Virtual Screening and X‑ray Crystallography Identify Non-Substrate Analog Inhibitors of Flavin-Dependent Thymidylate Synthase

Thymidylate synthase X (ThyX) represents an attractive target for tuberculosis drug discovery. Herein, we selected 16 compounds through a virtual screening approach. We solved the first X-ray crystal structure of Thermatoga maritima (Tm) ThyX in complex with a nonsubstrate analog inhibitor. Given the active site similarities between Mycobacterium tuberculosis ThyX (Mtb-ThyX) and Tm-ThyX, our crystal structure paves the way for a structure-based design of novel antimycobacterial compounds. The 1H-imidazo­[4,5-d]­pyridazine was identified as scaffold for the development of Mtb-ThyX inhibitors.