FIG 2.

Quercetin inhibits CtPilB. (A) An HTS identified quercetin as an inhibitor of MANT-ATP binding to CtPilB. Library compounds were screened by a high-throughput screen as described in the main text for chemicals that reduced the ability of PilB to associate with MANT-ATP. The red and green lines indicate the levels of fluorescence for the negative and positive controls, respectively. The former included MANT-ATP and CtPilB, while the latter included 20 μM ATP in addition. Compound 77, which is quercetin, was found to reduce the fluorescence to a level similar to that of ATP. The maximum value for the y axis here is 90, and compounds that emit significant fluorescence are cut off from this figure. (B) Quercetin reduces MANT-ATP binding to CtPilB. The fluorescence of triplicate samples containing constant concentrations of CtPilB and MANT-ATP with various concentrations of quercetin was analyzed. The three different sets of colored dots represent the averages from three independent experiments, with error bars omitted for clarity. Shown is the best fit of the data to an isotherm with an IC₅₀ of 2.12 μM (see M&M). The structure of quercetin is shown in the inset. (C) Quercetin inhibits the ATPase activity of CtPilB. The ATPase activity of CtPilB was determined in triplicates in the presence of quercetin at the indicated concentrations by endpoint assays. The three different sets of colored dots show the averages from three independent experiments, with errors bars omitted for clarity. The IC₅₀ of quercetin against CtPilB ATPase activity was determined to be 2.50 μM by curve fitting.