Drug resistance & Membrane Proteins (DRMP)

Equipe de recherche
RESPONSABLE
Pierre Falson
pierre.falson@ibcp.fr

CHAMPS D’EXPERTISE

Anticancer Drug resistance mediated by MDR ABC pumps (mainly ABCB1, ABCG2, ABCC1, ABCC2) and antifungal resistance mediated by yeast pumps (CDR1p, MDR1p);

ACTIVITES DE RECHERCHE / SERVICES

We study the efflux pumps Breast Cancer Resistance Protein(BCRP/ABCG2), Pleiotropic glycoprotein (P-gp/ABCB1), Multidrug Resistance Protein 1 and 2 (MRP1/ABCC1, MRP2/ABCC2), which confer resistance to multiple drugs (Multi-Drug Resistance, MDR) to cancer cells that overproduce them. BCRP and P-gp overexpression cause resistance to mitoxantrone of the acute myeloid leukemia; MRP1 is naturally overexpressed in neuroblastomas, and MRP2 is associated with resistance to cisplatin (Future Med Chem. 2015 7(15):2041). We also use the capacity of MRP1 to transport glutathione to over-sensitize to oxidative stress cells that overproduce it, and thus selectively induce their death ( Chem Rev. 2014 114 (11): 5753 ). We develop molecules that induce via MRP1 a massive efflux of GSH and promote apoptosis ( Eur J Med Chem 2017 130: 346 ; Biochem Pharmacol 2017 2017: 10 Curr Med Chem 2017 24 (12): 1186 ).

We also study the Candida Drug Resistance 1 (CDR1) and of the Multidrug Resistance 1 (MDR1) MDR efflux pumps, which give a complete resistance to azole antifungals, thanks to which Candida pass from opportunists to pathogens in case of gingival or vaginal mycosis, until deadly in nosocomial diseases, for immunocompromised, eg, after surgery or AIDS patients (Advances in Cellular and Molecular Otolaryngology 2014 2(1) ; Curr Med Chem. 2017 24(30):3242). We develop structural models of these and have identified critical residues for the transport of antifungals by mutating all the amino acids that constitute the membrane domain of these pumps. ( Biochim Biophys Acta 2016 1858 (11): 2858 ; Biochem J. 2016 473 (19): 3127 ). These proteins expel drugs because of their “polyspecific” nature, which allows them to transport molecules with no structural similarity, a mechanism we seek to establish the molecular bases. We succeeded in positioning the binding sites of 2 drugs that P-gp carries (FEBS Let 2014 281 (3): 673 ). More recently, we established that the drug-binding pocket of MDR1 is surrounded by peripheral zones providing this polyspecificity ( J Mol Biol 2018). Obtaining structural information at atomic resolution is absolutely fundamental to understanding the function and control it. Thanks to a collaboration with Geoffrey Chang in San Diego, USA, we also collaborated with an American team to solve the 3D structure of mouse P-gp in 2 conformations preceding drug binding (PNAS 2013 110 (33): 13386 ). We will release soon the x-ray and cryoEM structure of BmrA, an MDR ABC pump of Bacillus subtilis and after that of BCRP in complex with inhibitors that we develop. The membrane nature of these proteins is a constraint that leads us to develop tools and methods to solve them.

COMPETENCES / MOYENS TECHNIQUES

We develop cellular, enzymatic and structural models to understand how these pumps are organized and work. We develop inhibitors, in collaboration with chemists such as the MBLII141 which displays remarkable behavior and in vivo properties (Oncotarget 2014 5(23) :11957), and other compounds (J Med Chem. 2010 53(18):6720 ; J Med Chem. 2015 58(1):265 ; Bioorg. Med. Chem. 2018 26(2):421 ; Eur J Med Chem. 2016 122:408).

These pumps are membrane proteins, a type of proteins not stable in aqueous solutions. We have thus developed a first generation of calixarene-based detergents bearing 3 negative charges ( New J Chem 2008 32 (32): 1988 ). These detergents allowed to crystallize BmrA ( PLoS One 2011 6 (3): e18036). Two patents from these works ( WO2010116055 ; WO2009144419 ) have been licensed to the startup CALIXAR co-founded by Pierre Falson and Emmanuel Dejean in 2011. We just achieved a second generation of this type of detergents that displays a better interaction capacity, reinforced by hydrogen bonds provided by sugars. Some of them have quite remarkable properties of stabilization over time (more than 40 days at room temperature) and an increased resistance to thermal denaturation up of 30 °C (Angewandte Chemie, 2018 57(11):2948).

Our attempts to crystallize BmrA led us to optimize the size of the crown of detergent bound to the hydrophobic region of the membrane protein. We have developed and automated a method that uses the MALDI-TOF MS laser desorption mass spectrometry and validated it with several detergents and membrane proteins. With this method, it is now easy to model the detergent crown and predict its bulkiness ( Sci Rep. 2017 7: 41751 ).

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UMR CNRS-UCBL1 5086 Molecular Microbiology & Structural Biochemistry (MMSB)
UMR CNRS 5086, IBCP, 7, passage du Vercors
69367 LYON
http://mmsb.cnrs.fr/equipe/resistance-aux-medicaments-et-proteines-membranaires/