title: 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, FINDSITE(comb), 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.
authors: Center for the Study of Systems Biology, School of Biology, Georgia Institute of Technology, 950, Atlantic Drive, Atlanta, GA 30332, United States. Electronic address: bharath.srinivasan@biology.gatech.edu.
authors: Bharath
authors: B
authors: Srinivasan
authors: Center for the Study of Systems Biology, School of Biology, Georgia Institute of Technology, 950, Atlantic Drive, Atlanta, GA 30332, United States.
authors: Hongyi
authors: H
authors: Zhou
authors: Center for the Study of Systems Biology, School of Biology, Georgia Institute of Technology, 950, Atlantic Drive, Atlanta, GA 30332, United States.
authors: Sreyoshi
authors: S
authors: Mitra
authors: Center for the Study of Systems Biology, School of Biology, Georgia Institute of Technology, 950, Atlantic Drive, Atlanta, GA 30332, United States. Electronic address: skolnick@gatech.edu.
authors: Jeffrey
authors: J
authors: Skolnick
mesh_terms: D060746:Endoplasmic Reticulum-Associated Degradation / Q000187:drug effects*; D006801:Humans; D008024:Ligands; D062105:Molecular Docking Simulation; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000494:pharmacology*
chemical_list: D008024:Ligands; D054852:Small Molecule Libraries; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
keywords: Drug discovery; ERAD; N-glycanase 1; Thermal shift assay; Virtual ligand screening