Multiple Drug Transport Pathways through Human P-Glycoprotein

Abstract

P-glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11 to 12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methyl-pyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar is presented that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp.

Location of contact residues in pharmacologically distinct drug binding sites on P-glycoprotein

“…The candidate residues identified using this approach were in broad agreement with those identified from docking and MD simulation based studies [20,34,35]. Another investigation using a large number of P-gp isoforms adopted a different approach using arginine enhancer mutations to identify residues within the translocation pathway [36].…”

About P‐glycoprotein: a new drugable domain is emerging from structural data

“…Interestingly, the process by which Rhodamine‐123 interact with the NBD1 transmission interface may help to explain why several thioamide‐containing derivatives of Rhodamine‐123 can promote calcein‐AM uptake and inhibit vinblastine efflux with IC 50 s of ~2 μM . A different study also identified a possible site for tariquidar binding close to the ICL2/ICL3 region (TMD4/TMD9, site 1), together with two other sites located in the internal drug‐binding pocket (sites 2 and 3) . However, recent biochemical studies showed that tariquidar probably binds to sites 2 and 3 rather than to site 1, stabilizing the N ‐terminal domain, thus being able to block conformational changes leading to efflux and also rescue folding‐defective mutants …”

“…146 A different study also identified a possible site for tariquidar binding close to the ICL2/ICL3 region (TMD4/TMD9, site 1), together with two other sites located in the internal drug-binding pocket (sites 2 and 3). 147 However, recent biochemical studies showed that tariquidar probably binds to sites 2 and 3 rather than to site 1, stabilizing the N-terminal domain, thus being able to block conformational changes leading to efflux and also rescue folding-defective mutants. 148 The ability of drugs to spontaneously bind to the cytosolic P-gp domains was also addressed recently by several independent studies ( Figure 6).…”

In vitro and in silico evaluation of P‐glycoprotein inhibition through 99mTc‐methoxyisobutylisonitrile uptake

“…[14] 2.6 | Cellular experiments 99m Tc-MIBI is a substrate for the P-gp pump. and exhaustiveness was set to 8 for the docking procedure so as to include all 12 helices.…”