D 3 : DRUG DESIGN DAY Mardi 16 septembre, 9h

advertisement
PR2I « Santé et Sciences de la vie »
D3 : DRUG DESIGN DAY
Mardi 16 septembre, 9h-12h30, IMéRA
IMéRA, Institut d’Etudes Avancées de l’Université d’Aix-Marseille
2 place Le Verrier, 13004 Marseille
Organisateurs : Diane Braguer, Jean-Paul Borg, Faculté de Pharmacie, Marseille
Inscriptions : pascale.hurtado@univ-amu.fr
PROGRAMME ET ABSTRACTS
Accueil 9h
9h30-10h15
Jack Tuszynski, Cross Cancer Institute and University of Alberta, Edmonton, Canada
Computational Drug Design for Cancer Using Tubulin as a Target
10h15-10h45
Christine Contino-Pepin, IBMM, Avignon
Design of stable perfluorocarbon nanodroplets for theranostic applications
10h45-11h15
Philippe Roche, CRCM, Marseille
Drug Design Targeting Protein–Protein Interactions
11h15-11h45
Jean-Marie Pagès, UMR-MD1, Marseille
Tackling the bacterial antibiotic resistance: a new anti-resistance strategy
11h45-12h30 Round table
IMéRA - www.imera.univ-amu.fr – 04 13 55 21 52
“Computational Drug Design for Cancer Using Tubulin as a Target”
Jack Tuszynski
Allard Chair, Division of Experimental Oncology, Cross Cancer Institute and University of Alberta,
Edmonton, Canada
Abstract:
The ultimate goal of cancer research is to develop a drug or treatment regimen that will
target only cancer cells with minimal damage done to healthy tissues. The significance of
microtubules as a molecular target for chemotherapeutic treatments has been known for
decades. Tubulin, which makes up microtubules binds numerous small molecule ligands,
which result in the alteration of microtubule dynamics leading to cell cycle arrest and cell
death. Some of these ligands are currently used clinically for the treatment of several
types of cancer and include the drugs paclitaxel and vinblastine. These drugs bind to
several distinct binding sites within beta tubulin, which have been recently identified
through electron crystallography. The drawback of these drugs is their indiscriminate
binding to all cells leading to the death of both cancerous and healthy cells. Hence despite
the overall success of the vinca alkaloid and taxane drug families side effects such as
neurodegradation seriously impair the prognosis for many cancer patients treated with
them. Moreover, in many cases drug resistance develops in the course of chemotherapy.
We have focused on computational searches, optimization and testing new and repurposing old molecules that interfere with the formation of mitotic spindles during cell
division in tumors. To build the molecular models of our target tubulin, we used the
program Modeller that uses alignment of the sequences with known related structures to
obtain spatial restraints that the output structure must satisfy. Missing regions are
predicted by simulated annealing of a molecular mechanics model. The existence and
distribution of various tubulin isoforms is the basis for novel chemotherapeutic drug design
that can differentiate between different cell types to reduce side effects. The quality of the
resulting models for tubulin isoforms was investigated by an analysis of ten human beta
tubulin isoforms regarding their differences within ligand binding sites. New promising
colchicine derivatives have been designed and computationally tested for isoform
specificity. They have been synthesized and tested in our lab. The stabilities of these
derivatives have been computationally evaluated using quantum mechanical methods.
Small scale testing of these compounds on a number of tumour cell cultures has produced
promising results for their ability to selectively target specific cancer cells.
In collaboration with Dr. A. Ganguly (U of Calgary), we have tested some of our colchicine
derivatives for their effects on cell migration as an indicator of their effectiveness against
metastatic cancer cells. Our compounds CH-35 and CR-42-024 have shown nanomolar
cytotoxic activity in a breast cancer Xenograft model in nude mice. It was found that these
compounds exert cytotoxicity in tumour cells by blocking them in mitosis. Mitotic
abnormalities, such as an impaired spindle were also observed in the treated cells and
almost all the cells were blocked in prometaphase. The cytotoxicity of the colchicine
derivatives was further quantitated by utilizing clonogenic assays. It allowed us to
determine the lowest drug concentration needed to inhibit proliferation of different tumour
cell lines such as HELA, MCF7, MDA-MB231 etc. The cytotoxicity of the drugs was also
accessed in Chinese hamster ovary cell line which, previously showed unusual resistance
to drugs like colchicine when compared to human tumour cell lines. To determine the
effect of the drug on normal primary cells we also quantitated the mitotic index in primary
endothelial cells in presence of different concentrations of CH-35 and CR-42-024. It was
found that our colchicine derivatives were less toxic in primary human endothelial cells
when compared to the parent compound colchicine offering promise of fewer side effects.
