Discovery of Allosteric and Selective Inhibitors of Inorganic Pyrophosphatase from Mycobacterium tuberculosis

Inorganic pyrophosphatase (PPiase) is an essential enzyme that hydrolyzes inorganic pyrophosphate (PPi), driving numerous metabolic processes. We report a discovery of an allosteric inhibitor (2,4-bis(aziridin-1-yl)-6-(1-phenylpyrrol-2-yl)-s-triazine) of bacterial PPiases. Analogues of this lead compound were synthesized to target specifically Mycobacterium tuberculosis (Mtb) PPiase (MtPPiase). The best analogue (compound 16) with a Ki of 11 μM for MtPPiase is a species-specific inhibitor. Crystal structures of MtPPiase in complex with the lead compound and one of its analogues (compound 6) demonstrate that the inhibitors bind in a nonconserved interface between monomers of the hexameric MtPPiase in a yet unprecedented pairwise manner, while the remote conserved active site of the enzyme is occupied by a bound PPi substrate. Consistent with the structural studies, the kinetic analysis of the most potent inhibitor has indicated that it functions uncompetitively, by binding to the enzyme–substrate complex. ...