Protein-Protein Recognition : Specificity and Inhibition
This work was initiated in 2004 for the design of inhibitors targeting the Nef protein from the HIV virus, in collaboration with Dr. Yves Colette (INSERM-Marseille) and with a strong financial support from the National Agency of Research against AIDS (ANRS, 2 research grants and one PhD grant for the 2006-2010 period).
The Inhibition of protein-protein complexes is limited by several parameters such as biophysical parameters (size & depth of the interface), chemical parameters (chemical space available which does not correspond to what could inhibit a protein-protein interaction) and conceptual parameters (existence of ‘reference’ compounds to help in the discovery of new compounds).
The principal handicap relates to the latter where the absence of ‘reference’ molecules is a real impediment in the optimization of new lead compounds as biological probes/tools or potential drugs. To circumvent this problem we thus proposed a methodology in three stages which was called 2P2I (for Protein-Protein Interaction Inhibition) (Betzi et al., PNAS, 2007). We proposed to identify the first ‘hits’ using a combination of an original in silico screening protocol (Betzi et al., J. Chem. Inf. Model, 2006) coupled to NMR validation (Morelli & Rigby, Curr. Computer-Aided Drug Disc., 2007). The goal of this approach is to test a minimum number of molecules from a diverse database of selected chemical compounds.
Thanks to this approach we could highlight the first two inhibiting molecules of a complex involving the Nef protein from HIV (Betzi et al., PNAS, 2007 and Patent Publication n°: WO/2009/063412 International Application n°: PCT/IB2008/054741). This approach is now applied within our in silico screening platform (INT-3D) with several protein-protein complexes targeting the development of antivirals (SARS, HCV, measles virus, dengue virus…) and of anti-cancer compounds (prostate, brain and severe septic shock) (see § below for more details).
This research project is in collaboration with Dr. Y. Collette, Dr. ST. Arold (University of Texas, USA) and Pr. M. Huang from Harvard University & Fuzhou University, China. Two PhD students: Adrien Lugari (PhD grant from the National Agency of Research against AIDS) and Xiaoli Shi (PhD grant from the French Embassy in China) are involved in this project. ANRS also financed this project through a research grant (2008-2010).
Fyn SH3 domain interacts with the viral protein Nef but is also able to recognize other cellular proteins specifically, through the same three-dimensional mode of interaction. We identified, using double hybrid methodology (collaboration Dr. Y. Collette), multiple human cellular proteins which interact with Fyn SH3 domain via a specific recognition of the RT loop. We would like now to understand how this SH3 domain is able to recognize specifically these proteins by solving the 3D structure (x-ray diffraction) of these complexes and to compare the molecular basis in the understanding of the specific 3D molecular recognition of SH3 domains with their cellular or viral partners. This project is strongly supported by the ANRS which has financed a PhD grant (Mr. Adrien Lugari, third year PhD student) and a research project (2008-2010) as by the French Embassy in China which also granted a PhD grant on this project (Mrs. Xiaoli Shi, third year PhD student). We also obtained an Advanced Research program (PRA) that we got in collaboration with the laboratory of Professor Huang (Harvard Medical School, US & Fuzhou University, China) on this project. One research article has been already published on ALIX protein (Shi et al., Biochem. J., 2010).
In collaboration with Dr. Philippe Roche
As mentioned above, the success of the Nef-Fyn project has permitted us to present our 2P2I approach in numerous international conferences (26 invited conferences since 2006, 11 international – 15 national) and thus to extend the approach to other protein-protein complexes targeting viruses such as the Measles virus (Bernard et al., FEBS Letter, 2009), HCV virus (Betzi et al., antiviral research, 2009), SARS virus (Lugari et al., J. Biol. Chem, 2010), Dengue virus etc.. or cancer (Salmi-Smail et al., J. Med. Chem., 2010) such as prostate cancer (PAIR prostate grant just obtained for the next 3 years incollaboration with Dr. Palma Rocchi, INSERM-Marseille), brain cancer or severe septic shock (El Turk et al., Bioorg. Med. Chem., 2010). The increasing demand in anti-viral and anti-cancer research projects led Xavier Morelli & Philippe Roche to create a platform (INT-3D, director: X. Morelli; http://int3d.cnrs-mrs.fr) which handle these ‘side projects’. This platform has been already validated by the “cancéropôle PACA” and is a GIS-IBiSA validated platform through a strong collaboration with the PCML platform in Marseille-Luminy University (director: JC. Guillemot). We recently obtained a specific grant for the screening of the French National Chemical Library on the SARS coronavirus replication complex (Program Innovation Recherche PIR Thérapeutique, CNRS 2010) and also signed a collaborative project with a pharmaceutical company (2010-2011). We are now also part of the GDR “Chemoinformatics”.
