Biochemical Analysis of Multidrug Resistance-linked Transport Proteins

Division Of Basic Sciences - Nci

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Abstract

Clinical resistance to multiple chemically and functionally dissimilar drugs is multifaceted. P-glycoprotein (P-gp, ABCB1) and ABCG2 appear to contribute to this problem due to their broad and overlapping substrate specificities. Both transporters also play an important role in drug-drug interactions as well as in the bioavailability and pharmacokinetics of several drugs. We have designed a coordinated strategy using multidisciplinary approaches in order to understand the molecular basis of the polyspecificity exhibited by P-gp and the mechanism of drug transport. We focused on identifying residues on the homologous TMHs 6 and 12 that modulate the direction of transport from efflux to uptake. We also characterized second-site suppressor mutations in the ICH4 and the Q-loop of NBD1 that rescue the function of a non-functional mutant. Our studies focus on human P-gp and use Bac-Mam baculovirus HeLa cells for characterization of transport properties and the High Five insect cell-baculovirus expression system for biochemical and structural studies. In addition, we use in silico molecular docking and MD simulations to supplement the experimental data. 1. Mechanism of reversal of direction of P-gp-mediated drug transport from efflux to uptake. Our recent studies have focused on the characterization of the switch region in TMH6 and TMH12 of P-gp which controls the directionality of substrate transport. To fine-tune the switch region that regulates the direction of transport, we divided the mutations in TMH 12 into two halves to generate TMH 6,12-7AII (4 mutations in the TMH 12 upper half region- V974A/L975A/ V977A/F978A) and TMH 6,12- 8AII (5 mutations in the TMH 12 middle- lower region- S979A/V981A/ V982A/F983A/M986A), keeping the three mutations in TMH 6 constant. We characterized the transport properties of these mutants and found that 7A-II could import all 5 substrates, whereas 8A-II was able to import Rh123 at a low level. Interestingly, 8A-II was able to efflux flutax-1, Rhod-2 AM, and X-Rhod-1 AM at normal levels. For other substrates, 8A-II showed no or partial efflux, indicating the reversibility of the switch region. This indicates that the residues in the upper half region of TMH 12 are more important for the uptake of multiple substrates in addition to the general loss of efflux function. Recently, we generated a stable cell line-HEK293, lacking ABCB1, expressing WT and 7A-II mutant of P-gp. The hygromycin selected stable lines will be used to screen FDA approved library of anticancer drugs to identify substrates for the uptake. 2. The molecular basis of the polyspecificity of P-gp: The role of the access tunnel, or L-site, in the inhibitory activity of P-gp inhibitors. To assess the role of the access tunnel, or L-site, we used a mutational approach. Either five or nine residues, which line the access tunnel and interact with four inhibitors as observed in cryo-EM structures, were substituted with Ala to generate mutants named L-site 5A (W232A/L236A/F239A /L879A/F994A) and 9A (5A + N296A/I299A/F770A/V991A). These mutants, along with the WT P-gp, were expressed in HeLa cells using BacMam baculovirus. Cell surface and total cell expression of both the 5A and 9A mutants were at the same levels as WT P-gp. The 5A mutant had normal transport of eight of the ten fluorescent substrates tested and partial (<70%) transport of the other two, whereas the 9A mutant exhibited loss of transport of several substrates with only the efflux of Rhod 2-AM by this mutant remaining normal. Compared to the 5A mutant, 9A has an additional four residues substituted with Ala. We found that inhibitors including tariquidar, elacridar, and zosuquidar were able to inhibit transport of Rhod2-AM by these mutants, but with decreased affinity. In addition, zosuquidar, instead of inhibiting ATPase activity, stimulated activity of both 5A and 9A mutants consistent with its decreased affinity for these mutants. Another unexpected finding was that for 5A and 9A mutants, the ATP-dependent thermostability was abolished. The MD simulations data suggest that mutations in the L-site affect the conformation of ICH2 and its connection with NBD2 as well as the conformation of ICH4, which connects to NBD1. Thus, our findings suggest that inhibitors bind either to the SBP alone, and/or to alternate site(s) when the L-site is disabled by mutagenesis. 3. Mechanism of photodynamic priming-mediated regulation of P-gp and ABCG2. Photodynamic priming (PDP) represents an innovative strategy, harnessing sub-therapeutic photochemistry to induce sub-lethal effects in cancer cells. PDP affords precise spatial and temporal control, thereby mitigating the risk of off-target effects and reducing systemic toxicity. We decided to characterize the effect of PDP treatment on cellular energetics in TNBC parental MDA-MB-231 and vinblastine-selected P-gp-expressing drug-resistant VBL-MDA-MB-231 cells. As a first step, we determined whether there is a difference in the energetics of MDA-MB-231 and VBL-MDA-MB231 cells. The drug-resistant cells have higher glycolytic and mitochondrial ATP production rates compared to drug-sensitive cells, as they require more ATP to support the function of P-gp to efflux anticancer drugs and xenobiotics. Consistent with these results, analysis of total intracellular ATP content by an ATPLite luminescence assay showed higher intracellular ATP content in VBL-MDA-MB-231 cells. We then determined the optimal concentration of photosensitizer BPD and light dose PDP treatment and found that the PDP treatment significantly decreased mitochondrial and glycolytic ATP production rates in both drug-sensitive parental and P-gp-expressing drug-resistant cells. Th we screened four well-established BPD formulations-liposomal BPD (L-BPD), lyso-phosphatidylcholine-conjugated VP (lysoPC BPD), liposomal formulation of lysoPC VP (L-lysoPC BPD), and a self-assembled VP nanoaggregate (Nano-BPD), with a free-form of BPD as a control-for their ability to inhibit P-gp. Using a combination of in vitro intracellular BPD accumulation assays, P-gp substrate retention experiments, and Seahorse-based metabolic profiling, we identified Nano-BPD as the lead formulation for P-gp modulation in cancer cells. 4. Development of non-toxic natural products, repurposed drugs, and small molecules to modulate the resistance to anticancer drugs mediated by P-gp and ABCG2. 4a. Screening of A3 adenosine receptor nucleoside adenine ligands for modulation of human P-gp and ABCG2. We continue to work on synthesizing nucleoside adenine derivatives in order to identify potent A3 adenosine receptor ligands as modulators of both P-gp and ABCG2. So far, we have characterized the effects of 35 compounds, synthesized by our collaborator Kenneth Jacobson at NIDDK, NIH, on P-gp and ABCG2 by using ATPase assays. 4b. Characterization of tyrosine kinase inhibitors, orphan and re-purposed drugs, natural products, and small molecules for modulatory effects on P-gp and ABCG2. There is a significant overlap of substrate specificity between P-gp and ABCG2. We are screening tyrosine kinase inhibitors, small molecule libraries, and natural product compounds for their interactions with P-gp and ABCG2 with the goal of developing effective strategies against multidrug-resistant cancers. These studies also provide information about the modulatory effects of repurposed drugs that are either in clinical trials or have been approved by the FDA for the treatment of cancer. We found that epertinib, a potent inhibitor of EGFR and HER2 receptors, modulates the function of P-gp and ABCG2. Similarly, vodobatinib, a c-Abl kinase inhibitor overcomes resistance to anticancer drugs-mediated by both P-gp and ABCG2.