We have designed a coordinated strategy using multidisciplinary approaches to understand the molecular basis of polyspecificity 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 have devoted considerable effort to the screening and development of 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 previously have reported that the tyrosine rich 15Y P-gp mutant fails to transport large-size substrates. To understand how the 15Y mutant lost the ability to transport large size substrates, we decided first to make two mutants: one named 6Y with substitution of six residues in TMD1 (F72Y/F303Y/I306Y/F314Y/F336Y/L339Y) and another one termed 9Y with nine substitutions in TMD2 (F732Y/F759Y/F770Y/F938Y/F942Y/M949Y/L975Y/F983Y/F994Y). Out of six residues in TMD1 (6Y mutant), five are clustered in TMHs 5 and 6, whereas the nine residues in TMD2 (9Y mutant) are more widely dispersed throughout TMHs 7, 8, 11 and 12. Interestingly, the 6Y mutant partially transported BD-verapamil, but failed to transport all other tested substrates. On the other hand, the 9Y mutant transported all substrates, as did its parent 15Y mutant (including large-size substrates that are not transported by 15Y. This result indicated that certain 9Y residues were able to rescue the function of the 6Y mutant. To identify 9Y residues that can rescue the function of the 6Y mutant, we made two mutants in a 6Y background: one with 6Y plus three 9Y residues located in the upper leaflet (F732Y/F759Y/L975Y) and called it 6Y + ULY. The second mutant, termed 6Y + 3Y, contains 6Y plus three 9Y residues located in the lower leaflet of the membrane (F938Y/F942Y/F994). While the ULY mutant was able to transport eight out of ten substrates, the 6Y + 3Y mutant transported nine out of ten substrates. The 15Y, ULY and 6Y +3Y mutants failed to transport BD-vinblastine, showing that the mutation(s) in 6Y responsible for loss of BD-vinblastine transport are dominant mutations. The addition of ULY or 3Y substitutions from the 9Y mutant to WT P-gp did not have any effect on its expression or function. This is the first evidence of the presence of second site suppressor mutations in P-gp, although such mutations have been reported in other eukaryotic ABC transporters including CFTR (ABCC7), yeast PDR5 and Candida Cdr1. 2. The mechanism of the molecular basis of polyspecificity of P-g: (i) We tested the interaction of P-gp with various A3 adenosine receptor agonists that are being developed for the treatment of chronic diseases, including rheumatoid arthritis, psoriasis, chronic pain and hepatocellular carcinoma. Although compounds 3 and 8 displayed pronounced effects on P-gp function, we found that a BODIPY-conjugate of compound 8 (compound 24) was not transported by P-gp. The residues in the drug-binding pocket critical for interactions with adenosine analogs compound 3 and 8 were identified by in silico molecular docking. Molecular docking studies revealed that both compounds 3 and 8 bind in the same region of the drug-binding pocket as paclitaxel (Taxol). Collectively, these results indicate that nucleoside derivatives can exhibit varied modulatory effects on P-gp activity, depending on structural functionalization. This work was done in collaboration with Kenneth Jacobson, NIDDK. (ii) In addition, to study the transport function of P-gp, we synthesized a Bodipy-labeled fluorescent conjugate of cyclosporine A (BD-CsA). After synthesis and characterization of its chemical purity, BD-CsA was compared with the commonly used 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-CsA probe. In flow cytometry assays, the fluorescence intensity of BD-CsA was almost 10 times higher than that of NBD-CsA, enabling us to use significantly lower concentrations of BD-CsA to achieve the same fluorescence levels. We found that BD-CsA is recognized as a transport substrate by both human and mouse P-gp. In silico docking of BD-CsA and NBD-CsA to the human P-gp structure indicates that they both bind in the drug-binding pocket with similar docking scores and possibly interact with similar residues. Thus, we demonstrate that BD-CsA is a sensitive fluorescent substrate of P-gp that can be used to efficiently study the transporter's localization and function in vitro and in vivo. (This work was done in collaboration with Drs. Rolf Swenson and Raju Natarajan, Imaging Probe Development Center, NHLBI, NIH). (iii) Development of a thermal inactivation method for understanding the drug-substrate and ATP-dependent stability of P-gp. We are focusing on the thermal inactivation of ATPase activity and how it is affected by nucleotides, transported substrates and modulators. We developed a very simple assay consisting of heating insect cell membrane vesicles expressing P-gp to different temperatures ranging from 37C to 70C for 10 min and assessing the P-gp ATPase activity afterwards at 37C. Interestingly, in the absence of ATP, when the NBDs are separated from each other, the protein is highly susceptible to inactivation at higher temperature. In contrast, in the presence of ATP (under non-hydrolyzing condition), when the two NBDs are close together in an inward-closed conformation, the thermal stability increases over 20C. 3. Resolution of the three-dimensional structure of human Pgp: For structural studies it is important to obtain a large amount of purified functional protein. We compared three detergents (1,2-diheptanoyol-sn-glycero-3-phosphocholine, dodecyl maltoside and n-octyl-beta-D-glucopyranoside) used for solubilization and purification of human and mouse P-gp from insect High-Five cell membranes. P-gp purification was performed first using immobilized metal affinity chromatography, then followed by a second step of either anion exchange chromatography or size exclusion chromatography to yield protein in concentrations of 10 to 12 mg per ml. Size exclusion chromatography was the preferred method, as it allows separation of monomeric transporters from aggregates. We showed that the purified protein, when reconstituted in proteoliposomes and nanodiscs, exhibits both basal and substrate or inhibitor-modulated ATPase activity. We are currently using nickel-NTA followed by a size exclusion column for purification of P-gp and nanodiscs prepared with this protein are being used for cryo-EM studies. 4. Development of non-toxic natural product and small molecule modulators to overcome resistance mediated by P-gp and ABCG2: We continue to characterize the recently developed tyrosine kinase inhibitors, repurposed drugs, small molecules, natural products and synthetic derivations of curcumin for their effect on the function of P-gp and ABCG2. 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 the FLT3 inhibitor midostaurin selectively modulated the function of human P-gp. Similarly, glesatinib, a c-MET/SMO dual inhibitor also modulated the function of P-gp. On the other hand, the KIT and PDGFR-alfa inhibitor, avapritinib inhibited the function of both P-gp and ABCG2. Solensertib, an ASK1 inhibitor, sensitized P-gp- and ABCG2-expressing cells to anticancer drugs.
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