In collaboration with Dr. Philippe Roche
A detailed description of the chemical and topological spaces of protein-protein interfaces that can be disrupted by small chemical compounds was not available. Such a description would lead to a better definition of a potentially successful PPI target. To define the general parameters that characterize known PPIs targets, we analyzed 17 protein-protein hetero-complexes that have been structurally characterized by X-ray crystallography or NMR.
For each complex family, a three dimensional structure of one of the two partners bound to a small molecule inhibitor was also available, representing a total of 56 different small molecules inhibitors. We have then analyzed the protein/protein and protein/inhibitor interfaces in terms of geometrical parameters, atom and residue properties, and buried accessible surface (Bourgeas et al., PLoS ONE, 2010). The result of our study has permitted to expand the current knowledge with new data, focusing at the interface of protein/protein complexes (refer to review Betzi et al. Comb. Chem & HTS, 2009 or book chapter by Roche and Morelli, ‘In silico lead discovery’, Ed. Bentham, 2010). We have developed the 2P2I database (2P2IDB) containing the dataset used in this survey. This interacting structural database is publicly available and can be accessed and queried online at http://2P2Idb.cnrs-mrs.fr. The general biophysical, biochemical and structural properties of the interfaces found in 2P2IDB were compared to those of representative datasets of heterodimers complexes in their free, bound or ‘inhibited’ biological conformation. We have then established a characteristic profile for “druggable” protein-protein complexes (presence of minimum 2 pockets at the interface, structural rearrangement <2A, #segment at the interface, # of hydrogen bond per 100A² etc…). This characteristic profile will be used to screen the Protein Data Bank (PDB) in order to select the most promising target complexes that will be screened in the second phase of our program with our biological partners from neighbor institutes and in partnership with chemical providers (private or academic) and with pharmaceutical companies (already identified).
- #. Publication Title. Authors list. Journal. Pubmed ID: Publication ID number
- 43. Exploring Selective Inhibition of the First Bromodomain of the Human Bromodomain and Extra-terminal Domain (BET) Proteins. Raux B, Voitovich Y, Derviaux C, Lugari A, Rebuffet E, Milhas S, Priet S, Roux T, Trinquet E, Guillemot JC, Knapp S, Brunel JM, Fedorov AY, Collette Y, Roche P, Betzi S, Combes S, Morelli X. J Med Chem. 2016 Feb 25;59(4):1634-41. Pubmed ID: 26735842
- 42. Protein-Protein Interaction Inhibition (2P2I)-Oriented Chemical Library Accelerates Hit Discovery. Milhas S, Raux B, Betzi S, Derviaux C, Roche P, Restouin A, Basse MJ, Rebuffet E, Lugari A, Badol M, Kashyap R, Lissitzky JC, Eydoux C, Hamon V, Gourdel ME, Combes S, Zimmermann P, Aurrand-Lions M, Roux T, Rogers C, Müller S, Knapp S, Trinquet E, Collette Y, Guillemot JC, Morelli X. ACS Chem Biol. 2016 Jun 3. Pubmed ID: 27219844
- 41. 2P2Idb v2: update of a structural database dedicated to orthosteric modulation of protein-protein interactions.
Basse MJ, Betzi S, Morelli X, Roche P. Database (Oxford). 2016 Mar 15;2016. Pubmed ID: 26980515
- 40. Hamon V, Bourgeas R, Ducrot P, Theret I, Xuereb L, Basse MJ, Brunel JM, Combes S, Morelli X, Roche P. 2P2IHUNTER: a tool for filtering orthosteric protein-protein interaction modulators via a dedicated support vector machine. J R Soc Interface. 2013 Nov 6;11(90):20130860. Pubmed ID: 24196694
- 39. Breuer, S., Espinola, S., Morelli X., Torbett, B.E, Arold, ST., Engels, IH. A biochemical/biophysical assay dyad for the HTS-compatible triaging of inhibitors of the HIV Nef/Hck SH3 Interaction. Current Chemical Genomics and Translational Medicine, 2013, 7, 16-20.
