Structure based inhibitor design targeting glycogen phosphorylase b. Virtual screening, synthesis, biochemical and biological assessment of novel N-acyl-β-d-glucopyranosylamines

Glycogen phosphorylase (GP) is a validated target for the development of new type 2 diabetes treatments. Exploiting the Zinc docking database, we report the in silico screening of 1888 β- D-glucopyranose-NH-CO-R putative GP inhibitors differing only in their R groups. CombiGlide and GOLD docking programs with different scoring functions were employed with the best performing methods combined in a “consensus scoring” approach to ranking of ligand binding affinities for the active site. Six selected candidates from the screening were then synthesized and their inhibitory potency was assessed both in vitro and ex vivo. Their inhibition constants’ values, in vitro, ranged from 5 to 377 µM while two of them were effective at causing inactivation of GP in rat hepatocytes at low µM concentrations. The crystal structures of GP in complex with the inhibitors were defined and provided the structural basis for their inhibitory potency and data for further structure based design of more potent inhibitors

Synthetic, enzyme kinetic, and protein crystallographic studies of C-[béta]-D-glucopyranosyl pyrroles and imidazoles reveal and explain low nanomolar inhibition of human liver glycogen phosphorylase

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A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint

Misfolded glycoprotein recognition and endoplasmic reticulum (ER) retention are mediated by the ER glycoprotein folding quality control (ERQC) checkpoint enzyme, 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. The small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 “WY” conserved surface motif conserved across UGGTs but not present in other GT24 family glycosyltransferases. 5M-8OH-Q has a 47 μM binding affinity for CtUGGTGT24 in vitro as measured by ligand-enhanced fluorescence. In cellula, 5M-8OH-Q inhibits both human UGGT isoforms at concentrations higher than 750 μM. 5M-8OH-Q binding to CtUGGTGT24 appears to be mutually exclusive to M5-9 glycan binding in an in vitro competition experiment. A medicinal program based on 5M-8OH-Q will yield the next generation of UGGT inhibitors

Turning high-throughput structural biology into predictive inhibitor design

A common challenge in drug design pertains to finding chemical modifications to a ligand that increases its affinity to the target protein. An underutilized advance is the increase in structural biology throughput, which has progressed from an artisanal endeavor to a monthly throughput of hundreds of different ligands against a protein in modern synchrotrons. However, the missing piece is a framework that turns high-throughput crystallography data into predictive models for ligand design. Here, we designed a simple machine learning approach that predicts protein–ligand affinity from experimental structures of diverse ligands against a single protein paired with biochemical measurements. Our key insight is using physics-based energy descriptors to represent protein–ligand complexes and a learning-to-rank approach that infers the relevant differences between binding modes. We ran a high-throughput crystallography campaign against the SARS-CoV-2 main protease (MPro), obtaining parallel measurements of over 200 protein–ligand complexes and their binding activities. This allows us to design one-step library syntheses which improved the potency of two distinct micromolar hits by over 10-fold, arriving at a noncovalent and nonpeptidomimetic inhibitor with 120 nM antiviral efficacy. Crucially, our approach successfully extends ligands to unexplored regions of the binding pocket, executing large and fruitful moves in chemical space with simple chemistry

van der Waals interactions govern C-[beta]-D-glucopyranosyl triazoles' nM inhibitory potency in human liver glycogen phosphorylase

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Probing the [beta]-pocket of the active site of human liver glycogen phosphorylase with 3-(C-[beta]-D-glucopyranosyl)-5-(4-substituted-phenyl)-1, 2, 4-triazole inhibitors

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Nanomolar Inhibitors of Glycogen Phosphorylase Based on β-d-Glucosaminyl Heterocycles: A Combined Synthetic, Enzyme Kinetic, and Protein Crystallography Study

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Allosteric Inhibition of the SARS‐CoV‐2 Main Protease: Insights from Mass Spectrometry Based Assays**

Following translation of the SARS‐CoV‐2 RNA genome into two viral polypeptides, the main protease M pro cleaves at eleven sites to release non‐structural proteins required for viral replication. M Pro is an attractive target for antiviral therapies to combat the coronavirus‐2019 disease (COVID‐19). Here, we have used native mass spectrometry (MS) to characterize the functional unit of M pro . Analysis of the monomer‐dimer equilibria reveals a dissociation constant of K d = 0.14 ± 0.03 µM, revealing M Pro has a strong preference to dimerize in solution. Developing an MS‐based kinetic assay we then characterized substrate turnover rates by following temporal changes in the enzyme‐substrate complexes, which are effectively “flash‐frozen” as they transition from solution to the gas phase. We screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the catalytically active dimer, slow the rate of substrate processing by ~35%. This information was readily obtained and, together with analysis of the x‐ray crystal structures of these enzyme‐small molecule complexes, provides a starting point for the development of more potent molecules that allosterically regulate M Pro activity

Natural flavonoids as antidiabetic agents. The binding of gallic and ellagic acids to glycogen phosphorylase b

We present a study on the binding of gallic acid and its dimer ellagic acid to glycogen phosphorylase (GP). Ellagic acid is a potent inhibitor with K(i)s of 13.4 and 7.5 mu M, in contrast to gallic acid which displays K(i)s of 1.7 and 3.9 mM for GPb and GPa, respectively. Both compounds are competitive inhibitors with respect to the substrate, glucose-1-phoshate, and non-competitive to the allosteric activator, AMP. However, only ellagic acid functions with glucose in a strongly synergistic mode. The crystal structures of the GPb-gallic acid and GPb-ellagic acid complexes were determined at high resolution, revealing that both ligands bind to the inhibitor binding site of the enzyme and highlight the structural basis for the significant difference in their inhibitory potency. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved