Browse
Submit Data
Databases
API
Help

Dataset Information

15 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry.

ABSTRACT: The attachment of the sugar N-acetyl-D-glucosamine (GlcNAc) to specific serine and threonine residues on proteins is referred to as protein O-GlcNAcylation. O-GlcNAc transferase (OGT) is the enzyme responsible for carrying out the modification, while O-GlcNAcase (OGA) reverses it. Protein O-GlcNAcylation has been implicated in a wide range of cellular processes including transcription, proteostasis, and stress response. Dysregulation of O-GlcNAc has been linked to diabetes, cancer, and neurodegenerative and cardiovascular disease. OGA has been proposed to be a drug target for the treatment of Alzheimer's and cardiovascular disease given that increased O-GlcNAc levels appear to exert a protective effect. The search for specific, potent, and drug-like OGA inhibitors with bioavailability in the brain is therefore a field of active research, requiring orthogonal high-throughput assay platforms. Here, we describe the synthesis of a novel probe for use in a fluorescence polarization based assay for the discovery of inhibitors of OGA. We show that the probe is suitable for use with both human OGA, as well as the orthologous bacterial counterpart from Clostridium perfringens, CpOGA, and the lysosomal hexosaminidases HexA/B. We structurally characterize CpOGA in complex with a ligand identified from a fragment library screen using this assay. The versatile synthesis procedure could be adapted for making fluorescent probes for the assay of other glycoside hydrolases.

SUBMITTER: Borodkin VS

PROVIDER: S-EPMC7611149 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Screening-based discovery of drug-like O-GlcNAcase inhibitor scaffolds.

Project description:O-GlcNAcylation is an essential posttranslational modification in metazoa. Modulation of O-GlcNAc levels with small molecule inhibitors of O-GlcNAc hydrolase (OGA) is a useful strategy to probe the role of this modification in a range of cellular processes. Here we report the discovery of novel, low molecular weight and drug-like O-GlcNAcase inhibitor scaffolds by high-throughput screening. Kinetic and X-ray crystallographic analyses of the binding modes with human/bacterial O-GlcNAcases identify some of these as competitive inhibitors. Comparative kinetic experiments with the mechanistically related human lysosomal hexosaminidases reveal that three of the inhibitor scaffolds show selectivity towards human OGA. These scaffolds provide attractive starting points for the development of non-carbohydrate, drug-like OGA inhibitors.

| S-EPMC2828546 | biostudies-literature

Quantitative imaging of molecular order in lipid membranes using two-photon fluorescence polarimetry.

Project description:We present a polarimetric two-photon microscopy technique to quantitatively image the local static molecular orientational behavior in lipid and cell membranes. This approach, based on a tunable excitation polarization state complemented by a polarized readout, is easily implementable and does not require hypotheses on the molecular angular distribution such as its mean orientation, which is a main limitation in traditional fluorescence anisotropy measurements. The method is applied to the investigation of the molecular angular distribution in giant unilamellar vesicles formed by liquid-ordered and liquid-disordered micro-domains, and in COS-7 cell membranes. The highest order contrast between ordered and disordered domains is obtained for dyes locating within the membrane acyl chains.

| S-EPMC2776251 | biostudies-literature

Fragment-Based Lead Discovery Strategies in Antimicrobial Drug Discovery.

Project description:Fragment-based lead discovery (FBLD) is a powerful application for developing ligands as modulators of disease targets. This approach strategy involves identification of interactions between low-molecular weight compounds (100-300 Da) and their putative targets, often with low affinity (KD ~0.1-1 mM) interactions. The focus of this screening methodology is to optimize and streamline identification of fragments with higher ligand efficiency (LE) than typical high-throughput screening. The focus of this review is on the last half decade of fragment-based drug discovery strategies that have been used for antimicrobial drug discovery.

| S-EPMC9951956 | biostudies-literature

Fragment-Sized Thiazoles in Fragment-Based Drug Discovery Campaigns: Friend or Foe?

Project description:Thiazoles exhibit a wide range of biological activities and therefore represent useful and attractive building blocks. To evaluate their usefulness and pinpoint their liabilities in fragment screening campaigns, we assembled a focused library of 49 fragment-sized thiazoles and thiadiazoles with various substituents, namely amines, bromides, carboxylic acids, and nitriles. The library was profiled in a cascade of biochemical inhibition assays, redox activity, thiol reactivity, and stability assays. Our study indicates that when thiazole derivatives are identified as screening hits, their reactivity should be carefully addressed and correlated with specific on-target engagement. Importantly, nonspecific inhibition should be excluded using experimental approaches and in silico predictions. To help with validation of hits identified in fragment screening campaigns, we can apply our high-throughput profiling workflow to focus on the most tractable compounds with a clear mechanism of action.

| S-EPMC9743419 | biostudies-literature

Targeting metalloproteins by fragment-based lead discovery.

