologico, Università degli Studi di Siena, via
Aldo Moro 2,53100 Siena; bDipartimento di Neuroscienze, Università degli Studi di
Siena, via Aldo Moro, 53100 Siena
[email protected]
1. Introduction
The progress achieved over the past 15 years in understanding the action
mechanisms of cannabinoids has revived the therapeutic interest in these substances.
Selective cannabinoid type 2 receptor (CB2) ligands may be used to treat pain,
inflammation, osteoporosis, CB2-expressing malignant gliomas, tumors of immune
origin, and immunological disorders such as multiple sclerosis and Alzheimer’s
disease (AD), with no overt psychoactivity. The neuroprotective properties of
exogenous as well as endogenous cannabinoids have been known for years and the
underlying molecular mechanisms have been recently unveiled. In particular,
antioxidative, antiglutamatergic and antiinflammatory effects are now recognized as
derived from cannabinoid action and are known to be of common interest for many
neurodegenerative processes. Thus, these characteristics make cannabinoids attractive
candidates for the development of novel therapeutic strategies.1
Within a research program aimed at characterizing novel cannabinoid ligands, we
have described a family of quinolone-3-carboxamides bearing diverse substituents at
different positions of the aromatic ring as new CB2 ligands.2, 3 The tested compounds
exhibited high CB2 affinity and selectivity over the CB1 receptor subtype.
Neuroprotection studies on a ischemia/reperfusion model were carried out on
compound COR167 which had shown an agonist profile and analgesic activity in
vivo.
3. References
[1] Romero, J. et al. CNS&Neurological Disorders – Drug Targets 2009, 8, 440-450.
[2] Pasquini, S. et al. J. Med. Chem. 2008, 51, 5075-5084.
[3] Pasquini S. et al. J. Med. Chem. 2010, 53, 5915-5928.
21
Covalent modification of beta2-microglobulin induced by reactive
carbonyl species: high resolution mass spectrometric studies
L. Bertoletti,a L. Regazzoni,b A. Altomare,b R. Colombo,a G. Vistoli,b E. De Lorenzi,a
G. Aldini,b
a
Dipartimento di Scienze del Farmaco, Università degli Studi di Pavia;
b
Dipartimento di Scienze Farmaceutiche Pietro Pratesi, Università degli Studi di
Milano
[email protected]
1. Introduction
Beta2-microglobulin (β2m) is responsible for dialysis related amyloidosis (DRA), a
destructive osteoarticular disease that affects patients undergoing long-term
hemodialysis. Physicochemical and immunochemical analyses showed that one of the
constituents of DRA aggregates is an acidic form of β2m, which is generated through
covalent modifications induced by reactive carbonyl species (RCS) and glucose. The
resulting advanced lipoxidation and glycoxidation end products (ALEs and AGEs)
have been detected in long-lived β2m amyloid deposits mainly by
immunoistochemistry.1
Based on these evidences we investigated, by high resolution MS, the β2m nonenzymatic glycation and carbonylation, as induced by the main RCS.2
The identification of post-translational modifications was carried out by direct
infusion analysis of the intact protein (top-down proteomics) and NanoLC-ESIMS/MS analysis of the chymotryptic digest (bottom-up proteomics). After 24 hours
(the main half-life of β2m in DRA patients), under conditions similar to physiological,
we are able to identify two covalent adducts for dialdehydes (glyoxal and
methylglyoxal) occurring on Arg4. Neither cross-links/polymerization events nor
adducts for α,β-unsatured compounds were observed. These results give a deeper
insight on the covalently modified β2m and RCS as potential drug targets in DRA and
inflammation.
3. References
[1] Miyata T., Nephrology Dialysis Transplantation, 1996, 15, 25-28.
[2] Aldini G., Medicinal Research Reviews, 2007, 27, 817-868.
22
New potential antimalarials based on the
4-aminoquinoline scaffold
C. Camodeca, S. Gemma, S. Butini, G. Campiani
Dipartimento Farmaco Chimico Tecnologico, Università di Siena, via Aldo Moro 2,
53100 Siena, Italy and European Research Centre for Drug Discovery and
Development (NatSynDrugs)
[email protected]
1. Introduction
Malaria remains one of the world’s most tragic infectious diseases, afflicting
hundreds of millions and killing millions of people worldwide annually, especially in
developing countries. The efficacy of chloroquine (CQ), drug of choice for decades in
malaria treatment, has been compromised by the selection of CQ-resistant (CQ-R)
Plasmodium falciparum strains; so nowadays there is an urgent need to find new
classes of drugs with a low potential of developing resistance.1
The quinoline-based antimalarials are known to interfere with the detoxification of
free Fe(III)-heme which is released inside the acidic food vacuole. This leads to
intraparasitic accumulation of free heme that is highly toxic for the parasite.2
Consequently heme metabolism represents a valuable therapeutic target, since its
biochemistry in Plasmodium is completely different from the one of the host,
reducing the possibility to induce resistance under drug pressure.
2. Results
As a part of our continuous effort in the field of antiparasitic drug discovery,3-5 we
developed a new class of compounds based on the 4-aminoquinoline system. The
developed compounds showed activity against both CQ-R and CQ-sensitive strains.
3. References
[1]
[2]
[3]
[4]
[5]
World Health Organization (WHO 2008 report).
Egan, T. J. et al., Biochem. J., 2002, 365, 343-347.
Gemma, S. et al.; J. Med. Chem., 2007, 50, 595-598.
Gemma, S. et al.; J. Med. Chem., 2008, 51, 1278-1294.
Gemma, S. et al.; J. Med. Chem., 2009, 52,502-513.
23
Capillary Electrophoresis: an analytical strategy to evaluate the
effects of small molecules on the toxic aggregates of
Alzheimer’s disease
R. Colombo,a S. Butini,b G. Campiani,b A. Carotti,c M. Catto,c L. Verga,d E. De
Lorenzia
a
Dipartimento di Scienze del Farmaco, Università di Pavia; bDipartimento di
Chimica Farmaceutica e Applicata, Università di Siena; cDipipartimento FarmacoChimico, Università di Bari; dLaboratorio di Biotecnologie, Policlinico S. Matteo,
Pavia.
[email protected]
1. Introduction
Alzheimer’s disease (AD) is a form of amyloidosis, a disorder where the
conformational changes of a protein ultimately lead to the deposition of toxic
insoluble fibrils. In AD a small peptide (Ab42) is the main responsible for the
formation of amyloid fibrils in the brain. Fibrillogenesis is a dynamic process, where
the formation of different intermediate species at equilibrium includes non-covalently
associated soluble oligomers which are more neurotoxic than fibrils. Capillary
electrophoresis (CE) can be an excellent complementary tool to the variety of
spectroscopic techniques that are used to characterise this process, as it enables the
separation and the detection of oligomers during their formation and just before fibril
deposition.1
The CE method proved to be effective in monitoring over time the activity that coincubated small molecules with different structure have on the kinetics of Ab42
oligomer formation. An anthraquinone derivative with anticancer activity,2,3 a bistacrine derivative that inhibits human acetylcholinesterase and carnosine, a
physiological dipeptide with antioxidant properties have been tested. It was possible
to verify if these compounds inhibit, stabilise or slow down the formation of toxic
oligomers in a concentration-dependent manner. A correlation between this activity
and the presence of fibrils has been investigated by transmission electron microscopy.
Consistently with the results obtained their potential in the reduction of Ab42
neurotoxicity has been assessed.
3. References
[1] Sabella S. et al. Electrophoresis 2004, 25, 3186.
[2] Colombo R. et al. Electrophoresis 2009, 30, 1418.
[3] Colombo R. et al. Patent n. MI2008A000366.
24
Concentrations of HPLC-samples:
Current methods and future trends
J. Grebe,a F. de Lucab
a
Martin Christ Gefriertrocknungsanlagen GmbH, An der Unteren Söse 50, D-37520
Osterode/Germany; bDelchimica Scientific Glassware, V.le Gramsci, 18, I-80122
Napoli/Italy
[email protected]
1. Introduction
Very often concentration step turns out more and more as the bottleneck in the
whole synthesis and purification process. Target is the removal of low- and high
boiling organic solvents like ACN, Ethanol/Methanol, TFA, DCM, DMSO, NMP.
Current methods are Rotary Evaporators for large, single samples and multiple
sample systems like N2-Strippers, Vacuum-Vortexer and Rotational Vacuum
Concentrators (RVC).
Main advantages of RVC-systems are flexible use of existing sample vials,
deposition of sample pellets at the vial bottom, safe and controlled working
parameters (vacuum, energy input), automated cycles, minimized losses of sample
activities. Special systems with improved energy input by use of infrared light reduce
concentration times, see Figure1: Evaporation times electrical heating vs. IR-heating
and Figure 2: Typical RVC-System for fast solvent evaporation.
Future trends are going to a combination of primary evaporation in the liquid phasis
(to get rid of solvents) with a secondary sublimation step (freeze drying). This often
leads to a better shape of the substance (fluffy, porous, better to resolve) and increases
the total throughput of the concentrating/drying step.
Figure 1
Figure 2
25
Synthesis of Novel Endoperoxide Derivatives as Potential
Antimalarial Agents
S.Kunjir, S. Gemma, S. Butini, G. Campiani
European Research Centre for Drug Discovery and Development (NatSyn Drugs)
and Dipartimento Farmaco Chimico Tecnologico, Università di Siena, via Aldo Moro
2, Siena, Italy.
[email protected]
Malaria is one of the most ancient and unresolved afflictions of humankind.
According to the World Health Organization, malaria continues to affect upwards of
half a billion people worldwide and kills over 800,000 people each year1. Artemisinin,
a natural endoperoxide, and its semisynthetic derivatives represent promising
alternatives to quinoline antimalarials. However, artemisinin is highly expensive and
its synthesis is prohibitive for bulk production purposes. On the other hand, plakortin
is a natural endoperoxide exhibiting interesting antimalarial activity and characterized
by a simpler molecular scaffold2. In surge of novel antimalarials based on the
structure of plakortin we synthesized a novel series of simplified derivatives of
plakortin bearig various alkyl substituents at C-6 and in which the C-3 and C-4
substituents of the natural compound was fused together forming a bicyclic structure.
Compounds bearing methylcyclohexyl, methylcyclopentyl, and methyladamantyl
substituents at C-6 position showed promising activity against D10 and W2 strains.
References
[1] World Health Organization (WHO 2010 report)
[2] Fattorusso, C. et al: J. Med . Chem. 2006, 49, 7088-7094.
26
Latest innovations in vacuum technology and control1
C. Ruth
Area Sales Manager, VACUUBRAND GMBH + CO KG, Wertheim / Germany
[email protected]
1. Introduction
VACUUBRAND has 50 years of experience in the field of vacuum technology.
We offer a unique range of innovative, customer-oriented vacuum solutions for
laboratory applications.
Vacuum requirements spread from basic demand, like for filtration, over the entire
rough vacuum range to distillation processes, and finally into the fine vacuum range
for applications like lyophilisation. Different vacuum pump technologies are applied
to fulfil these needs. For rough vacuum applications like filtration, aspiration, vacuum
drying ovens, rotary evaporators, vacuum concentrators, and many more, the
diaphragm pump technology, with fully chemical resistant design, is the state-of-theart solution. Additional features like solvent recovery or vacuum control are available
to optimise process conditions. The rpm-controlled VARIO- line provides fully
automatic process control with maximum precision. For applications like
lyophilisation, molecular distillation, or Schlenk-line drying VACUUBRAND offers
a range of rotary vane pumps with fine vacuum performance, also available with cold
trap systems for pump-protection in chemistry use. The Chemistry Hybrid Pump RC 6
combines performance of a rotary vane unit with the capability of a chemistry
diaphragm unit in handling chemical vapours. The benefits are high performance in
the fine vacuum range, good capability of handling chemical vapours, and
outstandingly extended oil lifetime.
VACUUBRAND offers a wide range of solutions for laboratory use, designed to
improve application performance, minimise maintenance interruptions, optimise
productivity, and lead to long-term economical benefits.
3. References
[1] VACUUBRAND GMBH + CO KG
27
Mechanistic investigations on a ligand-exchange system operating
with a chiral mobile phase
B. Natalini, R. Sardella, F. Ianni, M. E. García Rubiño, A. Carotti, A. Macchiarulo,
R.Pellicciari
Dipartimento di Chimica e Tecnologia del Farmaco, Università di Perugia, Via del
Liceo 1, 0612-Perugia
[email protected]
1. Introduction
Owing to the exceptional sophistication of Chiral Ligand-Exchange
Chromatography systems operating with Chiral Mobile Phase (CMP) additives, only
few mechanistic investigations have been presented so far. Nevertheless, dedicated
computational protocols can furnish valuable information on the factors mainly
affecting the enantiorecognition event.
The accordance observed between QM calculations and crystallographic data led us to
use optimized ternary complexes carrying the chiral additive O-benzyl-(S)-serine [(S)OBS], to build up a computational model enabling to explain the enantiomer elution
order of amino acids with this enantioresolving agent1. Through the generation of a
“decision tree”, the descriptor “delta-Energy of solvation (delta-Esol)” was found to
correctly classify almost all the selected compounds according to the relative
chromatographic behaviour. The difference of the solvation energy between the
complexes of diastereomeric couples was identified of utmost importance for the
enantiorecognition event as a whole. With the same CMP system, the effect of the
copper(II) anion type was evaluated through QM calculations, by rationally changing
the following experimental chromatographic conditions: temperature, absolute
configuration of the chiral selector, and salt concentration. Interestingly, the
behavioral differences which turned out for the thermodynamic retention (k) and
separation (α) factor values seem to be correlated with the ESP (electrostatic
potential) balance values obtained by the ab initio approach.
3. References
[1] B. Natalini, N. Giacchè, R. Sardella, F. Ianni, A. Macchiarulo, R. Pellicciari, J. Chromatogr. A,
1217 (2010) 7523–7527.
28
Stereochemistry of 2-(6-fluorophenanthren-1-yl)propionic acid
unveiled by enantioselective HPLC, ECD spectroscopy, and TD-DFT
calculations
D. Tedesco,a R. Zanasi,b R. Ruzziconi,c M. Pistolozzi,a A. M. Di Pietra,a C. Bertuccia
a
Dipartimento di Scienze Farmaceutiche, Università di Bologna, Bologna, Italy;
b
Dipartimento di Chimica e Biologia, Università di Salerno, Fisciano (SA), Italy;
c
Dipartimento di Chimica, Università di Perugia, Perugia, Italy
[email protected]
1. Introduction
Owing to its peculiarities, fluorine has drawn an increasing
attention in the design of new drugs. Among them, fluorinated
profens have received particular consideration in reference to the
biological activity as successful non-steroidal anti-inflammatory
drugs (NSAIDs). In the present communication we report the
development of an enantioselective HPLC method for the resolution
of racemic 2-(6-fluorophenanthren-1-yl)propionic acid (1),1 and the
determination of the absolute configuration (AC) of its enantiomers through
electronic circular dichroism (ECD) and time-dependent density functional theory
(TD-DFT) computations.
The column (S,S)-Whelk-O®1 was efficient for the enantioresolution of profen 1.
The single enantiomeric fractions were collected and analyzed for determining their
enantiomeric excess and ECD spectra. The AC of the enantiomers of 1 was
determined by means of ECD theoretical calculations on (S)-1.2 Molecular mechanics
conformational analysis and DFT geometry optimization at the B3LYP/TZ2P level
isolated the 4 most populated conformers (a, 53.04%; b, 25.70%; c, 19.81%; d,
1.45%). TD-DFT calculations were performed at the B3LYP/TZ2P level and the
theoretical ECD spectrum was determined. The ECD spectrum of the second-eluted
enantiomer was found to be in excellent agreement with theoretical calculations on
(S)-1; therefore, the AC of the first- and second-eluted enantiomers were assessed as
(2R)-1 and (2S)-1, respectively.
3. References
[1] Ricci, G., and Ruzziconi, R., J. Org. Chem. 2005, 70, 611-623.
[2] Autschbach, J., Chirality 2009, 21, E116-E152.
29
CNT-based eletrochemiluminescent biosensor for palytoxin detection
V. A. Zamolo,a G. Valenti,b A. Tubaro,c F. Paolucci,b M. Prato a
a
Department of Pharmaceutical Sciences, University of Trieste, 34127 Trieste;
b
Department of Chemistry, University of Bologna, 40126 Bologna; cDepartment of
Materials and Natural Resources, University of Trieste, 34127 Trieste
[email protected]
Ostreopsis are marine Dinoflagellates involved in the production of palytoxins and
palytoxin-like compounds. Palytoxin (PLTX), isolated for the first time from the
coelenterate Palythoa toxica, is known as one of the most potent marine toxin: human
illness and fatalities have been reported after ingestion of PLTX-contaminated
tropical and subtropical fish and crabs.[1] Recently, Ostreopsis blooms have been
regularly registered also in some areas of the Mediterranean Sea, provoking in 2005
more than 200 cases of respiratory syndrome (20 % requiring hospitalization) in
patients exposed to marine aerosol by recreational or working activities on the beach
areas of Genoa, Italy.1 Furthermore, these toxins have also been detected in seafood
(mussels, clams and sea urchins) collected in Mediterranean Sea.2 In 2006 Ostreopsis
flowerings have been observed also in other Italian regions, comprising Friuli
Venezia Giulia, and in particular 10 km far from Trieste, in Canovella beach.
Due to high toxicological potential of PTX and its analogs, there is an urgent need to
realize a cheap and very sensitive system for their detection and quantification both in
seawater and in seafood.
To this aim, we have focused on the realization of a sensor device for PLTX detection
that takes advantage of the specificity towards analyte provided by selective
antibodies anti-PLTX, of the good chemical properties of carbon nanotubes (CNTs),3
suitable for interaction with biomolecules, and of the excellent sensitivity given by
electrochemiluminescence (ECL).4
The biosensor is assembled according to the ELISA5 sandwich principle, where the
toxin is caught by the capture antibody and recognized by a secondary antibody
labelled with a molecular marker.
First CNTs are functionalized covalently with monoclonal antibodies anti-PLTX,
acting as the capture moiety. An ITO electrode, necessary to start the
electrochemiluminescence process, is electrografted with polyacrylate, in order to
bind CNTs. The toxin solution is added, followed by a secondary polyclonal antibody
labeled with a ruthenium complex, which produces the electrochemical signal when
the proper potential is applied to the electrode.
Since the intensity of light emitted scales with the concentration of the label, and
therefore, the analyte, it is possible to measure quantitatively the concentration of the
antigene and the detection of PLTX is achieved.
References:
[1] Munday R., Occurrence and toxicology of palytoxin In: Botana L.M. (Ed), Seafood and Freshwater
Toxins, Marcel Dekker, New York, pp. 631-713, 2008
[2] Aligizaki K., Panagiota K., Nikolaidis G., Panou A., Toxicon 51, 418-427, 2008
[3] Iijima S., Nature 354, 56-58, 1991
[4] Richter M. M., Chem. Rev., 104 (6), 3003-3036, 2004
[5] Engvall E., Perlman P., "Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of
immunoglobulin G" Immunochemistry 8, 871–4, 1971
30
Non-Natural Macrocyclic Inhibitors of Histone Deacetylases
G. Battistuzzi,a G. Giannini,a W. Cabri,a L. Auzzas,b S. Hanessianc
a
Sigma-Tau R&D, Via Pontina Km 30.400, 00040 Pomezia, Roma, Italy; bIstituto di
Chimica Biomolecolare, CNR, 07100 Sassari, Italy; cDepartment of Chemistry,
Universite de Montreal, H3C3J7 Canada
[email protected]
1. Introduction
Nonpeptidic chiral macrocycles were designed on the basis of an analogue of
HDAC inhibitor suberoylanilide hydroxamic acid (SAHA, vorinostat) and evaluated
against 11 HDAC isoforms. The identification of critical amino acid residues highly
conserved in the cap region of HDACs guided the design of the suberoyl-based
macrocycles, which were expected to bear a maximum common substructure required
to target the whole HDAC panel.
Bearing in mind that recent papers show how HDAC profiling data need to be
evaluated with criticism,1 we found2 that macrocycles with two simple aromatic rings
in the cap region (R/S-9 for example) displayed a nanomolar activity profile against
all isoforms. As expected for hydroxamic acid-containing HDAC inhibitors, a
preferential activity against HDAC6 in the nanomolar range was observed for all
macrocycles. In particular, a relevant selectivity toward this isoform was detected
within the group of diamide-based macrocycles (R/S-36 for esample, 10-500-fold
over the other HDACs). An excellent cytotoxic activity was also found for selected
macrocycles against lung and colon cancer cell lines.2
3. References
[1] Bantscheff, M. et al Nature Biotechnology, Vol. adv. online publication, (January 2011)
[2] Auzzas, L. et al J. Med. Chem. 2010, 53, 8387–8399
31
Ligand-receptor interactions from NMR spectroscopy.
Application to cytotoxic agents binding to DNA
A. Carotenuto,a D. Brancaccio,a I. Gomez-Monterrey,a P. Campiglia,b A. Bertamino,a
P. Grieco,a E. Novellinoa
a
Department of Pharmaceutical Chemistry and Toxicology, University of Napoli, Via
Domenico Montesano 49, I-80131 Napoli, Italy; bDepartment of Pharmaceutical
Science, University of Salerno, I-84084 Fisciano, Salerno, Italy.
