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-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--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--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.
Gruol
D J
DJ
The Sidney Kimmel Cancer Center, San Diego, California 92121, USA. dgruol@skcc.org
Bernd
J
J
Phippard
A E
AE
Ojima
I
I
Bernacki
R J
RJ
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000037:antagonists & inhibitors* / Q000235:genetics; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D005260:Female; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D017239:Paclitaxel / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology*; D043823:Taxoids / Q000031:analogs & derivatives; D014407:Tumor Cells, Cultured; D000097202:ATP-Binding Cassette Sub-Family B Member 4
D018435:ATP Binding Cassette Transporter, Subfamily B; D000972:Antineoplastic Agents, Phytogenic; D008565:Membrane Proteins; D017239:Paclitaxel; D043823:Taxoids; D000097202:ATP-Binding Cassette Sub-Family B Member 4; C429967:tRA-96023