Biochemical Analysis of Multidrug Resistance-linked Transport Proteins

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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 are employing a novel approach of substituting multiple conserved residues with alanine in homologous transmembrane helices six and twelve 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 characterize the gain of function second-site suppressor mutations. We previously found that the 6Y mutant with substitution of six residues in TMD1 (F72Y/F303Y/I306Y/F314Y/F336Y/L339Y) partially transported BD-verapamil but failed to transport all other tested substrates. The atomic structure of P-gp in the ATP-bound conformation showed that intracellular helix four interacts with the Q-loop of NBD1. To test whether the substitution of the conserved F916 residue in intracellular helix four with tyrosine can rescue the function of the 6Y mutant, we added this residue to 6Y to produce the 6Y + F916Y mutant. To our surprise, we found that F916Y indeed rescues the function of the 6Y mutant. Consistent with the interaction between F916Y of intracellular helix four and F480Y from the Q-loop of NBD1, we found that addition of F480Y to the 6Y mutant also rescued the transport function of 6Y at least for selected substrates. We also added the residues Q535Y or Q1180Y from the signature region of NBD1 and NBD2 to 6Y to check whether these residues also rescue the function of the 6Y variant. However, the residues from the signature region of the NBDs did not have any effect on the function of the 6Y mutant. These results with second-site suppressor mutations demonstrate the connection between the drug-binding pocket and NBD1 of human P-gp. This is the first evidence of the presence of gain-of-function 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) Development of a thermal inactivation method for understanding the drug-substrate and ATP-dependent stability of P-gp. We observed that the thermostability of P-gp depends on the conformation of its NBDs. The effect of temperature on P-gp at the protein level can also be followed by quantifying the disappearance of the monomeric band of the transporter by SDS-PAGE. We used this approach to study the thermostability of P-gp mutants, which bind ATP to both NBDs (E556Q/E1201Q) or only to NBD2 (Y401A), but are unable to carry out ATP hydrolysis, to provide further support for the role of ATP-induced NBD dimerization during thermo-stabilization of P-gp. The inward-closed conformation that results from ATP-binding to both NBDs and their dimerization has remarkably higher thermostability when compared with the inward-open conformation in the absence of nucleotides. We also find significant differences in the thermostability of P-gp in the presence of transport substrates that stimulate ATP hydrolysis vs. inhibitors of both transport and ATPase activity. The inhibitors prevent the ATP-dependent dimerization of the NBDs, which is required for ATP hydrolysis, by stabilizing the inward-open conformation. These findings suggest that modulators, which bind in the TM regions, inhibit ATP hydrolysis and drug transport by preventing ATP-dependent dimerization of the NBDs of P-gp. Currently, we are testing 156 FDA approved cancer drugs for their effect on the thermal stabilization of P-gp and ABCG2. This will help us to identify potent inhibitors of P-gp and ABCG2. (ii) Various A3 adenosine receptor agonists are being developed by our collaborator Dr. Kenneth Jacobson (NIDDK, NIH) for the treatment of chronic diseases, including rheumatoid arthritis, psoriasis, chronic pain and hepatocellular carcinoma. We tested more than forty A3 adenosine receptor ligands and found that these ligands can have different modulatory effects on P-gp and ABCG2 activity, depending on structural functionalization. Although MRS 7343 (lead compound) inhibits the transport function of ABCG2, this adenosine receptor ligand showed no interaction with P-gp. Molecular modeling studies revealed that selected A3 adenosine receptor analogs interact with residues in the drug-binding pockets of both P-gp and ABCG2. Both P-gp and ABCG2 appear to affect the pharmacokinetics of A3 adenosine receptor ligands. MRS 7343 appears to be a good template to develop high affinity modulators for P-gp and ABCG2. 3. Resolution of the three-dimensional structure of human P-gp: 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 High-Five insect 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 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 selective class IIa histone deacetylase inhibitor TMP195 is a modulator of both P-gp and ABCG2, whereas the phosphodiesterase type 5 inhibitor MY-5445, the TKI inhibitor sitravatinib, the c-myc inhibitor tivatinib and licochalcone A modulate only the function of ABCG2. In addition, the PI3K/mTOR inhibitor samotolisib is a substrate for both P-gp and ABCG2. These studies were carried out in collaboration with Drs. Chung-Pu Wu (Chang Gung University, Taiwan) and Zhe-Sheng (Jason) Chen (St. John's University, NY).