- 38. Coursindel, T., Morelli X., Chaubet, G., Baraguey, C., Floquet, N., Betzi S., Collette, Y., Roche P., Isabelle Parrot.* & Martinez, J. Polyproline II Bio-inspired Folded Structures Based on Original Spirolactam Scaffolds. Organic & Biomolecular Chemistry. 2013 Jul 28;11(28):4719-26. doi: 10.1039/c3ob40643a. Pubmed ID: 23760068
- 37. Hamon, V., Brunel, J.M., Combes, S., Basse, M.J., Roche, P. and Morelli, X. 2P2Ichem: Focused Chemical Libraries Dedicated to Orthosteric Modulation of Protein-Protein Interactions. MedChemComm, 4. 2013. 4, 797-809
- 36. Ivarsson Y, Wawrzyniak AM, Kashyap R, Polanowska J, Betzi S, Lembo F, Vermeiren E, Chiheb D, Lenfant N, Morelli X, Borg JP, Reboul J, Zimmermann P. Prevalence, specificity and determinants of lipid-interacting PDZ domains from an in-cell screen and in vitro binding experiments. PLoS One. 2013;8(2):e54581. Pubmed ID: 23390500
- 35. 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 Acid Research, 2013 Jan; 41: 41, D824-827. Pubmed ID: 23203891
- 34. Morelli, X*., Hupp, T. Searching for the holy grail; protein-protein interaction analysis and modulation
EMBO Rep. 2012 Oct;13(10):877-9. doi: 10.1038/embor.2012.137. Pubmed ID: 22986552
- 33. Shi X, Betzi S, Lugari A, Opi S, Restouin A, Parrot I, Martinez J, Zimmermann P, Lecine P, Huang M, Arold ST, Collette Y, Morelli X. Structural recognition mechanisms between human Src homology domain 3 (SH3) and ALG-2-interacting protein X (Alix). FEBS Lett. 2012 Jun 21;586(13):1759-64. Pubmed ID: 22641034
- 32. Stocker, P., Brunel, JM., de Rezende, L., A.T.-do Amaral, Morelli, X., Roche, P., Vidal, N., Giardina, T., Perrier, J. Aminoacylase 1-catalysed deacetylation of bioactives epoxides mycotoxin-derived mercapturates ; 3,4-epoxyprecocenes as models of cytotoxic epoxides. Biochimie (2012), 2012 Aug;94(8):1668-75. doi: 10.1016/j.biochi.2012.01.006 Pubmed ID: 22349737
- 31. Lugari, A., Breuer, S., Coursindel, T., Opi, S., Shi, X., Nazabal, A., Torbett, B.E., Martinez, J., Collette, Y., Parrot, I., Arold, ST.* & Morelli, X.* A Protein-Protein Disorder Catalyzer Targeting The HIV-1 Nef Protein. Bioorg. Med. Chem. 2011 Dec 15;19(24):7401-6. doi: 10.1016/j.bmc.2011.10.051. Pubmed ID: 22061824
- 30. Morelli, X., Bourgeas, R. & Roche, P. Chemical and Structural Lessons from recent successes in Protein-Protein Interaction Inhibition (2P2I) Curr. Opin. Chem. Biol. 2011 Aug;15(4):475-81.
- 29. Roche, P. & Morelli, X*. Protein-Protein Interaction Inhibition (2P2I): Mixed Methodologies for the Acceleration of Lead Discovery In In silico lead discovery, Miteva, M., Ed. Bentham: 2011; pp 167-200. (book, chapter 7)
- 28. Lugari, A., Betzi, S., Decroly, E., Bonnaud, E., Hermant, A., Guillemot, JC., Debarnot, C., Arana, C., Borg, JP., Connolly, J., Bouvet, M., Canard, B., Morelli, X*. and Lécine, P*. Molecular Mapping of the RNA Cap 2'-O-Methyltransferase Activation Interface between SARS Coronavirus nsp10 and nsp16. J. Biol. Chem. 2010 Oct 22;285(43):33230-41.
- 27. Shi, X., Lugari, A., Opi, S., Restouin R., Coursindel, T., Parrot, I., Perez, J., Zimmerman, P., Huang, M., Arold ST., Collette Y. and Morelli, X*. Identification and Biophysical Assessment of the molecular recognition mechanisms between the Human Hemopoietic Cell Kinase Src Homology Domain 3 and ALG-2-Interacting protein X. Biochem. J. 2010 Sep 14;431(1):93-102
- 26. El Turk, F., Fauvet, B., Ouertatani-sakouhi, Lugari, A., Betzi, S., Roche, P., Morelli, X*. and Lashuel, H*. An integrative in silico methodology for the Identification of modulators of Macrophage migration Inhibitory Factor (MIF)Tautomerase Activity. Bioorg. Med. Chem. 2010 Jul 15;18(14):5425-5440.
