Methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase as a target for bis-indole alkaloids with antibacterial activities

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.

FIGURE 1.

T. pachastrelloides collected from South Africa used in this study to purify MRSA PK inhibiting bis-indole alkaloids.

FIGURE 2.

Structures of bis-indole alkaloids with MRSA PK inhibitory activities purified from T. pachastrelloides in this study.

FIGURE 3.

SDS-PAGE of purified MRSA PK and human PK isoforms. 5-μg aliquots of MRSA PK, human M1, M2, R, and L PK proteins purified to homogeneity through the nickel-nitrilotriacetic acid chromatography step as described under “Experimental Procedures” were applied to lanes 1–5, respectively. Low molecular weight (LMW) standards (GE Healthcare) were applied as size markers. Proteins were stained with Coomassie Brilliant Blue dye.

FIGURE 4.

Inhibition of MRSA and human PK isoforms enzymatic activities by bis-indole alkaloids. A, bis-indoles (A, C, and D) exhibited potent inhibitory activity against MRSA PK at 10 μm. B, two bis-indoles (C and D) exhibited potent selective inhibitory activity against MRSA PK versus human PK isoforms at 5 μm. PK enzymatic activity was assayed as described under “Experimental Procedures.” The data shown are mean ± S.D. of inhibitory effects (%) from three independent experiments each performed in triplicate.

FIGURE 5.

Potency of inhibition of catalytic activity of MRSA PK by cis-3,4-dihyrohamacanthin B (left) and bromodeoxytopsentin (right). Potency of inhibition of MRSA PK was determined as described under “Experimental Procedures” with 10 μm P-enolpyruvate and 2 mm ADP substrates. The data are derived from one of three experiments with similar results each performed in triplicate and analyzed with Prism GraphPad software (single-site model). Error bars indicate the range of values within the single experiment shown. The final concentration of MRSA PK protein used in these studies was 15 nm.

FIGURE 6.

Antibacterial activities of two bis-indole compounds C (left) and D (right) with potent PK inhibitory activities against S. aureus strain RN4220.

FIGURE 7.

Cytotoxicity assessment of the PK inhibiting bis-indole compound against HEK 293T cells. Cytotoxicity of compound C was determined using HEK 293T cells through a 1-day incubation assay as described under “Experimental Procedures.” The data presented are representative of three experiments performed in triplicate.

FIGURE 8.

Structure of S. aureus PK in complex with compound C. A, tetrameric structure of PK illustrating the ligand density at the small interface. Position of active and effector sites are for reference only. B, interface binding pocket. Refined ligand position shown with ligand omit map (green). Anomalous map calculated from data collected at bromine edge (purple) indicates the position of the ligand bromine atoms. Residues lining the binding pocket from chain A are labeled. Hydrogen bonds between Ser³⁶² and indole nitrogens are marked in red. C and D, comparison of S. aureus (C) and representative human PK structure (PDB 1ZJH) (D) illustrating closer packing of helix 340–350 and differences in the primary structure of this helix and the interface binding pocket, which contribute to selectivity.