β-Lactamase (penicillinase) renders early, natural β-lactams like penicillin G useless against methicillin-resistant Staphylococcus aureus (MRSA), which also expresses PBP2a, responsible for resistance to semisynthetic, penicillinase-insensitive β-lactams like oxacillin. Antimicrobial discovery is difficult, and resistance exists against most treatment options. Enhancing βlactams against MRSA would revive its clinical utility. Most research on antimicrobial enhancement against MRSA focuses on oxacillin due to βlactamase expression. Yet, Moreillon and others have demonstrated that penicillin G is as potent against a β-lactamase gene knockout strain, as vancomycin is against wild-type MRSA. Penicillin G overcame PBP2a because β-lactamase activity was blocked. Additionally, animals treated with a combination of direct β-lactamase inhibitors like sulbactam and clavulanate with penicillin G developed resistant infections, clearly demonstrating that direct inhibition of β-lactamase is not a good strategy. Here, we show that 50 μM pyrimidine-2-amines (P2As) reduce the minimum inhibitory concentration (MIC) of penicillin G against MRSA strains by up to 16fold by reducing β-lactamase activity but not by direct inhibition of the enzyme. Oxacillin was not enhanced due to PBP2a expression, demonstrating the advantage of penicillin G over penicillinase-insensitive β-lactams. P2As modulate an unknown global regulator but not established antimicrobial-enhancement targets Stk1 and VraS. P2As are a practical implementation of Moreillon's principle of suppressing β-lactamase activity to make penicillin G useful against MRSA, without employing direct enzyme inhibitors.

We have ascertained that phenylindolylmethyldiaminopyrimidines (PIDAPs), stop the growth of USA300 MRSA at low micromolar concentrations. The controls, penicillin G and vancomycin, are able to stop the growth of MRSA at ~765 μM (256 μg/mL) and ~1.38 μM (2 μg/mL) respectively. We have also found out that PIDAPs are bactericidal at or close to the MIC. No activity was observed against Gram-negative pathogens. Other Gram-positive pathogens have not yet been tested. Based on a search through the ChEMBL database, PIDAPs are a novel class of chemicals with antimicrobial properties. A limited structure-function study suggests that the diaminopyrimidine is part of the pharmacophore. Unfortunately, we also detected potential dose-limiting toxicity on human cell lines. Further, detailed studies are needed.

We have ascertained that phenylindolylmethyldiaminopyrimidines (PIDAPs), stop the growth of USA300 MRSA at low micromolar concentrations. The controls, penicillin G and vancomycin, are able to stop the growth of MRSA at ~765 μM (256 μg/mL) and ~1.38 μM (2 μg/mL) respectively. We have also found out that PIDAPs are bactericidal at or close to the MIC. No activity was observed against Gram-negative pathogens. Other Gram-positive pathogens have not yet been tested. Based on a search through the ChEMBL database, PIDAPs are a novel class of chemicals with antimicrobial properties. A limited structure-function study suggests that the diaminopyrimidine is part of the pharmacophore. Unfortunately, we also detected potential dose-limiting toxicity on human cell lines. Further, detailed studies are needed.

β-lactamase (penicillinase) renders early β-lactams like penicillin G useless against methicillin-resistant Staphylococcus aureus (MRSA). Antimicrobial discovery is difficult, and resistance exists against most treatment options. Enhancing β-lactams against MRSA would revive their clinical utility. Moreillon and others have demonstrated that penicillin G is as potent against a β-lactamase gene knockout strain, as vancomycin is against wild-type MRSA. Yet, direct β-lactamase inhibitors like sulbactam and clavulanate gave rise to penicillin G resistance. Instead, 50 μM pyrimidine-2-amines (P2A) reduce the minimum inhibitory concentration (MIC) of penicillin G against MRSA strains by up to 64-fold by reducing β-lactamase expression. PBP2a prevented oxacillin enhancement, demonstrating the advantage of penicillin G over penicillinase-insensitive β-lactams. P2As modulate an unknown global regulator, but not established antimicrobial-enhancement targets Stk1 and VraS. P2As are a practical implementation of Moreillon’s principle of suppressing β-lactamase activity to make penicillin G useful against MRSA, without employing direct enzyme inhibitors.

β-lactamase (penicillinase) based resistance renders early β-lactams like penicillin G useless against methicillin-resistant Staphylococcus aureus (MRSA). Finding novel antimicrobials is difficult, and resistance has been observed against most treatment options. Increasing the susceptibility of MRSA to early β-lactams would help bring back the clinical utility of a powerful and safe class of antimicrobials. Moreillon and coworkers have already demonstrated that β-lactamase inhibition can render penicillin G more powerful than oxacillin. Yet, they also highlighted that direct β-lactamase inhibitors are not the best tool for the job. Here, we reveal that certain pyrimidin-2-amines (P2A) reduce the minimum inhibitory concentration (MIC) of penicillin G against resistant S. aureus strains reliably by up to 64-fold, when present at 50 μM. P2As do not inhibit β-lactamases directly, but reduce its expression; This is in stark contrast to direct penicillinase inhibitors like sulbactam and clavulanate that covalently bind the secreted protein. The MIC of penicillinase-insensitive oxacillin were not altered, clearly because these cannot overcome PBP2a; This demonstrates the advantage of penicillin G over penicillinase-insensitive β-lactams. Gene knockout experiments show that factors commonly associated with decreased resistance to cell wall-active antimicrobials, like Stk1 and VraS, are not the target of P2As. Yet, multiple gene knockouts were resistant to P2A activity, suggesting these chemicals act through an as-yet-unknown central controller. A preliminary structure-activity relationship has also provided insights into pharmacophoric features. We have demonstrated that Moreillon’s principle, that penicillin G is useful against MRSA upon cessation of β-lactamase activity, can be practically implemented by suppression of penicillinase expression instead of direct β-lactamase inhibition.