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