Discovery and Exploration of Mechanistically Distinct FtsZ Compounds Inhibiting Mycobacterial Cell Division

Abstract The Mycobacterium tuberculosis (Mtb) complex causes disease in humans and animals, underscoring the need for antitubercular agents with novel mechanisms. We investigated FtsZ, a conserved essential component of the mycobacterial divisome, by screening anti-TB compounds for FtsZ binding, GTPase inhibition, and impact on cell morphology. Hits were triaged as thermal stabilizers or destabilizers of MtbFtsZ based on thermal-shift profiles and validated using CRISPRi ftsZ knockdown in Mycobacterium bovis BCG. Bacterial cytological profiling and orthogonal biochemical and cell-based assays, including hydrogen-deuterium exchange mass spectrometry, dynamic light scattering, and Z-ring imaging, supported that the compounds had distinct effects on FtsZ and the divisome. We explored several chemotypes through analog synthesis and used the GAC15 series to investigate cellular mechanisms. Amide hydrolysis and nitro-group reduction were implicated in loss of GAC15 activity in BCG. These compounds provide tools for dissecting FtsZ dynamics and divisome function and a framework for discovering novel anti-TB agents.