Discovery of a small-molecule inhibitor targeting human GMP synthetase.

Guanosine monophosphate synthetase (GMPS) catalyzes the ATP-dependent conversion of xanthosine monophosphate (XMP) to guanosine monophosphate (GMP), a key step in de novo purine biosynthesis. Dysregulation of GMPS expression has been implicated in multiple cancers, underscoring its potential as a therapeutic target. Here, we identified a novel small molecule GMPS inhibitor, G18, through large-scale virtual screening of 1.27 million compounds. Biochemical validation using an inorganic phosphatase (IPP1)-coupled colorimetric assay demonstrated that G18 inhibits GMPS with an IC50 of 73.8 μM. Isothermal titration calorimetry (ITC) confirmed direct and thermodynamically favorable binding (Kd = 6.94 μM). Moreover, G18 suppressed HeLa cell proliferation with an IC50 of 73.3 μM. Structural modeling and 500-ns molecular dynamics simulations revealed that G18 binds within the ATPase domain, forming stable hydrogen-bonding and hydrophobic interactions that stabilize the enzyme-inhibitor complex. Together, these results identify G18 as a promising lead compound for GMPS-targeted anticancer drug discovery and provide structural insights for further optimization.