OpenTox Virtual Conference 2023 

Efflux mechanism and conformational landscape of P-glycoprotein to better rationalize the prediction of inhibitors involved in drug-drug interactions

P-glycoprotein (P-gp) is one of the most studied ABC transporters which plays a key role in the ADMET (Absorption, Distribution, Metabolism, Excretion, Toxicity) properties of very diverse xenobiotics and drugs. Moreover, P-gp is of major interest in cancer treatment because of its involvement in the multidrug resistance (MDR) phenotype by expelling anticancer drugs out of tumor cells, thus reducing their therapeutic activity. The inhibition of P-gp also causes drug-drug interactions (DDI) which have an impact on drug efficacy/toxicity. The ability to identify substrates and inhibitors of P-gp is therefore crucial to prevent DDI and adverse drug reactions (ADR) during the clinical stages of the drug development process. 

To ensure its (drug) efflux functions, P-gp uses the energy provided by ATP binding and hydrolysis to transit between open states (inward-facing, IF) allowing compound binding; and closed states (outward-facing, OF) allowing its eviction from the cell. The IF and OF 3D structures of human P-gP were solved by cryo-EM (Kim and Chen, 2018; Nosol et al., 2020; Urgaonkar et al., 2022). However, these structures give only a partial information and do not elucidate the efflux mechanism involving large conformational changes in order to expulse very diverse drug substrates. 

Recently, we developed an original enhanced sampling method for molecular dynamics (MD), namely kinetically excited targeted MD, that allowed us to reveal the transitions between the IF and OF states and the translocation pathway in BCRP, another important ABC transporter (Dudas et al., 2022). Here, we optimized this approach to generate transitory conformations along the gating cycle of P-gp and to unveil the mechanisms of substrate efflux and inhibitor interactions. Our simulations provided for the first time exploration of the P-gp transition pathway, and the studied conformational landscape revealed some crucial features about its functions and dynamics. Such data are subsequently useful to i) better define the binding sites, ii) characterize their interaction modes with known active molecules and iii) rationalize the prediction of new inhibitors and substrates. 

Dudas, B., Decleves, X., Cisternino, S., Perahia, D., Miteva, M.A., 2022. ABCG2/BCRP transport mechanism revealed through kinetically excited targeted molecular dynamics simulations. Comput. Struct. Biotechnol. J. 20, 4195–4205. https://doi.org/10.1016/j.csbj.2022.07.035 

Kim, Y., Chen, J., 2018. Molecular structure of human P-glycoprotein in the ATP-bound, outward-facing conformation. Science 359, 915–919. https://doi.org/10.1126/science.aar7389 

Nosol, K., Romane, K., Irobalieva, R.N., Alam, A., Kowal, J., Fujita, N., Locher, K.P., 2020. Cryo-EM structures reveal distinct mechanisms of inhibition of the human multidrug transporter ABCB1. Proc. Natl. Acad. Sci. 117, 26245–26253. https://doi.org/10.1073/pnas.2010264117 

Urgaonkar, S., Nosol, K., Said, A.M., Nasief, N.N., Bu, Y., Locher, K.P., Lau, J.Y.N., Smolinski, M.P., 2022. Discovery and Characterization of Potent Dual P-Glycoprotein and CYP3A4 Inhibitors: Design, Synthesis, Cryo-EM Analysis, and Biological Evaluations. J. Med. Chem. 65, 191–216. 

https://doi.org/10.1021/acs.jmedchem.1c01272