Rational Design of Diaryl Ether-Based Dual Inhibitors Targeting Successive Essential Enzymes HadAB and InhA in Mycobacterium tuberculosis

The emergence of drug-resistant Mycobacterium tuberculosis underscores the need for innovative therapeutic strategies targeting essential metabolic pathways. We designed, synthesized, and evaluated a series of dual inhibitors targeting two key enzymes of the mycobacterial FAS-II system, HadAB and InhA. Using a diaryl ether scaffold, six thiosemicarbazone derivatives and their aldehyde intermediates were prepared and tested for enzymatic and antimycobacterial activity. Thiosemicarbazone derivatives and aldehyde intermediates both strongly inhibited InhA, and the thiosemicarbazones additionally potentially inhibited HadAB through covalent interaction with the HadA subunit, supporting the dual-target approach. Several compounds showed low micromolar to submicromolar activity against drug-susceptible and clinical M. tuberculosis strains, including an ethA -deficient mutant. Crystallographic structures of InhA–ligand complexes revealed key binding interactions and clarified inhibition mechanisms. Despite some cytotoxicity concerns, these findings provide a promising basis for developing optimized dual-target inhibitors of the M. tuberculosis FAS-II pathway.