[email protected]
1. Introduction
NMR-based screening has emerged as a potent technique for the identification of
small molecules that interact with a macromolecule target. NMR-based screening can
be performed by monitoring either the macromolecule target signals or the ligand
signals. Unlike macromolecule-observed, ligand-observed screening is not limited by
the size of the macromolecule and does not require isotope-labeled samples. Several
methods based on the ligand observation have been proposed in the literature, among
these WaterLOGSY (water-ligand observed via gradient spectroscopy) [1], and STDNMR (saturation transfer difference) experiments [2]. Here, we apply these NMR
techniques for the evaluation of the DNA interactions of a new series of thiophennaphthoquinones (TNQ) with interesting cytotoxic activity.
Two active and one inactive TNQ derivatives were tested to see if they interact with
DNA by WaterLOGSY and STD-NMR techniques. The spectra acquired in the
presence of poly(dG-dC)•poly(dG-dC) copolymer as DNA target, demonstrated that
active compounds/DNA interactions did occur while inactive compound does not
interact with DNA. Furthermore, experimental (differential frequency STD) and
computational methods allowed to clarify the binding modes of these compounds to
DNA.
3. References
[1] DALVIT, C., et al., 2001. WaterLOGSY as a method for primary NMR screening: practical aspects
and range of applicability. J. Biomol. NMR, 21, 349-359.
[2] MAYER, M., et al., 1999. Characterization of ligand binding by Saturation Transfer Difference
NMR spectroscopy. Angew. Chem. Int. Ed., 38, 1784-1788.
32
Synthesis and biological evaluation of a new thiazole derivative,
inducing histone hypoacetylation and apoptosis
in human leukemia cells
S. Carradori
Dipartimento di Chimica e Tecnologie del Farmaco, Università di Roma La Sapienza,
P.le A. Moro 5, 00185, Rome, Italy.
[email protected]
1. Introduction
We previously synthesized and demonstrated that some thiazole derivatives, and in
particular
3-methylcyclopentilidene-[4-(4’-chlorophenyl)thiazol-2-yl]hydrazone
(CPTH6), reduced Gcn5-mediated histone acetylation and acetyltransferase (HAT)
activity in a model of Saccharomyces cerevisiae yeast.1
Concentration- and time- dependent inhibition of cell proliferation, paralleled by
accumulation of cells in the G0/G1 phase and depletion from the S/G2M phases was
observed. Apoptosis was induced through both intrinsic and extrinsic pathways, as
evidenced by cleavage of caspase 8, caspase 9, caspase 3, and PARP. Also the role of
mitochondrial pathway on CPTH6-induced apoptosis was demonstrated, being a
decrease of mitochondrial membrane potential and the release of cytochrome c, from
mitochondria to cytosol, induced by CPTH6. Solid tumor cell lines from several
origins were demonstrated to be differently sensitive to CPTH6 treatment in terms of
cell proliferation, and a clear correlation between the inhibitory efficacy on H3/H4
histones acetylation and cytotoxicity was found. Differentiating effect on leukemia
and neuroblastoma cell lines was also induced by CPTH6. These results make CPTH6
a suitable tool for discovery of molecular targets of HATs and, potentially, for the
development of new anticancer therapies, which warrants further investigations.2
3. References
[1] Carradori, S. et al. A novel histone acetyltransferase inhibitor modulating Gcn5 network:
cyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl)hydrazone. J. Med. Chem. 2009, 52, 530-536.
[2] Del Bufalo, D. et al. CPTH6, a thiazole-derivative, induces histone hypoacetylation and apoptosis
in human leukemia cells. Leukemia, submitted.
33
Covalent antibody-carbon nanotubes conjugates for
improved anticancer therapy
C. Fabbro,a C. Guo,b E. Venturelli,c T. Da Ros,a K. Kostarelos,b A. Bianco,c M. Pratoa
a
Dipartimento di Scienze Chimiche e Farmaceutiche, Università di Trieste, Trieste,
34127 (Italy); bNanomedicine Laboratory, Centre for Drug Delivery Research, The
School of Pharmacy, University of London, London, WC1N 1AX (UK); cCNRS,
Institut de Biologie Moléculaire et Cellulaire, Laboratoire d’Immunologie et Chimie
Thérapeutiques, Strasbourg, 67000 (France)
[email protected]
Anticancer therapy, though being often very effective, still suffers of some severe
drawbacks, which render vital and urgent the need for further research. One of the
main limitations of almost every cancer treatment so far is the lack of selectivity for
tumor. In fact cytotoxic drugs (or other therapies) do not exert their action solely on
cancer cells, but also on healthy organs, resulting in severe side effects for patients,
therefore limiting their compliance and the maximum administrable dose.
In the present work we studied the possibility to combine the technologies of
carbon nanotubes (CNTs) and antibodies (Ab), with the aim of overcoming these
limitations. One of the main possible applications of CNTs is in fact in drug delivery.
They could be used as carriers, exploiting their capability to host diagnostic or
therapeutic molecules and to enter cells. Moreover, a nanovector could be conceived
to be targeted towards cancer, either only in a non-specific way (Enhanced
Permeability and Retention effect), or with a specific targeting agent attached to the
carrier itself. It is important to underline that the main drawback of carbon nanotubes
for this application is their lack of solubility in physiological solvents. As a
consequence, we need to chemically treat them to improve this aspect, before
proceeding with the conjugation to the Ab. To do so, we decided to use a covalent
approach, that should guarantee a good stability to the final constructs, avoiding an in
vivo dissociation of the Ab due to its possible displacement by other biological
macromolecules.
Antibodies have remarkable selectivity and picomolar potency, and this quality
could render them an outstanding antineoplastic agent, since they can target key
regulators in the development of cancer. Nevertheless, to date, they have not been
used for intracellular targets as they do not readily cross cell membranes. The
conjugation of antibodies to CNTs should enable their use in an intracellular
environment, addressing one of the important limitations of antibody-based cancer
therapy, and paving the way to a variety of unexplored therapeutic possibilities. With
a different approach, Ab can also serve as targeting agents. If we conjugate them to
CNTs, they should specifically direct the system and thus they will enable a targeted
drug delivery.
With the aim of studying these different opportunities we prepared different kinds of
CNTs-Ab constructs. A detailed description of the Ab conjugation to the CNTs,
together with the full characterization of the constructs will be presented.
34
Structure-activity relationships of new anticancer agents based on
naphthalene diimide scaffold
A. Milelli,a A. Minarini,a M.L. Micco,a G. Zuccari,a L. Raffaghello,b C. Stefanelli,c C.
Sissi,d F. Diaz,e C. Melchiorre,a V. Tumiattia
a
Dipartimento di Scienze Farmaceutiche, di bBiochimica Università di Bologna;
c
Laboratorio di Oncologia, Istituto Giannina Gaslini, Genova; dDipartimento di
Scienze Farmaceutiche, Università di Padova; eCentro de Investigaciones Biológicas,
Madrid
[email protected]
1. Introduction
A class of anticancer agents recently developed is represented by the 1,4,5,8Naphthalentetracarboxylic diimide derivatives. Several compounds characterized by
this scaffold have been reported able to exhibit bis-threading intercalating ability, to
enhance the stabilization of DNA triplex, to stabilize or alkylate G-quadruplex DNA
structure. We recently published a paper describing the design and synthesis of new
substituted naphthalene diimides as anticancer agents.1
H
N
OCH3
n
O
O
N
N
O
O
H
N
n
OCH3
1: n = 1 2: n = 2
Compounds 1 and 2 were the most interesting derivatives showing a promising
anticancer biological profile. The aim of the present work is to investigate the role of
the different substituents on the benzyl moieties and for this purpose several
analogues of 1 and 2 were synthesized. Their biological activities will be discussed in
detail.
This research was supported by grants from MIUR, the University of Bologna, and
Polo Scientifico-Didattico di Rimini. We thank the National Cancer Institute for the
anticancer assays.
3. References
[1] Tumiatti V. et al. J. Med. Chem. 2009, 23, 7873-7.
[2] Milelli A. et al. manuscript in preparation.
35
Structure-Activity Relationship Study of New FK228 Analogues as
Antitumor Agents
S. Di Maro,a R.-C. Pong,b J.-T. Hsieh,b J.-M. Ahn,b E. Novellinoa
a
Department of Pharmacological and Toxicological Chemistry, University of Naples
“Federico II”, Naples, Italy; bDepartment of Urology, University of Texas
Southwestern Medical Center at Dallas, Dallas, TX 75390, USA; cDepartment of
Chemistry, University of Texas at Dallas, Richardson, TX 75080, USA;
[email protected]
1. Introduction
Aberrant recruitment and expression of HDACs have been correlated to
tumorigenesis process and provided a rationale for the use of HDAC inhibitors to treat
cancers. Among many compounds under preclinical investigation, a bicyclic
depsipeptide FK228 is considered as the most potent HDAC inhibitor in vitro and in
vivo; however, its synthetic challenges have made in-depth SAR studies difficult,
which would facilitate the understanding of structural requirements for antitumor
activity of the natural product1.
Herein, we report novel FK228 analogues in which the most synthetically challenging
unit, (3S,4E)-3-hydroxy-7- mercapto-heptenoic acid was modified by simple isosteric
substitutions.2 More than 60 compounds were synthesized to explore the roles of LVal, Z-Dhb and D-Val of the original FK228 structure with a variety of side chain
functional groups. To examine anticancer activity, the prepared FK228 analogues
were evaluated on various cancer cells, and new potent FK228 analogues were
identified (IC50 values between 20-50 nM).
3. References
[1] Li, K.; Wu, J.; Xing, W.; Simon, J. A. Total Synthesis of the Antitumor
Depsipeptide FR-901228. J. Am. Chem. Soc. 1996, 118, 7237-7238.
[2] a) Di Maro S, Pong RC, Hsieh JT, Ahn JM. J. Med. Chem. 2008, 51(21), 6639-41. b) Di Maro S,
Ahn JM. Adv. Exp. Med. Biol. 2009, 611, 17-8
36
Ruthenium-Porphyrin Conjugates with Cytotoxic and Phototoxic
Antitumor Activity
C. Spagnul,a T. Gianferrara,a A. Bergamo,b I. Bratsos,c B. Milani,c G. Sava,b,d E.
Alessioc
a
Dipartimento di Scienze Chimiche e Farmaceutiche, P.le Europa 1, Università di
Trieste, bCallerio Foundation Onlus, Via A. Fleming 22-31, 34127 Trieste, Italy,
c
Dipartimento di Scienze Chimiche e Farmaceutiche, Via Giorgieri 1, Università di
Trieste, dDipartimento di Scienze della Vita, Via Giorgieri 5-9-10, Università di
Trieste
[email protected]
1. Introduction
Natural and synthetic water-soluble porphyrins and metalloporphyrins are are
extensively investigated – and in some cases clinically applied – for their potential
biological and biomedical applications, mainly as photosensitizers in the
photodynamic therapy of cancer (PDT). An emerging strategy to obtain water-soluble
porphyrins is that of binding charged metal fragments to the periphery of the
porphyrinic chromophore, mainly at the meso positions. The conjugates are expected
to combine the phototoxicity and the tumor-localization properties of the porphyrin
chromophore with the cytotoxicity of the metal fragment for additive antitumor effect.
We report here the preparation of two different classes of porphyrin-ruthenium
conjugates with potential applications in PDT. Ruthenium was chosen because several
Ru(II) compounds have shown promising anticancer activity. The conjugation with
the porphyrin moiety was accomplished either through peripheral pyridyl rings (e.g.
meso-4'- or meso-3'-tetrapyridylporphyrin, 4'TpyP or 3'TPyP) or through bipyridine
chelating units connected to the chromophore through a flexible hydrophilic spacer
(Figure 1).1
N
N
N
N
N
N
H
H
N
O
O
N
N
H
sp ac
er
N
H
N
H
N
N
N
N
H
N
N
H
H
N
er
sp ac
H
N
O
N
N
H
N
H
N
N
N
H
N
N
r
sp ace
O
O
O
sp a
cer
O
O
N
= Ru(II) fr agment
The most water-soluble conjugates were investigated for in vitro tumour cell
inhibition and also the phototoxic effect was evaluated. All conjugates have IC50
values in the low micromolar range, that decrease of one order of magnitude upon
irradiation of cell cultures with visible light.2 The most active compounds are
phototoxic at low light and drug doses and are promising sensitizers for the PDT of
tumours.
3. References
[1] Gianferrara, T.; Bratsos, I.; Iengo, E.; Milani, B.; Oštrić, A.; Spagnul, C.; Zangrando, E.; Alessio, E.
Dalton Trans. 2009,10742–10756. [2] Gianferrara, T.; Bergamo, A.; Bratsos, I.; Milani, B.; Spagnul,
C.; Sava, G.; Alessio, E. J. Med. Chem. 2010, 53, 4678–4690.
37
Targeting tyrosine kinases: anti-proliferative and anti-angiogenic
properties of 4-anilinoquinazolines
F. Tonus, I. Zanusso, L. Urbani, A. Chilin, M.T. Conconi, A. Guiotto
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Padova
[email protected]
1. Introduction
Inhibition of Tyrosine Kinases (TKs) blocks multiple intracellular signaling
pathways involved in tumor growth, progression and angiogenesis. Therefore, the
development of small ATP-mimetic Tyrosine Kinase Inhibitors (TKIs) resulted very
attractive as anti-tumoral and anti-angiogenic tools.1 A major goal in the field of TKIs
development is to find compounds able to inhibit more than one TK, in order to
overcome the drug resistance onset. In this way, the blockade of several signaling
pathways involved in tumor growth, as EGFR and VEGFR, has a strong rationale for
development of multi-targeted TKIs.2
Several 4-anilinoquinazolines derivatives have been synthesized.3 The compounds
have been submitted to specific biological assay on a panel of six different TKs and
on tumor cell lines. The most active compounds were submitted to other biological
evaluations in order to investigate the mechanism of action. The title derivatives
induced cell death through activation of the pro-apoptotic pathway. Almost all the
compounds showed very interesting anti-angiogenic activity. The effect on cell
growth and cell migration induced was also evaluated in Human Umbilical Vein
Endothelial Cells (HUVEC).4
3. References
[1] Dowell, J., Minna, J. D., Kirkpatrick, P., Nat. rev. drugs discov., 2005, 4, S14-S15.
[2] Ryan, A. J., Wedge, S. R., BJC, 2005, 92, S6-S13.
[3] Marzaro, G., Guiotto, A., Pastorini, G., Chilin, A., Tetrahedron, 2010, 66, 962-968.
[4] Conconi, M.T. et al, Invest New Drugs, 2010, Epub ahead of print.
38
Synthesis and Evaluation of 3-Methyl-4-oxo-1,5,6,7-tetrahydroindole2-carboxylic acid ester derivatives as potential anticancer agents
P. Trifiro,a A. Abate,a R. Amici,a M. R. Cera,a G. Fagà,a P. De Wulf,b M. Varasia
a
Drug Discovery Program, Istituto Europeo di Oncologia, IFOM-IEO Campus,
Milan, Italy; bDepartment of Experimental Oncology, Istituto Europeo di Oncologia,
IFOM-IEO Campus, Milan, Italy
[email protected]
1. Introduction
Molecules that bind tubulin affect dynamic instability of microtubules causing
mitotic arrest of proliferating cells, leading to cell death and tumor growth inhibition.
Polymerization and depolymerization of microtubules occur in a dynamic equilibrium
disrupted by tubulin-targeting chemotherapeutics.1 Several classes of antitubulin
products have been effectively used as antitumoral agents. Despite their wide use and
clinical success, however, approved therapies have several limitations that have
driven significant efforts toward the discovery of novel antitubulin agents.
Among the most critical limitations of antitubulin agents are their sensitivity to
multidrug resistance pumps and the development of drug resistance.2 During a
screening of commercially available chemical libraries, 3-Methyl-4-oxo-1,5,6,7tetrahydroindole-2-carboxylic acids esters emerged as novel antitubulin agents.3 The
compounds identified showed depolymerisation of mitotic spindle and gave apoptosis
when cells were treated in metaphase, and inhibitory activity on tumor resistant cell
lines.
We here present the modifications made on the scaffold
mainly directed towards modification of R1, R2, Ar and X
(see figure 1), in order to fully exploit initial hits obtained
from HTS. All the new compounds have been tested for their
ability to inhibit tubulin polimerization and to inhibit
proliferation of chemo-resistant tumour cells.
X
O
N
Ar
Figure 1
R1
O
R2
3. References
[1] Dumontet, C., Jordan, M.A. Nature Rev. Drug Discov. 9, 790-803 (2010)
[2] Kavallaris, M., Nature Rev. Cancer 10, 194-204 (2010)
[3] Screpanti, E., et al. PLoS ONE, 2010, vol 5 (7), e11603
39
Isoxazolo[5,4-e]isoindole a new ring system
with potent antitumor activity
P.Barraja, V. Spanò, P. Diana, A. Montalbano, A. Carbone, G. Cirrincione
Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari (STEMBIO),
Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo.
[email protected]
Antimitotic agents, one of the major classes of cytotoxic drugs for cancer
treatment, have gained in recent years great attention. They cause the mitotic arrest in
eukaryotic
cells
by
interfering
with
the
normal
microtubule
polymerization/depolymerization process.1
Taxol, vincristine, colchicine and combretastatins, are leading examples of this class
of compounds, acting in three different binding regions of microtubule.2 In particular
those targeting the colchicines–binding site are considered important lead structures
for the development of new antitumor agents. Combretastatin A4 phosfate (CA4p),3 a
water soluble prodrug of CA4, is currently in phase III clinical trials for the treatment
of cancer and early results are very promising. To date many efforts have been done
to discover new combretastatin analogues replacing the alkenyl bridge of the natural
CA-4. Among these a number of analogues of CA-4 were prepared using fivemembered heterocycles such as imidazole, pyrazole, oxazole and isoxazole as
linkers.4,5
In our search for new antitumor agents and considering our interest in pyrrolo fused
heterocycles we focused our attention on the synthesis of the new ring system
isoxazolo[5,4-e]isoindole encouraged by the evidence that the isoxazole nucleus is
part of many drugs with antitumor activity. Our synthetic project consisted in the
annellation of the isoxazole core on the isoindole ring, by means of suitable
substrates. A first series of thirteen derivatives was synthetized and evaluated for
antitumor activity at the NCI of Bethesda, and two of them (mean pGI50 5.80 and
6.68) were selected for Hollow Fiber Assay. Considering the good results, new
compounds were prepared properly modificated from their original structure.
Fourteen out of twenty four compounds were evaluated against the full panel of 60
tumor cell lines at the NCI . Results will be discussed.
N O
R2
NR
R1
References
[1] Shi, Q.; Chen, K.;Morris-Natschke, S. L.; Lee, K.-H. Curr. Pharm. Des. 1998, 4, 219. [2] Q. Li, H.
L. Ham, S. H. Rosemberg, In Annual reports in Medicinal Chemistry; Doherty, A. M. Ed.; Academic
Press: San Diego, 1999, vol. 34, 139-148; McGown, A. T.; Fox, B. W. Cancer Chemother.
Pharmacol.1990, 26, 79. [3] Pettit, G. R.; Temple, C., Jr.; Narayanan, V. L.; Varma, R.; Simpson, M.
J.; Boyd, M. R.; Rener, G. A.; Bansal, N. Anticancer Drug Des. 1995, 10, 299; Chaplin, D. J.; Pettit, G.
R.; Hill, S. A. Anticancer Res. 1999, 19, 189. [4] L. Wang, K. W. Woods, Q. Li, K. J. Barr, R. W. Mc
Croskey, S. M. Hannick, L. Gherke, R. B. Credo, Y. –H. Hui, K. Marsh, R. Warner, J. Y. Lee, N.
Zielinski-Mozng, D. Frost, S. H. Rosemberg, H. L. Sham, J. Med. Chem., 2002, 45, 1697; K. Ohsumi,
T. Hatanaka, K. Fujita, R. Nakagawa, Y. Fukuda, Y. Nihei, Y. Morinaga, Y. Akiyama, T. Tsuji,
Bioorg. Med. Chem., 1998, 8, 3153. [5] C-M. Sun, L-G. Lin, Yu, H-J., C.-Y. Cheng, Y-C Tsai, C-W.
Chu, Y-H Din, Y-P. Chau, M-J. Don, Bioorg. Med. Chem., 2007, 17, 1078; J. Kaffy, R. Pontikis, D.
Carrez, A. Croisy, C. Monneret, J. C. Florent, Bioorg. Med. Chem., 2006, 14, 4067.
40
A new weapon in the fight against HIV. Discovery and synthesis of
new inhibitors of the human RNA helicase DDX3
G. Casaluce,a M. Radi,a F. Falchi,a L. Botta, a F. Manetti,a G. Magab, A. Garbelli,b A.
Samuele,b S. Zanoli,b E. Zucca,b M. Bottaa
a
Dip. Farmaco Chimico Tecnologico, Università di Siena, Via Alcide de Gasperi 2,
53100 Siena, Italia, fax +39-0577-234333; bIstituto di Genetica Molecolare, IGMCNR, Via Abbiategrasso 207, I-27100 Pavia
[email protected]
The Human DEAD-box protein is an ATP-dependent RNA helicase which has a
role in processes regulating gene expression, including transcription, splicing, mRNA
export and translation. Recently DDX3 has been identified as an essential cofactor for
HIV-1 RNA export from the nucleus. Any drug targeting this step will act at an
earlier level than protease inhibitors, shutting down the viral proliferation before
structural proteins have been produced in the infected cells. Targeting a cellular
factor, rather than a viral protein, could represent a more efficient antiviral approach
to overcome the drug-resistance associated with the approved drugs.1
A computational protocol based on pharmacophoric
modeling and molecular docking calculation lead to the
discovery of the first small molecule able to inhibit Hiv-1
replication by targeting the human cellular cofactor DDX3
(Fig 1). Herein we describe a hit optimization protocol
applied to the design and synthesis of second-generation
DDX3 inhibitors with better inhibitor activity towards
cellular DDX3 enzyme and HIV-1 replication. The
Figure 1.
described compounds represent a significant advancement
in the quest of potential novel drugs targeting HIV-1 host cellular cofactor.2
References
[1] Maga, G.; Falchi, F.; Garbelli, A.; Belfiore, A.; Witvrouw, M.; Manetti, F.; Botta, M. J. Med.
