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 [IC 50 ] 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.

Central Role of Pyruvate Kinase in Carbon Co-catabolism of Mycobacterium tuberculosis

“…The most common metabolite that activates PK in other organisms is fructose 1,6-bisphosphate. Fructose 1,6-bisphosphate enhances the flux through glycolysis, and increases glucose uptake and catabolism (24,25,38). To measure the potential allosteric regulation of PK in Mtb we cloned, expressed, and purified PK MTB in E. coli, and measured the kinetic parameters of the enzyme.…”

“…In humans, during tumor development, proliferating malignant cells up-regulate the expression of a less active PK, PKM2, which leads to the accumulation of upstream glycolytic metabolites that serve as precursors for the synthesis of phospholipids and nucleotides needed for the replicating cells (23). In bacteria, inhibitors designed against PK from Staphylococus aureus showed bacteriocidal effects against methicillin-resistant S. aureus (MRSA) strains and a wide range of both Gram-positive and Gram-negative bacteria, thus suggesting PK as a potential antibacterial drug target (24).…”

Targeting fundamental pathways to disrupt Staphylococcus aureus survival: clinical implications of recent discoveries

“…Glycolysis occurs in all eukaryotes (and is a component of host defense against S. aureus, ref. 29); however, a specific inhibitor of S. aureus pyruvate kinase, which mediates the final glycolysis step, has been developed (30,31). As patients with uncontrolled diabetes have increased levels of circulating glucose, specific inhibition of S. aureus glycolysis may prevent the pathogen from leveraging this metabolic imbalance, especially if the glycolytic dependence of S. aureus occurs to the same degree in humans and mice.…”

“…S. aureus produces a glutamate dehydrogenase (GudB) and an acetate kinase that can catabolize free amino acids from the host (32). Additionally, S. aureus produces a pyruvate kinase and glucose transporters that facilitate glycolysis (27)(28)(29)(30)(31) and is able to scavenge free lipoic acid (33). (B) Numerous critical steps have been identified for the acquisition of host metals by S. aureus.…”

Novel inhibitors of the methicillin-resistant Staphylococcus aureus (MRSA)-pyruvate kinase

“…PK has recently been discovered as an essential hub protein in the interactome of MRSA 20, 21 . PK plays a central role in the carbohydrate metabolism.…”