β-Lactamase Suppression as a Strategy to Target Methicillin-Resistant Staphylococcus aureus: Proof of Concept

Abstract

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.

New Weapons to Fight against Staphylococcus aureus Skin Infections

“…Another strategy that has been proposed to overcome resistance is the blocking of β-lactamases. In this regard, Thomas et al used pyrimidine-2-amines (P2As) as a new inhibitor that, through an unknown regulator, prevents blaZ expression and therefore suppresses β-lactamase activity in vitro [13]. The presence of P2As enhanced penicillin G sensitivity and reduced the MIC of Amp against MRSA USA300 from 256 µg/mL to 64 µg/mL [13].This approach has the advantage of not employing direct enzyme inhibitors such as sulbactam and clavulanate that can contribute to the development of resistance [14].…”

A benzoxazolylurea inhibits VraS and enhances antimicrobials against vancomycin intermediate-resistantStaphylococcus aureus

“…We have identified a powerful VraS inhibitor, chemical 1 ( We ran a screen for vancomycin enhancers against Mu50 (8), an archetypal VISA strain (see (31) and Supplementary Methods for details). Mu50 was intermediate-resistant (vancomycin MIC 4 μg/mL) as expected (9).…”