Tubulin aggregation was also observed in cells, an effect that was not found in cells
treated with colchicine. We have determined that the colchicine derivatives control the
migration of vascular endothelial cells for additional therapeutic benefits. This indicates
that metronomic therapy should be effective at controlling the growth of primary tumor by
preventing the migration of endothelial cells. It was also found that the dynamics and cell
migration became inhibited at 1/50th to 1/100th of the cytotoxic concentration. In
conclusion, we have shown that a class of novel colchicine derivatives: (a) can inhibit
migration in primary endothelial cells, (b) can selectively induce cytotoxicity in rapidly
dividing cells, (c) in mouse models can cause anti-angiogenic effects. We will also report
the results of in vivo studies of our lead compound in xenograft mouse tests for efficacy
and in healthy rats for toxicity.
Acknowledgements:
Funding for this work was provided by ACB, the Allard Foundation, NSERC and MITACS
and the US Army. Oncovista of San Antonio, Texas, provided samples of colchicine
derivatives for our tests. Collaboration on the project with Dr. R.F. Ludueña is gratefully
acknowledged.
References:
J. Y. Mane, V. Semenchenko, R. Perez-Pineiro, D. Wishart, and J. A. Tuszynski
Experimental and computational study of the interaction of novel colchicinoids with a
recombinant human αI/βI-tubulin heterodimer" Chemical Biology & Drug Design 82 (1): 60
(2013)
C-Y Tseng, J. Y. Mane, P. Winter, L. Johnson, T. Huzil, E. Izbicka, R. F. Luduena and J. A.
Tuszynski, Application of the maximum entropy method to estimate the expression levels
of tubulin isotypes in cytotoxicity assays against colchicine derivatives, Molecular Cancer
(2010), 9:131doi:10.1186/1476-4598-9-131
J. T, Huzil, P. Winter, L. Johnson, A. L. Weis, T. Bakos, A. Banerjee, R. F. Luduena, S.
Damaraju and J. A. Tuszynski,Modification of colchicine cytotoxicity and selectivity through
the rational design of novel derivatives, Chemical Biology and Drug Design (2010)
75(6):541-50.
D.Bartusik, B. Tomanek, E. Lattová, H. Perreault, J. Tuszynski, and G. Fallone, Derivatives
of thiocolchicine and its applications to CEM cells treatment using 19F magnetic resonance
ex vivo, Bioorganic Chemistry (2010) 38(1):1-6.
D. Bartusik, B. Tomanek, E. Lattová, H. Perreault , J.A. Tuszynski, and G. Fallone, The
efficacy of new Colchicine derivatives and viability of the T-Lymphoblastoid cells in the
three-dimensional culture using 19F MRI and HPLC-UV ex vivo, Bioorganic Chemistry
(2009) 37(6):193-201.
“Design of stable perfluorocarbon nanodroplets for theranostic applications"
Contino-Pépin C.1, Desgranges S.1, Astafyeva K.2, Somaglino L.2, Urbach W.3, Taulier N.2
(1) Avignon Université/ CNRS/ Montpellier 1/ Montpellier 2, IBMM UMR5247, Equipe Chimie
BioOrganique et Systèmes Amphiphiles, F-84000, Avignon, France. E-mail: christine.pepin@univ-
avignon.fr
(2) Laboratoire d'Imagerie Paramétrique, UPMC Paris – CNRS, UMR 7623, Paris, France.
(3) Laboratoire de Physique Statistique de l'ENS, UMR 8550 CNRS, 24 rue Lhomond, 75005,
Paris, France.
Chemotherapy still remains a major cancer treatment which effectiveness is hampered by
several factors including genomic instability of tumor cells, multidrug resistance, and
toxicity on healthy tissues. Thus, there is an urgent need to imagine and develop
alternatives to conventional chemotherapy leading to personalized and efficient therapy
with lower side effects. Among all the explored options including cancer prevention, it
appears that detecting cancer earlier and monitoring changes in response to therapy are
key components to improve treatment efficacy and reduce the global burden of the
disease.1 In this aim, we recently exploited the tumor microvasculature to design
sophisticated tools for both early detection of tumor development and controlled therapy.
