High-throughput screening and Bayesian machine learning for copper-dependent inhibitors of Staphylococcus aureus.

One potential source of new antibacterials is through probing existing chemical libraries for copper-dependent inhibitors (CDIs), i.e., molecules with antibiotic activity only in the presence of copper. Recently, our group demonstrated that previously unknown staphylococcal CDIs were frequently present in a small pilot screen. Here, we report the outcome of a larger industrial anti-staphylococcal screen consisting of 40 771 compounds assayed in parallel, both in standard and in copper-supplemented media. Ultimately, 483 had confirmed copper-dependent IC50 values under 50 µM. Sphere-exclusion clustering revealed that these hits were largely dominated by sulfur-containing motifs, including benzimidazole-2-thiones, thiadiazines, thiazoline formamides, triazino-benzimidazoles, and pyridinyl thieno-pyrimidines. Structure-activity relationship analysis of the pyridinyl thieno-pyrimidines generated multiple improved CDIs, with activity likely dependent on ligand/ion coordination. Molecular fingerprint-based Bayesian classification models were built using Discovery Studio and Assay Central, a new platform for sharing and distributing cheminformatic models in a portable format, based on open-source tools. Finally, we used the latter model to evaluate a library of FDA-approved drugs for copper-dependent activity in silico. Two anti-helminths, albendazole and thiabendazole, scored highly and are known to coordinate copper ions, further validating the model's applicability.

Pyrazolopyrimidinones, a novel class of copper-dependent bactericidal antibiotics against multi-drug resistant S. aureus.

The treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections poses a therapeutic challenge as even last resort drugs become increasingly ineffective. As the demand for antibiotics with novel modes of action is growing, new approaches are needed to probe a greater spectrum of antimicrobial activities for their potential efficacy against drug-resistant pathogens. The use of copper (Cu) by the innate immune system to mount an antimicrobial response against bacterial invaders has created an opportunity to explore a role for Cu in antimicrobial therapy. Here we describe pyrazolopyrimidinones (PZP) as novel copper-dependent inhibitors (CDI) of S. aureus. 5-Benzyl-3-(4-chlorophenyl)-2-methyl-4H,7H-pyrazolo[1,5-a]pyrimidin-7-one (PZP-915) showed potent bactericidal properties at sub-micromolar concentrations and activity against clinical MRSA isolates and biofilms cultures. This cupricidal activity is founded on the molecule's ability to coordinate Cu and induce accumulation of Cu ions inside S. aureus cells. We demonstrate that exposure to 915 + Cu led to an almost instantaneous collapse of the membrane potential which was accompanied by a complete depletion of cellular ATP, loss of cell-associated K+, a substantial gain of cell associated Na+, and an inability to control the influx of protons in slightly acidic medium, while the integrity of the cell membrane remained intact. These findings highlight PZP-915 as a novel membrane-directed metalloantibiotic against S. aureus that is likely to target a multiplicity of membrane associated protein functions rather than imposing physical damage to the membrane structure.