Studying Target–Engagement of Anti-Infectives by Solvent-Induced Protein Precipitation and Quantitative Mass Spectrometry

Antimicrobial resistance (AMR) poses a serious threat to global health. The rapid emergence of resistance contrasts with the slow pace of antimicrobial development, emphasizing the urgent need for innovative drug discovery approaches. This study addresses a critical bottleneck in early drug development by introducing integral solvent-induced protein precipitation (iSPP) to rapidly assess the target-engagement of lead compounds in extracts of pathogenic microorganisms under close-to-physiological conditions. iSPP measures the change in protein stability against solvent-induced precipitation in the presence of ligands. The iSPP method for bacteria builds upon established SPP procedures and features optimized denaturation gradients and minimized sample input amounts. The effectiveness of the iSPP workflow was initially demonstrated through a multidrug target-engagement study. Using quantitative mass spectrometry (LC-MS/MS), we successfully identified known drug targets of seven different antibiotics in cell extracts of four AMR-related pathogens: the three Gram-negative bacteria <i>Escherichia coli</i>, <i>Klebsiella pneumoniae</i>, <i>Pseudomonas aeruginosa</i> and the Gram-positive bacterium <i>Staphylococcus aureus</i>. The iSPP method was ultimately applied to demonstrate target-engagement of compounds derived from target-based drug discovery. We employed five small molecules targeting three enzymes in the 2-<i>C</i>-methyl-d-erythritol 4-phosphate (MEP) pathway─a promising focus for anti-infective drug development. The study showcases iSPP adaptability and efficiency in identifying anti-infective drug targets, advancing early-stage drug discovery against AMR.