Site occupancy calibration of taxane pharmacology in live cells and tissues

Drug receptor site occupancy is a central pharmacology parameter that quantitatively relates the biochemistry of drug binding to the biology of drug action. Taxanes and epothilones bind to overlapping sites in microtubules (MTs) and stabilize them. They are used to treat cancer and are under investigation for neurodegeneration. In cells, they cause concentration-dependent inhibition of MT dynamics and perturbation of mitosis, but the degree of site occupancy required to trigger different effects has not been measured. We report a live cell assay for taxane-site occupancy, and relationships between site occupancy and biological effects across four drugs and two cell lines. By normalizing to site occupancy, we were able to quantitatively compare drug activities and cell sensitivities independent of differences in drug affinity and uptake/efflux kinetics. Across all drugs and cells tested, we found that inhibition of MT dynamics, postmitotic micronucleation, and mitotic arrest required successively higher site occupancy. We also found interesting differences between cells and drugs, for example, insensitivity of the spindle assembly checkpoint to site occupancy. By extending our assay to a mouse xenograft tumor model, we estimated the initial site occupancy required for paclitaxel to completely prevent tumor growth as 80%. The most important cellular action of taxanes for cancer treatment may be formation of micronuclei, which occurs over a broad range of site occupancies.

Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli

ABSTRACTThe AcrB protein ofEscherichia coli, together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions—Y49S, V127A, V127G, D153E, and G288C—mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions—F453C and L486W—were mapped to transmembrane (TM) helices 5 and 6, respectively. The nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux ofN-phenyl-1-naphthylamine, the TM suppressors did not improve the nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain.IMPORTANCEThe AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure-function plasticity of the AcrB protein pertaining to its drug efflux activity.