Among the several tools likely to reach this goal, commonly presented as “theragnostics”,
we focused on nanodroplets of liquid perfluorocarbon stabilized in water thanks to a shell
resulting from the self-assembling of fluorinated surfactants called “F-TAC”.2
When exposed to variable intensities of incident ultrasound waves, these nano-emulsions
can be used as ultrasound-mediated theranostics with “two in one” medical applications:3
(i) low intensity ultrasound waves provide echogenicity according to the difference in
acoustic impedance between the perfluorocarbon encapsulated within the shell and the
surrounding tissues,
(ii) high-power ultrasound waves focused at a specific site like a solid tumor or its
surrounding vasculature trigger the disruption of the nano-emulsions. If a hydrophobic
cytotoxic agent is encapsulated into the liquid core, it can hence be selectively delivered at
its site of action. A thalidomide derivative developed by our team4 was chosen as a model
drug for encapsulation studies. We report herein how nano-emulsions with appropriate
properties in terms of stability size, drug encapsulation were generated thanks to the
optimization of F-TAC structural parameters.
References
1- E. S. Kawasaki, T. A. Player. Nanomedecine 1, 101-109, 2005.
2- C. Contino-Pépin, J. C. Maurizis, B. Pucci. Curr. Med. Chem. 2, 645-665, 2002.
3- R. Diaz-Lopez, N. Tsapis, D. Libong, P. Chaminade, C. Connan, M.M. Chehimi, R. Berti,
N. Taulier, W. Urbach, V. Nicolas, E. Fattal. Biomaterials 30, 1462-1472, 2009.
4- Contino-Pépin, C.; Parat, A.; Patinote, C.; Karlik, S. J.; Pucci, B. ChemMedChem 5,
2057-2064, 2010
“Drug Design Targeting Protein-Protein Interactions”
Philippe Roche CRCM, Marseille
Due to the implication of protein-protein interactions (PPIs) in numerous diseases,
modulators of this class of targets are certainly the next generation of highly innovative
drugs that will reach the market [1]. However, in silico design of such compounds still
remains challenging mainly due to the structural complexity of the protein-protein
interfaces and to the inadequacy of chemical libraries dedicated to this chemical space. We
have developed 2P2IDB, a hand-curated structural database dedicated to PPI with known
orthosteric inhibitors ([2,3], http://2p2idb.cnrs-mrs.fr). Analysis of the small molecule
inhibitors present in 2P2IDB led us to define a characteristic profile for PPI inhibitors [4].
On average, PPI modulators are relatively hydrophobic, rigid, large (high MW), non-planar
and non-linear compounds with multiple aromatic residues that differentiate them from
standard drugs. Using dedicated support vector machine approaches, we have developed
2P2IHUNTER, a tool for filtering potential orthosteric PPI modulators from large collection
of compounds [5]. This algorithm has been applied to a set of 8.3 million compounds from
the “big vendors” to design 2P2I3D, a medicinally oriented diverse PPI-focused chemical
library [6]. This 2P2I3D library composed of 1664 compounds has been plated and tested
against several structurally diverse protein–protein targets, including PDZ domains,
bromodomains and protein-peptide interactions. The design and molecular properties of
the chemical library and results from the experimental screening will be discussed during
the presentation.
1- Zhang, X., Betzi, S., Morelli, X. and Roche, P. Focused chemical libraries - design and
enrichment: an example of protein-protein interaction chemical space. Future
Med Chem. 2014, 6, 1291-1307.
2- Basse, M.J., Betzi, S., Bourgeas, R., Bouzidi, S., Chetrit, B., Hamon, V., Morelli, X. and
Roche, P. 2P2Idb: a structural database dedicated to orthosteric modulation of
protein-protein interactions. Nucleic Acids Res, 2013, 41, D824-827.
3- Bourgeas R, Basse M-J, Morelli X, Roche P: Atomic Analysis of Protein-Protein
Interfaces with Known Inhibitors: The 2P2I Database. PLoS ONE 2010, 5, e9598.