- 25. Bourgeas, R., Basse, MJ. Morelli, X*. and Roche P*. Atomic Analysis of Protein-Protein Interfaces with Known Inhibitors: The 2P2I Database. PLoS ONE. 2010 5, e9598.
- 24. Salmi-Smail, C., Fabre, A., Dequiedt, F., Restouin, A., Roche, P., Morelli, X., Brunel JM., and Collette, Y. Structure-activity relationships of a series of SAHA analogues histone deacetylase inhibitors. J. Med. Chem. 2010 Apr 22;53(8):3038-47.
- 23. Betzi, S., Guerlesquin, F. and Morelli, X*. Protein Protein Interaction Inhibition (2P2I):Less and Less Undruggable Targets Comb. Chem. & HTS. 2009 Dec;12(10):968-983.
- 22. Betzi, S., Eydoux, C., Busseta, C., Blemont, M., Leyssen, P., Debarnot, C., Ben-Rahou, M., Haiech J, Hibert M, Gueritte F, Grierson DS, Romette, JL., Guillemot, JC., Neyts, J., Alvarez, K., Morelli, X.*, Dutartre, H.* and Canard, B. Identification of allosteric inhibitors blocking the hepatitis C virus polymerase NS5B in the RNA synthesis initiation step. Antiviral Research 2009 Oct;84(1):48-59. Epub 2009 Jul 7.
- 21. Bernard, C., Gely, S., Bourhis, J.M., Morelli, X., Longhi, S. and Darbon, H. Interaction between the C-terminal domains of N and P proteins of measles virus investigated by NMR FEBS Letter. 2009 Apr 2;583(7):1084-9. Epub 2009 Mar 9.
- 20. Betzi, S., Restouin, A., Arold, ST., Parrot, I., Guerlesquin, F., Morelli, X*. and Collette, Y*. Protein-Protein Interaction Inhibition (2P2I) combining High Throughput and Virtual Screening: Application to the HIV-1 Nef protein Proc Natl Acad Sci U S A. 2007 Dec 4;104(49):19256-61. Epub 2007 Nov 27.
- 19. Huang, W., Yuan, C., Ansaldi, M., Morelli, X., Meehan,E.J., Chen,L., Huang M. Expression, Purification, Crystallization and Molecular Replacement Studies of TorI, an Inhibition Protein of Tor System. Chinese Journal of Structural Chemistry. 2007 26(5):594-598.
- 18. Morelli, X., Rigby, A.C. Acceleration of the Drug Discovery Process: A Combinatorial Approach Using NMR Spectroscopy and Virtual Screening. Current Computer-Aided Drug Design. 2007 March;3(1):33-49.
- 17. Betzi, S., Suhre, K., Chetrit, B., Guerlesquin, F., Morelli, X*. GFscore: A General Non-Linear Consensus Scoring Function for High-Throughput Docking. Journal of Chemical Information and Modeling. 2006 Jul-Aug;46(4):1704-12
- 16. Nouailler, M., Morelli, X., Bornet, O., Chetrit, B., Dermoun, Z., Guerlesquin, F. Solution structure of HndAc: A thioredoxin-like domain involved in the NADP-reducing hydrogenase complex. Protein Science. 2006 Jun;15(6):1369-78.
- 15. Pieulle, L, Morelli X., Gallice, P., Lojou, E., Barbier, P., Czjzek, M., Bianco, P., Guerlesquin, F., Hatchikian, EC. The type I/type II cytochrome c3 complex: an electron transfer link in the hydrogen-sulfate reduction pathway. Journal of Molecular Biology. 2005 Nov 18;354(1):73-90. Epub 2005 Sep 29.
- 14. ElAntak, L., Ansaldi, M., Guerlesquin, F., Mejean, V., Morelli X*. Structural and genetic analyses reveal a key role in prophage excision for the TorI response regulator inhibitor. Journal of Biological Chemistry. 2005 Nov 4;280(44):36802-8. Epub 2005 Aug 2.
