Abstract
The Endoplasmic Reticulum (ER) glycoprotein folding Quality Control (ERQC) machinery aids folding of glycoproteins in the ER. Misfolded glycoprotein recognition and ER-retention is mediated by the ERQC checkpoint enzyme, the 170 kDa UDP-Glucose glycoprotein glucosyltransferase (UGGT). UGGT modulation is a promising strategy for broad-spectrum antivirals, rescue-of-secretion therapy in rare disease caused by responsive mutations in glycoprotein genes, and many cancers, but to date no selective UGGT inhibitors are known. Towards the generation of selective UGGT inhibitors, we determined the crystal structures of the catalytic domain of Chaetomium thermophilum UGGT (CtUGGTGT24), alone and in complex with the inhibitor UDP-2-deoxy-2-fluoro-D-glucose (U2F). Using the CtUGGTGT24 crystals, we carried out a fragment-based lead discovery screen via X-ray crystallography and discovered that the small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 ‘WY’ conserved surface motif that is not present in other GT24 family glycosyltransferases. The 5M-8OH-Q molecule has a 613 µM binding affinity for human UGGT1in vitro as measured by saturation transfer difference NMR spectroscopy. The 5M-8OH-Q molecule inhibits both human UGGT1and UGGT2 activity at concentrations higher than 750 µM in modified HEK293-6E cells. The compound is toxic in cellula and in planta at concentrations higher than 1 mM. A few off-target effects are also observed upon 5M-8OH-Q treatment. Based on an in silico model of the interaction between UGGT and its substrate N -glycan, the 5M-8OH-Q molecule likely works as a competitive inhibitor, binding to the site of recognition of the first GlcNAc residue of the substrate N -glycan.
Significance Statement When a candidate drug target is the product of a housekeeping gene - i.e. it is important for the normal functioning of the healthy cell – availability of inhibitors for tests and assays is of paramount importance. One such housekeeping protein is UGGT, the enzyme that makes sure that only correctly folded glycoproteins can leave the endoplasmic reticulum for further trafficking through the secretory pathway. UGGT is a potential drug target against viruses, in certain instances of congenital rare disease, and against some cancers, but no UGGT inhibitors are known yet. We discovered and describe here a small molecule that binds human UGGT1 in vitro and inhibits both isoforms of human UGGT in cellula. The compound paves the way to testing of UGGT inhibition as a potential pharmacological strategy in a number of medical contexts.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
The authors declare no competing interests here.
* During the synthesis of the N-linked glycan precursor, ALG6 appends the first glucose to the Man9GlcNAc2 carbohydrate - before it is built into a triglucosylated form by other ALG enzymes at the ER membrane. The Glc3Man9GlcNAc2 glycan is then appended to newly synthesized proteins and trimmed to a monoglucosylated state by glucosidases I and II, providing the ligand necessary for binding to the lectin chaperones calnexin and calreticulin (37). During glycan maturation in wild type cells, ER lectins binding can occur after the glycan is trimmed from a Glc3Man9GlcNAc2 to a GlcMan9GlcNAc2 form, or through glucosylation of a Man9GlcNAc2 carbohydrate by the UGGTs (36).
