Discovery and optimization of a new class of pyruvate kinase inhibitors as potential therapeutics for the treatment of methicillin-resistant Staphylococcus aureus infections

A novel series of 2,2′-bridged bis-indoles has been designed and synthesized as potent and selective allosteric inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). Based on examination of the X-ray structure of the 3,3′-bridged bis-indole natural product cis-3-4-dihydrohamacanthin B (4) bound to MSRA PK (IC50=16nM and MIC=12.5μg/ml), a 2,2′-bridged bis-indole series was envisaged and was optimized for enzyme and cell growth inhibition activity to identify compounds such a 10d with low nanomolar potency (IC50=2.0nM) for inhibition of MRSA PK, high selectivity over human PK isoforms, low mammalian cell toxicity and excellent antibacterial activity against MRSA (MIC=0.5μg/ml). A novel series of bis-indoles derived from naturally occurring marine alkaloid 4 were synthesized and evaluated as inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK is not only critical for bacterial survival which would make it a target for development of novel antibiotics, but it is reported to be one of the most highly connected ‘hub proteins’ in MRSA, and thus should be very sensitive to mutations and making it difficult for the bacteria to develop resistance. From the co-crystal structure of cis-3-4-dihydrohamacanthin B (4) bound to S. aureus PK we were able to identify the pharmacophore needed for activity. Consequently, we prepared simple direct linked bis-indoles such as 10b that have similar anti-MRSA activity as compound 4. Structure–activity relationship (SAR) studies were carried out on 10b and led us to discover more potent compounds such as 10c, 10d, 10k and 10m with enzyme inhibiting activities in the low nanomolar range that effectively inhibited the bacteria growth in culture with minimum inhibitory concentrations (MIC) for MRSA as low as 0.5μg/ml. Some potent PK inhibitors, such as 10b, exhibited attenuated antibacterial activity and were found to be substrates for an efflux mechanism in S. aureus. Studies comparing a wild type S. aureus with a construct (S. aureus LAC Δpyk::ErmR) that lacks PK activity confirmed that bactericidal activity of 10d was PK-dependant.