Chem. 2008, 51, 6635-6638
[2] Botta et al. submitted for publication
41
Molecular modelling studies on arylthioindoles as potent inhibitors of
tubulin polymerization
A.Colucciaa, A. Brancaleb
Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Chimica e
Tecnologie del Farmaco, Sapienza Università di Roma, Piazzale Aldo Moro 5, I00185 Roma, Italy; bWelsh School of Pharmacy, Cardiff University, King Edward VII
Avenue, Cardiff, CF10 3NB, UK.
[email protected]
The crucial role played by microtubules in the life of eukaryotic cell makes
tubulin an important route for the anticancer therapy. The ArilThioIndoles (ATIs)1-4
along with the corresponding ketone and methylene compounds are potent tubulin
assembly inhibitors. We are here reporting the result of a series of docking and
molecular dynamics experiments on this series of compounds. The results obtained
from our in silico studies not only provided us with an insight on the nature of the
binding of the ATIs to tubulin, but were also at the core of the design of a new series
of potent inhibitors of tubulin polymerization.
References
[1] La Regina G, Coluccia A, Silvestri R et al. J. Med. Chem. 2004 47:6120-6123.
42
The role of N-terminal domain in the regulation of “constitutively
active” conformation of protein kinase CK2α: new insights from a
molecular dynamics investigation
A Cristiania,b, G. Costa,b G. Cozza,c F. Meggio,c L. Scapozza,a S. Morob
a
LCT-Biochimie Pharmaceutique, Section des Sciences Pharmaceutiques, Université
de Genève, Quai E. Ansermet 30, 1211 Genève (SWITZERLAND); bMolecular
Modelling Section (MMS), Department of Pharmaceutical Sciences, University of
Padova, Via Marzolo 5, 35131 Padova (ITALY); cDepartment of Biological
Chemistry, University of Padova, Padova 35121 (ITALY).
[email protected]
Protein kinase CK2α is defined as a “constitutively active” protein kinase in
contrast with most of the other protein kinases characterized by the presence of
distinct conformations associable with the active and the inactive states of the kinase.1
As previously demonstrated by in vitro mutation studies,2 CK2 activity is
substantially regulated by the interaction between the N-terminal tail and the kinase
domain. In fact, progressive deletions of the N-terminal tail show a decrease in the
activity of the kinase. In particular, deletions D2-12 and D2-30 progressively reduce
the basal activity of CK2α, even if the detrimental effects of D2-12 deletion can be
partially controverted by the addition of CK2b subunit.
In this work, molecular dynamics (MD) simulations been carried out on wild-type
(wt), Δ2-12 and Δ2-30 deleted mutants of CK2α in order to explore the role of the Nterminal tail on the CK2α conformational behaviour. Furthermore, classical MD
simulations have been anticipated to conformational impact of a novel suggested
mutants of CK2α, such as the triple mutant “Y206F, R10A, Y261F” and the single
one Y125F.
References
[1] Cozza, G. et al, Protein kinase CK2 in health and disease: Cellular functions of protein kinase
CK2: a dynamic affair. Cell Mol Life Sci, 2009. 66((11-12)): p. 1830-1839.
[2] Sarno S. et al, Unique activation mechanism of protein kinase CK2. The Journal of Biological
Chemistry, 2002. 277(25): p. 22509-22514.
43
SeRAPhiC: an ideal benchmark for in silico fragment-based drug
design
A. D. Faviaa, G. Bottegonia, I. Nobeli,b A. Cavallia,c
a
D3 Dept., Istituto Italiano di Tecnologia (IIT) Genoa, IT; bCrystallography, Institute
for Structural and Molecular Biology, Dep. of Biological Sciences, Birkbeck College,
University of London, UK; cDip. di Scienze Farmaceutiche, Università di Bologna, IT
[email protected]
1. Introduction
Fragment-based (FB) approaches are firmly established tools in drug discovery
campaigns in which low molecular weight molecules (100-250 Da) are screened
against biological targets of pharmaceutical interest1. Because of their low binding
affinities for macromolecules, fragments can be assayed only by highly sensitive
biophysical techniques that are expensive and quite hard to fully automatize2. To this
purpose it is then tempting to prioritize fragments with well established computational
techniques, such as molecular docking, before experimental testing. However, since
current algorithms are typically tuned for bigger molecules with drug-like features,
some issues emerge about their prompt applicability in the fragment-based field.
The main objective of this study was to build (see Figure) a set of high quality
protein-fragment complexes to test the performance of standard docking softwares
when dealing with fragments. Eventually the dataset was challenged by means of selfand cross-docking procedures to address several issues such as: a) can molecular
docking reproduce correctly the experimentally solved structures? b) How thorough
must be the sampling in order to reproduce the experimental data? c) Are standard
scoring functions able to discriminate between the native pose and the other energy
minima?
The dataset, named SERAPhiC (Selected Fragment Protein Complexes), in a
ready-to-dock format is freely available (http://www.iit.it/it/drug-discovery-anddevelopment/seraphic.html) to offer computational scientists worldwide a reliable test
set to work with
3. References
[1] Chessari, G.; Woodhead, A. J. From fragment to clinical candidate--a historical perspective.
Drug Discov Today 2009, 14, 668.
[2] Carr, R. A.; Congreve, M.; Murray, C. W.; Rees, D. C. Fragment-based lead discovery: leads by
design. Drug Discov Today 2005, 10, 987.
44
Swimming into peptidomimetic chemical space using
pepMMsMIMIC
M. Floris,a J. Masciocchi,b M. Fanton,c S. Moroc
a
CRS4 – Bioinformatics Laboratory, Parco Sardegna Ricerche, Pula (CA) 09010,
Italy; bCBiB, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076
Bordeaux Cedex, France; cMolecular Modeling Section (MMS), Department of
Pharmaceutical Sciences, University of Padova, PD 35131, Italy
[email protected]
pepMMsMIMIC is a novel web-oriented peptidomimetic virtual screening tool
based on a multi-conformers 3D- similarity search strategy. Key to the development
of pepMMsMIMIC has been the creation of a library of 17 million of conformers
calculated from 3.9 million of commercially available chemicals collected in the
MMsINC® database. Using as input the three-dimensional structure of a peptide
bound to a protein, pepMMsMIMIC suggests which chemical structures are able to
mimic the protein-protein recognition of this natural peptide using both
pharmacophore and shape similarity techniques. We hope that the accessibility of
pepMMsMIMIC (freely available at http://mms.dsfarm.unipd.it/pepMMsMIMIC) will
encourage medicinal chemists to de-peptidize protein-protein recognition processes of
biological interest, thus increasing the potential of in silico peptidomimetic screening
of known small molecules to expedite drug development.
45
Computer-aided drug discovery of p-TEFB inhibitors as
anti-HIV agents
N. Iraci,a L. Sancineto,a S. Massari,a A. Marcello,b V. Cecchetti,a O. Tabarrinia
a
Dipartimento di Chimica e Tecnologia del Farmaco, Universita’ di Perugia, Via del
Liceo,1 06123 Perugia (Italy), bInternational Centre for Genetic Engineering and
Biotechnology (ICGEB), Laboratory of Molecular Virology, Padriciano 99, 34012
Trieste, Italy.
[email protected]
1. Introduction
Remarkable progress has been made in the treatment of HIV infection, however, the
emergence of multidrug resistant viral strains along with the inability of the current
drug regimen to completely eradicate the virus in the HIV infected individuals,
demand new drugs capable of interfering with other targets or steps of the viral
replicative cycle. An appealing strategy could be the interference with host factors
essential for viral replication. In this context, we have planned to inhibit the P-TEFb
(Positive Transcriptional Elongation Factor b), composed of the cyclin dependent
kinase CDK9 associated with the regulatory subunit Cyclin-T1, which plays a pivotal
role in HIV transcription. P-TEFb is recruited by viral protein Tat to the nascent stem
loop RNA TAR, resulting in resumption of productive elongation after
phosphorylation, by mean of CDK9, of both the RNA pII CTD and NELFs (Negative
transcriptional ELongation Factors).
Using as target the recently published crystallographic structure of P-TEFb1 in
complex with flavopiridol (the prototypical CDK inhibitor), a multistep virtual
screening was performed on vendor, in-house, as well as on fragment-based library.
The latter was conceived keeping in mind the chemical accessibility for the
successive lead optimization phase. The virtual hits were tested for their ability to
inhibit the kinase activity of P-TEFb and four of them showed IC50 values in the
micromolar range. It is worth noting that two out of four real hits belong to the
fragment-based library. Structural information of the fragment hits will be used to
design larger molecules that pick-up additional protein-ligand interaction, hopefully
resulting in improved affinity for the target.
P-TEFb druggability, the multi-step virtual screening and preliminary biological
results, will be the object of this presentation.
3. References
[1] Baumli, S.; Lolli, G.; Lowe, E. D.; Troiani, S.; Rusconi, L.; Bullock, A. N.; Debreczeni, J. E.;
Knapp, S.; Johnson, L. N. The structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol
and regulation by phosphorylation. Embo J 2008, 27, 1907-18.
46
Structure-based design of EGFR covalent inhibitors
A. Lodola,a C. Carmi,a S. Rivara,a A. Cavazzoni,b E. Galvani,b P. G. Petronini,b M.
Mora
a
Dipartimento Farmaceutico, Università degli Studi di Parma; bDipartimento di
Medicina sperimentale,Università degli Studi di Parma
[email protected]
Irreversible EGFR inhibitors can circumvent resistance to first-generation
reversible, ATP competitive inhibitors in the treatment of non-small-cell lung cancer,
including gefitinib. They contain a driver group, which assures target recognition, and
a warhead, generally an acrylamide, group (as for PD168393) that binds covalently to
Cys797 of EGFR.1
Employing the information encoded in the X-ray structure of the EGFR-PD168393
covalent adduct,2 we designed new irreversible EGFR inhibitors carrying different
cysteine-reactive groups at position 6 of a 4-anilinoquinazoline scaffold.
Docking runs revealed that all the inhibitors could be accommodated within EGFR
with a binding mode resembling that of PD168393. Furthermore, in all the
simulations, the reactive center of the designed warhead was placed at distances
allowing the formation of a covalent bond with Cys797.
The newly synthesized 4-(3-bromoanilino)quinazoline derivatives were tested as
EGFR inhibitors in enzyme-based and cell-based assays. We found that all the
compounds inhibit EGFR with IC50 values in the nanomolar range. Furthermore,
some of them irreversibly inhibited EGFR kinase activity in A549 lung cancer cell
lines.
Crucially, at concentrations significantly lower than gefitinib, some of the novel
compounds inhibited EGFR and downstream signalling pathways, suppressed
proliferation, and induced apoptosis in gefitinib-resistant NSCLC H1975 cells,
harboring the T790M mutation in EGFR.3
References
[1] Kwak, E. et al, Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 7665–7670.
[2] Blair, J. A. et al, Nat. Chem. Biol. 2007, 3, 229–238.
[3] Carmi, C. et al, J Med Chem, 2010, 53, 2038-2050.
47
Rational drug design of DNA G-quadruplex binders using
structure-based computational approaches
S. Alcaro, A. Artese, S. Distinto, F. Moraca, F. Ortuso
Dipartimento di Scienze Farmacobiologiche, Università degli Studi “Magna
Græcia”, Campus “S. Venuta” 88100, Catanzaro.
[email protected]
1. Introduction
Human telomeric DNA G-Quadruplex is considered as an attractive target for
cancer therapeutic intervention.1 In this study we developed pharmacophore models
using structure-based computational approach. The models were validated and a
virtual screening of several commercial databases was carried out in order to identify
novel G-Quadruplex binders.
We considered four telomeric G-Quadruplex
complexes (PDB codes: 3CE5, 2JWQ, 1NZM,
3CDM). Based on structural and literature data, we
modified original pharmacophore hypothesis for
each complex. The best models, derived from the
validation procedure, were chosen for virtual
screening of ~ 2.500.000 compounds available from
different vendors. Screening results identified a set
of 88.141 compounds that was filtered considering a
pharmacophore fit value cut off. This process led us to obtain a hit list of 2.580
compounds on which further docking studies will be performed. The molecular
recognition study will take into account the Quadruplex polimorfism.2,3 Compounds
with the best score will be finally subjected to biological testing.
3. References
[1] Zhang, Z. et al. Nucleic Acids Res 2010, 38, 1009-1021
[2] Dai, J. et al Biochimie 2008, 90, 1172-1183
[3] Alcaro, S. et al ChemMedChem, 2010, 5, 575-583
48
From computer to in vivo: computational/experimental evidences for
designing efficient nanovectors for gene therapy
P. Posocco,a L. Peng,b J.-P. Behr,c D. K. Smith,d D. W. Pack,e S. Pricla
a
Department of Industrial Engineering and Information Technology, University of
Trieste, Trieste, Italy; bDepartment of Chemistry, CNRS, Marseille, France; cFaculté
de Pharmacie, Université Louis Pasteur, Strasbourg, France; dDepartment of
Chemistry, University of York, United Kingdom; eDepartment of Chemical and
Biomolecular Engineering, University of Illinois, Urbana, USA
[email protected]
While gene therapy has attracted extensive attention and showed significant
promise for the study, diagnosis and treatment of important human diseases,
delivering nucleic acid therapeutics specifically and efficiently into cells in vivo
remains a great challenge.1 Delivery barriers, which arise mainly from the routes of
administration associated with complex physiochemical microenvironments of the
human body, hinder the development of RNA-interference (RNAi)/DNA-based
therapeutics in clinical practice. However, in available delivery systems, non-viral
synthetic polymeric and dendrimeric vectors are showing huge potential.2
Recent efforts3 evidence that molecular modeling investigations support the
development of new nanovectors from their conception to the application in vivo. In
this study, we combine simulation/experimental approaches to define the mode and
the molecular requirements of the interaction of gene-based therapeutics and
dendrimeric delivery agents. Thus, critical molecular parameters for optimizing and
de novo designing nanovectors for tissues and tumor specific uptake can be
determined. This would provide valuable information to devise optimal delivery
modalities that would increase the efficacy of gene therapy in cells and laboratory
animals and move them toward clinical applications.
References
[1] Majoros, I. J. Progress in Molecular Biology and Translational Science (2010), 95, 193.
[2] Bai, S. et al. Crit Rev Ther Drug Carrier Syst (2006), 23, 437.
[3] a) Pricl, S. et al. Nat. Rev. Clin. Oncol. (2011), doi:10.1038; b) Peng, L. et al. ACS Nano (2010)
(accepted manuscript)
49
Synthesis and biological evaluation of multi-target directed ligands
against prion diseases
S. Bongarzone,a,b H. N. Ai Tran,c M. Roberti,d A. Cavalli,d P. Carloni,e G. Legname,c,b
M. L. Bolognesid
a
Statistical and Biological Physics Sector, Scuola Internazionale Superiore di Studi
Avanzati, Trieste; bSISSA-Unit, Italian Institute of Technology, Trieste; cNeurobiology
Sector, Scuola Internazionale Superiore di Studi Avanzati (SISSA), Trieste;
d
Dipartimento di Scienze Farmaceutiche, Università di Bologna; eGerman Research
School for Simulation Sciences GmbH, Forschungzentrum Jülich GmbH - RWTH
Aachen University
[email protected]
1. Introduction
Prion diseases are a group of invariably fatal disorders, for which there is not cure.
The molecular mechanisms of these diseases involve a complex variety of processes
operating simultaneously and synergistically, including: (i) protein aggregation; (ii)
oxidative stress; (iii) reduced levels of potent free-radical scavengers; (iv) unbalance
of metal ions. Therefore, the challenges in devising effective therapies for
neurodegenerative diseases are likely to be related to such multifactorial nature.
In an effort to rationally develop antiprion lead
compounds, we envisaged the planar 2,5-bis-diaminobenzoquinone (BQ) scaffold as privileged motifs in
modulating protein-protein interactions. In particular, we
reported that the 1 exhibited remarkable antiprion activity
in a cellular model (EC50 = 0.87 mM).1 Starting from these
results, a further library featuring a central BQ nucleus,
with two spacers in position 2 and 5 connected to two
terminal moieties, was designed. Notably, 6-chloro1,2,3,4-tetrahydroacridine 2 displayed an EC50 of 0.17 µM,
which was lower than that of the reference compound quinacrine (EC50 = 0.40 mM).
More importantly, 2 possessed the capability to contrast prion fibril formation and
oxidative stress, together with a low cytotoxicity. These results validated our design
rationale as a viable strategy for the identification of novel lead compounds with more
than one activity against prion diseases.2
3. References
[1] Tran, H. N. A.; Bongarzone, S.; Carloni, P.; Legname, G.; Bolognesi, M. L. Bioorg. Med. Chem.
Lett. 2010, 20, 1866-1868.
[2] Bongarzone, S.; Tran, H. N. A.; Cavalli, A.; Roberti, M.; Carloni, P.; Legname, G.; Bolognesi, M.
L. J. Med. Chem. 2010, 53, 8197–8201.
50
In silico exploration of the Embryonic Lethal Abnormal Vision
protein-mRNA interaction followed by in vitro bioassays for the
identification of peptides potentially useful in neurodegenerative
diseases treatment
A. Carnevale Baraglia,a M. Amadio,a A. Pascale,a S. Govoni,a E. Laurini,b S. Pricl,b
D. Rossi,a O. Azzolina,a S. Collinaa
a
Dipartimento di Scienze del Farmaco, Università di Pavia, viale Taramelli 12,
27100, Pavia-I; bDipartimento di Ingegneria Industriale e dell'Informazione,
Università di Trieste, Via A. Valerio, 10, 34127, Trieste-I
[email protected]
1. Introduction
The Embryonic Lethal Abnormal Vision (ELAV) proteins are RNA-Binding Proteins
that, in response to intra- and extracellular signals, bind preferentially to the Adenineuracile Rich Elements (ARE) of target mRNAs affecting their stability and rate of
translation.
Recently, we studied for the first time the ELAV-mRNA system from a medicinal
chemistry point of view by evaluating the effect of four peptides-corresponding to the
sequences of ELAV proteins primarily involved in the binding to ARE-mRNA- on
the stability of VEGF and NOVA-1 transcripts. Biological results clearly showed
ELAV-like properties for the equimolar mixture of the peptides.1
To explore the chemical features directly
involved in the binding of the ELAV protein
HuD with target mRNAs (Figure 1), we studied
in silico the HuD-mRNA complex by means of
molecular dynamics simulations. In vitro
bioassay results, obtained testing peptides either
individually or in couples, confirmed the
molecular modelling predictions.
Figure 1 Snapshot extracted from
MD simulation of HuD-mRNA
complex
3. References
[1] Rossi D. et al. J Med Chem 2009, 52, 5
51
A small focused chemical library of 2-aminoimidazole derivatives as
BACE-1 inhibitors
G. Chiriano,a A. De Simone,b F. Mancini,b D. I. Perez,c A. Cavalli,b,d M. L.
Bolognesi,b G. Legname,a A. Martinez,c V. Andrisano,b P. Carloni,e M. Robertib
a
Scuola Internazionale Superiore degli Studi Avanzati (SISSA) di Trieste;
b
Dipartimento di Scienze Farmaceutiche, Università di Bologna; cInstituto de
Quimica Medica-CSIC di Madrid; dIstituto Italiano di Tecnologia di Genova;
e
German Research School for Simulation Sciences di Julich
e-mail [email protected]
1. Introduction
Alzheimer’s Disease (AD) is a progressive and fatal brain disorder. One of its
major characteristic and pathological hallmarks is represented by the senile plaques,
whose main component is the amyloid-β peptide (Aβ). The β-secretase APP cleaving
enzyme (BACE-1) catalyzes the rate limiting step in the production of Aβ, thus, its
inhibition is considered a key therapeutic strategy for the development of diseasemodifying drugs in AD1.
We report a rational structure-based approach aimed at the
discovery of new 2-aminoimidazoles as BACE-1 inhibitors.
Taking advantage of a microwave-assisted synthetic protocol, a
small library of derivatives was obtained and biologically
evaluated. Remarkably, two compounds showed low
micromolar activities in both enzymatic and cellular assays.
Moreover, one of them (1) exhibited the capability to cross the
blood brain barrier in the parallel artificial membrane
permeability assay. Overall, 1 emerges as a promising BACE-1
inhibitor hit for the development of new anti-AD lead
candidates.
3. References
[1] M. Citron, Nat Rev Drug Discov 2010, 9, 387.
