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Identification of pyruvate kinase in methicillin-resistant Staphylococcus aureus as a novel antimicrobial drug target.

ABSTRACT: Novel classes of antimicrobials are needed to address the challenge of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). Using the architecture of the MRSA interactome, we identified pyruvate kinase (PK) as a potential novel drug target based upon it being a highly connected, essential hub in the MRSA interactome. Structural modeling, including X-ray crystallography, revealed discrete features of PK in MRSA, which appeared suitable for the selective targeting of the bacterial enzyme. In silico library screening combined with functional enzymatic assays identified an acyl hydrazone-based compound (IS-130) as a potent MRSA PK inhibitor (50% inhibitory concentration [IC50] of 0.1 ?M) with >1,000-fold selectivity over human PK isoforms. Medicinal chemistry around the IS-130 scaffold identified analogs that more potently and selectively inhibited MRSA PK enzymatic activity and S. aureus growth in vitro (MIC of 1 to 5 ?g/ml). These novel anti-PK compounds were found to possess antistaphylococcal activity, including both MRSA and multidrug-resistant S. aureus (MDRSA) strains. These compounds also exhibited exceptional antibacterial activities against other Gram-positive genera, including enterococci and streptococci. PK lead compounds were found to be noncompetitive inhibitors and were bactericidal. In addition, mutants with significant increases in MICs were not isolated after 25 bacterial passages in culture, indicating that resistance may be slow to emerge. These findings validate the principles of network science as a powerful approach to identify novel antibacterial drug targets. They also provide a proof of principle, based upon PK in MRSA, for a research platform aimed at discovering and optimizing selective inhibitors of novel bacterial targets where human orthologs exist, as leads for anti-infective drug development.

SUBMITTER: Zoraghi R

PROVIDER: S-EPMC3088274 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Identification of pyruvate kinase in methicillin-resistant Staphylococcus aureus as a novel antimicrobial drug target.

Zoraghi Roya R See Raymond H RH Axerio-Cilies Peter P Kumar Nag S NS Gong Huansheng H Moreau Anne A Hsing Michael M Kaur Sukhbir S Swayze Richard D RD Worrall Liam L Amandoron Emily E Lian Tian T Jackson Linda L Jiang Jihong J Thorson Lisa L Labriere Christophe C Foster Leonard L Brunham Robert C RC McMaster William R WR Finlay B Brett BB Strynadka Natalie C NC Cherkasov Artem A Young Robert N RN Reiner Neil E NE

Antimicrobial agents and chemotherapy 20110228 5

Novel classes of antimicrobials are needed to address the challenge of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). Using the architecture of the MRSA interactome, we identified pyruvate kinase (PK) as a potential novel drug target based upon it being a highly connected, essential hub in the MRSA interactome. Structural modeling, including X-ray crystallography, revealed discrete features of PK in MRSA, which appeared suitable for the selective targeti ...[more]

PMID: 21357306

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Project description: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 ?g/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.

Project description:The chemotherapeutic options for methicillin-resistant Staphylococcus aureus (MRSA) infections are limited. Due to the multiple resistant MRSA, therapeutic failure has occurred frequently, even using antibiotics belonging to different categories in clinical scenarios, very recently. This study aimed to investigate the interactions between 11 antibiotics representing different mechanisms of action against MRSA strains and provide therapeutic strategies for clinical infections. Susceptibilities for MRSA strains were determined by broth microdilution or agar dilution according to CLSI guideline. By grouping with each other, a total of 55 combinations were evaluated. The potential synergism was detected through drug interaction assays and further investigated for time-killing curves and an in vivo neutropenic mouse infection model. A total of six combinations (vancomycin with rifampicin, vancomycin with oxacillin, levofloxacin with oxacillin, gentamycin with oxacillin, clindamycin with oxacillin, and clindamycin with levofloxacin) showed synergistic activity against the MRSA ATCC 43300 strain. However, antibacterial activity against clinical isolate #161402 was only observed when vancomycin combined with oxacillin or rifampicin in time-killing assays. Next, therapeutic effectiveness of vancomycin/oxacillin and vancomycin/rifampicin was verified by an in vivo mouse infection model inoculated with #161402. Further investigations on antimicrobial synergism of vancomycin plus oxacillin and vancomycin plus rifampicin against 113 wild-type MRSA strains were evidenced by combined antibiotic MICs and bacterial growth inhibition and in vitro dynamic killing profiles. In summary, vancomycin/rifampicin and vancomycin/oxacillin are the most potential combinations for clinical MRSA infection upon both in vitro and in vivo tests. Other synergetic combinations of levofloxacin/oxacillin, gentamycin/oxacillin, clindamycin/oxacillin, and clindamycin/fosfomycin are also selected but may need more assessment for further application.

Dataset Information

Identification of pyruvate kinase in methicillin-resistant Staphylococcus aureus as a novel antimicrobial drug target.

Publications

Identification of pyruvate kinase in methicillin-resistant Staphylococcus aureus as a novel antimicrobial drug target.

Similar Datasets

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