Novel small molecule binders of human N-glycanase 1, a key player in the endoplasmic reticulum associated degradation pathway

Abstract

Peptide:N-glycanase (NGLY1) is an enzyme responsible for cleaving oligosaccharide moieties from misfolded glycoproteins to enable their proper degradation. Deletion and truncation mutations in this gene are responsible for an inherited disorder of the endoplasmic reticulum-associated degradation pathway. However, the literature is unclear whether the disorder is a result of mutations leading to loss-of-function, loss of substrate specificity, loss of protein stability or a combination of these factors. In this communication, without burdening ourselves with the mechanistic underpinning of disease causation because of mutations on the NGLY1 protein, we demonstrate the successful application of virtual ligand screening (VLS) combined with experimental high-throughput validation to the discovery of novel small-molecules that show binding to the transglutaminase domain of NGLY1. Attempts at recombinant expression and purification of six different constructs led to successful expression of five, with three constructs purified to homogeneity. Most mutant variants failed to purify possibly because of misfolding and the resultant exposure of surface hydrophobicity that led to protein aggregation. For the purified constructs, our threading/structure-based VLS algorithm, FINDSITEcomb, was employed to predict ligands that may bind to the protein. Then, the predictions were assessed by high-throughput differential scanning fluorimetry. This led to the identification of nine different ligands that bind to the protein of interest and provide clues to the nature of pharmacophore that facilitates binding. This is the first study that has identified novel ligands that bind to the NGLY1 protein as a possible starting point in the discovery of ligands with potential therapeutic applications in the treatment of the disorder caused by NGLY1 mutants.

Chemical space of Escherichia coli dihydrofolate reductase inhibitors: New approaches for discovering novel drugs for old bugs

“…In practice, the predictions from FINDSITE comb have been experimentally assessed on a significant number of medically relevant target proteins belonging to different fold-classes and coming from several different organisms 55 and achieves good enrichment in identifying active small-molecules. The methods predicted low nanomolar binders for the enzyme dihydrofolate reductase from Escherichia coli and also predicted micromolar binders for several different protein targets such as the phosphatase domain of protein tyrosine phosphatase delta (from rat) and omega (from Homo sapiens ), tryptophanyl tRNA synthetase from H. sapiens , ubiquitin-conjugating enzyme from P. falciparum , nucleosome assembly protein 1 from P. knowlesi , thioredoxin peroxidase 2 from P. falciparum , the catalytic domain of cAMP dependent protein kinase from H. sapiens 55 and N-glycanase 1 59 .…”

A Natural Compound Containing a Disaccharide Structure of Glucose and Rhamnose Identified as Potential N-Glycanase 1 (NGLY1) Inhibitors

“…N-Glycanase 1 (NGLY1) is a de-N-glycosylating enzyme that catalyzes the hydrolysis of the amide bond between the proximal N-acetylglucosamine (GlcNAc) residue and the Asn side chain to which it is attached, removing N-glycans from glycosylated proteins in the cytosol [1][2][3]. Discovered in 1993, NGLY1 is known to participate in clearing misfolded glycoproteins during the process of glycoprotein synthesis through the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway [4].…”