52
Successful optimization of human carbonic anhydrase inhibitors as
neuroprotective agents
F. M. Damiano,a R. Gitto,a L. De Luca,a C. T. Supuran,b A. Innocenti,b A. Chimirria
a
Dipartimento Farmaco-Chimico, Università di Messina; bDipartimento di Chimica,
Università di Firenze.
[email protected]
1. Introduction
Fifteen human carbonic anhydrase (hCA) isoforms have been identified. They
differ widely in cellular localization and play a key role into a series of physiological
processes linked to the catalytic hydratation of CO2. These enzymes are also
therapeutic targets to treat several disorders suggesting the development of selective
inhibitors (CAIs) as new potential drugs. The most active CAIs, currently in therapy,
generally lack of isoform selectivity thus producing unwanted effects. We previously
identified some isoquinoline-sulfonamides that showed inhibitory effects against
attractive hCA isoforms1-3. Particularly, some of these new CAIs targeted selectively
hCAVII and hCA XIV isozymes, localized in a number of brain tissues suggesting
that these sulfonamides could be useful as neuroprotective agents.
The research reported here represents a continuation of our efforts to develop
potent and selective CAIs and obtain further SAR information. Thus, we introduced
some structural modifications on isoquinoline scaffold and synthesized new
analogues. All synthesized compounds were tested for their inhibitory effects against
selected isoforms (hCA II, hCA VII, hCA XIV) and the obtained results highlighted
an improvement of affinity and selectivity. We also performed docking studies in
order to explore the chemical features controlling the catalytic site interactions.
3. References
[1]Gitto, R.; Agnello, S.; Ferro, S.; De Luca, L.; Vullo, D.; Brynda, J.; Mader, P.; Supuran, CT.;
Chimirri, A. J. Med. Chem., 2010, 53, 2401.
[2]Gitto, R.; Agnello, S.; Ferro, S.; Vullo, D.; Supuran, CT.; Chimirri, A. ChemMedChem, 2010, 5,
823.
[3]Gitto, R.; De Luca, L.; Ferro, S.; Agnello, S.;Damiano, F.; Supuran, C.T.; Brynda, J.; Mader, P.;
Chimirri, A. Drugs Future, 2010, 35, 131.
Research supported by MIUR and University of Messina (PRIN 2008, PRA2008/09)
53
Aminothienopyridazine inhibitors of tau aggregation
F. Piscitelli,a C. Ballatore,b,c K.R. Brunden,c M.J. James,c A. Crowe,3 Y. Yao,3 E.
Hyde,c J.Q. Trojanowski,c V.M.-Y. Lee, b A.B. Smith IIIb
a
Dip. di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, piazzale
Aldo Moro 5, 00185 Roma; bDepartment of Chemistry, School of Arts and Sciences,
University of Pennsylvania, 231 South 34th St., Philadelphia, PA 19104-6323; cCenter
for Neurodegenerative Diseases Research, Institute on Aging, University of
Pennsylvania, 3600 Spruce Street, Philadelphia, PA 19104-6323
[email protected]
Protein aggregates comprising hyperphosphorylated tau constitute the typifying
lesions of different neurodegenerative disease known as tauopathies.1 Tau is an
endogenous MT-stabilizing agent that is highly expressed in the axons of neurons.
The MT-stabilizing function of tau is essential for the axonal transport of proteins,
neurotransmitters and other cellular constituents. Under pathological conditions, tau
misfolding and aggregation results in axonal transport deficits that appear to have
deleterious consequences for the affected neurons, leading to synapse disfunction and,
ultimately, neuronal loss. As a result agents capable of preventing the self-assembly
of tau comprise an attractive strategy for the prevention and/or treatment of
neurodegenerative tauopathies.
We reported the discovery of the aminothienopyridazine (ATPZ) class of tau
aggregation inhibitors2 and now describe a series of new analogues that are both
effective inhibitors of tau fibrillization and display significant brain-to-plasma
exposure ratios after administration to mice. Further, two of the most promising
examples were found to reach significant brain exposure levels following oral
administration. Taken together, these results suggest that examples from the ATPZ
class hold promise as candidates for in vivo efficacy studies in animal models of
neurodegenerative tauopathies.
References
[1] Ballatore, C. et al. Tau-mediated neurodegeneration in Alzheimer's disease and related disorders.
Nature Rev Neurosci. 2007, 8, 663-672
[2] Crowe, A. et al. Identification of aminothienopyridazine inhibitors of tau assembly by quantitative
high-throughput screening. Biochemistry 2009, 48, 7731-7735.
54
New Leads for the Inhibition of Monoammino-Oxidase B
M. L. Sanna,a R. Meleddu,a M. C. Cardia,a S. Distinto,b S. Alcaro,b E Maccionia
a
Dipartimento Farmaco Chimico Tecnologico, University of Cagliari, Via Ospedale,
72 Cagliari (CA), Italy; b Dipartimento di Scienze Farmaco Biologiche, University
“Magna Graecia” of Catanzaro, Campus “S. Venuta”, 88100 Catanzaro (CZ), Italy
[email protected]
1. Introduction
Neurodegenerative disorders are a wide and heterogeneous class of diseases.1
Although the involvement of apoptosis in neurodegenerative pathologies is
controversial, some evidences suggest that cell-death mediators can play a critical role
in initiating the process.2 From this point of view the pivotal role of monoamineoxidase (MAO) in controlling the formation of both neuro-toxic compounds and
oxygen reactive species has been reported.3 So far MAO inhibitors can be useful as
therapeutic agents in the early phases of neurodegenerative disorders.
Aiming at the identification of new leads for the selective inhibition of the B
isoform of MAO, we have recently reported on the design, the synthesis, and the
biological properties of a wide selection of differently substituted heterocyclic nuclei.4
Generally both activity and selectivity are dependent on the nature and the relative
position of the substituents on the heterocyclic core. Moreover, in the case of chiral
compounds, the resolution of the racemic mixture has been performed and
stereochemistry revealed to be crucial in determining the biological properties. This
was particularly evident in a series of 3-acetyl-2,5-diaryl-2,3-dihydro-1,3,4oxadiazoles whose activity and selectivity towards MAO-B is extremely encouraging.
3. References
[1] Lin M. T. and Flint Beal M., Nature, 443, (2006), 787-795.
[2] Jellinger, K. A., J. Neural Transm., Suppl. (2003), 101-144.
[3] Arguelles, S., Herrera, A. J., Carreno-Muller, E., de Pablos, R. M., Villaran, R. F., Espinosa-Oliva,
A. M., Machado, A., and Cano, J., Neurotoxicology, 31, (2010) 55-66.
[4] Maccioni, E., Alcaro, S., Orallo, F., Cardia, M.C., Distinto, S., Costa, G., Yañez, M., Sanna, M.L.,
Vigo, S., Meleddu, R., Secci D., Eur.J.Med.Chem., 45, (2010), 4490-4498, and references therein cited.
55
Protein-protein interaction disruptors.
Design and synthesis of thienylpyridyl oligomers
as non-peptidic alpha helix mimetics
M. De Giorgi,a,b A.S. Voisin-Chiret,a J. Sopková-de Oliveira Santos,a M. Muraglia,b
F. Corbo,b C. Franchini,b S. Rault a
a
Université de Caen Basse-Normandie, U.F.R. des Sciences Pharmaceutiques,
CERMN, UPRES EA-4258, FR CNRS INC3M, Bd Becquerel - 14032 CAEN Cedex,
France; bDipartimento Farmaco-chimico - Università degli Studi di Bari Aldo Moro
– Via Orabona 4, 70125, BARI, Italia.
[email protected] or [email protected]
Protein-protein interactions (PPIs) are attractive targets because they control
numerous cellular processes. Consequently their misregulation can result in numerous
disease states such as HIV, cancer, diabetes and neurodegenerative diseases.1
Recognition between proteins is often facilitated by secondary structure elements and
between them alpha helices are often found at the interface of PPIs.2 Hamilton and coworkers described small inhibitors of protein-protein interactions as terphenyl and
terpyridyl scaffolds in which substituents on aromatic rings present functionalities in a
spatial orientation that are able to mimic residues on an alpha-helix.3 Our laboratory is
interested to the synthesis of non-peptidic alpha-helix mimetic protein-protein
interaction disruptors. So, as continuation of our recent works,4 we envisaged to
synthesize new compounds less hydrophobic than these described by Hamilton by
changing the nature of (het)aromatic units. So, owing to the versatile chemistry of
thiophene, we decided to introduce the thienyl moiety to obtain new pyridylthienyl
oligomers.
R
R
S
S
N
References
[1]
Davis, J.M.; Tsou, L.K.; Hamilton, A.D. Chem. Soc. Rev. 2007, 36, 326-334 ; [2] Kutzki, O. et al. J. Am. Chem.
Soc. 2002, 124, 11838; [3] Che, Y. et al. J. Comp. Aid. Mol. Des. 2006, 20, 109; [4] a) Voisin-Chiret, A. S.;
Bouillon, A.; Burzicki, G.; Célant, M.; Legay, R.; El-Kashef, H.; Rault, S., Tetrahedron 2009, 65, 607-612; (b)
Burzicki, G.; Voisin-Chiret, A. S.; Sopkovà-de Oliveira Santos, J.; Rault, S., Tetrahedron 2009, 65, 5413-5417; (c)
Burzicki, G.; Voisin-Chiret, A. S.; Sopkovà de Oliveira Santos, J.; Rault, S. Synthesis 2010, 16, 2804-2810; (d)
Voisin-Chiret, A. S.; Muraglia, M. ; Burzicki, G.; Perato, S.; Corbo, F.; Sopkovà de Oliveira Santos, J.; Franchini,
C.; Rault, S., Tetrahedron 2010, 66, 8000-8005 ; (e) De Giorgi, M. ; Voisin-Chiret, A. S.; Sopkovà de Oliveira
Santos, J.; Franchini, C.; Rault, S., J. Org. Chem. 2011, submitted.
56
Targeting protein-protein interactions: a promising anti-HIV drug
discovery strategy
L. De Luca,a S. Ferro,a R. Gitto,a M.L. Barreca,b Z. Debyser,c A Chimirria
a
Dipartimento Farmaco-Chimico, Università di Messina, bDipartimento di Chimica e
Tecnologia del Farmaco, Università di Perugia, cMolecular Medicine, Katholieke
Universiteit Leuven and IRC KULAK.
[email protected]
1. Introduction
AIDS is the most challenging pandemic of the 21st century. Despite its increasing
incidence, the prognosis has improved significantly due to the development of an
antiretroviral arsenal. In recent years, HIV-1 integrase (IN) has become an attractive
target and in 2007, the FDA approved raltegravir, the first IN inhibitor signaling the
advent of a new generation of anti-HIV drugs. However, the development of viruses
resistant to raltegravir led to continuous effort to design inhibitors targeting different
steps in the integration process. An increased understanding of IN structural biology
has opened up novel approaches to inhibiting IN, such as targeting the protein–protein
interactions (PPIs) with nuclear protein lens epithelium growth factor (LEDGF/p75),
which plays an important role in the integration.
On this basis, our research group has developed a pharmacophore model for
potential small-molecule inhibitors of HIV-1 IN–LEDGF/p75 interactions, then used
for virtual screening of our in-house database, CHIME, leading to the identification of
some interesting hits 1-2.
Application of docking simulations and different computational approaches led to the
investigation of the possible binding mode of our hits and to the discovery of new
findings helpful for their optimization.
New small molecules were designed and synthesized proving to be able to interfere
with the IN–LEDGF/p75 interactions at micromolar concentration. This study can be
considered a valid starting point for the identification of PPIs with a different
mechanism of action from currently anti-HIV agents.
3. References
[1] De Luca L.; Barreca M.L. et al. ChemMedChem, 2009, 4, 1311.
[2] De Luca L.; Ferro S. et al. Bioorg Med Chem, 2010, 18, 7515.
Research supported by THINC project (HEALTH-F3-2008-201032)
57
Molecular Dynamics as tool to investigate conformational transitions
of MDM2 in response to ligand binding
N. Giacchè, A. Macchiarulo, R. Pellicciari
Dipartimento di Chimica e Tecnologia del Farmaco, Università degli Studi di
Perugia, via del Liceo 1, 06123 Perugia.
[email protected]
1. Introduction
MDM2 is an oncogenic protein that regulates the activity and stability of p53. A
large body of experiments have validated MDM2 as therapeutically important target
for the development of anticancer drugs.1-2 Accordingly, during the last decade,
diverse small molecules have been disclosed as able to disrupt the interaction between
MDM2 and p53. On this basis, experimental and computational approaches have been
developed to study the interaction of small molecules within the binding cleft of
MDM2 in order to facilitate the bench to bedside translation of new potent and
selective chemical entities for novel anticancer therapies.3
As a continuation of our efforts in the field,4-5 I will present the results of molecular
dynamic simulations carried out to study the different conformational transitions of
MDM2 in response to small molecule binding. Our study points out a conditional
active role of the N-terminal region (1-25 residues) of MDM2 in the molecular
recognition of inhibitors, that is dependent
on the specific chemical class of the
ligand. While useful to drive medicinal
chemistry in designing new MDM2
inhibitors with improved potency and
selectivity, the results obtained shed
additional lights on the binding mode of
small molecule inhibitors to MDM2.
3. References
[1] Vogelstein, B.; et al. Nature 2000, 408, 307-310
[2] Toledo, F.; et al. Nat. Rev. Cancer 2006, 6, 909-923
[3] Patel, S.; et al. Expert Opin. Investig. Drugs 2008, 17, 1865-1882
[4] Macchiarulo, A.; et al. J. Chem. Inf. Model. 2008, 48, 1999-2009
[5] Carotti, A.; et al. Proteins 2009, 77, 524-535.
58
Chemical genetics approach to drug discovery by Diversity-Oriented
Synthesis (DOS) of peptidomimetics
A. Trabocchi,a I. Stefanini,b D. Cavalieri,b A. Guarnaa
a
Department of Chemistry “Ugo Schiff”, University of Florence, via della Lastruccia
13, Sesto Fiorentino, Florence, Italy; bDepartment of Pharmacology “Mario Aiazzi
Mancini”, University of Florence, viale Pieraccini 6, 50139, Florence, Italy
[email protected]
Chemical genetics, which relies on selecting small molecules for their ability to
induce a biological phenotype or to interact with a particular gene product, is an
emerging powerful tool for lead generation in drug discovery. Accordingly, DiversityOriented Synthesis (DOS) of small molecule peptidomimetics gives access to
collections of new chemotypes bearing high structural diversity. Moreover, biological
evaluation using cell growth as a phenotypic screening on Saccharomyces cerevisiae
deletant strains is a powerful tool to identify new chemotypes as hit compounds in the
discovery of new antifungal and anticancer agents, and also in the dissection of their
mode of action.
Our efforts in this field are focused on the generation of diversity-oriented
molecular probes of peptidomimetic nature, and on the evaluation of their ability to
induce phenotypic effects with functional implications on a panel of strains of the
budding yeast Saccharomyces cerevisiae.1 Accordingly, we describe the synthetic
strategies towards the generation of morpholine-based scaffolds from amino acid and
sugar derivatives in a diversity-oriented fashion, and the successful application of a
library of these molecules on a panel of Saccharomyces cerevisiae strains, which
provided the identification of new chemotypes involved in mitochondria metabolism
and respiration.2
References
[1] (a) Trabocchi, A.; Menchi, G.; Guarna, F.; Machetti, F.; Scarpi, D.; Guarna, A. Synlett 2006, 331353. (b) Lalli, C.; Trabocchi, A.; Sladojevich, F.; Menchi, G.; Guarna A. Chem. Eur.J. 2009, 15, 78717875. (c) Stefanini, I.; Trabocchi, A.; Marchi, E.; Guarna, A.; Cavalieri, D. J. Biol. Chem. 2010, 285,
23477 – 23485 (d) Trabocchi, A.; Cavalieri, D.; Guarna, A. Pure Appl. Chem. 2011, 83, 687 – 698.
[2](a) Trabocchi, A.; Stefanini, I.; Morvillo, M.; Ciofi, L.; Cavalieri, D.; Guarna, A. Org.
Biomol.Chem. 2010, 8, 5552 – 5557. (b) Ciofi, L.; Morvillo, M.; Sladojevich, F.; Guarna, A.;
Trabocchi, A. Tetrahedron Lett. 2010, 51, 6282 – 6285.
Acknowledgements: financial support from Fondazione Roma and CINMPIS.
59
Structural conformational analysis of E-cadherin germline missense
mutations in two sporadic gastric cancer patients
M. Garziera,a S. Geremia,b L. Caggiari,a R. Cannizzaro,c V. De Re
a
Experimental and Clinical Pharmacology Unit, CRO Aviano, PN, Italy, bDepartment
of Chemical Sciences, University of Trieste, TS, Italy, cGastroenterology Unit, CRO
Aviano, PN, Italy.
[email protected]
1. Introduction
E-cadherin is a transmembrane glycoprotein responsible for cell to cell adhesion in
epithelial cells. Several germline mutations of its gene (CDH1) are associated with
familial gastric cancer. We report the finding of two missense germline mutations in
two sporadic gastric cancer cases. One mutation was novel while the other variant
was previously reported and his pathogenic role confirmed. By a multivariate in silico
analysis we want to predict its structural conformational and consequently its possible
pathogenetic effect in sporadic gastric cancer.1
The mutational screening for CDH1
revelead 2 missense variants in two distinct
patients, both localized in the EC2 domain of
the E-cadherin. The novel mutation (820 G>A)
in exon 6 lead to a Gly to Ser exchange at
codon 120 in mature protein. G120S is not
dramatic for the local structure; however
Ser120 may interfere with functional calcium
sites and with EC1 domain of another dimer
interface. Moreover, Ser could be involved in
post translation modification.
The second missense variant (892 G>A) in exon7, is an Ala to Thr change
(A144T). Ala144 is near to the interaction region between the EC1 and EC2 domains.
Thr is sterically obtrusive because interact with Asp136 and Asp138 that are directly
involved in Ca++.2
3. References
[1] Keller G et al., J Med Genet 2004; 41:e89.
[2] Brooks-wilson AR et al., J Med Genet 2004;41:508-17.
60
From single- to multi-target tyrosine kinases inhibitors: using the
“old-age” kinases inhibitors data to face the “new-age”
G. Marzaro, F. Tonus, P. Manzini, A. Guiotto, A. Chilin
a
[email protected]
1. Introduction
The cell cycle regulatory systems alterations may cause cancer onset and
progression and metastasis. Among the proteins involved in signal recognition,
transduction and amplification, tyrosine kinases (TKs) plays a key role. In the last
years several efforts have been made to discover potent and selective TKs inhibitors.1
However, it has been recently demonstrated that compounds with high selectivity
inhibition profile may undergo to drug resistance onset. On the contrary, compounds
with a broad TKs inhibition profile could be more useful for anticancer therapy.2 All
this findings have caused a renewal in the TKIs development approach. The nonserendipitous based discovery of multi-target TKIs thus is a field of major interest, as
well as the development of novel approaches to the rational design of compounds
with specific inhibitory profile.
Herein we present an overview of the chemoinformatic tools developed in our
research group to screen and design novel potential TKs inhibitors. Several QSAR
models have been developed starting from literature
data and have been validated through both external
validation sets and novel compounds evaluation. A
comparison between classical and modern approaches
(i.e. a novel clusterization-based method and use of
artificial neural network) are also presented. Finally, the
preliminary results based on docking studies will be
discussed.
3. References
[1] J. Mendelsohn, J Clin Oncol 2002, 20, 1S.
[2] A. Petrelli, S. Giordano, Curr Med Chem 2008, 15, 422.
61
Posters
62
Bioactive lipids metabolites in amanita virosa
F. Cateni,a M. Zacchigna,a G. Procidab, B. Doljakc, M. Anderluhc, A. Piltaverc
a
Dipartimento di Scienze Chimiche e Farmaceutiche, Università di Trieste, P.zle
Europa, 1, 34127 Trieste; bDipartimento di Ingegneria Industriale e
dell’Informazione, Università di Trieste, Via A. Valerio 1, 34127 Trieste; cFaculty of
Pharmacy, University of Ljubljana, Slovenia.
[email protected]
1. Introduction
Amanita fungi are the main lethal toadstool among the 1000 poisonous mushrooms
known in the world. Thrombin is the key serine proteinase of the coagulation cascade
and therefore a suitable target for inhibition of blood coagulation. An extract of
Amanita virosa considerably inhibited thrombin (48%) and showed no inhibitory
activity on trypsin. On the basis of inhibition selectivity between thrombin and trypsin
and potency of thrombin inhibition, Amanita virosa constituted a good starting
material for isolation of further compounds that are active against thrombin.
In a preliminary study, 95 selected
mushroom species have been screened in order
to find novel specific non-peptidic thrombin
inhibitors. The extract of Amanita virosa
considerably inhibited thrombin (48% at
concentration of 120 µg/mL).1
A bioassay oriented fractionation of the extract
of Amanita virosa has led to the isolation of active compounds. On the basis of
spectroscopic data, chemical reactions and GC-MS analysis, complex mixtures of
triglycerides, monoacylglycerols, free fatty acids and ergosterol have been isolated
and identified.
28
22
21
24
23
12
19
1
11
26
20
18
25
17
27
13
16
9
14
8
2
HO
10 H
5
3
4
H
15
7
6
3. References
[1] B. Doljak, M. Stegnar, U. Urleb, S. Kreft, A. Umek, M. Ciglaric, B. Strukelj, T. Popovic.
