11 research outputs found
O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry
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 <i>Clostridium perfringens</i>, <i>Cp</i>OGA, and the
lysosomal hexosaminidases HexA/B. We structurally characterize <i>Cp</i>OGA 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
O-GlcNAc transferase invokes nucleotide sugar pyrophosphate participation in catalysis
Protein O-GlcNAcylation is an essential post-translational modification on hundreds of intracellular proteins in metazoa, catalyzed by O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) using unknown mechanisms of transfer and substrate recognition. Through crystallographic snapshots and mechanism-inspired chemical probes, we define how human OGT recognizes the sugar donor and acceptor peptide and uses a new catalytic mechanism of glycosyl transfer, involving the sugar donor α-phosphate as the catalytic base as well as an essential lysine. This mechanism seems to be a unique evolutionary solution to the spatial constraints imposed by a bulky protein acceptor substrate and explains the unexpected specificity of a recently reported metabolic OGT inhibitor. © 2012 Nature America, Inc. All rights reserved
A mutant O-GlcNAcase enriches Drosophila developmental regulators
YesProtein O-GlcNAcylation is a reversible post-translational modification of serines/threonines on
nucleocytoplasmic proteins. It is cycled by the enzymes O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase
(O-GlcNAcase or OGA). Genetic approaches in model organisms have revealed that protein O-GlcNAcylation is
essential for early embryogenesis. Drosophila melanogaster OGT/supersex combs (sxc) is a polycomb gene,
null mutants of which display homeotic transformations and die at the pharate adult stage. However, the identities
of the O-GlcNAcylated proteins involved, and the underlying mechanisms linking these phenotypes to embryonic
development, are poorly understood. Identification of O-GlcNAcylated proteins from biological samples is
hampered by the low stoichiometry of this modification and limited enrichment tools. Using a catalytically inactive
bacterial O-GlcNAcase mutant as a substrate trap, we have enriched the O-GlcNAc proteome of the developing
Drosophila embryo, identifying, amongst others, known regulators of Hox genes as candidate conveyors of OGT
function during embryonic development.Wellcome Trust Investigator Award (110061); MRC grant (MC_UU_12016/5); and Royal Society Research Grant
Discovery of New Bromodomain Scaffolds by Biosensor Fragment Screening
[Image: see text] The discovery of novel bromodomain inhibitors by fragment screening is complicated by the presence of dimethyl sulfoxide (DMSO), an acetyl-lysine mimetic, that can compromise the detection of low affinity fragments. We demonstrate surface plasmon resonance as a primary fragment screening approach for the discovery of novel bromodomain scaffolds, by describing a protocol to overcome the DMSO interference issue. We describe the discovery of several novel small molecules scaffolds that inhibit the bromodomains PCAF, BRD4, and CREBBP, representing canonical members of three out of the seven subfamilies of bromodomains. High-resolution crystal structures of the complexes of key fragments binding to BRD4(1), CREBBP, and PCAF were determined to provide binding mode data to aid the development of potent and selective inhibitors of PCAF, CREBBP, and BRD4
A novel allosteric inhibitor of the uridine diphosphate N-acetylglucosamine pyrophosphorylase from Trypanosoma brucei.
Uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the final reaction in the biosynthesis of UDP-GlcNAc, an essential metabolite in many organisms including Trypanosoma brucei, the etiological agent of Human African Trypanosomiasis. High-throughput screening of recombinant T. brucei UAP identified a UTP-competitive inhibitor with selectivity over the human counterpart despite the high level of conservation of active site residues. Biophysical characterization of the UAP enzyme kinetics revealed that the human and trypanosome enzymes both display a strictly ordered bi−bi mechanism, but with the order of substrate binding reversed. Structural characterization of the T. brucei UAP−inhibitor complex revealed that the inhibitor binds at an allosteric site absent in the human homologue that prevents the conformational rearrangement required to bind UTP. The identification of a selective inhibitory allosteric binding site in the parasite enzyme has therapeutic potential