A solid-phase glycosyltransferase assay for high-throughput screening in drug discovery research

Glycosyltransferases mediate changes in glycosylation patterns which, in tu rn, may affect the function of glycoproteins and/or glycolipids and, furthe r downstream, processes of development, differentiation, transformation and cell-cell recognition. Such enzymes, therefore, represent valid targets fo r drug discovery. We have developed a solid-phase glycosyltransferase assay for use in a robo tic high-throughput format. Carbohydrate acceptors coupled covalently to po lyacrylamide are coated onto 96-well plastic plates. The glycosyltransferas e reaction is performed with recombinant enzymes and radiolabeled sugar-nuc leotide donor at 37 degrees C, followed by washing, addition of scintillati on counting fluid, and measurement of radioactivity using a 96-well beta-co unter. Glycopolymer construction and coating of the plastic plates, enzyme and substrate concentrations, and linearity with time were optimized using recombinant Core 2 beta 1-6-N-acetylglucosaminyltransferase (Core 2 GlcNAc- T). This enzyme catalyzes a rate-limiting reaction for expression of polyla ctosamine and the selectin ligand sialyl-Lewis(x) in Omega-glycans. A glyco polymer acceptor for beta 1-6-N-acetylglucosaminyltransferase V was also de signed and shown to be effective in the solid-phase assay. In a high-throughput screen of a microbial extract library, the coefficient of variance for positive controls was 9.4%, and high concordance for hit v alidation was observed between the Core 2 GlcNAc-T solid-phase assay and a standard solution-phase assay. The solid-phase assay format, which can be a dapted for a variety of glycosyltransferase enzymes, allowed a 5-6 fold inc rease in throughput compared to the corresponding solution-phase assay.