'Screening for selective thrombin inhibitors in mushrooms' Blood Coagul. Fibrinolysis 2001, 12, 123128.
63
Ellagic acid and its derivates as catalytic inhibitors of human
topoisomerase II
V. Furlanetto, S. Stragliotto, G. Zagotto, S. Moro, B. Gatto
[email protected]
Ellagitannins (ETs) are natural compounds abundant in some fruits and nuts with a
large variety of molecular structures. They are among the main pharmacologically
active substances in several traditional herbal products used in folk medicine,1 and
their metabolites are reported by several authors to be responsible for anti-viral, antioxidant, anti-peroxidating, anti-foodborn pathogens, anti-mutagenic, antiproliferative
and proapoptotic activities.2-4 The main product of the ETs degradation is the ellagic
acid (EA), which is further biodegradated in urolithins by the colonic microflora.5
EA inhibits the human topoisomerase II4 but its mechanism of action has not been
elucidated in details. To elucidate its distinct antitumor properties and to obtain
structure-activity relationship (SAR) information, we have characterized the effects of
EA and several synthetic derivates on human topo II using enzymatic assays, and on
cellular culture using the two lines MCF-7 and HT-29.
Four bilactones (EA, 1179, 1183 and 1186) and two urolithins (1182 and 1185)
were highly active on the human topo II, both in isoforms α and β, with IC50 ≤1µM.
The results for these six active compounds point out to the importance of the number
and relative position of hydroxyl groups on both EA and urolithin scaffold to gain
activity on human topo II. These six potent inhibitors of the human topos II were
slightly cytotoxic on MCF-7 breast cancer cells. The data collected with the RPHPLC about the LogPow show that all the compounds with activity on human topo II
had the lowest LogP, suggesting low penetration of the cell membrane.
Further competitive inhibition tests aimed at elucidating the mechanism of
inhibition toward topo II, demonstrated that ATP competitively and dose-dependently
influences the inhibition of active compounds on human topo II. The results suggest
that ellagic acid and its derivates are catalytic inhibitors of the human enzyme, and
may share a common binding site with ATP.
References
[1] Quideau, S. and K.S. Feldman, Ellagitannin Chemistry. Chem Rev, 1996. 96(1): p. 475-
504.
[2] Bell, C. and S. Hawthorne, Ellagic acid, pomegranate and prostate cancer -- a mini
review. J Pharm Pharmacol, 2008. 60(2): p. 139-44.
[3] Losso, J.N., et al., In vitro anti-proliferative activities of ellagic acid. J Nutr Biochem,
2004. 15(11): p. 672-8.
[4] Constantinou, A., et al., The dietary anticancer agent ellagic acid is a potent inhibitor of
DNA topoisomerases in vitro. Nutr Cancer, 1995. 23(2): p. 121-30.
[5] Gonzalez-Barrio, R., et al., Bioavailability of anthocyanins and ellagitannins following
consumption of raspberries by healthy humans and subjects with an ileostomy. J Agric Food
Chem, 2010. 58(7): p. 3933-9.
64
Selective recognition of nucleic acids by peptidyl-anthraquinones
A. Sosic,a M. Casatti,a V. Santagada,b M. Palumbo,a C. Sissi,a B. Gattoa
a
Dipartimento di Scienze Farmaceutiche, Università di Padova, Via Marzolo, 5,
35131 Padova, Italy
b
Dipartimento di Chimica Farmaceutica e Tossicologica, Università degli Studi di
Napoli “Federico II”, Via D. Montesano, 49, 80131, Napoli, Italy
[email protected]
Natural and synthetic derivatives characterized by the presence of an
anthraquinone nucleus represent an important class of antineoplastic agents whose
mechanism of action is related to recognition of DNA and inhibition/poisoning of
nucleic acid tracking enzymes. We report here the biological evaluation of
anthraquinone-peptide conjugates where simple peptide sequences represent the basic
elements for specific recognition of DNA regions of relevant importance in
antineoplastic or antiviral chemotherapy.
A first physiological relevant example is the nucleic acid multistrand structure
assumed by G-rich DNA and RNA sequences, represented by G-quadruplex. These
can be found at the end of each chromosome as well as in the promoter regions of
several oncogenes. The selective stabilization of these peculiar arrangements can stop
replication of cancerous cells thus finding application in chemotherapy.
Anthraquinones (AQ) with side chains at the 2,6 positions have been previously found
to bind preferentially to G-quadruplex structures.
Several nucleic acid sequences and structures play also fundamental roles in early
steps of viral replication. In particular, HIV-1 reverse transcriptase (RT) uses double
stranded RNA sequences as well as DNA/RNA hybrids to perform its catalytic
activities. The “R” regions of HIV-genome contain also highly structured RNA and
DNA sequences (TAR and cTAR), which are selectively recognized by peptidylanthraquinones. Increasing the nucleic acid stability would decrease the efficiency of
strand transfer steps by RT and eventually the completion of reverse transcription:
selective recognition of these sequences could lead to inhibition of HIV-1 replication.
In this line of research we describe here the DNA binding of a series of 2,6
substituted anthraquinones toward G quadruplexes as well as toward HIV-1 relevant
nucleic acids structures, by using the same technological platform, i.e. Fluorescence
Quenching Assays (FRET probes coupled to thermal melting experiments). Our
results demonstrate that anthraquinones exhibit different selectivity toward different
targets, namely the human telomeric sequence formed by repeats of the
hexanucleotide (TTAGGG), the Primer Binding Sequence (PBS) and the polypurine
tract (PPT) of HIV-1, thus establishing the basis for the exploitation of these
compounds as leads for different classes of chemotherapeutics.
65
Virtual ligand screening studies
on an intermediate activation state of CDK5
G. Bottegoni,a M. Veronesi,a C. Dalvit,a A. Cavallia,b
a
Dipartimento di Ricerca e Sviluppo dei Farmaci, Istituto Italiano di Tecnologia
(IIT), via Morego 30, 16163, Genova, IT; bDipartimento di Scienze Farmaceutiche,
Università di Bologna, via Belmeloro 6, 40127, Bologna, IT
[email protected]
1. Introduction
Cyclin-Dependent Kinase 5 (CDK5) is a ubiquitously expressed kinase that has
been identified as a potential target in the treatment of several neurodegenerative
conditions, such as Amyotrophic Lateral Sclerosis and Alzheimer’s Disease.
Recently, an intermediate metastable state of CDK5, structurally different from
both the kinase active and inactive rearrangements, has been isolated by
computational means.1 According to the idea that selectivity can be achieved
exploiting the intrinsic kinase flexibility,2 a VLS campaign was carried out to identify
novel molecular entities that specifically target the intermediate conformation.
A targeted database of 40.000 molecules underwent a stepwise screening
procedure largely based on the 4D docking technique.3 25 molecules were actually
purchased and tested for activity by means of n-FABS (n Fluorine Atoms for
Biochemical Screening),4 a powerful and reliable NMR-based functional assay. The
easy set-up along with the reduced number of generated artifacts are the main
advantages of n-FABS technology when compared to other methodologies used in
HTS. Moreover the direct NMR characterization of the screened compounds with the
NMR-based SPAM Filter5 allows easy identification of false positives and false
negatives.
3. References
[1] Berteotti A. et al. JACS 2009, 131, 244-50
[2] Cavalli A. et al. Proteins 2001, 45, 478-485
[3] Bottegoni G. et al. J Med Chem 2009, 52, 397-406
[4] Dalvit C. et al. JACS, 2003, 125, 14620-14625
[5] Dalvit C. et al. Curr. Drug Disc. Tech., 2006, 3, 115-124.
66
Investigating the conduction mechanism of the herg potassium
channel
L. Ceccarini,a M. Masetti,a A. Cavalli,a,b M. Recanatinia
a
Dipartimento di Scienze Farmaceutiche, Università di Bologna, via Belmeloro 6, I40126 Bologna, Italy; bDrug Discovery and Development Dept., Istituto Italiano di
Tecnologia, via Morego 30, I-16163 Genoa, Italy
[email protected]
1. Introduction
hERG is a voltage-gated K+ channel expressed in different cells. However its
major role is played in cardiomyocites where it is responsible for the rapid delayed
rectifier current (Ikr), which is important for cardiac action potential repolarization.
Since its malfunctioning was found out to be involved in some forms of Long QT
Syndrome, there is a great interest in the comprehension of its role and its
functionality. This work is aimed at investigating at the atomistic level the mechanism
of ion conduction in hERG channel by means of computational techniques.
Umbrella sampling simulations were used to reproduce the K+ ion conduction and
to obtain a quantitative definition of the free energy surface along this pathway. In
particular the path collective variables protocol was used, where the initial guessed
path is a trajectory describing a complete cycle of
permeation: a K+ ion enters the Selectivity Filter
from the intracellular cavity and another one exits
from the other side. The resulting PMF is showed in
Fig1. The achieved results can be useful to compare
K+ conduction of the wild-type channel and
mutants. In particular we focused on one mutation
(G628S), which has been found to cause congenital
Long QT Syndrome.
3. References
[1] Recanatini, M. Expert Opin. Drug. Metab. Toxicol. 2009, 5(9):1005-21
[2] Branduardi, D. J. Chem. Phys. 2007, 126(5):054103
[3] Zhou, Z. CT. J. Biol. Chem. 1998, 273(33):21061-6.
67
Simplification of ellagic acid as a strategy for the identification of
new powerful, stable, and selective inhibitors of protein kinase CK2
G. Cozza,a,b A. Gianoncelli,a P. Bonvini,c E. Zorzi,c A. Rosolen,c L. A. Pinna,b F.
Meggio,b G. Zagotto,a S. Moroa
a
Molecular Modeling Section (MMS), Dipartimento di Scienze Farmaceutiche,
Università di Padova, via Marzolo 5, Padova, Italy; bDipartimento di Chimica
Biologica, Università di Padova, Padova, Italy; cClinica di Oncoematologia
Pediatrica, Azienda Ospedaliera, Università di Padova, Padova, Italy.
[email protected]
Protein kinase CK2 is a multifunctional serine/threonine protein kinase that
specifically phosphorylates residues located in acidic sequences of more than 300
substrates.1 Its high constitutive activity is implicated in growth homeostasis through
the modulation of the rates of cell proliferation and death. This observation in
conjunction with CK2 association with a number of pathologies including
inflammation, infection, and tumorigenesis2 raised a general interest in developing
potent and selective inhibitors against this kinase.
The recent discovery of ellagic acid as a potent inhibitors of CK23 prompted us to
undertake a computer-aided ligand optimization aimed at the simplification of
scaffold which in mammalian tissues might quickly undergo metabolic degradation to
urolitins.4 This analysis led to the discovery of the inhibitory properties of urolithin A
(IC50 = 0.3µM), a tricyclic natural metabolite of ellagic acid. Variably substituted
derivatives of urolithin A were also synthesized and exploited to perform a structureactivity relationship study. Among these derivatives a number of new powerful CK2
inhibitors were identified and subjected to a partial screening of selectivity. Two
compounds in particular, a bromo- and a nitroderivative of urolithin A, were found to
be competitive with respect to the phosphodonor substrate ATP, displaying Ki values
in the low nanomolar range (15 and 7 nM).
References
[1] Salvi M. et al. (2009) Biochim Biophys Acta 1793:847-859.
[2] Guerra B. and Issinger OG (2008) Curr Med Chem 15: 1870-1886.
[3] Cozza G. et al. (2006) J Med Chem 49: 2363-2366.
[4] Seeram N.P. et al. (2006) J Nutr. 136, 2481-2485.
68
Binding mode investigation and SAR studies of HIV-1 integrase
inhibitors
S. De Grazia,a L. De Luca,a R. Gitto,a S. Ferro,a Z. Debyser,b A. Chimirria
a
Dipartimento Farmaco-Chimico, Università-Messina, Italy. bMolecular Medicine,
Katholieke Universiteit-Leuven, Belgium.
[email protected]
1. Introduction
Many antiretroviral drugs are currently available to treat human immunodeficiency
virus type 1 (HIV-1) infection. However, their success is often limited by drug-related
toxicities, drug–drug/food interactions, and by the emergence of multidrug resistant
viral strains. Therefore, there is a continuous effort to design newer inhibitors
operating with different mechanisms. In recent years, HIV-1 integrase (IN) has
become an attractive target for designing antiretroviral agents because it has no
known human homologue, and thus there is a lower risk of disrupting normal cellular
processes.
Our research group has been recently engaged in
the stucture-function study of IN and in the
development of its inhibitors. Different 3D
pharmacophore models were performed and were
exploited to guide the indentification of new antiviral
agents.1-2
Herein we report an expanded SAR investigation of a
new series of benzylindoles which allowed us to
evidence the main chemical fragments involved in the
IN strand-transfer inhibition. The crystal structure of
the foamy virus IN in complex with its cognate DNA
(PFV-IN/DNA),3 was used to obtain more details about the binding mode of our new
compounds.
3. References
[1] Ferro S, De Luca L et al. J. Med. Chem. 2009, 52, 569-573.
[2] De Luca L, De Grazia S et al. Eu J Med Chem , 2011, 46, 756-764.
[3] Hare S, Gupta S S et al. Nature 2010, 464, 232-236.
Research supported by THINC project (HEALTH-F3-2008-201032)
69
Identification of Acridones as New Inhibitors of HCV NS5B
Polymerase Through Scaffold Hopping Strategy
F. Gosetto,a M. L. Barreca,a G. Manfroni,a S. Sabatini, a V. Cecchettia
Dipartimento di Chimica e Tecnologia del Farmaco, Università di Perugia, Via del
Liceo 1, 06123, Perugia
[email protected]
1. Introduction
Chronic infection by hepatitis C virus (HCV) often leads to severe liver disease such as
cirrhosis and hepatocellular carcinoma. Despite more than 20 years after the HCV
identification are elapsed, the current standard of care for treating HCV is based on
aspecific therapy often associated with severe side effects and low sustained virological
response. The identification of more effective and tolerated therapies are therefore urgently
needed. Among all the HCV proteins involved in the viral replication, the NS5B
polymerase is recognized as key target for therapeutic intervention. This enzyme offers a
wide range of possibilities for the discovery of new molecular entities as anti-HCV agents
since it has at least five druggable allosteric binding sites. Although a broad range of nonnucleoside inhibitors (NNIs) have been reported in literature,1 drugs active against NS5B
polymerase have not yet been approved by the FDA. Our research efforts are thus
addressed towards the identification of new chemotypes as allosteric NS5B polymerase
inhibitors, with a special focus on the development of Palm Site I NNIs (PSI-NNIs). Data
literature report detailed structural analysis of the ligand/protein contacts for different
inhibitors belonging to the hydroxyquinoline-benzothiazine (BTZ) and -benzothiadiazine
(BTDZ) classes, which are the most investigated PSI-NNIs.
2. Results
Virtual screening of in-house library suggested the rational design of a series of acridone
derivatives as potential bioisosteric replacement for the BTZ and BTDZ moieties. The work
hypothesis was validated by performing automated docking studies of the designed
compounds. The synthesis of the most promising derivatives was realized and the results of
biological evaluation on both enzymatic and cellular assays will be presented.2
3. References
[1]. Li, H.; Shi, S. T. Non-nucleoside inhibitors of hepatitis C virus polymerase: current progress and
future challenges. Future Med. Chem. 2010, 2(1), 121-141.
[2] This work is sponsored by Fondazione Cassa di Risparmio di Perugia.
70
Structure and function of human aromatase investigated by
molecular simulations
J. Sgrignani, A. Magistrato
CNR-IOM-Democritos National Simulation Center at SISSA Trieste (Italy) and
International School for Advanced Studies (SISSA), Trieste (Italy)
[email protected]
Aromatase is an enzyme of crucial biological importance in the biosynthesis of
estrogens, located in the endoplasmatic reticulum (ER) membrane. High levels of
estrogens are related with important pathological diseases. Recently the structure of
aromatase has been solved using X-Ray diffraction,1 however some structural aspects
related with its membrane bound nature are still unsolved.
This study aims at exploring the mechanism of this important enzyme via
molecular simulations. In particular we are exploring (1) the enzyme-membrane
interactions, (2) the molecular species (androstenedione and oxygen) involved in the
enzymatic reaction, and (3) the enzymatic reaction, using force field based molecular
dynamics simulations and hydrid quantum-classical (QM/MM) metadynamics
calculations.
To date our studies have provided, for the
first time, a molecular model of aromatase/ER
membrane interactions.
The structural features of the model have
been validated against the mutagenic data
available in the literature.
The QM/MM metadynamics calculations
necessary to characterize the aromatase reaction
mechanism pathway are currently in progress.
Filkckaksdag Fig 1. X‐ray structure of human aromatase. 3. References
[1] Ghosh D, Griswold J, Erman M, Pangborn W (2009) Nature 457:219-223.
71
Conventional and MW assisted synthesis and selective inhibition of
human monoamine oxidases of
(4,5-substituted-thiazol-2-yl)hydrazones
M. D’Ascenzio,a P. Chimenti,a D. Secci,a A. Bolasco,a S. Carradori,a M. Yáñezb
a
Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma
“La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy.
b
Departamento de Farmacología and Instituto de Farmacia Industrial, Universidad
de Santiago de Compostela, E-15782 Santiago de Compostela, Spain.
e-mail: [email protected]
1. Introduction
Monoamine oxidases are flavoenzymes involved in the control of intracellular
concentration of monoaminergic neurotransmitters and neuromodulators. They exist
in two isoforms: MAO-A and MAO-B. By inhibiting MAOs, it is possible to increase
the synaptic availability of such monoamines and enhance their physiological
functions. In previous studies conducted by our group of research, several substituted
(thiazol-2-yl) hydrazones have been studied as potent and selective hMAO
inhibitors.1-3
(4,5-Substituted-thiazol-2-yl)hydrazone derivatives were synthesized in high
yields. The potential inhibitory effects of the test drugs on hMAO activity were
investigated using microsomal MAO isoforms. Tested compounds showed to be
active at micromolar concentrations. All derivatives bearing a naphthalene moiety
possessed a greater inhibitory activity on hMAO-A rather than on hMAO-B. In fact,
they better adapt themselves to the active site of hMAO-A, which is characterized by
a single large hydrophobic cavity. The conventional synthesis was then compared to
the MW-assisted one, at different conditions of temperature, power, pressure, and
catalysis.4
3. References
[1] Chimenti, F. et al. J. Med. Chem. 2008, 51, 4874-4880.
[2] Chimenti, F. et al. J. Med. Chem. 2010, 53, 6516-6520.
[3] Chimenti, F. et al. Bioorg. Med. Chem. 2010, 18, 5715-5723.
[4] Chimenti, F.; D’Ascenzio, M. et al. Med. Chem. Commun. 2010, 1, 61-72.
72
Novel chiral sigma 1 receptor ligand potentiating NGF-induced
neurite outgrowth in PC12 cells
A. Pedrali,a F. Reversi,b D. Curti,b D. Rossi,a O. Azzolina,a S. Collinaa
a
Department of Drug Sciences, University of Pavia, Viale Taramelli 12, I-27100
Pavia; bDepartment of Legal Medicine, Forensic and Pharmaco-toxicological
Sciences, Lab. of Cellular and Molecular Neuropharmacology, University of Pavia,
via Ferrata 9, 27100, Pavia
[email protected]
1. Introduction
Sigma1 receptors (σ1-Rs) are highly expressed in CNS. Literature evidences
suggest that σ1-R agonists stimulate neurite outgrowth in PC12 cells and also in
motoneurons in organotypic spinal cord cultures.1 Moreover, σ1-R and their
modulation play a role in many different pathophysiological conditions including
psychosis, such as anxiety, schizophrenia, depression, drug addiction,
neurodegenerative diseases and cancer.
Our research has been recently focused on the synthesis and pharmacological
evaluation of σ1-R ligands based on arylalkenyl- and
arylalkylaminic scaffolds. The affinities of all
compounds towards σ, k-, µ-opioid and NMDA
receptors were evaluated.2 (R/S)-1 resulted the most
interesting compound.
The effect of (R/S)-1 on NGF-induced neurite outgrowth in PC12 cells was evaluated
and σ1-R agonist/antagonist profile was drawn.
In the mean time, our efforts have been addressed towards the preparation of 1 pure
enantiomers. To this aim, both direct chromatographic chiral resolution of the racemic
mixture and asymmetric synthesis were investigated. A chiral HPLC method suitable
for both analytical and preparative purposes was developed.
3. References
[1] Guzmán-Lenis, M.S. et al. Neuroscience, 2009, 162, 31-38.
[2] Collina, S. et al Bioorg. Med. Chem., 2007, 15, 771-783.
73
New σ Receptor Ligands with Neuroprotective Effect: Synthesis and
Resolution of cis-(±)-Methyl (1R,2S/1S,2R)-2-[(4-Hydroxy-4phenylpiperidin-1-yl)methyl]-1-(4methylphenyl)cyclopropanecarboxylate [(±)-PPCC]
O. Prezzavento,a A. Campisi,b C. Parenti,c S. Ronsisvalle,a G. Aricò,c E. Arena,a G.
M. Scoto,c A. Vanella,b G. Ronsisvallea
a
Department of Pharmaceutical Sceinces, Medicinal Chemistry Section, bDepartment
of Biological Chemistry, Medical Chemistry and Molecular Biology, cDepartment of
Pharmaceutical Sceinces, Pharmacology Section, University of Catania, V.le A.
