Antimicrobial Susceptibility Testing
The 18 compounds were then tested for their ability to suppress the growth of N. gonorrhoeae FA19 using the disc diffusion zone method. Eleven compounds did not suppress growth and were excluded (data not shown). For the remaining 7 compounds, the minimal inhibitory concentrations (MICs) were determined against three N. gonorrhoeae strains: FA19, a penicillin-susceptible strain [28]; FA6140, a penicillin-resistant but cephalosporinsusceptible strain [29]; and 35/02, a strain that exhibits intermediate resistance to cephalosporin (CephI)[30]. The IC50 values and MICs of the seven compounds are shown in Table 2 and their respective structures are shown in Fig. 5. The MIC values ranged from 2?2 mg/ml and all except for compound 4 were higher in resistant strains compared to FA19. The best compound overall (7) exhibited an MIC of 2 mg/ml (5.6 mM) against FA19 and 8 mg/ml (23.4 mM) against both FA6140 and 35/02.
assay was determined during initial optimization and after the HTS assay. Pc is the mean FP signal of PBP 2-bound tracer control, Nc is the mean FP signal of free Bocillin-FL, and Dc is the FP signal in the presence of 100 mm penicillin G. S/N (signal-to-noise) and the Z factors are as defined in Material and Methods. (n = number of measurements). See also Fig. S1). 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?000 mM) with fixed concentrations of PBP 2 (1 mM) and Bocillin-FL (1 mM) 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 6 standard deviation of four replicate experiments at each concentration of penicillin G. bonds are observed between Asp346 and the secondary amine of the thiazolidine ring, and between the hydroxyl of Tyr544 and the carbonyl of compound 7. Additionally, there are arene-hydrogen interactions from the thiazolidine ring to both Thr347 and Ser362. The bromine atom appears to be totally solvated and does not interact with the protein directly. Overall, the docking results suggest that all of the seven compounds can bind within the active site of PBP 2, although precise details of binding can only be revealed by experimental structural studies.
Discussion
As important targets for b-lactams and against the background of increasing clinical resistance to existing antibiotics, much effort has been directed toward developing new inhibitors of PBPs. Some approaches have sought to adapt the b-lactam moiety by, for instance, incorporating elements of the peptide substrates onto one of the R1 or R2 side chains [37,38,39], while others have synthesized compounds that mimic the tetrahedral intermediates of the reaction, including phosphonates and boronates [40,41,42,43]. Given the wealth of structural information for several clinically important PBPs, in silico docking has also been employed in some systems [44]. To contribute to this effort, we have developed a high-throughput assay for PBPs that measures the fluorescence polarization (FP) of Bocillin-FL [27] and used it to screen for potential inhibitors of N. gonorrhoeae PBP 2 from a library of 50,080 compounds. After eliminating those that acted pro-miscuously, did not demonstrate a concentration-dependent inhibition, or were b-lactams, 18 compounds were examined in more detail, and 7 of these exhibited antimicrobial activity against N. gonorrhoeae, including PenR and CephI strains. To the best of our knowledge, this is the first report of high-throughput screening (HTS) against a PBP. Development of the assay was made possible by using fluorescence polarization to distinguish between the fluorescent penicillin (Bocillin-FL) remaining in solution from that bound to the PBP target, an approach first suggested by Zhao et al. [27]. The robustness and sensitivity of the assay is supported by the large assay window and Z factor. As a safeguard against any false positives arising from the FP-based assay, an SDS-PAGE competition assay was used to confirm inhibition of PBP 2. The identification of a cephalosporin with an IC50 of 3 mM against PBP2 was demonstration of the validity of the assay. Out of the seven compounds that exhibited the lowest IC50 values for PBP 2 (, 50 mM) and good anti-gonococcal activity, two of these (compounds 4 and 5) contain a sulfonamide group between two aromatic rings. Interestingly, a sulfonamide of similar structure was identified recently as an inhibitor of PBP2a from methicillin-resistant Staphylococcus aureus (MRSA) [45]. Similar to compounds 4 and 5, compound 1 has two aromatic rings joined by a bridging group reminiscent of the sulfonamide inhibitors and showed reasonable antimicrobial activity against FA19, but less so against 6140 and 35/02. 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 mM penicillin G), denoted by open circles, represent the percent of inhibition based on the average of four FP measurements. Fifty-two plates with 96610X cocktails each and one plate with eight610 X cocktails were screened. The box indicates the 58 cocktails that exhibited $80% inhibition of Bocillin-FL binding to PBP 2. nitrobenzaldehyde group. This compound demonstrated moderate antibacterial activity against FA19, with higher MIC values against FA610 and 35/02. It was the most successful compound during the docking simulations and in this model there is an interaction with the serine nucleophile of PBP 2 (Ser310). Compound 6 exhibited the lowest IC50 value (49 mM) with good MICs against FA19 (4 mg/ml) and the two antibiotic-resistant strains (both were 8 mg/ml). Compound 7 showed rather Table 2. IC50 values and antimicrobial activities of seven compounds identified by high-throughput screening against PBP 2 from N. gonorrhoeae strain FA19.