Biochemical Analysis of Multidrug Resistance-linked Transport Proteins

Division Of Basic Sciences - Nci

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Abstract

We have designed a coordinated strategy using multidisciplinary approaches to understand the molecular basis of the polyspecificity exhibited by P-gp and the mechanism of P-gp-mediated drug transport. Our approaches include several biochemical and biophysical assays, cell-based transport assays, purification and reconstitution in lipid nanodiscs for structural studies using cryo-EM, medicinal chemistry to synthesize a large number of compounds to assess their structure-activity relationships, in silico molecular modeling and MD simulations to extend our understanding of the mechanistic aspects and the structure-function relationships of ABC drug transporters. In addition, we are employing a novel approach of substituting multiple conserved residues with alanine in homologous transmembrane helices six (TMH 6) and twelve (TMH 12) to elucidate the transport mechanism of P-gp. Furthermore, we are devoting considerable effort to the screening and development of tyrosine kinase inhibitors (TKIs) and small molecule modulators of P-gp and ABCG2 that are used in the clinic for treatment of various types of cancers. 1. Elucidation of the catalytic cycle of ATP hydrolysis and transport pathway of P-gp: We continue to study the catalytic cycle of P-gp, specifically correlating the structural conformations with the various steps that occur during ATP hydrolysis. Cryo-EM single particle studies have revealed two major conformations, one the inside-open (IO) conformation, in which the NBDS are separated and the drug-binding cavity is accessible for interaction of substrates or inhibitors. The second conformation, inside-closed (IC) is observed in the ATP-bound E556Q/E1201Q (EQ) mutant, in which the NBDs are dimerized (closed) and the drug-binding cavity is collapsed. The IC conformation of the EQ mutant was used as evidence to suggest that the substrate is effluxed from the cell before ATP is hydrolyzed. However, such an interpretation is controversial, as previous findings suggested that the transport occurs after ATP is hydrolyzed, calling into question whether the ATP-bound EQ mutant P-gp structure actually represents the pre-hydrolysis state of wild-type (WT) P-gp. To address this question, we compared trypsin sensitivity and the binding of the Fab of the conformation-sensitive human P-gp-specific antibody UIC2 under different conditions during ATP hydrolysis to WT and the EQ mutant. The trypsin sensitivity of WT and EQ mutant P-gp in the presence and absence of ATP was similar, indicating that the overall conformation of the EQ mutant is the same as WT protein. We found that purified WT and EQ mutant P-gps in nanodiscs exhibit differences in binding of UIC2-Fab under certain conditions. Our data on UIC2-Fab binding and the thermal stability results suggest that the EQ mutant conformation might include elements from both published cryo-EM structures. The extracellular region seems to resemble the IO conformation reported by the Locher group, whereas the NBDs are dimerized, similar to the structure of the IC conformation solved by the Chen group. Taken together, the published structures and our findings suggest that WT P-gp and EQ mutant P-gp do not appear to transition through the same conformational changes, which can have major implications for our understanding of the ATP hydrolysis cycle. 2. The reversal of the direction of the transport mediated by P-gp: We used a novel approach of introducing multiple mutations in homologous transmembrane helices, biochemical and cell-based transport assays, and molecular modeling to investigate the mechanism of drug transport by P-gp. A variant of P-gp termed 14A having 14 mutations in TMH 6 and TMH 12, which line the central cavity of the drug-binding pocket, lost the ability to export most of the substrates tested, but surprisingly gained the ability to import four substrates, including Rhodamine123 (Rh123) and Flutax-1 (a Taxol derivative). Similar to the efflux function of wild type P-gp, we found that uptake by the 14A mutant is ATP hydrolysis-, substrate concentration- and time-dependent. The mutant P-gp also increases sensitivity of Hela cells to Rh123 by 2 to 2.5-fold. Further mutagenesis identified residues from both TMHs 6 and 12 that synergistically form a switch in the central region of these two helices that governs whether a given substrate is pumped outside (efflux) or inside (influx) the cell. Additional biochemical and kinetic analyses showed a similar overall and ATP-dependent conformational change of the 14A mutant as occurs in wild type P-gp. Molecular dynamics simulations and in silico modeling indicated the presence of a spacious cavity in the 14A mutant, and suggest how the mutations may allow for uptake, as the path is open for substrates to enter the lumen from outside the cell, leading to the reversal of the direction of transport by the 14A mutant. These findings reflect an important and paradigm-altering discovery that an ABC exporter, human P-gp, can be converted to an importer. This represents a significant advance in the basic understanding of this medically relevant ABC drug transporter. Until now, the reversal of the direction of an ABC transporter had not been demonstrated. The use of the uptake function of a P-gp variant to hypersensitize cancer cells to drugs by mediating their uptake provides a novel approach to selectively kill P-gp-expressing cancer cells. This work also opens the door to the interesting possibility that drug uptake pumps could be designed to deliver specific cancer therapeutics. 3. Resolution of the three-dimensional structure of human P-gp: For structural studies it is important to reconstitute purified P-gp in lipidic nanodiscs. In collaboration with Dr. Maria Moreno (Coimbra University, Portugal), we have analyzed the lipidomics of the membranes of High-Five insect cells expressing P-gp. We found that phosphatidylethanolamines are the most abundant phospholipids, followed by phosphatidylcholines. The High-Five cell membranes are also enriched in negatively charged lipids (phosphatidylserines, phosphatidylinositol's and phosphatidylglycerols), and contain small amounts of sphingomyelin, ceramides and monoglycosilatedceramides. The most abundant acyl chains are monounsaturated (from oleic and palmitoleic acids), with significant amounts of saturated chains (from stearic and palmitic acids). The characterization of the phospholipids by GC-MS allowed identification of the combination of acyl chains, with palmitoyloleioyl being the most representative for all major phospholipid classes except for phosphatidylserines, which are mostly saturated. The analysis of the lipid composition of the High-Five cell membranes will help us to generate lipidic nanodiscs of same lipid composition that allows optimal activity of the purified P-gp. 4. Development of non-toxic natural product and small molecule modulators to overcome resistance mediated by P-gp and ABCG2: We continue to characterize recently developed TKIs, repurposed drugs, small molecules, natural products and indeno[1,2]indoles for their effect on the function of P-gp and ABCG2. These studies were carried out in collaboration with Drs. Glaucio Valdameri (Federal University of Parana, Brazil), Chung-Pu Wu (Chang Gung University, Taiwan) and Zhe-Sheng Chen (St. John's University, NY). Our goal is to characterize the effect of these clinically important modulators to help us to understand the polyspecificity of these transporters. We found that BMS-599626, a highly selective Pan-HER kinase inhibitor modulates ABCG2-mediated drug resistance. Similarly, the third-generation EGFR inhibitors almonertinib and branebrutinib, a Bruton's tyrosine kinase inhibitor, were found to modulate the function of P-gp. As these are repurposed drugs, they are potentially useful to overcome clinical drug resistance.