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Structural recognition of tubulysin B derivatives by multidrug resistance efflux transporters in human cancer cells.

ABSTRACT: Multidrug resistance (MDR) is a major hindrance to curative chemotherapy of various human malignancies. Hence, novel chemotherapeutics must be evaluated for their recognition by MDR efflux transporters. Herein we explored the cytotoxic activity of synthetic tubulysin B (Tub-B, EC1009) derivatives (Tub-B-hydrazide/EC0347 and Tub-B bis-ether/EC1820), and their recognition by the MDR efflux transporters P-glycoprotein 1 (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP). Originally isolated from Myxobacteria, tubulysins exhibited potent cytotoxic activity via microtubule depolymerization, and evaded recognition by these MDR efflux pumps. We show that subtle modifications in the natural Tub-B structure enhance its cytotoxicity and drug efflux efficiency. Whereas increasing the lipophilicity of Tub-B drugs enhanced their diffusion into the cell and consequently decreased the IC50 values (≥ 0.27 nM), increasing drug polarity enhanced their recognition by P-gp (>200-fold resistance in P-gp-overexpressing cells). Furthermore, restricting drug exposure time to the clinically relevant 4 h pulse, markedly enhanced efflux by P-gp, resulting in a 1000-fold increased resistance, which was further enhanced upon increased P-gp levels (i.e. an additional 3-fold increase in P-gp levels resulted in >6,000-fold resistance). The unique ability of EC1009 to evade recognition by MDR efflux pumps warrants drug development of tubulysin B derivatives as potent antitumor agents which overcome MDR in cancer.

SUBMITTER: Stark M

PROVIDER: S-EPMC5564821 | biostudies-other | 2017 Jul

REPOSITORIES: biostudies-other

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Project description:UnlabelledThe AcrAB-TolC system in Escherichia coli is an intrinsic RND-type multidrug efflux transporter that functions as a tripartite complex of the inner membrane transporter AcrB, the outer membrane channel TolC, and the adaptor protein AcrA. Although the crystal structures of each component of this system have been elucidated, the crystal structure of the whole complex has not been solved. The available crystal structures have shown that AcrB and TolC function as trimers, but the number of AcrA molecules in the complex is now under debate. Disulfide chemical cross-linking experiments have indicated that the stoichiometry of AcrB-AcrA-TolC is 1:1:1; on the other hand, recent cryo-electron microscopy images of AcrAB-TolC suggested a 1:2:1 stoichiometry. In this study, we constructed 1:1-fixed AcrB-AcrA fusion proteins using various linkers. Surprisingly, all the 1:1-fixed linker proteins showed drug export activity under both acrAB-deficient conditions and acrAB acrEF double-pump-knockout conditions regardless of the lengths of the linkers. Finally, we optimized a shorter linker lacking the conformational freedom imparted by the AcrB C terminus. These results suggest that a complex with equal amounts of AcrA and AcrB is sufficient for drug export function.ImportanceThe structure and stoichiometry of the RND-type multidrug exporter AcrB-AcrA-TolC complex are still under debate. Recently, electron microscopic images of the AcrB-AcrA-TolC complex have been reported, suggesting a 1:2:1 stoichiometry. However, we report here that the AcrB-AcrA 1:1 fusion protein is active for drug export under acrAB-deficient conditions and also under acrAB acrEF double-deficient conditions, which eliminate the aid of free AcrA and its close homolog AcrE, indicating that the AcrB-AcrA 1:1 stoichiometry is enough for drug export function. In addition, the AcrB-AcrA fusion protein can function without the aid of free AcrA. We believe that these results are very important for considering the structure and mechanism of AcrAB-TolC-mediated multidrug export.

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Structural recognition of tubulysin B derivatives by multidrug resistance efflux transporters in human cancer cells.

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