@article {Gruol104, author = {Donald J. Gruol and Josh Bernd and Alba E. Phippard and Iwao Ojima and Ralph J. Bernacki}, title = {The Use of a Novel Taxane-Based P-Glycoprotein Inhibitor to Identify Mutations That Alter the Interaction of the Protein with Paclitaxel}, volume = {60}, number = {1}, pages = {104--113}, year = {2001}, doi = {10.1124/mol.60.1.104}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {Murine thymoma cell lines expressing mutated forms of themdr1b P-glycoprotein were isolated using a novel taxane-based P-glycoprotein inhibitor tRA-96023 (SB-RA-31012). The selection strategy required resistance to a combination of tRA-96023 and colchicine. Five mutations were identified (N350I, I862F, L865F, L868W, and A933T) that reduce the capacity of tRA-96023 to inhibit P-glycoprotein-dependent drug resistance. These mutations also result in a loss of paclitaxel resistance ranging from 47 to 100\%. Four mutations are located in the second half of the protein, within or near the proposed transmembrane segment (TMS) 10{\textendash}11 regions. The fifth mutation (N350I) is within the first half of the protein, proximal (cytoplasmic) to TMS 6. The variant cell line expressing the L868W mutation was subjected to a second round of selection involving tRA-96023 and the toxic drug puromycin. This resulted in the isolation of a cell line expressing a P-glycoprotein with a double mutation. The additional mutation (N988D) is located within TMS 12 and conveys further decreases in resistance to paclitaxel and the capacity of tRA-96023 to inhibit drug resistance. Taken together, the results indicate a significant contribution by the TMS 10{\textendash}12 portion of the protein to the recognition and transport of taxanes and give evidence that the cytoplasmic region proximal to TMS 6 also plays a role in taxane interactions with P-glycoproteins. 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. TMStransmembrane segment5βPodo5β-pregnane-17ol-3,20-dionePCRpolymerase chain reactionPgpP-glycoprotein}, issn = {0026-895X}, URL = {https://molpharm.aspetjournals.org/content/60/1/104}, eprint = {https://molpharm.aspetjournals.org/content/60/1/104.full.pdf}, journal = {Molecular Pharmacology} }