Figure 1.
Optimization of the fluorescence polarization (FP) assay.
A. Fluorescence polarization (mP) of free Bocillin-FL at various concentrations from 0.002 to 4 µM. B. Binding experiments with Bocillin-FL (1 µM) and PBP 2 at various protein concentrations from 0.2 to 4 µM. Fluorescence of Bocillin-FL – PBP2 was measured in relative fluorescence units (RFU) (open circles). Maximum specific binding (triangles), i.e. assay window (ΔmP), was determined by FP. Assay window is defined as the difference between FP of protein-tracer sample and free-tracer, i.e. ΔmP = mPs – mPfree. In all experiments, the data points represent the mean ± standard deviation of four replicate experiments at each concentration of Bocillin-FL or PBP 2.
Table 1.
Statistical parameters of the FP assay.
Figure 2.
Inhibition of FP of Bocillin-FL and PBP 2 by increasing amounts of penicillin G.
Assays contained variable concentrations of penicillin G (0.05–1000 µM) with fixed concentrations of PBP 2 (1 µM) and Bocillin-FL (1 µM) with or without 10% DMSO. The solid line represents data for PBP 2 without DMSO and the dashed line is PBP 2 with DMSO. In all experiments, the data points represent the mean ± standard deviation of four replicate experiments at each concentration of penicillin G.
Figure 3.
Initial HTS of 50,080 small lead compounds from DIVERSet ChemBridge Library was performed using cocktails of 10 different compounds (10X cocktails).
Each plate contained duplicate samples of 10X cocktails with PBP 2 and Bocillin-FL. Data points denoted by the black dots represent the percent of inhibition for each 10X cocktail determined using the average of two sample FP measurements, and the means of free Bocillin-FL (Nc) and bound Bocillin–FL (Pc) controls recorded in quadruplicate for each corresponding plate. Data points of the displaced tracer controls (Dc - the FP of the Bocillin-FL - protein at 100 µM penicillin G), denoted by open circles, represent the percent of inhibition based on the average of four FP measurements. Fifty-two plates with 96×10X cocktails each and one plate with eight×10 X cocktails were screened. The box indicates the 58 cocktails that exhibited ≥80% inhibition of Bocillin-FL binding to PBP 2.
Table 2.
IC50 values and antimicrobial activities of seven compounds identified by high-throughput screening against PBP 2 from N. gonorrhoeae strain FA19.
Figure 4.
IC50 values of three representative hits from HTS for inhibition of acylation of PBP 2 by Bocillin-FL.
IC50s of the inhibitors were determined by SDS-PAGE-based concentration-response assays using a 0.05–1000 µM concentration range for the inhibitors with 1 µM PBP 2 and 10 µM of Bocillin-FL. Triton X-100 (0.01%) was included to eliminate promiscuous inhibitors. A. Compound 2 (in Table 2). B. Compound 4. C. Compound 7. In all experiments, the data points represent the mean ± standard deviation for two replicate (compounds 4 and two) or four replicate (compound 7) experiments at each concentration of inhibitor.
Figure 5.
Structures of seven compounds that exhibited antimicrobial activity against N. gonorrhoeae.
Compound numbers correspond with those in Table 2.
Figure 6.
Docking of two compounds into the structure of N. gonorrhoeae penicillin-binding protein 2.
The main chain of PBP 2 is shown as a grey ribbon, residues of the active site motifs of PBPs are shown with green bonds and additional amino acids that are predicted to form interactions with the ligand are shown in blue. A. compound 2. B. compound 7. Figure prepared with MOE 2011.10 (Chemical Computing Group Inc.).