Doria 6, 95125 Catania, Italy,
[email protected]
Sigma receptors are distributed in both central nervous system and peripheral
tissues and are involved in a variety of physiological activities. Maurice at all. showed
that activation of σ1 prevents neuronal death caused by excitotoxicity.1 It has also
been shown that hyperstimulation of glutamate receptors provoke a neuronal loss.2
Previous study of Campisi et all. demostrate that glutamate expoisure of differentiated
astricytes provokes an increase of [Ca2+], a decrease of ATP concentration and ROS
production, mithocondrial impairment and activation of a variety of calciumdependent proteins.3 Campisi et all. also showed how the recovery of cellular redox
state given by antioxidants, is closely linked to a contemporary and dose-dependent
reduction of transglutaminase-2 (TG2) up-regulation induced by glutamate.4 We have
previous reported the synthesis and biological profiles of σ selective ligand (±)-PPCC
and we demonstrated that this compound was able to modulate TG2 expression in
primary rat astroglial cell cultures.5,6 We here report the synthesis of pure
diastereoisomers and their capability of restoring the astroglial oxidative status
modified by glutamate, counteracting also TG2 overexpression.7 To assess protective
effects of sigma ligands on DNA damages induced by glutamate, we are investigating
and reporting some data of p21 expression.
References
[1] Maurice, T. et all. Pharmacol Ther. 2009, 124, 195-206
[2] Matute, C. et all. Eur. J. Pharmacology 2002, 447, 239-246
[3] Campisi et all. FEBS Lett. 2004, 578, 80-84
[4] Campisi et all. Brain Res. 2003, 978, 24-30
[5] Prezzavento O. et all. J. Med. Chem. 2007, 50, 951-961
[6] Przzavento O. et all. Life Sci. 2008, 82, 49-53
[7] Prezzavento O. et all. J. Med. Chem. 2010, 53, 5881-5885
74
Synthesis and preliminary biological activity study of a novel class
nicotinic acetylcholine receptors (nAChRs) ligands structurally
related to anatoxin-a
C. Rullo,a D. Simoni,a R. Rondanina, P. Marchettia, R. Baruchello, a G. Grisolia,a G.
Barbato,a S. Mangiola,a R. Giovannini,b# C. Marchioro,b G. P. Borea,c S. Merighi,c D.
Donatib*
a
Department of Pharmaceutical Sciences, University of Ferrara, Via Fossato di
Mortara 17/19, 44100, Ferrara, Italy; bMedicines Research Centre, GlaxoSmithKline
spa, Via Fleming, 4, 37135 Verona; #BI Research Italia S.r.l. Via Lorenzini n. 8 –
20139 Milano, Italy; *Medicinal Chemistry Department,Oncology Business Unit,
Nerviano Medical Sciences srl, Viale Pasteur, 10, 20014 Nerviano (MI); cDepartment
of Clinical and Experimental Medicine, University of Ferrara, Via Fossato di
Mortara 17/19, 44100, Ferrara, Italy
[email protected]
Neuronal nicotinic receptors (nAChRs) are still object of extensive studies since of
their pharmacological potentialities for treatment of CSN disorders such as Parkinson’
disease, Alzheimer disease and pain.1 Anatoxin-a 1, an alkaloid extracted from a
filamentous freshwater blue-green algae, Anabaena flosaquae, has been recently the
subject of structural modifications in the search of novel agents characterized by high
selectivity toward different Ach receptor subtypes.2
Here, we give details of a synthetic
R
N
strategy that allowed us to obtain a
O
R
O
small series of derivatives structurally
N
related to anatoxin-a. Focusing mainly
O
NH
NH
NH
our attention on the methylketone side
chain, two groups of compounds were
obtained, one bearing an isoxazole ring (+)-Anatoxin-a, 1
2
3
at the side chain 2, and the other with a
conformationally restricted isoxazole-based motif 3.
All compounds were tested as receptor ligands for nAchRs by inhibition of (3H)
citysine.
References
[1] Daly, J. W. Cell. and Mol. Neurobiology, 2005, 25, 3/4, 513-552.
[2] Aráoz, R., Mólgo, J., Tandeau de Marsac, N. Toxicon, 2010, 56, 813-828.
75
Synthesis and antiproliferative activity of indazole derivatives
D. Raffa,a B. Maggio,a M.V. Raimondi,a S. Cascioferro,a F. Plescia,a M. Tolomeo,b G.
Daidonea
a
Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari e bCentro
Interdipartimentale di Ricerca in Oncologia Clinica, Università degli Studi di
Palermo
[email protected]
1. Introduction
Indazole nucleus represents a very attractive scaffold to obtain new molecules
endowed with antineoplastic activity.1 On the basis of these literature data we have
designed some indazole derivatives such as N-indazolylbenzamides and N-indazolylN’-phenylureas as potential CDK1 inhibitors. In fact the above compounds contain
the structural feature, common to the majority of CDK inhibitors, requested to make
hydrogen bonds with the molecular fork present in the hinge region of CDKs. 2
The N-indazolylbenzamides 1 were obtained by reacting aminoindazoles and
substituted benzoylchlorides. By reacting 3-aminoindazoles and phenylisocyanates,
the derivatives 2, 3 and 4 were obtained. Among the synthesized compounds, some
derivatives 1 resulted to be CDK1 inhibitors showing IC50 values in the range 4.410.0 µM. Some representative compounds of type 2, 3 and 4 were evaluated for their
in vitro antiproliferative activity against the full NCI tumor cell lines panel. The most
active of the tested compounds showed a mean GI50 value of 1.90 µM.
H
N
N
O
N
H
R
N
1
HN
N
H
O
N
R2
NH2
R3
N
O
HN
H
N
H
N
O
N
H
N
H
N
R1
R2
R2
N
O
R2
3
4
2
3. References
[1] L. Huang et al. Bioorg. Med. Chem. 2006, 14, 528-536.
[2] F. Candurri et al. Current Computer-Aided Drug Design 2005, 1, 53-64.
76
Antiproliferative activity and mechanism of action of Peniocerol, a
sterol isolated from Myrtillocactus geometrizans
A. Braga,1 L. Dalla Via,1 A. García-Argáez,1 M. Martínez-Vázquez,2 A. Toninello3
1
Department of Pharmaceutical Sciences, University of Padova, Italy; 2Chemistry
Institute, National Autonomous University of Mexico; 3Department of Biological
Chemistry, University of Padova, Italy.
[email protected]
Several natural compounds can modulate apoptosis (programmed cell death)
pathways that are frequently altered in human cancers. In this connection, these
compounds may provide novel opportunities for the development of new anti-cancer
drugs.1
Myrtillocactus geometrizans is a mexican plant known as “garambullo”. Mexican folk
medicine attributes to the aerial parts of this species anti-inflammatory property and
actually some natural compounds from Myrtillocactus geometrizans exhibited antiinflammatory and cytotoxic activity.
From roots and aerial parts of Myrtillocactus geometrizans was isolated the sterol,
peniocerol (Pen).2
Cholest-8-ene-3β,6α-diol (Pen)
In this study we report the antiproliferative activity of Pen and investigate the
mechanism of action responsible of its cytotoxicity.
In particular, the sterol Pen showed a significant capacity to inhibit cell growth on
human tumor cell lines (A-431, HeLa and Hep-G2).
As it is well-known, apoptosis can be triggered by extrinsic or intrinsic pathway.
Due to the crucial role played by mitochondria on intrinsic programmed cell death, it
was evaluated the activity of Pen on rat liver mitochondria (RLM). Results show that
this sterol behaves as a typical mitochondrial permeability transition inducer in RLM
by means of reactive oxygen species (ROS) production with consequent oxidative
stress establishment and release of pro-apoptotic factors.
References
[1] Fulda, S., 2010. Modulation of apoptosis by natural products for cancer therapy. Planta Med 76:
1075-1079.
[2] Salazar, J.R. et al., 2011. Cytotoxic and anti-inflammatory activities of Chichipegenin, Peniocerol
and Macdougallin isolated from Myrtillocactus geometrizans. Nat Prod Res. In press.
77
Optimization of glucocorticoids sensitivity in vitro assay
E. Cuzzoni, S. De Iudicibus, R. Franca, G. Decorti
Department of Life Science, University of Trieste
[email protected]
1. Introduction
With the actual risk-adapted poly-chemotherapeutic approaches, more than 80% of
patients affected by acute lymphoblastic leukemia (ALL) achieves a successful
remission. In vivo response to a 7-day steroid pre-phase has emerged as the strongest
prognostic factor of outcome.1 In vitro prediction of lymphocytes intrinsic sensitivity
to chemoterapics is usually determined by MTT-test, but this assay is scarcely
reproducible for glucocorticoids (GCs). The aim of this study was to establish a cell
viability assay based on the [3H] thymidine incorporation in order 1) to evaluate
steroid sensitivity in the normal population (by means of methylprednisolone (PDN)
and dexamethasone (DXM) IC50); 2) to establish cut-off values for classifying
subjects as GC resistant or sensitive; 3) to determine the role of polymorphisms of
genes involved in GC pharmacokinetics and pharmacodynamics in GCs response. The
final goal is to transpose this methodological approach to ALL blasts and get a
relaible tool to improve the clinical management of the disease.
2. Results and discussion
GC-inhibition in concanavalin-A stimulated peripheral blood mononuclear cell
(PBMC) proliferation from 27 healthy blood donors was investigated. Median IC50 of
PDN and DXM were used as reference values to categorize subjects into good or poor
responders. Comparison of individual profile revealed a very similar pattern of
response to both steroids. The [3H] thymidine incorporation assay was performed also
on PBMC of 20 patients affected by inflammatory diseases, whose clinical steroid
response was already known. Data were collected only for PDN. A significant
difference in IC50 values was observed between patients responding to GCs and those
who were resistant (p=0.0098, Mann-Whitney t-test). Genetic polymorphisms did not
show any significant association with IC50. These results suggest that this in vitro cell
viability test could be useful for physicians to predict precociously the GC response in
leukemia and other diseases, but larger numbers are required to assess cut-off
reference values of sensitivity or resistance.
3. References
[1] Pui CH, Carroll WL, Meshinchi S, Arceci RJ. J Clin Oncol. 2011 Feb 10;29(5):551-65.
78
The long and winding road of the c-Kit juxtamembrane domain
V. Dal Col,a E. Laurini,a S. Pricl,a P. Posocco,a M. A. Pierotti,b P. Casali,b S. Pilotti,b
E. Tamborinib
a
Dipartimento di Ingegneria industriale e dell’ Informazione tecnologica, Università
di Trieste, Italia; bDipartimento di Patologia sperimentale, Istituto Nazionale per lo
Studio e la Cura dei Tumori, Milano, Italia
[email protected]
1. Introduction
Gastrointestinal stromal tumors (GISTs) are the most common primary
mesenchymal tumors of the human gastrointestinal tract, and most GISTs express
constitutively activated c-Kit oncoproteins.1 Several clinical studies demonstrate that
tumors showing mutation in exon 11 (encoding the juxtamembrane domain) respond
better than all the other GIST tumoral genotypes. The KIT wild-type sequence folds
into a series of β- hairpin structures. The structure, stability and folding of β- hairpin
structures has been the object of many studies. Recently, several works reported
successful simulations of reversible hairpin folding of different peptides in explicit
water at native folding conditions through self-guided molecular dynamics (SGMD)
simulations.2
Using SGMD simulations we study the reversible
folding events of wild-type and mutated c-Kit JMX
domains. The mutations considered were the two-residue
deletion Δ559-560 (Fig.1) and the missense point mutation
V560G, both known to constitutively activate c-Kit in
GISTs. This work aids to support the clinical evidence of a
better response to Imatinib, an ATP-competitive TKIs, of
KIT exon 11 mutant in comparison with WT receptor and
provides the first atomistic description of the output of
several clinical studies of GIST treated with Imatinib.
Fig.1
3. References
[1] Hirota S, Isozaki K, Moriyama Y, Hashimoto K, Nishida T, Ishiguro S et al. , Science, 1998; 279:
577-580
[2] Wu X-W, Brooks BR Byophys J, 2004; 86: 1946-1958
79
Design and synthesis of multifunctional ligands endowed with
antiproliferative activity
E. Giacomini,a A. Gambuzzi,a M. Recanatini,a M. Tolomeob, M. Robertia
a
Dipartimento di Scienze Farmaceutiche, Università di Bologna, via Belmeloro 6,
40126 Bologna, Italy. bCentro Interdipartimentale di Ricerca in Oncologia Clinica,
Università di Palermo, Via del Vespro 129, 90127 Palermo, Italy.
[email protected]
1. Introduction
Over the past years, we have been engaged in a project aimed at identifying novel
biologically active small molecules, which can be either antitumor leads candidates,
or valuable chemical tools to study molecular pathways in cancer cell. As a part of
this project, in view of the multifactorial mechanistic nature of cancer, we planned to
develop some hybrid compounds using the multitarget-directed drug design strategy.
The desired new chimeric molecules were obtained by linking together
Suberoylanilide Hydroxamic Acid analogues, targeting histone deacetylases, and
variously substituted stilbene, biphenyl or terphenyl derivatives able to interfere with
cell cycle progression.1,2 The distinct synthons were separately synthesized following
different protocols previously described by us,1,2 and linked via amide bond taking
advantage of appropriate coupling conditions. The new compounds are currently
under biological study.
3. References
[1] Pizzirani D., et al., ChemMedChem, 3, 345 (2008)
[2] Pizzirani D., et al. J. Med Chem, 52, 3012 (2009)
80
Functionalized Fullerenes as nanocarriers for anticancer drugs
M. Lucafò,a S. Pacor,a C. Fabbro,b S. Zorzet,a T. Da Ros,b M. Prato,b G. Savaa
a
Dipartimento di Scienze della Vita, bDipartimento di Scienze Chimiche e
Farmaceutiche, Università di Trieste.
[email protected]
1. Introduction
The use of nanomaterials to diagnose and treat diseases has been a goal for scientists
ever since the advent of methods to manipulate molecules at the nanometer scale.1
Fullerenes (C60) are among the nanomaterials being investigated for diagnostic and
therapeutic modalities. In particular they might be suitable vectors for drug delivery
due to their small size, large surface area and characteristics of solubility.2 However,
the interactions of C60 derivatives with biological systems and cells are not yet
understood. The cytotoxicity of different fullerenes, the process of cell recognition
and the mechanism of cellular uptake are the subject of this work.
Functionalized fullerenes were synthesized by 1,3-dipolar cycloaddition of
azomethine ylides to the C60 cage. This is a versatile reaction to modify C60 in terms
of water-solubility and to offer an anchor point for the attachment of other moieties.
Cell cytotoxicity on human mammary carcinoma cell lines, evaluated with the MTT
test and further confirmed by a flow cytometry approach with DiOC6 probe and PI
analyses, did not show any necrotic nor apoptotic effect even at the higher
concentration used (µM) after a long lasting cell exposure (72h) to twice of the tested
compounds. Cell uptake and internalization of the less cytotoxic, fluorescein-labeled,
compound, evaluated by flow cytometry and confirmed by confocal microscopy,
suggests an interesting uptake of this compound by cells and, so far, it can be
considered a suitable vector to deliver anticancer agents to tumour cells.
3. References
[1]Dellinger A et al. Uptake and distribution of fullerenes in human mast cells. Nanomedicine: NBM
2010;6:575-581.
[2]Zhanga LW et al. Endocytic mechanisms and toxicity of a fullerene in human cells. Tox. Lett.
2009;191:149-157.
81
Following the indole brick road to identify small molecule cell
permeable activators of protein arginine methyltransferase 4
(PRMT4/CARM1)
C. Milite,a S. Castellano,a M. Viviano,a A. Yamani,a A. Spannhoff,b D. Cheng,b R.
Ragno,c E. Novellino,d A. Mai,c M.T. Bedford,b G. Sbardellaa
a
Dipartimento di Scienze Farmaceutiche e Biomediche, Università degli Studi di
Salerno; bThe University of Texas, M.D. Anderson Cancer Center; cIstituto Pasteur –
Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco,
Sapienza Università di Roma; dDipartimento di Chimica Farmaceutica e
Tossicologica, Università di Napoli “Federico II”
[email protected]
1. Introduction
In 2004, a small molecule screening study led some of us to the identification of
the symmetric urea derivative arginine methyltransferase inhibitor 1 (AMI-1) as a
potent arginine methylation specific inhibitor. By molecular modeling studies, we
proposed that AMI-1 spans both the SAM and Arg binding sites without fully
occupying them.1 As the structure of AMI-1 is related to suramin-like sulfonated
ureas, we supposed that it is likely poorly bioavailable. Therefore, we designed and
synthesized carboxy analogues of AMI-1(c-AMI-1) that are nearly as potent as the
parent compound while retaining PRMT selectivity.2
Here we report the replacement of
the hydroxynaphthalene moiety with
the bioisosteric indole framework, a
“privileged”
structural
motif
commonly found in pharmaceutical
drugs and natural products. Following
this “indole brick road”, we
unpredictably identified the first selective and cell-permeable PRMT4/CARM1
activators (i-AMA).
3. References
[1] Ragno, R.; Simeoni, S.; Castellano, S.; Vicidomini, C.; Mai, A.; Caroli, A.; Tramontano, A.;
Bonaccini, C.; Trojer, P.; Bauer, I.; Brosch, G.; Sbardella, G. J. Med. Chem. 2007, 50, 1241-1253.
[2] Castellano, S.; Milite, C.; Ragno, R.; Simeoni, S.; Mai, A.; Limongelli, V.; Novellino, E.; Bauer, I.;
Brosch, G.; Spannhoff, A.; Cheng, D.; Bedford, Mark T.; Sbardella, G. ChemMedChem 2010, 5, 398414.
82
Phenanthroline-based metal complexes: further insight into the
recognition of G-quadruplex structures
C. Musettia, S. Biancoa, G. Marsona, A.P. Krapchob, M. Palumbo,a C. Sissia
a
Dipartimento di Scienze Farmaceutiche, Università di Padova, via Marzolo 5, 35100
Padova; bChemistry Department, University of Vermont, Cook Science Building,
Burlington 05405 (Vermont)
[email protected]
G-quadruplexes are special secondary structures formed by the folding of guanine
rich nucleic acid tracts. Since their formation is well established in important regions
of the genome, such as telomeres and the regulatory sequences of many genes, these
structures are likely to play important roles in the regulation of many biological
events in the cell. Specifically, G-quadruplexes have become valid targets in the
development of anticancer drugs.
Recently, a number of metal ion complexes have been evaluated as G-quadruplex
binders with the goal of finding new compounds that selectively interacts to this
particular structure with little affinity for the double helix.
In previous works, we focused on the phenanthroline scaffold and we investigated if
G-quadruplex DNA recognition can be a result of metal ion-driven ligand assembly
through coordination1,2. Herein, we further analyzed the G-quadruplex interaction
properties of phenanthroline derivatives substituted with aromatic moieties at position
2. By using circular dicroism, UV spectroscopy and microcalorimetric techniques we
show that metal ion coordination can constrain the system to a planar arrangement
thus incrementing selectivity of the ligand for G-quadruplex structures in comparison
to dsDNA. This likely reflects the possible stacking of the resulting extended aromatic
surface on a G-quartet. On the other hand, free ligand can adopt several not planar
conformations which allows proper fitting in the dsDNA.
Our data yield useful information to rationally design new agents for effective and
selective recognition of defined G-quadruplex arrangements.
References
[1] Musetti, C.; Lucatello, L.; Bianco, S.; Krapcho, A. P.; Cadamuro, S. A.; Palumbo, M.; Sissi, C.;
Dalton Trans., 2009, 19, 3657-3660
[2] Bianco, S.; Musetti, C.; Waldeck, A.; Sparapani, S.; Seitz, J. D.; Krapcho, A. P.; Palumbo, M.;
Sissi, C.; Dalton Trans., 2010, 39, 5833-5841
83
Molecular modeling investigation of EGFR mutations and their
response to Iressa
F. Santese a, E. Laurini a, S. Pricl a, M. A. Pierottib, S. Pilottib, E. Tamborinib
a
Dipartimento di Ingegneria Industriale e dell'Informazione, Università degli Studi di
Trieste, Via A. Valerio, 10 34127 Trieste; bDipartimento di Patologia Sperimentale,
Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano
[email protected]
1. Introduction
The importance of EFGR in driving tumor genesis in NSCLC, as well as other types
of cancer, has been clinically validated and inhibitors of EGFR have been approved
for treatment of a series of cancer. ATP competitive inhibitors of the TKD domain
include gefitinib (Iressa®).1 However, mutations in the tyrosine kinase domain have
been shown to be strongly associated with the response of NSCLC patients to
gefitinib treatment. To improve the understanding of the acquired resistance
mechanism, molecular modeling is a good tool for this purpose. Accurate
computation of free energies of binding remains a key challenge for computer-aided
drug design.2-3
In this work, we performed simulations of three different point mutation of EGFR in
complex with gefitinib: L858R, T790M and R831H.
The first two mutations are the most frequent in patients
with NSCLC; L858R was found to be a responsive
mutation and T790M a resistance mutation, confirming
experimental data. The results of R831H (Fig.1), a less
common and recently discovered mutation, show a
good responsive behaviour but less than L858R. The
agreement of our results with clinical data proves the
reliability of molecular simulations to predict the
behavior of this kind of complex.
