Classic Galactosemia is a rare genetic metabolic disorder (1/60,000 births) that is characterized by decreased production of galactose-1-phosphate uridyltransferase (GALT), an enzyme responsible for the conversion of galactose-1-phosphate (gal-1-p) to glucose-1-phosphate. The resulting elevated intracellular concentrations of gal-1-p are believed to be the major pathogenic mechanism in Classic Galactosemia that leads to a myriad of secondary symptoms and, if untreated, death of the patient. Galactokinase (GALK) is an upstream enzyme in the Leloir pathway that is responsible for conversion of galactose to gal-1-p. Therefore, it was hypothesized that the identification of a small-molecule inhibitor of GALK would act to decrease levels of gal-1-p and allow for a novel entry into therapies for this disorder. In collaboration with Professor Kent Lai of the University of Utah, a quantitative high-throughput screening (qHTS) assay has been performed at the NIH Chemical Genomics Center (NCGC) and has identified a potent and selective inhibitor of GALK bearing a 1,4-dihydropyrimidine core. Several rounds of medicinal chemistry were performed and generated a small molecule capable of inhibiting GALK with an IC50 of 1.0μM. The probe ML152 (CID-664331; SID-87550830) is selective for GALK against CDP-ME, and thus represents an advancement over previously reported inhibitors. The probe was the best analog for inhibition of GALK in a purified enzyme assay. Its mechanism of action, as determined via substrate competition and kinetic assays, are consistent with it being ATP competitive.