Enzymatic and Structural Analysis of Inhibitors Designed against Mycobacterium tuberculosis Thymidylate Kinase

Abstract

The chemical synthesis of new compds. designed as inhibitors of Mycobacterium tuberculosis TMP kinase (TMPK) is reported. The synthesis concerns TMP analogs modified at the 5-position of the thymine ring as well as a novel compound with a six-membered sugar ring. The binding properties of the analogs are compared with the known inhibitor azido-TMP, which is postulated here to work by excluding the TMP-bound Mg2+ ion. The crystallog. structure of the complex of one of the compds., 5-CH2OH-dUMP, with TMPK has been determined at 2.0 Å. It reveals a major conformation for the hydroxyl group in contact with a water mol. and a minor conformation pointing toward Ser99. Looking for a role for Ser99, we have identified an unusual catalytic triad, or a proton wire, made of strictly conserved residues (including Glu6, Ser99, Arg95, and Asp9) that probably serves to protonate the transferred PO3 group. The crystallog. structure of the com. available bisubstrate analog P1-(adenosine-5')-P5-(thymidine-5')-pentaphosphate bound to TMPK is also reported at 2.45 Å and reveals an alternative binding pocket for the adenine moiety of the mol. compared with what is observed either in the Escherichia coli or in the yeast enzyme structures. This alternative binding pocket opens a way for the design of a new family of specific inhibitors. P226