Fig.1
3. References
[1] [email protected]://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm (accessed
Jan 22, 2009)
[2] Gohlke, H.; Klebe, G. Angew. Chem., Int. Ed. 2002, 41, 2645–2676
[3] Jorgensen, W. L. Science 2004, 303, 1813–1818.
84
Synthesis and biological evaluation of Δ 2-isoxazoline derivatives as
DNA methyltransferase 1 (DNMT1) inhibitors
M. Viviano,a S. Castellano,a P. Conti,b L. Tamborini,b A. Pinto,b S. Sicher,b D. Kuck,c
C. De Micheli,b J.L. Medina-Franco,d F. Lyko,c G. Sbardellaa
a
Dipartimento Scienze Farmaceutiche e Biomediche, Università degli Studi di
Salerno,Italy; bDipartimento di Scienze Farmaceutiche ‘Pietro Pratesi’, Università
degli Studi di Milano, Italy; cDivision of Epigenetics, Deutsches
Krebsforschungzentrum, Germany; dTorrey Pines Institute for Molecular Studies, FL
34987, USA.
[email protected]
1. Introduction
Being responsible of DNA methylation, DNA methyltransferases (DNMTs) are
recognized as valuable epigenetic targets for the development of anti-cancer therapies.
Recently we synthesized a series of constrained analogues of procainamide,1 a
specific inhibitor of the maintenance methyltransferase DNMT-1.2 A few of them
resulted more effective in inhibiting the activity of the target.
Taking advantage of microwave and continuous–flow techniques,3 we replaced the
oxazoline ring (1-4) with the more stable and versatile 2-isoxazoline (5a-k) and
introduced
a
new
N
N
N
N O
conformational restriction
a, R = Ph
O
b, R = p-OCH Ph
R
(6a-k). Docking studies and
5a-k
O
c, R = p-NO Ph
X
1a,b
d, R = p-OTsPh
N
2a,b
biological assays were
N
e, R = p-COOHPh
H
f, R = p-NH Ph
N
H
N
N
performed to evaluate the
g, R = p-OHPh
procainamide
O
O
h, R = PhCH
N
potency of such derivatives
i, R = p-OCH PhCH
X
3a,b
j, R = p-NO PhCH
R
X = NO , NH
N
in inhibiting the activity of
4a,b
k, R = p-NH PhCH
6a-k
Figure 1
DNMT-1.
3
2
2
2
2
3
2
2
2
2
2
2
2
3. References
[1] Castellano, S.; Kuck, D.; Sala, M.; Novellino, E.; Lyko, F.; Sbardella, G. J. Med. Chem. 2008, 51,
2321.
[2] Lee, B. H; Yegnasubramanian, S.; Lin, X.; Nelson, W. G. J. Biol. Chem. 2005, 280, 40749.
[3] Castellano, S.; Tamborini.; Viviano, M.; Pinto, A.; Sbardella, G.; Conti, P. J. Org. Chem., 2010, 75
(21), 7439–7442
85
The therapeutic potential of p53 reactivation by MDM2 antagonists
in human glioblastoma multiforme cells
S. Bendinelli,a B. Costa,a P. Gabelloni,a E. Da Pozzo,a I. Gomez-Monterrey,b A.
Bertamino,c P. Campiglia,c E. Novellino,b C. Martinia
a
Dip. Psich. Neurobiol. Farmacol. e Biotecnol., Università di Pisa, bDip. Chim.
Farm. e Tossicol., Università di Napoli “Federico II”; cDepartment of
Pharmaceutical Science, University of Salerno, I-84084 Fisciano, Salerno, Italy
[email protected]
1. Introduction
Protein p53 is the key element of a network of signalling pathways essential for
cell growth regulation and apoptosis. The levels and activity of p53 are controlled in
large part by MDM2, an E3 ubiquitin-ligase enzyme that promotes its degradation.1 In
the human glioblastoma multiforme (GBM) both overexpression and excessive
nuclear accumulation of MDM2 have been evidenced. Such data prompted us to
investigate whether the nutlin-3, a small molecule that specifically blocks the
interaction of MDM2 with p53,2 and the novel MDM2 antagonist, ISA27, affected the
proliferation/viability of the human GBM cell line, U87MG.
The MDM2 antagonists, nutlin-3 and ISA27, inhibited the viability/proliferation of
U87MG cells in a dose and time-dependent manner, suggesting the specificity of the
effect observed. The cytofluorimetric analyses demonstrated that these MDM2
antagonists were able to induce growth cell arrest. Specifically, nutlin-3 caused
accumulation of cells in the G1 phase after any tested incubation time. ISA27 caused
inhibition of G1/S transition after 24h and inhibition of G2/M transition after 72h. In
addition, ISA27 induced apoptotic cell death. We evidenced that ISA 27 had noncytotoxic effects on untransformed circulating cells, as tested in human
lymphomonocytes. Experiments are on-going to evaluate the influence of these
MDM2 antagonists on expression of specific genes regulated by p53.
3. References
[1] Lyubomir T. Vassilev, 2006, review, Trends in Molecular Medicine
[2] Sanjeev Shangary and Shaomeng Wang, 2009, Annu Rev Pharmacol Toxicol
86
Folding vs. charge: understanding selective target recognition by the
thrombin aptamer
G. Marson, M. Palumbo, C. Sissi
Department of Pharmaceutical Sciences, via Marzolo 5, 35131 Padova, Italy
[email protected]
The use of nucleic acids as drugs is a consistently growing approach. Different
therapeutical strategies can take advantage of the biological and biophysical
properties of DNA and RNA to properly modulate a selected cellular pathway.
A peculiar characteristic of these molecules is their structural flexibility which
allows them to assume distinct foldings depending on their sequence or
environment.1
During the last twenty years, this has led to the theoretical and experimental
development of oligonucleotide aptamers, short sequences that can recognize a
target with affinity and specificity comparable to antibodies. A leading example is
provided by the Thrombin Binding aptamer (TBA), a 15-mer DNA selected by its
high affinity for the coagulation factor.2 The very stable complex formation is the
result of a combination of complementary protein-DNA recognition promoted by
the aptamer sequence and folding as well as of electrostatic interactions generated
by the charge balance at the binding site.
This DNA sequence represented the starting milestone for the design of a series
of new Trombin Binding Aptamers that shares a common G-quadruplex structure
but that have different binding sites on thrombin.3 The recognition of distinct
thrombin exosite is supposed to be largely driven by the DNA sequence which
forms loops of variable bases composition.
Herein, we investigate the relative role of the DNA sequence and charge in
defining the biological properties of the resulting aptamer-thrombin complexes.
Thus, we compared the thrombin binding modes and inhibition properties of
selected TBAs to those of unrelated single stranded oligonucleotides, that do not
adopt a defined folded structure. Upon changing the length and the sequence of
these linear sequences we can attribute an unequivocal role to the peculiar TBAs'
conformation in defining the specifity of thrombin-DNA interaction. The relevance
of aptamer folding rather then charge density for effective and selective recognition
of Thrombin exosites emerged. Additionally, a correlation between the aptamers
loop sequence and their localization on the protein surface could be drown.
References
[1] Nagatoihi, S., Circular dichroism spectra demonstrate formation of the thrombin binding DNA
aptamer G-quadruplex under stabilizing-cation-deficient conditions. Biochem. Biophys. Res. Commun.
(2007), 352, 812–817
[2] Tasset, D., M., Oligonucleotide Inhibitors of Human Thrombin that Bind Distinct Epitopes. J. Mol.
Biol. (1997), 272, 688-698
[3] Smirnov, I., Effect of Loop Sequence and Size on DNA Aptamer Stability. Biochemistry (2000),
39, 1462-1468
87
Photoactive bait peptides for identifying enzyme-substrate and
protein-protein interactions
D. Rotili,a B. M. Kessler,b C. J. Schofield,c A. Maia
a
Dipartimento di Chimica e Tecnologie del Farmaco, “Sapienza” Università di
Roma, P.le Aldo Moro 5, 00185 Roma, Italia, bNuffield Department of Medicine,
University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK, cDepartment of
Chemistry, University of Oxford, Mansfield Road, Oxford, OX1 3TA, UK
[email protected]
1. Introduction
Significant advances in the study of protein-protein interactions have been enabled
by linking affinity purification techniques to mass spectrometric analyses. However, it
is difficult to maintain transient interactions during purification, particularly when
working at, often low, endogenous levels. One solution is to irreversibly cross-link
molecules, e.g. by a photoactivated process.1
Here, we report a relatively simple strategy that involves photoactivated crosslinking of “bait” peptide fragments of proteins to other proteins in crude cell lysates.
We tested this method by studies on the role of post-translational hydroxylation in the
human hypoxic response system.2
Overall, our results reveal the potential of a photoactivated peptide cross-linking
approach for “capturing” enzyme-substrate and protein-protein interactions that are
dependent on post-translational modifications.
3. References
[1] Salisbury CM, Cravatt BF. J. Am. Chem. Soc., 2008,130,2184-2194.
[2] Rotili D, Altun M, Hamed RB, Loenarz C, Thalhammer A, Hopkinson RJ, Tian YM, Ratcliffe PJ,
Mai A, Kessler BM, Schofield CJ, Chem. Commun., 2011,47,1488-1490.
88
New non steroidal agonists of estrogen receptor β
F. Minutolo,a S. Bertini,a, A. De Cupertinis,a T. Tuccinardi,a A. Martinelli,a K. E.
Carlson,b J. A. Katzenellenbogen,b M. Macchiaa
a
Dipartimento di Scienze Farmaceutiche, Via Bonanno 6, 56126, Pisa, Tel.
05052219500 – Fax 0502219605; bDept. of Chemistry, University of Illinois, 600 S.
Mathews Avenue, Urbana, IL 61801, USA.
[email protected]
ERβ selective agonists (SERBAs) are considered as potential active agents in
therapeutic areas such as prostate hyperplasia and cancer, bone loss, arthritis, and
intestinal inflammation; the beneficial estrogen effects produced by a highly selective
ERβ stimulation are not associated with the undesired proliferative effects on breast
and uterus (mediated by the ERα-subtype).
The ERβ pharmacophoric model is characterized by a central “core” (represented
by a phenyl, an heterocycle or an alkene), a phenolic ring, and a para-hydroxyphenyl
function opposite the phenolic ring (Figure 1). Accordingly to the ERβ
pharmacophore, a series of monoaryl-substituted salicylaldoximes were synthesized
by our research group. The salicylaldoxime moiety was introduced as a bioisosteric
replacement of the estrogen phenolic ring, because it can form, through an intramolecular H-bond, a six-membered pseudo-cycle with an exocyclic hydroxyl group.
Compound 1, the simplest member of this series, showed a good level of ERβselectivity, although its absolute affinity was not satisfactory. Then, we explored the
effect due to the inversion of the position between the phenolic and oxime functions
by synthesizing derivative 2, which, although less ERβ-affine and ERβ-selective than
compound 1, is able to participate to a peculiar interaction with a threonine residue of
the binding cavity of ERβ, as highlighted by docking studies. The introduction of a
chloro substituent in the central scaffold (compound 3) led to the best results in terms
of ERβ-affinity (RBA = 130% with respect to estradiol) and a noticeable degree of
ERβ-selectivity. Furthermore, the introduction of a meta-fluorine atom in the
peripheral 4-aryl substituent of 3 (compound 4) led to an increased in ERβ-selectivity
together with a parallel slight decrease in ERβ-affinity (RBA = 87%).
F
OH
substituent
a
HO
central
core
HO
ERβ Pharmacophoric
model
Cl
HO
N
H
c
Cl
F
H
N
O
OH
b
a
RBA ERα = 0.007%
1 RBA ERβ = 0.553%
β/α = 79
OH
Cl
c
OH
HO
O
RBA ERα = 1.88 %
4 RBA ERβ = 87.11 %
β/α = 46
N
H
HO
N
H
b
O
RBA ERα = 0.064%
2 RBA ERβ = 2.64 %
β/α = 41
OH
O
RBA ERα = 4.46%
3 RBA ERβ = 130.3 %
β/α = 30
Figure 1. Compounds 1-4 deriving from ERβ Pharmacophore. The relative binding affinity (RBA) are reported as percentages
(%) of that of estradiol, which is set at 100%. Kd of estradiol is 0.2 nM for ERα and 0.5 nM for ERβ. Ki values for the new
compounds can be readily calculated by using the formula: Ki = (Kd[estradiol]/RBA) × 100.
References
[1] F. Minutolo, R. Bellini, S. Bertini, I. Carboni, A. Lapucci, L. Pistolesi, G. Prota, S. Rapposelli, F.
Solati, T. Tuccinardi, A. Martinelli, F. Stossi, Kathryn E. Carlson, B. S. Katzenellenbogen, J. A.
Katzenellenbogen, and M. Macchia, J. Med. Chem. 2008, 51, 1344–1351.
[2] F. Minutolo, S. Bertini, C. Granchi, T. Marchitiello, G. Prota, S. Rapposelli, T. Tuccinardi, A.
Martinelli, J. R. Gunther, K. E. Carlson, J. A. Katzenellenbogen, and M. Macchia, J. Med. Chem. 2009,
52, 858-867.
89
Regulation of angiogenesis by α 1 and β adrenoceptors
G. Fornaciari,b D. Pascual Sahuquilloa, P. D’Ocon Navaza a
a
Departamento de Farmacologia, Universitat de València Av. Vicente Andrés s/n
46100 Burjassot, València; bDipartimento di Scienze Farmaceutiche, Università di
Pisa, Via Bonanno 6, 56126 Pisa.
[email protected]
1. Introduction
Angiogenesis, the process of formation of new capillaries from preexisting blood
vessels, is a key aspect of endothelial function. The presence of α1 and β
adrenoceptors (α1-AR; β-AR) in vascular endothelium1 suggests their possible
participation in the regenerative phenomenon by regulating the endothelial cell
responses involved. With this work we planned to investigate the uncleared ARs role
in the angiogenic process, and in particular the modulator properties exerted by
Carvedilol, α1 and β-AR antagonist. Actually, despite the wide clinical use as first
choice drug for many heart diseases, its mechanism of action is not still completely
cleared.
2. Materials and methods
Rat aorta explants were cultured in a
three-dimensional extracellular matrix
(Matrigel ®) for 7 days in the presence or
absence of α1 and β agonists, and/or with
Carvedilol.
The cultures were examined by microscope and image analysis software allowed the
new vessels growth quantification. (Figure 1)
Our preliminary results confirm a proangiogenic activity for the α1-AR agonist
Phenylephrine, in accordance with previous studies allowing how α1-ARs could
activate growth factors pathways or stimulate their production.3 Carvedilol also
appears to stimulate the angiogenesis but it is not able to reverse the inhibitory
activity exerted by the selective β1-AR agonist Dobutamine. Furthermore, a selective
β2-AR agonist, Salbutamol, leds to contradictory results.
4. References
[1] Daly CJ et al., Fluorescent ligand binding reveals heterogeneous distribution of adrenoceptors and
'cannabinoid-like' receptors in small arteries. Br J Pharmacol. 2010; 159(4):787-96.
[2] Ciccarelli M. et al., Endothelial alpha1-adrenoceptors regulate neo-angiogenesis. Br J Pharmacol.
2008; 153(5):936-46.
[3] Weil J et al. Norepinephrine upregulates vascular endothelial growth factor in rat cardiac myocytes
by a paracrine mechanism. Angiogenesis. 2003; 6(4):303-9.
90
Synthesis and biological evaluation of a series of benzotriazole ureas
as inhibitors of the endocannabinoid metabolism
L. Morera,a G. Labar,b G. Ortar,a D. M. Lambert b
a
Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma;
b
Université catholique de Louvain, LDRI Louvain Drug Research Institut, Medicinal
Chemistry Dpt, Brussels, Belgium
[email protected]
1. Introduction
Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) are two
enzymes from the serine hydrolase superfamily that degrade the endocannabinoids 2arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide, AEA),
respectively. Inhibitors of these enzymes constitute important pharmacological tools
to examine the endocannabinoid system and could also be developed as a new
promising class of analgesic drugs.1 Considering that MAGL and FAAH are both
inhibited by carbamates bearing a N-piperidine/piperazine group,2,3 we synthesized a
series of compounds by using the benzotriazole as the leaving group and by
substituting the carbamic moiety for an urea.
On the basis of the pharmacological evaluation of the compounds on recombinant
human MAGL and FAAH, we found that these derivatives can be tuned for MAGLor FAAH-selectivity as well as for dual MAGL-FAAH inhibition by attachment of
appropriate groups on the piperazine ring nitrogen. As general trend, we observed that
the phenyl piperazyl moiety is better for the FAAH inhibition, while the benzyl
piperazyl ureas are more potent MAGL inhibitors.
O
X
N
n
R
N
N
N
X = N, CH
n = 0,1
3. References
[1] Petrosino S., Di Marzo V., Curr. Opin. Investig. Drugs. 2010, 1, 51-62.
[2] Long J. Z., Jin X., Adibekian A., Li W., Cravatt B. F., J. Med. Chem. 2010, 53, 1830–1842.
[3] Sanofi Aventis, PCT Int. Appl. WO2008145843, 2008.
91
Structural investigations on a novel class of 1,2,4-triazolo [1,5c]pyrimidines as adenosine receptor antagonists
S. Federico,a S. Paoletta,b S. Lee Cheong,c G. Pastorin,c B. Cacciari,d K.-N. Klotz,e S.
Moro,b G. Spallutoa
a
Università di Trieste, Piazzale Europa 1, I-34127 Trieste, Italy,b Università di
Padova, via Marzolo 5, I-35131 Padova, Italy,c National University of Singapore, 3
Science Drive 2, Singapore 117543,d Università degli Studi di Ferrara, via Fossato di
Mortara 17-19, I-44100 Ferrara, Italy,e Universität of Würzburg, Versbacher Str. 9,
D-97078 Würzburg, Germany
[email protected]
Several classes of heterocyclic derivatives have been reported as AR antagonists
with high levels of both affinity and selectivity. In particular, in recent years we
investigated in depth the nucleus of triazolo-pyrazolo-pyrimidine as a basis for
designing ARs antagonists. Nevertheless, this class of compounds, like other tricyclic
structures, was subject to poor water solubility and more importantly complicated
synthetic routes. In consideration of these problems, medicinal chemists recently
focused their attention on the synthesis of more simplified heterocyclic derivatives, in
particular bicyclic systems. The 1,2,4-triazolo[1,5-c]pyrimidines possesses a low
molecular weight and less nitrogen atoms than pyrazolo-triazolo-pyrimidines, thus it
may be a scaffold with promising pharmacokinetics properties. A preliminary
investigation on these molecules was carried out introducing arylacetyl or
arylcarbamoyl moieties at the N5 position, which enhanced affinity at the hA2B and
hA3 ARs, when utilized on the pyrazolo-triazolo-pyrimidine nucleus. In addition, in
order to better investigate the possible substitutions on this scaffold, also moieties
without a carbonyl group (alkyl and aryl moieties) were introduced at N5 position.
Surprisingly, compound bearing the methylamino substitution at the 5 position
displays high affinity (KihA3=4.14 nM) and selectivity (236, 25, 592-fold vs hA1,
hA2A and hA2B ARs) at the hA3AR subtype . The analogue was docked in a homology
model of the hA3AR based on the crystallographic structure of the hA2AAR,
confirming the interactions with residues important to have potency at the hA3AR.
References
[1]Moro, S., et al. Med. Res. Rev. 2006, 26 (2), 131-159.
[2]Cacciari, B., et al. Purinergic Signalling 2007, 3, 183-193.
92
Design, synthesis and biological evaluation of resveratrol analogues
on influenza A virus infection
G. Cuzzucoli Crucitti,a L. Pescatori,a L. Nencioni,b A.T. Palamara,b R. Fioravanti,a R.
Costi,a R. Di Santoa
a
Dipartimento di Chimica e Tecnologie del Farmaco, bDipartimento di Sanità
Pubblica, Sapienza Università di Roma, P.le Aldo Moro 5, 00185 Roma
[email protected]
1. Introduction
Influenza is caused by a group of enveloped RNA viruses that belong to the
Orthomyxoviridae family. Three types of influenza viruses are known: A, B and C,
based on the antigenicity of nucleoprotein (NP) and matrix protein (M1).1 Among
these, the influenza A is responsible for annual seasonal epidemics worldwide, and
novel virus strains emerge sporadically as pandemic viruses. It has two subtypes
important for humans: A(H3N2) and A(H1N1), of which the former is currently
associated with most deaths.
Dietary polyphenols, such as resveratrol (found
in red wine) and curcumin (found in curry
powder), have been used safely for centuries as
traditional medicines. These compounds were
reported
to
posses
anticancer,
antiinflammatory, and antiviral properties. In our
recent study,2 we have reported the inhibition of
influenza A virus replication by resveratrol, so
to continue our research in this field, and to find
inhibitors of influenza A virus, we designed,
synthesized, and tested polyphenols, including
chalcones, and curcumin analogs.
3. References
[1] Tscherne, D.M.; Garcia-Sastre, A. J. Clin. Inv 2011, 121, 6-13
[2] Palamara, A.T.; Nencioni, L.; Aquilano,K.; De Chiara, G.; Hernandez, L.; Cozzolino, F.; Ciriolo,
M.R.; Garaci, E. J Infect Dis. 2005, 191, 1719-1729.
