High‐Throughput Approaches in Carbohydrate‐Active Enzymology: Glycosidase and Glycosyl Transferase Inhibitors, Evolution, and Discovery

Abstract

Carbohydrates are attached and removed in living systems through the action of carbohydrate‐active enzymes such as glycosyl transferases and glycoside hydrolases. The molecules resulting from these enzymes have many important roles in organisms, such as cellular communication, structural support, and energy metabolism. In general, each carbohydrate transformation requires a separate catalyst, and so these enzyme families are extremely diverse. To make this diversity manageable, high‐throughput approaches look at many enzymes at once. Similarly, high‐throughput approaches can be a powerful way of finding inhibitors that can be used to tune the reactivity of these enzymes, either in an industrial, a laboratory, or a medicinal setting. In this review, we provide an overview of how these enzymes and inhibitors can be sought using techniques such as high‐throughput natural product and combinatorial library screening, phage and mRNA display of (glyco)peptides, fluorescence‐activated cell sorting, and metagenomics.

Glycoengineering of Therapeutic Antibodies with Small Molecule Inhibitors

“…Where can improved inhibitors come from? Two promising routes are the identification of new inhibitor chemotypes from screening [ 106 ], and, facilitated by the growing availability of structural information for glycosyltransferases, structure-based rational design [ 107 ]. Chemical glycoengineering may also take inspiration from other fields where glycosyltransferase inhibitors have long been sought after, such as anti-cancer and anti-bacterial drug discovery.…”

Glycoenzyme Tool Development: Principles, Screening Methods, and Recent Advances†

“…Analytical approaches such as fluorometric, chromogenic, or enzyme‐linked immunosorbent assay (ELISA) are all commonly used for glycoenzyme variant screening. [ 78 ] The Thorson lab reported the use of plate reading screening based on a fluorescent surrogate acceptor substrate to expand the promiscuity of a natural product glucosyltransferase, oleandomycin (OleD), via directed evolution. OleD was previously reported to be weakly active toward the fluorophore coumarin 4‐methylumbelliferone (MU).…”

Beyond GalNAc! Drug delivery systems comprising complex oligosaccharides for targeted use of nucleic acid therapeutics

“…169,170 GTs are classied into families according to sequence homology and structure, which are highly conserved within the family along with its mechanism, but can hold enzymatic activities for different glycosyl donor and acceptors. 131,171 A wealth of information has been acquired on GTs over the last few decades, but much is still unknown, 172 with the most recent advances published within the Carbohydrate-Active enZYmes database (https://www.cazy.org) including information on families, structures, and activities of GTsa set of 114 families currently (February 2022). Use of these enzymes is arguably favourable to the chemosynthetic route due to the reactions being easier, cleaner, and greener given the milder conditions used.…”