Binding Modes And Mechanism Research Of P-gp Substrates And Inhibitors

P-glycoprotein(P-gp),an ATP-binding cassette(ABC)multidrug transporter,can actively transport a broad spectrum of chemically diverse anticancer drugs out of cells,which is the predominant mechanism of typical multidrug resistance(MDR)in tumors.The inhibition of P-gp-mediated drug efflux is considered a promising approach for overcoming MDR in clinical cancer therapy.Therefore,a comprehension of the binding modes and functional mechanisms of P-gp substrate and inhibitor will be of great importance in the exploration of the molecular mechanisms of cancer MDR and the development of anticancer drugs.So far,the intrinsic molecular aspects of the substrate binding and translocation process remain largely unknown,due to the absence of high-resolution three-dimensional crystal structures of human P-gp and the limitation of current experimental techniques.In this paper,the computational techniques,such as quantitative structure-activity relationship(QSAR),molecular docking,molecular dynamics(MD)simulation were used to systematically explore the molecular recognition,binding modes,binding pathways and mechanism research of P-gp substrates and inhibitors.The main results and conclusions are summarized as follows:(1)Emerging chemical pattern(ECP)combined by CAEP(Classification by aggregating emerging patterns)algorithm was utilized to predict P-gp substrates as well as their potential binding sites.An optimal ECP model using only 3 descriptors was established with prediction accuracies of 0.80,0.81 and 0.74 for 803 training samples,120 test samples,and 179 independent validation samples,respectively.Hierarchical cluster analysis(HCA)of the ECPs of P-gp substrates derived 2 distinct ECP groups(ECPGs).Interestingly,HCA of the P-gp substrates based on ECP similarities also showed 2 distinct classes,which happened to be dominated by the 2 ECPGs,respectively.In the light of available experimental proofs,the 2 distinct ECPGs were proved to be closely related to the binding profiles of R-and H-site substrates,respectively.The present study demonstrates,for the first time,a successful ECP model,which can not only accurately predict P-gp substrates,but also identify their potential substrate-binding sites.(2)Emerging chemical pattern(ECP)combined by CAEP(Classification by aggregating emerging patterns)algorithm was used to predict P-gp inhibitors.On the basis of a training set with only 7 positive and 27 negative samples,an optimal ECP model using only 3 descriptors was established with prediction accuracies of 0.80 for the primary 857 training samples,and 0.86 for the 418 test samples.The best model was further evaluated by 2 independent validation sets and achieved relatively high accuracies of 0.70 and 0.80,respectively,which confirms good extrapolative capability of the ECP model.ECP analysis showed that HTv(H total index weighted by atomic van der Waals volume)is a key feture to distinguish P-gp inhibitors and non-inhibitors.In addition,most P-gp inhibitors tend to bind to R-site.Further comparison of the distributions of 6 molecular properties in 666 P-gp inhibitors and 441 substrates reveals that inhibitors are significantly more hydrophobic than substrates while substrates tend to have more H-bond acceptors than inhibitors,which are informative features for differentiating P-gp inhibitors from substrates.(3)To explore the binding modes of P-gp substrates and inhibitors,666 inhibitors and 441 substrates were docked into 2 apo crystal structures of mouse P-gp(PDB ID: 4M1 M,4Q9H)by using Surflex-Dock method.Results showed that no significant differences were observed in the total scores between substrates and inhibitors.Also,for the same receptor conformation of P-gp,no significant difference was observed in the locations of binding sites between substrates and inhibitors.Moreover,inhibitors tend to non-specifically interact with hydrophobic residues while more electrostatic and polar interactions occurred between P-gp and substrates.In addition,the docking results of 8 substrates with known binding sites indicated significant difference in the physical locations of H-and R-sites between different P-gp conformations.These findins support the idea of more than one H-or R-sites existing in the binding cavity of P-gp transmembrane regions.(4)The time-independent partial nudged elastic band(PNEB)molecular dynamics simulations were performed to characterize the binding pathway from the membrane to the binding pocket of P-gp for a substrate(Rhodamine-123)and a cyclopeptide inhibitor(QZ-Leu),respectively.PNEB simulations showed that both Rhodamine-123 and QZ-Leu can enter the the internal pocket of P-gp from the membrane,through the entrance gate formed by TM4 and TM6.The free energy profiles along the binding pathway calculated by using MM/GBSA method indicated that in both cases the binding processes are spontaneous.The MD trajectories showed that TM4 underwent a significant conformational change when the bulky QZ-Leu passed the entrance gate,indicating that the flexibility of TM4 plays a regulatory role in the access of large molecules to the binding sites.Also,a obvious rotation of the side chain of Phe339(TM6)was observed in the binding processes of Rhodamine-123 and QZ-Leu,suggesting that Phe339 may function as a gatekeeper to dominate the opening and closure of the binding pathway from membrane to the binding cavity of P-gp.(5)Molecular dynamics(MD)simulations were performed to explore the effects of substrates and inhibitors binding on the conformational dynamics of P-gp.The results showed that the C? distances between the conserved residues of Walker A and signature motifs in each ATP-binding site decreased moderately in Rhodamine-123(substrate)bound state,suggesting that the binding of substrates tends to result in the NBD1 and NBD2 moving toward each other,which benefits the dimerization of NBDs and the formation of the ATPase sites.By contrast,a significant increase of the corresponding distances was observed in the QZ-Leu(inhibitor)bound state,suggesting that inhibitors may impede ATP-hydrolysis by keeping the NBDs apart.These findings disclose significant differences in conformational dynamics between substrates and inhibitors bound states.Further binding free energy calculations by MM/GBSA method indicate that the binding affinity of QZ-Leu is higher than that of Rhodamine-123.It can be inferred that the high affinity of inhibitors is in favour of the separation of the NBDs and the inhibition of ATP-hydrolysis,while the relatively lower affinity of substrates supports the following transport process.In summary,the findings in our researches can provide key information for the virtual screening of P-gp substrates and inhibitors and the development of anticancer drugs,and offer important clues for the deep mechanism researches of P-gp-mediated MDR.