Fig. 4.

Analogs of the pyranopyridine EPI MBX2319 exhibit improved efflux pump inhibition. The bactericidal activity of a minimally bactericidal dose of ciprofloxacin (0.01 μg/mL) is potentiated against E. coli by MBX2319 (A), MBX3132 (B), and MBX3135 (C). The concentrations of MBX3132 and MBX3135 used are 10-fold lower than the concentration of MBX2319. Inhibition of efflux pump activity is shown in a cell-based assay that measures accumulation of the fluorescent dye H33342 in the presence of MBX2319 (D), MBX2931 (E), MBX3132 (F), and MBX3135 (G). Effect of 10 nM MBX2319 (H), MBX3132 (I), and MBX3135 (J) on the Michaelis–Menten kinetics of AcrAB-TolC using the nitrocefin efflux assay (31). CIP, ciprofloxacin; Cmpd, MBX compound alone; C_p, periplasmic concentration of nitrocefin (micromolar); RFU, relative fluorescence unit; V_e, efflux pump velocity (in nanomoles per second per milligram).