- 13. Pieulle, L., Nouailler, M., Morelli, X., Cavazza, C., Gallice, P., Blanchet, S., Bianco, P., Guerlesquin, F. and Hatchikian, C. Multiple orientations in a physiological complex: the pyruvate-ferredoxin oxidoreductase-ferredoxin system. Biochemistry. 2004 Dec 14;43(49):15480-93.
- 12. Grant, M. A., Morelli, X. J. & Rigby, A. C. Conotoxins and structural biology: a prospective paradigm for drug discovery. Current Protein and Peptide Science 5, 235-48
- 11. Huang, M., Rigby, A. C., Morelli, X., Grant, M. A., Huang, G., Furie, B., Seaton, B. & Furie, B. C. (2003). Structural basis of membrane binding by Gla domains of vitamin K-dependent proteins. Nature Structural Biology 10, 751-6
- 10. ElAntak, L., Morelli, X., Bornet, O., Hatchikian, C., Czjzek, M., Dolla, A. & Guerlesquin, F. (2003). The cytochrome c3-[Fe]-hydrogenase electron-transfer complex: structural model by NMR restrained docking. FEBS Letter Jul; 31;548(1-3):1-4
- 9. Czjzek, M., ElAntak, L., Zamboni, V., Morelli, X., Dolla, A., Guerlesquin, F. & Bruschi, M. The Crystal Structure of the Hexadeca-Heme Cytochrome Hmc and a Structural Model of Its Complex with Cytochrome c(3). Structure (Cambridge). Dec; 10(12):1677-86
- 8. ElAntak, L., Bornet, O., Morelli, X., Dolla, A., Guerlesquin, F. Sequential NMR Assignment of the ferri-cytochrome c3 from Desulfovibrio vulgaris Hildenborough. Journal of Biomolecular NMR. 2002 May;23(1):69-70
- 7. Renisio, JG., Ferrat, G., Morelli, X., Cambillau, C., Darbon H. Solution structure and docking on H14 of PepH14, a peptide modelled on the basis of HCG, an antigen of H14. Biochemical Journal. 2002 May 7
- 6. Morelli, X.J.*, Palma, P.N., Guerlesquin, F., Rigby AC. A novel approach for assessing macromolecular complexes combining soft-docking calculations with NMR data. Protein Science. 2001 Oct;10(10):2131-7.
- 5. Chapon, V., Simpson, H.D., Morelli, X., Brun, E., Barras, F. Alteration of a single tryptophan residue of the cellulose-binding domain blocks secretion of the Erwinia chrysanthemi Cel5 cellulase (ex-EGZ) via the type II system. Journal of Molecular Biology. 2000 Oct 20;303(2):117-23
- 4. Morelli, X., Czjzek, M., Hatchikian, C.E., Bornet, O., Fontecilla-Camps, J.C., Palma, N.P., Moura, J.J., Guerlesquin, F. Structural model of the Fe-hydrogenase/cytochrome c553 complex combining transverse relaxation-optimized spectroscopy experiments and soft docking calculations. Journal of Biological Chemistry. 2000 Jul 28;275(30):23204-10.
- 3. Morelli, X., Dolla, A., Czjzek, M., Palma, P.N, Blasco, F., Krippahl, L., Moura, J.J., Guerlesquin, F. Heteronuclear NMR and soft docking: an experimental approach for a structural model of the cytochrome c553-ferredoxin complex. Biochemistry. 2000 Mar 14;39(10):2530-7.
- 2. Morelli, X., Guerlesquin, F. Mapping the cytochrome c553 interacting site using 1H and 15N NMR. FEBS Letter. 1999 Oct 22;460(1):77-80.
- 1. Morelli, X., Dolla, A., Toci, R., Guerlesquin, F. 15N-labelling and preliminary heteronuclear NMR study of Desulfovibrio vulgaris Hildenborough cytochrome c553. European Journal of Biochemistry. 1999 Apr;261(2):398-404.
- 1) Baty D., Collette Y., Guerlesquin F., Morelli X., Parot I., Arold S., Benichou S. "Peptides inhibiteurs de l'interaction entre la protéine Nef de VIH-1 et le domaine SH3 de la protéine Hck". 2005 "Nouveaux peptides et leurs applications biologiques". PATENT NUMBER : WO2007066018
2) Betzi, S., Restouin, A., Guerlesquin, F., Morelli, X.***, and Collette, Y***. « Inhibiteurs chimiques de l'interaction entre la protéine Nef du VIH-1 et le domaine SH3 de la protéine Hck ». Publication n°: WO/2009/063412 International Application n°: PCT/IB2008/054741