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Reconstitution of P‐glycoprotein in Nanodiscs using the MSP1D1 Scaffold Protein for Biochemical Inhibitor Screens

Many cancers become multidrug resistant due to overexpression of the transmembrane efflux pump P‐glycoprotein (P‐gp). Compounds inhibiting P‐gp function may therefore have the potential to improve the effectiveness of chemotherapeutic drugs. Using in silico methods, we have been able to successfully identify a number of putative P‐gp inhibitor compounds. In cell culture assays, many of these compounds were shown to re‐sensitize cancers to chemotherapeutics. However, when tested with purified P‐gp, some of these compounds showed little effect on the catalytic activity of the isolated protein. The relatively high concentrations of detergents needed to solubilize P‐gp likely reduced the potency of the inhibitor compounds. To overcome this issue, purified P‐gp was inserted into nanostructures (nanodiscs) which much more closely resemble natural cell membrane. We and others have observed that this stabilizes P‐gp and stimulates its catalytic activity. However, previous assays of inhibitor compounds with medium size MSP1E3D1 nanodiscs still did not show clear effects on catalytic activity of the protein despite the fact that the inhibitors were effective in cell culture. We hypothesize that the previously used MSP1E3D1 nanodiscs still may have been too large, leaving hydrophobic space into which the inhibitors could partition, again reducing the effective concentration of the inhibitors. To improve on this protocol, we here show our efforts to use a shorter membrane scaffold protein, known as MSP1D1, for the assembly of smaller nanodiscs. After optimization of conditions for assembly and insertion, and tests for biological activity, we will assay our potential inhibitor hit compounds for their ability to affect catalytic ATP hydrolysis activities of P‐gp.