93
Design, sythesis and biological activities of new active agents against
Mycobacterium tuberculosis
L. Pescatoria D. De Vita,a G. Cuzzucoli Crucitti,a P. Mellini,a B. Saint Joanis,b R.
Brosh,b S. Tortorella,a L. Scipione,a R. Costi,a R. Di Santoa
a
Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma,
P.le Aldo Moro, 5 00185 Roma; bInstitut Pasteur, Pathogenomique Mycobacterienne
Integree, 25 Rue du Dr. Roux, 75724 Paris
[email protected]
Tuberculosis (TB), infectious disease mainly caused by Mycobacterium
tuberculosis (MTB), is a major public health problem and remains a leading cause of
death worldwide. The 2009 report of World Health Organization (WHO), indicates
that there were 9.3 million incident cases, with 86% of incident cases in Africa and
Asia.1 The report also reveals 1.3 million deaths from TB among HIV-negative
people, and an additional 456 000 deaths among HIV-positive TB cases. The need for
new shorter therapeutic regimens active against on MTB, drive public and private
pharmaceutical research to accelerate in the development process of new anti-TB
drugs; new promising molecules have been developed and new molecular target for
anti-TB drugs were identified.2 To this aim, we designed and synthesized a series of
cycloserine derivatives and a series of pyrrole derivatives related to RDS 63, an hit
found active against M. tuberculosis.3 These two series of compounds have been
tested on the M. tuberculosis strains H37Rv. Results of the antimycobacterial assays
will be shown and discussed.
Cl
R1
H
N
O
O
O
O
OH
N R2
N
Cl
Cycloserine derivative
RDS 63
References
[1] WHO Report 2009 - Global Tuberculosis Control. Epidemiology, Strategy, Financing. Geneva,
Switzerland: WHO. http://www.who.int/entity/tb/publications/global_report/2009/pdf/full_report.pdf.
[2] V. Bhowruth, L.G.Dover, G.S.Besra, Tuberculosis Chemotherapy: recent developments and future
perspectives. Progress in Medicinal Chemistry, 2007, 45,169-203.
[3] Di Santo, R.; Costi, R.; Artico, M.; Massa, S.; Lampis, G.; Deidda, D.; Pompei, R. Pyrrolnitrin and
related pyrroles andowed with antibacterial activities against Mycobacterium tuberculosis. Bioorg.
Med. Chem. Lett., 1998, 8, 2931-2936.
94
Activity of drugs against dormant mycobacterium tuberculosis
L. Pescatori,a D. De Vita,a P. Mellini,a L. Scipione,a S. Tortorella,a R. Di Santo,a G.
Cuzzucoli Crucitti,a R. Costi,a L. Fattorini,b G. Piccarob
a
Dipartimento di Chimica e Tecnologia del Farmaco, Sapienza Università di Roma;
b
Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore
di Sanità, Roma.
[email protected]
1. Introduction
Actually, 2 billion people are estimated to be latently infected with Mycobacterium
tuberculosis (Mtb), and 10% of them will develop clinical manifestations of active TB
during their lifetime, often for poorly understood reasons.1 Chemoprophylaxis with
isoniazid can reduce the risk of reactivation by as much 90% but does not eliminate
the development of TB; furthermore this treatment is very long (9 months): thus the
search for drugs that kill dormant Mtb is an urgent need.2
As a part of our research program
we focused our attention to the
synthesis of metronidazole analogs
considering that metronidazole kills
dormant bacilli under anaerobic
conditions and has effect on actively
growing aerobic cultures when
associated
with
first
line
2
antimycobacterial
drugs.
The
synthesized compounds were tested,
using the Wayne methods, and 1 and
2 resulted to be more active then
metronidazole.
N
N
O
N
O
N
O
N
O
NO2
NO2
N
CF3
(1)
(2)
O
N
F
N
O
N
H
O
N
NO2
(3)
3. References
[1] Barry C.E. 3rd, H.I. Boshoff, V. Dartois, T. Dick, S. Ehrt, J. Flynn, D. Schnappinger, R.J.
Wilkinson, and D. Young. Nat Rev Microbiol. 2009; 7:845-55.
[2] Filippini P, Iona E, Piccaro G, Peyron P, Neyrolles O, Fattorini L. Antimicrob Agents Chemother.
2010;54:2712-5.
95
Synthesis and biological evaluation of new heterocyclic derivatives
with potential antimycobacterial activity: systematic approach to
lead compound optimization
M. Biava,a G. C. Porretta,a G. Poce,a S. Alfonso,a C. Battilocchio,a A. De Logu,b F.
Manetti,c M. Bottac
a
Dipartimento di Chimica e Tecnologie del Farmaco, Università Sapienza, P.le A
Moro 5, 00185 Rome, Italy; bCattedra di Microbiologia Applicata, Facoltà di Scienze
Matematiche Fisiche Naturali, Università degli Studi di Cagliari, Via Porcell 4,
09124 Cagliari, Italy; cDipartimento Farmaco Chimico Tecnologico, Università degli
Studi di Siena, Via Aldo Moro, 5310 Siena, Italy
[email protected]
1. Introduction
From the last WHO (World Healt Organization) report on Tuberculosis (TB), it’s
clear, that, among bacterial diseases, TB still represents the major challenge in
pharmaceutical research.1 Starting from 1,5-diarylpyrrole derivatives endowed with
better activity against Mycobacterium Tuberculosis2-4 previously achieved, new
heterocyclic compounds were synthesized.
The pyrrole core was substituted with the bioisoster imidazole and the C-3
thiomorpholinyl-methyl
moiety
with
the
piperidinyl-methyl
and
tetrahydrothiopyranyl-methyl ones, in order to evaluate how these substitutions can
affect the activity of compounds. Among the synthesized derivatives, one compound
active against M. tuberculosis 103471 (MIC <0.125 µg/mL) and with a Protection
Index (PI = CC50 / MIC) higher than 400, was identified.
3. References
[1]
World Health Organization Report 2010: Global Tuberculosis Control.
[2]
Biava, M.; Porretta, G.C.; Poce, G.; De Logu, A.; Saddi, M.; Meleddu, R.; Manetti, F.; De
Rossi, E.; Botta M. J. Med. Chem. 2008, 51, 3644-48.
[3]
Biava, M.; Porretta, G.C.; Poce, G.; De Logu, A.; Meleddu, R.; De Rossi, E.; Manetti, F.;
Botta M. Eur. J. Med. Chem. 2009, 44, 4734–38.
[4]
Biava, M.; Porretta, G.C.; Poce, G.; Battilocchio, C.; Alfonso, S.; De Logu, A.; Serra, N.;
Manetti, F.; Botta, M. Bioorg. & Med. Chem. 2010, 18(22), 8076-84.
96
Author Index
97
Author
Abate A.
Ahn J.-M.
Ai Tran H. N.
Alcaro S.
Alcaro S.
Aldini G.
Alessio E.
Alfonso S.
page number1
39
36
50
55
48
22
37
16
96 (PA)
22
51
39
63
52
16
74
74
13 (PA)
48
31
51
73
54
75
40
57
70
74
96
16 (PA)
31 (PA)
82
49
86 (PA)
37
86
32
89
22 (PA)
7 (PA)
29
34
83
16
96
72
50, 52
SC
SC
SC
SC
SC
SC
SC
SC
P
SC
SC
SC
P
SC
SC
P
P
SC
SC
SC
SC
P
SC
P
SC
SC
P
P
P
SC
SC
P
SC
P
SC
P
SC
P
SC
ML
SC
SC
P
SC
P
P
SC
Altomare A.
Amadio M.
Amici R.
Anderluh M.
Andrisano V.
Anzini M.
Arena E.
Aricò G.
Arista L.
Artese A.
Auzzas L.
Azzolina O.
Ballatore C.
Barbato G.
Barraja P.
Barreca M. L.
Baruchello R.
Battilocchio C.
Battilocchio C.
Battistuzzi G.
Bedford M.T.
Behr .J-P.
Bendinelli S.
Bergamo A.
Bertamino A.
Bertini S.
Bertoletti L.
Bertucci C.
Bianco A.
Bianco S.
Biava M.
Bolasco A.
Bolognesi M. L.
1
PL: Plenary Lecture; ML: Main Lecture; SC: Short Communication; P: Poster; PA: Presenting author
98
Bongarzone S.
Bonvini P.
Borea G. P.
Botta L.
Botta M.
SC
P
P
SC
SC
P
SC
P
P
SC
SC
ML
SC
P
SC
SC
SC
SC
P
SC
SC
SC
SC
SC
P
P
SC
SC
SC
SC
P
SC
SC
SC
SC
SC
P
SC
P
SC
P
P
P
P
SC
SC
ML
SC
P
SC
Bottegoni G.
Braga A.
Brancaccio D.
Brancale A.
Branduardi D.
Bratsos I.
Brosh R.
Brunden K.R.
Butini S.
Cabri W.
Cacciari B.
Caggiari L.
Calderone V.
Camodeca C.
Campiani G.
Campiglia P.
Campisi A.
Cannizzaro R.
Carbone A.
Cardia M. C.
Carloni P.
Carlson K. E.
Carmi C.
Carnevale Baraglia A.
Carotenuto A.
Carotti A.
Carradori S.
Casadó V.
Casali P.
Casaluce G.
Casatti M.
Cascioferro S.
Castellano S.
Cateni F.
Catto M.
Cavalieri D.
Cavalli A.
Cavazzoni A.
50 (PA)
68
75
41
41
96
44
65 (PA)
77 (PA)
32 (PA)
42
9
37
94
54
20, 23, 24, 26
31
15
92
60
16
23 (PA)
20, 23, 24, 26
32
86
74
60
40
55
50, 52
89
47
51
32
24, 28
33 (PA)
72
14
79
41 (PA)
65
76
82, 85
63 (PA)
24
59
9 (PA)
44, 50, 52
66, 67
47
99
Ceccarini L.
Cecchetti V.
P
SC
P
SC
P
SC
SC
P
SC
P
SC
SC
ML
SC
P
SC
SC
SC
P
SC
SC
P
SC
PL
P
SC
P
SC
SC
P
P
P
P
P
P
SC
P
P
SC
P
P
P
SC
ML
SC
SC
P
SC
P
P
Cera M. R.
Cheng D.
Cheong S. L.
Chilin A.
Chimenti P.
Chimirri A.
Chiriano G.
Cirrincione G.
Colizzi F.
Collina S.
Colombo R.
Coluccia A.
Conconi M.T.
Conti P.
Corbo F.
Corelli F.
Costa B.
Costa G.
Costantino G.
Costi R.
Cozza G.
Cristiani A.
Crowe A.
Curti D.
Cuzzoni E.
Cuzzucoli Crucitti G.
D’Ascenzio M.
D’Ocon Navaza P.
Da Pozzo E.
Da Ros T.
Daidone G.
Dal Col V.
Dalla Via L.
Dalvit C.
Damiano F. M.
Daniele S.
Trincavelli M.L.
De Cupertinis A.
De Giorgi M
De Grazia S.
De Iudicibus S.
67 (PA)
46
70
39
82
15
38, 61
72
53, 57
69
52 (PA)
40
9
51
73
22, 24 (PA)
42 (PA)
38
84
56
21
86
43
3 (PA)
93, 94, 95
43
68 (PA)
43 (PA)
54
73
78 (PA)
93 (PA), 94, 95
72 (PA)
90
86
34
81
76
19
79 (PA)
77
66
53 (PA)
6
14.(PA)
14
89 (PA)
56 (PA)
69 (PA)
78
100
De Logu A.
De Lorenzi E.
de Luca F.
De Luca L.
P
SC
SC
SC
P
P
SC
SC
P
SC
SC
P
P
SC
SC
P
SC
SC
SC
P
P
SC
P
SC
SC
SC
P
SC
SC
P
SC
P
P
SC
P
P
SC
SC
P
P
SC
P
SC
SC
SC
P
SC
P
SC
SC
De Micheli C.
De Re V.
De Simone A.
De Vita D.
De Wulf P.
Debyser Z.
Decorti G.
Di Maro S.
Di Pietra A. M.
Di Santo R.
Diana P.
Diaz F.
Distinto S.
Doljak B.
Donati D.
Fabbro C.
Fagà G.
Falchi F.
Fanton M.
Fattorini L.
Favia A. D.
Federico S.
Ferro S.
Fioravanti R.
Floris M.
Fornaciari G.
Franca R.
Franchini C.
Frosini M.
Furlanetto V.
Gabelloni P.
Galvani E.
Gambuzzi, A.
Gao Z.-G.
Garbelli A.
García Rubiño M. E.
García-Argáez A.
Garziera M.
Gatto B.
Gemma S.
Geremia S.
96
22, 24
25
57 (PA), 53
69
85
60
52
94, 95
39
57
69
78
36 (PA)
29
93, 94, 95
40
35
48, 55
63
75
34 (PA)
81
39
41
45
95
44 (PA)
15 (PA)
92
57
69
91
45 (PA)
90 (PA)
78
56
21
64 (PA)
86
47
80
15
41
28
77
60 (PA)
64, 65
20, 23, 26
60
101
Ghelardini C.
Giacchè N.
Giacomini, E.
Gianferrara T.
Giannini G.
Gianoncelli A.
Giordani A.
Giovannini R.
Gitto R.
SC
SC
P
SC
SC
P
SC
P
SC
P
SC
SC
P
P
SC
SC
SC
P
SC
SC
SC
SC
SC
SC
SC
SC
SC
SC
SC
SC
SC
P
P
SC
P
SC
P
P
SC
P
P
SC
SC
P
P
SC
SC
SC
P
P
Gmeiner P.
Gomez-Monterrey I.
Gosetto F.
Govoni, S.
Grebe J.
Grieco P.
Grisolia G.
Guarna A.
Guiotto A.
Guo G.
Hanessian S.
Hsieh J.-T.
Huebner H.
Hyde E.
Ianni F.
Innocenti A.
Iraci N.
Jacobson K. A.
James M.J.
Kastrup J.S.
Katzenellenbogen J. A.
Kessler B. M.
Klotz K.-N.
Kostarelos K.
Krapcho A.P.
Kuck D.
Kunjir S.
Labar G.
Lambert D. M.
Lassiani L.
Laurini E.
Lee Cheong S.
Lee V.M.-Y.
Legname G.
Lodola A.
Lucafò M.
Lyko F.
16
58 (PA)
80 (PA)
37
31
68
16
75
53, 57
69
14
32
86
70 (PA)
51
25 (PA)
32
75
59
38, 61
34
31
36
14
54
28
53
46 (PA)
15
54
20
89
88
15
92
34
83
85
26 (PA)
91
91
18
19 (PA), 51
79, 84
92
54
50, 52
47
81 (PA)
85
102
Macchia M.
SC
P
SC
SC
SC
P
P
P
SC
SC
SC
P
P
P
SC
SC
SC
P
P
P
P
SC
P
ML
SC
P
SC
SC
P
SC
P
SC
P
SC
SC
P
P
SC
ML
SC
SC
P
SC
ML
P
SC
SC
SC
P
SC
Macchiarulo A.
Maccioni E.
Maga G.
Maggio B.
Magistrato A.
Mai A.
Mamolo M. G.
Mancini F.
Manetti F.
Manfroni G.
Mangiola S.
Manzini P.
Maramai S.
Marcello A.
Marchetti P.
Marchioro C.
Marson G.
Martinelli A.
Martinez A.
Martínez-Vázquez M.
Martini C.
Marzaro G.
Masciocchi J.
Masetti M.
Massari S.
Medina-Franco J.L.
Meggio F.
Melchiorre C.
Meleddu R.
Mellini P.
Merighi S.
Micco M.L.
Micheli F.
Milani B.
Milelli A.
Milite C.
Minarini A.
Mini E.
Minutolo F.
Montalbano A.
Mor M.
Moraca F.
Morera L.
Moro S.
14
89
28, 58
55
41
76
71
82, 88
19
52
41
96
70
75
61
20 (PA)
46
75
75
83, 87 (PA)
89
52
77
6 (PA)
14
86
61 (PA)
45
67
46
85
43
68
35
55
94, 95
75
35
10 (PA)
37
35 (PA)
82 (PA)
35
8 (PA)
89
40
47
48 (PA)
91 (PA)
15, 43, 45
103
P
SC
SC
P
SC
SC
SC
P
SC
SC
P
SC
SC
P
SC
SC
P
ML
P
P
SC
P
SC
SC
P
SC
P
SC
SC
P
ML
SC
P
SC
SC
SC
P
SC
P
SC
P
P
P
P
P
SC
SC
P
SC
P
Mugnaini C.
Muraglia M.
Musetti C.
Natalini B.
Navarro N.
Nencetti S.
Nencioni L.
Nobeli I.
Novellino E.
Nuti E.
Orlandini E.
Ortar G.
Ortuso F.
Pack D. W.
Pacor S.
Padova A.
Palamara A.T.
Palumbo M.
Paoletta S.
Paolino M.
Paolucci F.
Parenti C.
Pascale A.
Pascual Sahuquillo D.
Pasquini S.
Pastorin G.
Patel J.
Pavan M. V.
Pedrali A.
Pellicciari R.
Peng L.
Perez D. I.
Pescatori L.
Petronini P. G.
Piccaro G.
Pickering D.S.
Pierotti M. A.
Pilotti S.
Piltaver A.
Pinna L. A.
Pinto A.
Piscitelli F.
Pistolozzi M.
Plescia F.
Poce G.
64, 68, 92
21
56
83 (PA)
28
14
14
93
44
20, 32, 36
82, 86
14
14
91
48
49
81
11 (PA)
93
65, 83, 87
15
92
17 (PA)
30
74
51
90
21
15
92
9
18 (PA)
73 (PA)
28, 58
49
52
93, 94, 95 (PA)
47
95
20
79, 84
79, 84
63
68
85
54 (PA)
29
76
16
96
104
Pong R.-C.
Porretta G. C.
SC
SC
P
SC
P
PL
SC
P
P
SC
P
P
SC
P
SC
P
P
SC
ML
P
SC
P
SC
SC
P
P
P
P
P
SC
SC
P
P
P
SC
SC
P
P
SC
SC
SC
P
P
SC
SC
SC
P
P
SC
P
Posocco P.
Prato M.
Prezzavento O.
Pricl S.
Procida G.
Radi M.
Raffa D.
Raffaghello L.
Ragno R.
Raimondi M.V.
Rault S.
Recanatini M.
Regazzoni L.
Reversi F.
Rivara S.
Roberti M.
Rondanin R.
Ronsisvalle G.
Ronsisvalle S.
Rosolen A.
Rossello A.
Rossi D.
Rotili D.
Rullo C.
Ruth C.
Ruzziconi R.
Sabatini S.
Saint Joanis B.
Samuele A.
Sancineto L.
Sanna L.S.
Santagada V.
Santese F.
Sardella R.
Sautebin L.
Sava G.
Sbardella G.
Scapozza L.
Schofield C. J.
36
16
96
19, 49 (PA)
79
4 (PA)
30, 34
81
74
19, 49, 51
79, 84
63
41
76
35
82
76 (PA)
56
9
67, 80
22
73
47
50, 52
80
75
74
74 (PA)
68
14
51
73
88 (PA)
75 (PA)
27 (PA)
29
70
94
41
46
55 (PA)
65
84 (PA)
28 (PA)
16
37
81
82, 85
43
88
105
Scipione L.
Scoto G. M.
Secci D.
Sgrignani J.
Sicher S.
Siegel J.
Simoni D.
Sissi C.
P
P
P
P
P
SC
P
SC
P
SC
SC
SC
P
SC
SC
P
P
SC
SC
SC
SC
P
SC
SC
P
P
SC
P
P
P
SC
P
SC
SC
ML
SC
SC
P
SC
SC
SC
SC
P
SC
SC
SC
SC
SC
P
SC
Smith D. K.
Smith III A.B.
Sopková-de Oliveira Santos J.
Sosic A.
Spagnul C.
Spalluto G.
Spannhoff A.
Spanò V.
Stefanelli C.
Stefanini I.
Stragliotto S.
Supuran C. T.
Tabarrini O.
Tamborini E.
Tamborini L.
Tedesco D.
Tolomeo M.
Tolomeo M.
Toninello A.
Tonus F.
Tortorella S.
Trabocchi A.
Trifiro P.
Trincavelli M.L.
Trojanowski J.Q.
Tubaro A.
Tuccinardi T.
Tumiatti V.
Urbani L.
Valenti S.
Valenti G.
Vanella A.
Varasi M.
Varnavas A.
Venskutonytė R.
Venturelli E.
Verga L.
Veronesi M.
Vio L.
94, 95
74
72
71
85
15
75
35
65, 83, 87
49
54
56
65 (PA)
37 (PA)
15
92 (PA)
82
40 (PA)
35
59
15
64
53
46
79, 84
85
29 (PA)
76
80
77
38 (PA) 61
94, 95
59 (PA)
39 (PA)
6
54
30
89
35
38
20
30
74
39
18
20
34
24
66
19
106
Vistoli G.
Viviano M.
Voisin-Chiret A.S.
Yamani A.
Yáñez M.
Yao Y.
Zacchigna M.
Zagotto G.
Zamolo V. A.
Zampieri D.
Zanasi R.
Zanoli S.
Zanusso I.
Zorzet S.
Zorzi E.
Zucca E.
Zuccari G.
SC
P
SC
P
P
SC
P
P
SC
SC
SC
SC
SC
P
P
SC
SC
22
82, 85 (PA)
56
82
72
54
63
64, 68
30 (PA)
19
29
41
38
81
68
41
35
107

abstract tot - 5° NPCF Nuove Prospettive in Chimica Farmaceutica

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