Journal of Glycobiology
Open Access

Discovery of glycosyltransferase inhibitors of GALNT3 using the assay optimization and a transcreener UDP2 assay and orthogonal pooled screening

Glycobiology World Congress

August 10-12, 2015 Philadelphia, USA

Tom Zielinski

Posters-Accepted Abstracts: J Glycobiol

Abstract :

Glycosyltransferase enzymes participate in diverse metabolic and regulatory roles by catalyzing the transfer of sugars to
protein, lipid and carbohydrate acceptors as well as to other endogenous and xenobiotic molecules. Of more than 200
human glycosyltransferases (GTs), there are over 20 distinct polypeptide N-acetylgalactosaminyltransferases (GALNTs) that
catalyze the initial step of O-glycosylation by transferring GalNac to Thr or Ser residues on multiple targets including mucins.
Abnormal post-translational glycosylation of mucin is a driver of cancer-associated changes diversely affecting growth and
survival of cancer cells and their ability to invade and metastasize. GALNT3 overexpression and dysregulation has been
directly linked to multiple cancers including gastric, pancreatic, ovarian, lung and others making it a compelling target for
drug discovery. Development of an HTS workflow for GALNT3 is described here. Recombinant First, GALNT3 enzyme
activity robustness was first evaluated tested and optimized in the Trasncreener UDP2 Assay with the donor and acceptor
substrates UDP-GalNac and Mucin 10 (153-165) EA2 peptide. Second, in order to perform a pilot screen, optimal GALNT3
concentration and Km values for both substrates were determined. Thirdly, A pilot screen was run using the TR-FRET-based
assay with a with a diverse, pre-screened filtered 8,000 compound OPS orthogonally pooled compound set library from LCGC
from the Lankenau Institute for Medical Research (LIMR) Chemical Genomics Center which allowed screening of 8,000
compounds in duplicate in just five 384-well plates. Hits were confirmed followed by dose-response curves of all potential hits
measurements with the primary screening assay and then further validated with a FP based Transcreener UDP2 Assay second
assay format. Finally, two confirmed hits were further evaluated for the longevity of target engagement by doing jump dilution
performing rapid dilution experiments to assess measure dissociation rates residence time and reversibility.

Biography :

Tom Zielinski has worked at Bellbrook Labs for over 8 years where he is the Manager of Biochemical assays. Over 20 years, he has been an R&D Scientist who has
helped develop numerous cellular and biochemical assays for enzymes in diverse classes such as GPCRs, kinases, methyltransferases and glyosyltransferases.