The use of a novel taxane-based P-glycoprotein inhibitor to identify mutations that alter the interaction of the protein with paclitaxel

Murine thymoma cell lines expressing mutated forms of the mdr1b P-glycoprot ein were isolated using a novel taxane-based P-glycoprotein inhibitor tRA-9 6023 (SB-RA-31012). The selection strategy required resistance to a combina tion of tRA-96023 and colchicine. Five mutations were identified (N3501, I8 62F, L865F L868W, and A933T) that reduce the capacity of tRA-96023 to inhib it P-glycoprotein-dependent drug resistance. These mutations also result in a loss of paclitaxel resistance ranging from 47 to 100%. Four mutations ar e located in the second half of the protein, within or near the proposed tr ansmembrane:segment (TMS) 10-11 regions. The fifth mutation (N3501) is with in the first half of the protein, proximal (cytoplasmic) to TMS 6. The vari ant cell line expressing the L868W mutation was subjected to a second round of selection involving tRA-96023 and the toxic drug puromycin. This result ed in the isolation of a cell line expressing a P-glycoprotein with a doubl e mutation. The additional mutation (N988D) is located within TMS 12 and co nveys further decreases in resistance to paclitaxel and the capacity of tRA -96023 to inhibit drug resistance. Taken together, the results indicate a s ignificant contribution by the TMS 10-12 portion of the protein to the reco gnition and transport of taxanes and give evidence that the cytoplasmic reg ion proximal to TMS 6 also plays a role in taxane interactions with P-glyco proteins. Interestingly, mutations within TMS 6 and 12 were found to cause a partial loss of PSC-833 inhibitor activity, suggesting that these regions participate in the interactions with cyclosporin and its derivatives.