Abstract
Novel classes of antimicrobials are needed to address the emergence of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). We have recently identified pyruvate kinase (PK) as a potential novel drug target based upon it being an essential hub in the MRSA interactome (Cherkasov, A., Hsing, M., Zoraghi, R., Foster, L. J., See, R. H., Stoynov, N., Jiang, J., Kaur, S., Lian, T., Jackson, L., Gong, H., Swayze, R., Amandoron, E., Hormozdiari, F., Dao, P., Sahinalp, C., Santos-Filho, O., Axerio-Cilies, P., Byler, K., McMaster, W. R., Brunham, R. C., Finlay, B. B., and Reiner, N. E. (2011) J. Proteome Res. 10, 1139-1150; Zoraghi, R., See, R. H., Axerio-Cilies, P., Kumar, N. S., Gong, H., Moreau, A., Hsing, M., Kaur, S., Swayze, R. D., Worrall, L., Amandoron, E., Lian, T., Jackson, L., Jiang, J., Thorson, L., Labriere, C., Foster, L., Brunham, R. C., McMaster, W. R., Finlay, B. B., Strynadka, N. C., Cherkasov, A., Young, R. N., and Reiner, N. E. (2011) Antimicrob. Agents Chemother. 55, 2042-2053). Screening of an extract library of marine invertebrates against MRSA PK resulted in the identification of bis-indole alkaloids of the spongotine (A), topsentin (B, D), and hamacanthin (C) classes isolated from the Topsentia pachastrelloides as novel bacterial PK inhibitors. These compounds potently and selectively inhibited both MRSA PK enzymatic activity and S. aureus growth in vitro. The most active compounds, cis-3,4-dihyrohyrohamacanthin B (C) and bromodeoxytopsentin (D), were identified as highly potent MRSA PK inhibitors (IC(50) values of 16-60 nM) with at least 166-fold selectivity over human PK isoforms. These novel anti-PK natural compounds exhibited significant antibacterial activities against S. aureus, including MRSA (minimal inhibitory concentrations (MIC) of 12.5 and 6.25 μg/ml, respectively) with selectivity indices (CC(50)/MIC) >4. We also report the discrete structural features of the MRSA PK tetramer as determined by x-ray crystallography, which is suitable for selective targeting of the bacterial enzyme. The co-crystal structure of compound C with MRSA PK confirms that the latter is a target for bis-indole alkaloids. It elucidates the essential structural requirements for PK inhibitors in "small" interfaces that provide for tetramer rigidity and efficient catalytic activity. Our results identified a series of natural products as novel MRSA PK inhibitors, providing the basis for further development of potential novel antimicrobials.
Highlights
Methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK) has been recently identified as a potential novel antimicrobial drug target
In Vitro Screening of the Marine Extract Library—During the course of our search for potent and selective MRSA PK inhibitors from marine organisms, purified recombinant MRSA PK (Fig. 3) was used as a target enzyme to examine a total of 968 crude benthic marine invertebrate extracts collected from Papua New Guinea, Indonesia, Dominica, Brazil, British Columbia, South Africa, and Norway
All extracts were used at a concentration of 5 g/ml, with a substrate concentration of 10 mM P-enolpyruvate, which is close to the MRSA PK Km (e.g. 6.6 mM) [4], so that the IC50 values should approximate the Ki
Summary
Methicillin-resistant Staphylococcus aureus (MRSA) PK has been recently identified as a potential novel antimicrobial drug target. We have recently identified pyruvate kinase (PK) as a potential novel drug target based upon it being an essential hub in the MRSA interactome Identification of new targets for antibacterial development based upon novel scaffolds with unique mechanisms of action are critically needed [3] To this end, we recently identified pyruvate kinase (PK), an evolutionarily conserved, highly connected essential hub protein in MRSA, with structural features distinct from the mammalian orthologs, as a novel candidate drug target [1, 4, 5]. In this study, we describe for the first time the structural features of S. aureus PK obtained by x-ray crystallography and highlight significant structural differences in the bacterial enzyme compared with human PKs in the small interface located between the adjacent PK subunits This region was important for the preferential binding of bis-indole alkaloids to MRSA PK during co-crystallization of compound C with MRSA PK. The results of these studies may allow for the design of potent and specific bacterial PK inhibitors
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