Fragment Based Drug Discovery by Microscale Thermophoresis Targeting Klebsiella pneumoniae and Escherichia coli IspE

Danica J Walsh,1 Daan Willocx,1 Rawia Hamid,1,2 Mostafa M Hamed,1 Anna KH Hirsch1,2 1Department of Drug Design and Optimization, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) – Helmholtz Centre for Infection Research (HZI), Saarbrücken, 66123, Germany; 2Department of Pharmacy, Saarland University, Saarbrücken, 66123, GermanyCorrespondence: Anna KH Hirsch, Helmholtz Institute for Pharmaceutical Research (HIPS), Helmholtz Centre for Infection Research (HZI), Saarland University, Campus E8.1, Saarbrücken, 66123, Germany, Email [email protected]: Antimicrobial drug discovery urgently requires innovative strategies to combat the growing threat of multidrug-resistant pathogens. The 2-C-methylerythritol 4-phosphate (MEP) pathway, essential in many pathogenic bacteria yet absent in humans, represents an attractive source of selective antibacterial targets. Within this pathway, the enzyme IspE is a promising candidate for inhibitor development.Methods: We screened a halogen-enriched fragment library to identify inhibitors of IspE from Klebsiella pneumoniae and Escherichia coli. We validated our hits through biochemical assays, followed by structure–activity relationship (SAR) studies to optimize the fragments inhibitory activity. Mode-of-inhibition experiments were conducted to determine the mechanism of enzyme inhibition.Results: Screening identified a fragment-like compound 1 and several additional hits that inhibited K. pneumoniae IspE and E. coli IspE at micromolar concentrations. SAR analysis generated optimized fragments with enhanced potency. Mode-of-inhibition assays revealed that these fragments act as competitive inhibitors with respect to the natural substrate, 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME).Conclusion: This newly identified fragment class provides a promising foundation for the development of IspE inhibitors. The findings highlight the utility of fragment-based screening approaches for discovering novel antimicrobial agents targeting the MEP pathway.Keywords: IspE, fragment based drug discovery, Klebsiella pneumoniae, Escherichia coli, MEP pathway