Optimization and structure–activity relationships of a series of potent inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase as novel antimicrobial agents

A novel series of hydrazones were synthesized and evaluated as inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK has been identified as one of the most highly connected ‘hub proteins’ in MRSA. PK has been shown to be critical for bacterial survival which makes it a potential target for development of novel antibiotics and the high degree of connectivity implies it should be very sensitive to mutations and thus less able to develop resistance. PK is not unique to bacteria and thus a critical requirement for such a PK inhibitor would be that it does not inhibit the homologous human enzyme(s) at therapeutic concentrations. Several MRSA PK inhibitors (including 8d) were identified using in silico screening combined with enzyme assays and were found to be selective for bacterial enzyme compared to four human PK isoforms (M1, M2, R and L). However these lead compounds did not show significant inhibitory activity for MRSA growth presumably due to poor bacterial cell penetration. Structure–activity relationship (SAR) studies were carried out on 8d and led us to discover more potent compounds with enzyme inhibiting activities in the low nanomolar range and some were found to effectively inhibit bacteria growth in culture with minimum inhibitory concentrations (MIC) as low as 1 μg/mL. These inhibitors bind in two elongated flat clefts found at the minor interfaces in the homo-tetrameric enzyme complex and the observed SAR is in keeping with the size and electronic constraints of these binding sites. Access to the corresponding sites in the human enzyme is blocked.

In silico and in vitro enzyme screening identified a benzimidazole hydrazone (8d) as a lead inhibitor of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK) but without significant antibacterial activity. Structural and structure–activity studies have shown that this series binds in a novel allosteric binding site unique to bacterial PK. The original series was optimized for enzyme inhibition activity to identify indole analogs, such as 7g, with nanomolar potency as inhibitors of MRSA PK, with high selectivity over human PK isoforms and which exhibit minimum inhibitory concentrations (MIC) for MRSA as potent as 1 μg/ml.Figure optionsDownload as PowerPoint slide