eNTRy rule optimization of phenotypic hits for Klebsiella pneumoniae activity under in vivo relevant conditions (360G-Wellcome-349097_Z_26_Z)

As part of the Gr-ADI consortium this proposal aims to address a critical gap in Gram-negative antibiotic discovery: poor intracellular accumulation of phenotypic hits. Following the eNTRy rules (1), we will upcycle phenotypic hits identified against an efflux-impaired Klebsiella pneumoniae strain to increase activity against wild-type strains, thereby expanding the pool of chemical starting points for further drug discovery efforts. Using an expanded AI/ML-optimized Entryway analysis1, hits will be scored and prioritized for synthesis based on predicted accumulation, with the objective to build-in intracellular accumulation through minimal computational filtering/medicinal chemistry rounds. Hit accumulation will be inferred by improved activity against wild-type K. pneumoniae relative to the efflux impaired strain, through a multiplex assay that will be developed to assess activity against both strains simultaneously. This assay will also be used for de novo screening of additional compound libraries under in vivo relevant conditions, accelerating identification and prioritization of promising novel hits potentially acting through inhibition of in vivo essential targets. The major outcomes of this project are novel hits against wild-type K. pneumoniae for further progression and validated methodologies for improving intracellular accumulation of hits. These methodologies could also boost antibiotic discovery efforts against other Gram-negative WHO priority pathogens.