4- Morelli X, Bourgeas R, Roche P: Chemical and structural lessons from recent
successes in protein-protein interaction inhibition (2P2I). Curr Opin Chem Biol
2011, 15, 475-481.
5- Hamon, V., Bourgeas, R., Ducrot, P., Theret, I., Xuereb, L., Basse, M.J., Brunel, J.M.,
Combes, S., Morelli, X. and Roche, P. 2P2IHUNTER: a tool for filtering orthosteric
protein-protein interaction modulators via a dedicated support vector machine.
J R Soc Interface, 2014, 11, 20130860.
6- Hamon, V., Brunel, J.M., Combes, S., Basse, M.J., Roche, P. and Morelli, X. 2P2Ichem:
Focused Chemical Libraries Dedicated to Orthosteric Modulation of ProteinProtein Interactions. MedChemComm, 2013, 4, 797-809.
“Tackling the bacterial antibiotic resistance : a new anti-resistance strategy”
Jean-Marie Pagès
UMR_MD1, Aix Marseille Université, IRBA, 13385, Marseille, France
Abstract
Today, Bacterial multidrug resistance is an health priority issue. In Gram-negative bacteria,
inner and outer membranes provide a efficient barrier protecting the cell against clinically
used antibiotics. Regarding the bacterial susceptibility towards antibacterial agents, the
membrane permeability is a prominent part of the early bacterial defense favoring the
acquisition of additional resistance mechanisms. A main challenge is to determine in cellulo
the parameters modulating the activity of antibiotics: it is absolutely required to
understand membrane permeation and intracellular concentration of antibiotics in clinical
isolates: passing the membrane barrier to reach the threshold concentration inside the
bacterial volume is a pivotal step of antibacterial action.
Different strategies could be proposed to bypass the bacterial membrane barrier in order
to increase the concentration of antibiotics inside resistant bacteria. Regarding Influx, from
pharmacochemistry and appropriate molecular protocol we can stimulate the drug
penetration through the bacterial membrane using a combination of defined antibiotic with
a permeabilizer (as escort molecule). For Efflux, we can collapse the efflux pumps activity
by developing combinations with rationally synthesized efflux blockers or inhibitors
targeting the expel mechanism and preserving the intra-bacterial drug concentration.
UMR_MD1 is studying different pharmacomodulations and combinations to combat
membrane-associated mechanisms of resistance; we are testing some molecules and
strategies in order to circumvent the antibiotic resistance in MDR isolates of Gram-negative
bacteria.
Recent publications
Kaščáková S, Maigre L, Chevalier J, Réfrégiers M, Pagès JM. Antibiotic transport in
resistant bacteria: synchrotron UV fluorescence microscopy to determine antibiotic
accumulation with single cell resolution. PLoS One. 2012;7(6):e38624. doi:
10.1371/journal.pone.0038624. Epub 2012 Jun 12.
Huguet F, Melet A, Alves de Sousa R, Lieutaud A, Chevalier J, Maigre L, Deschamps P,
Tomas A, Leulliot N, Pagès JM, Artaud I. Hydroxamic acids as potent inhibitors of Fe(II)
and Mn(II) E. coli methionine aminopeptidase: biological activities and X-ray structures of
oxazole hydroxamate-EcMetAP-Mn complexes. ChemMedChem. 2012 Jun;7(6):1020-30.
Pagès JM, Kascàkovà S, Maigre L, Allam A, Alimi M, Chevalier J, Galardon E, Réfrégiers M,
Artaud I. New Peptide-based antimicrobials for tackling drug resistance in bacteria: singlecell fluorescence imaging. ACS Med Chem Lett. 2013 May 7;4(6):556-9.
Handzlik J, Szymańska E, Alibert S, Chevalier J, Otrębska E, Pękala E, Pagès JM, KiećKononowicz K. Search for new tools to combat Gram-negative resistant bacteria among
amine derivatives of 5-arylidenehydantoin. Bioorg Med Chem. 2013 Jan 1;21(1):135-45.
Artaud I, Allam A, Alves-de-sousa R, Galardon E, Maigre, Laure; Pagès JM, Hessani A,
Alimi M. New Peptides with Metal Binding Abilities and Their Use as Drug Carriers.
Bioconjugate Chemistry, 2014, in revision
Download