Studying Target–Engagement\nof Anti-Infectives\nby Solvent-Induced Protein Precipitation and Quantitative Mass Spectrometry

Antimicrobial resistance (AMR) poses a serious threat\nto global\nhealth. The rapid emergence of resistance contrasts with the slow\npace of antimicrobial development, emphasizing the urgent need for\ninnovative drug discovery approaches. This study addresses a critical\nbottleneck in early drug development by introducing integral solvent-induced\nprotein precipitation (iSPP) to rapidly assess the target–engagement\nof lead compounds in extracts of pathogenic microorganisms under close-to-physiological\nconditions. iSPP measures the change in protein stability against\nsolvent-induced precipitation in the presence of ligands. The iSPP\nmethod for bacteria builds upon established SPP procedures and features\noptimized denaturation gradients and minimized sample input amounts.\nThe effectiveness of the iSPP workflow was initially demonstrated\nthrough a multidrug target–engagement study. Using quantitative\nmass spectrometry (LC-MS/MS), we successfully identified known drug\ntargets of seven different antibiotics in cell extracts of four AMR-related\npathogens: the three Gram-negative bacteria Escherichia\ncoli, Klebsiella pneumoniae, Pseudomonas aeruginosa and the Gram-positive\nbacterium Staphylococcus aureus. The\niSPP method was ultimately applied to demonstrate target–engagement\nof compounds derived from target-based drug discovery. We employed\nfive small molecules targeting three enzymes in the 2-<i>C</i>-methyl-d-erythritol 4-phosphate (MEP) pathwaya\npromising focus for anti-infective drug development. The study showcases\niSPP adaptability and efficiency in identifying anti-infective drug\ntargets, advancing early-stage drug discovery against AMR.