Biochemical Analysis of Multidrug Resistance-linked Transport Proteins

Division Of Basic Sciences - Nci

Investigators

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Abstract

We have designed a coordinated strategy using multidisciplinary approaches to understand the molecular basis of the polyspecificity exhibited by the ATP-binding cassette (ABC) drug transporter P-glycoprotein (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 structure-activity relationships, in silico molecular modeling and MD simulations to extend our understanding of the mechanistic aspects and structure-function relationships. In addition, we are employing a novel approach of substituting multiple conserved residues with alanine in homologous transmembrane helices (TMHs) 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 both 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 being the inward-open (IO) conformation, in which the NBDs are separated, and the drug-binding cavity is accessible for interaction with substrates or inhibitors. The second conformation, inward-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. Orthovanadate is a transition-state analog of inorganic phosphate that inhibits the ATPase activity of P-gp and other ABC transporters. We tested several polyoxyvanadate compounds for their effect on the drug transport and ATPase activity of P-gp. Interestingly, we found that decavanadate inhibits both the drug transport and ATPase activity of P-gp with the same potency as orthovanadate. Molecular docking studies indicate that decavanadate does not appear to bind to the drug-binding pocket, suggesting that most likely it interacts with the ATP sites. Moreover, decavanadate binding to P-gp is conformation-sensitive. We plan to exploit this property of decavanadate to learn more about conformational changes associated with the catalytic cycle. 2. Reversal of the direction of 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 gained the ability to import four substrates, including Rhodamine123 and Flutax-1 (a Taxol derivative). By generating several variants with substitution of residues in both TMH 6 and 12, we found that the switch that controls the direction of transport resides in the center region. Further characterization of a series of mutants indicated that three residues (V334, F336 and F336) from TMH 6 and six residues (F978, S979, V981, V982, F983 and M986) from TMH 12 contribute to maximal accumulation of four substrates. Interestingly, residues F978, S979, V981, V982 and F983 are critical for modulation of the direction of drug transport. To assess the role of residues in TMH 4 and TMH 10, we generated a TM4,10-14A mutant in which seven residues from TMH 4 and seven from TMH 10 were substituted with Ala. This mutant P-gp did not mediate accumulation of any tested substrate and also failed to efflux all tested substrates. These data demonstrate that the residues in TMH 4 and 10 are critical for the transport function of P-gp. 3. Mechanism of photodynamic regulation of P-gp and ABCG2: We have begun to elucidate the molecular mechanism of photo dynamic therapy (PDT)-mediated regulation of ABC drug transporters. PDT is a photochemistry-based tool that involves light activation of photosensitizers to generate reactive oxygen species. In the clinic, PDT has been used to treat various diseases such as actinic keratosis, non-small cell lung cancer, and head and neck cancer. ATPase activity and in silico molecular docking analyses show that the photosensitizer benzoporphyrin derivative (BPD) binds to ABCB1 and ABCG2 with micromolar half-maximal inhibitory concentrations in the absence of light. Light activation of BPD generates singlet oxygen to further reduce the ATPase activity of ABCB1 and ABCG2 by up to 12-fold in an optical dose-dependent manner. Gel electrophoresis and Western blotting revealed that light-activated BPD induces aggregation of these transporters by covalent crosslinking. Thus, PDT affects the function of ABCB1 and ABCG2 by modulating the ATPase activity and protein integrity of these transporters. Insights gained from this study concerning the photodynamic manipulation of ABC drug transporters could aid in the development and application of new optical tools to overcome the multidrug resistance that often develops after cancer chemotherapy. To improve the efficiency of PDT for drug-resistant cancer, we devised a photoimmunoconjugate formulation combining hydrophobic BPD photosensitizers and a conformation-sensitive UIC2 monoclonal antibody to identify P-gp expression on triple negative breast cancer (TNBC) cells. We found that a UIC2-BPD conjugate can be used to fluorescently label P-gp in live TNBC cells, indicating its potential use for fluorescence imaging of tumors expressing this multidrug transporter. These studies were carried out in collaboration with Huang-Chiao (Joe) Huang as a part of partnership program between CCR, NCI and the University of Maryland. 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 including SKLB610, ensartinib, and OTS964, repurposed drugs, small molecules (phenylfurocoumarin derivative and ubiquitin-activating enzyme inhibitor TAK-243), and natural products 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), Zhe-Sheng Chen (St. John's University, NY) and Shinobu Ohnuma (Tohoku University Graduate School of Medicine, Japan). A3 adenosine receptor agonists have been developed for the treatment of chronic diseases such as rheumatoid arthritis, psoriasis, chronic pain, and hepatocellular carcinoma. Previously we demonstrated that various agonists and antagonists of adenosine receptor modulate the function of P-gp. We expanded these studies to test whether A3 adenosine receptor ligands, both adenine nucleoside and nucleobase derivatives, interact with ABCG2, modulating the absorption of drugs in the intestine. We synthesized 63 compounds and tested them for their effect on the ATPase activity of ABCG2. Of these, compound 60, a 7-deaza-5'-ester derivative with low adenosine receptor affinity, was identified as a high-affinity ligand for ABCG2 (in collaboration with Kenneth Jacobson). We found that A3 adenosine receptor ligands can exhibit *TRUNCATED*