Project description:It has been estimated that nearly one-third of functional proteins contain a metal ion. These constitute a wide variety of possible drug targets including metalloproteinases, dehydrogenases, oxidoreductases, hydrolases, deacetylases, or many others in which the metal ion is either of catalytic or of structural nature. Despite the predominant role of a metal ion in so many classes of drug targets, current high-throughput screening techniques do not usually produce viable hits against these proteins, likely due to the lack of proper metal-binding pharmacophores in the current screening libraries. Herein, we describe a novel fragment-based drug discovery approach using a metal-targeting fragment library that is based on a variety of distinct classes of metal-binding groups designed to reliably anchor the fragments at the target's metal ions. We show that the approach can effectively identify novel, potent and selective agents that can be readily developed into metalloprotein-targeted therapeutics.

| S-EPMC3135788 | biostudies-literature

Fragment-based inhibitor discovery against β-lactamase.

Project description:The production of β-lactamase is one of the primary resistance mechanisms used by Gram-negative bacterial pathogens to counter β-lactam antibiotics, such as penicillins, cephalosporins and carbapenems. There is an urgent need to develop novel β-lactamase inhibitors in response to ever evolving β-lactamases possessing an expanded spectrum of β-lactam hydrolyzing activity. Whereas traditional high-throughput screening has proven ineffective against serine β-lactamases, fragment-based approaches have been successfully employed to identify novel chemical matter, which in turn has revealed much about the specific molecular interactions possible in the active site of serine and metallo β-lactamases. In this review, we summarize recent progress in the field, particularly: the identification of novel inhibitor chemotypes through fragment-based screening; the use of fragment-protein structures to understand key features of binding hot spots and inform the design of improved leads; lessons learned and new prospects for β-lactamase inhibitor development using fragment-based approaches.

| S-EPMC4111090 | biostudies-literature

Fragment-Based Discovery of Novel MUS81 Inhibitors

Project description: Not available

| S-EPMC11247637 | biostudies-literature

Integrated biophysical approach to fragment screening and validation for fragment-based lead discovery.

Project description:In fragment-based drug discovery, the weak affinities exhibited by fragments pose significant challenges for screening. Biophysical techniques are used to address this challenge, but there is no clear consensus on which cascade of methods is best suited to identify fragment hits that ultimately translate into bound X-ray structures and provide bona fide starting points for synthesis. We have benchmarked an integrated biophysical approach for fragment screening and validation against Mycobacterium tuberculosis pantothenate synthetase. A primary screen of 1,250 fragments library was performed by thermal shift, followed by secondary screen using one-dimensional NMR spectroscopy (water ligand observed gradient spectroscopy and saturation transfer difference binding experiments) and ultimate hit validation by isothermal titration calorimetry and X-ray crystallography. Our multibiophysical approach identified three distinct binding sites for fragments and laid a solid foundation for successful structure-based elaboration into potent inhibitors.

| S-EPMC3740835 | biostudies-literature

Crystallographic fragment-based drug discovery: use of a brominated fragment library targeting HIV protease.

Project description:A library of 68 brominated fragments was screened against a new crystal form of inhibited HIV-1 protease in order to probe surface sites in soaking experiments. Often, fragments are weak binders with partial occupancy, resulting in weak, difficult-to-fit electron density. The use of a brominated fragment library addresses this challenge, as bromine can be located unequivocally via anomalous scattering. Data collection was carried out in an automated fashion using AutoDrug at SSRL. Novel hits were identified in the known surface sites: 3-bromo-2,6-dimethoxybenzoic acid (Br6) in the flap site and 1-bromo-2-naphthoic acid (Br27) in the exosite, expanding the chemistry of known fragments for development of higher affinity potential allosteric inhibitors. At the same time, mapping the binding sites of a number of weaker binding Br-fragments provides further insight into the nature of these surface pockets.

| S-EPMC3898673 | biostudies-literature

Ligand and Target Discovery by Fragment-Based Screening in Human Cells

Project description:Advances in the synthesis and screening of small-molecule libraries have accelerated the discovery of chemical probes for studying biological processes. Still, only a small fraction of the human proteome has chemical ligands, and the full complement of proteins with potential to be targeted by small molecules remains unknown. Here, we describe a platform that marries fragment-based ligand discovery with quantitative chemical proteomics to map thousands of reversible small molecule-protein interactions directly in human cells, many of which can be site-specifically determined. We advance this knowledge to create ligands that affect the activity of proteins heretofore lacking chemical probes and to identify by phenotypic screening small molecules that promote adipocyte differentiation by engaging the poorly characterized membrane protein PGRMC2. Fragment-based screening in human cells thus provides an extensive proteome-wide map of protein ligandability and facilitates the coordinated discovery of bioactive small molecules and their molecular targets.

2017-01-24 | GSE90731 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data