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High-Throughput Screening for Human Galactokinase Inhibitors

Department of Pediatrics, University of Miami

Kent Lai

Department of Pediatrics, University of Miami, klai{at}med.miami.edu

Peter Buchwald

Department of Molecular & Cellular Pharmacology and Diabetes Research Institute, University of Miami

Manshu Tang

Department of Biochemistry & Molecular Biology, Leonard M. Miller School of Medicine, University of Miami, Miami, Florida

Inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) can result in a potentially lethal disorder called classic galactosemia. Although the neonatal lethality associated with this disease can be prevented through early diagnosis and a galactose-restricted diet, the lack of effective therapy continues to have consequences: developmental delay, neurological disorders, and premature ovarian failure are common sequelae in childhood and adulthood. Several lines of evidence indicate that an elevated level of galactose-1-phosphate (gal-1-p), the product of galactokinase (GALK), is a major, if not sole, pathogenic mechanism in patients with classic galactosemia. The authors hypothesize that elimination of gal-1-p production by inhibiting GALK will relieve GALT-deficient cells from galactose toxicity. To test this hypothesis, they obtained human GALK using a bacterial expression system. They developed a robust, miniaturized, high-throughput GALK assay (Z' factor = 0.91) and used this assay to screen against libraries composed of 50,000 chemical compounds with diverse structural scaffolds. They selected 150 compounds that, at an average concentration of 33.3 µM, inhibited GALK activity in vitro more than 86.5% and with a reproducibility score of at least 0.7 for a confirmatory screen under identical experimental conditions. Of these 150 compounds, 34 were chosen for further characterization. Preliminary results indicated that these 34 compounds have potential to serve as leads to the development of more effective therapy of classic galactosemia. ( Journal of Biomolecular Screening 2008:415-423)

Key Words: classic galactosemia • galactose-1-phosphate uridyltransferase (GALT) • galactokinase (GALK) • galactose-1-phosphate • GHMP small-molecule kinase • high-throughput screening • small-molecule inhibitors

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