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14 items found (0 items not approved), displaying 11 to 14.[First/Prev] 1, 2 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I04-1-Macromolecular Crystallography (fixed wavelength)	Development of Specific, Irreversible Inhibitors for a Receptor Tyrosine Kinase EphB3 Alvin Kung , Ying-chu Chen , Marianne Schimpl , Feng Ni , Jianfa Zhu , Maurice Turner , Henrik Molina , Ross Overman , Chao Zhang Journal Of The American Chemical Society 138, 10554 - 10560 DOI: 10.1021/jacs.6b05483 Show Abstract ▼ Abstract: Erythropoietin-producing human hepatocellular carcinoma (Eph) receptor tyrosine kinases (RTKs) regulate a variety of dynamic cellular events, including cell protrusion, migration, proliferation, and cell-fate determination. Small-molecule inhibitors of Eph kinases are valuable tools for dissecting the physiological and pathological roles of Eph. However, there is a lack of small-molecule inhibitors that are selective for individual Eph isoforms due to the high homology within the family. Herein, we report the development of the first potent and specific inhibitors of a single Eph isoform, EphB3. Through structural bioinformatic analysis, we identified a cysteine in the hinge region of the EphB3 kinase domain, a feature that is not shared with any other human kinases. We synthesized and characterized a series of electrophilic quinazolines to target this unique, reactive feature in EphB3. Some of the electrophilic quinazolines selectively and potently inhibited EphB3 both in vitro and in cells. Cocrystal structures of EphB3 in complex with two quinazolines confirmed the covalent linkage between the protein and the inhibitors. A “clickable” version of an optimized inhibitor was created and employed to verify specific target engagement in the whole proteome and to probe the extent and kinetics of target engagement of existing EphB3 inhibitors. Furthermore, we demonstrate that the autophosphorylation of EphB3 within the juxtamembrane region occurs in trans using a specific inhibitor. These exquisitely specific inhibitors will facilitate the dissection of EphB3’s role in various biological processes and disease contribution.	Aug 2016
I04-1-Macromolecular Crystallography (fixed wavelength)	The active site of O-GlcNAc transferase imposes constraints on substrate sequence Shalini Pathak , Jana Alonso , Marianne Schimpl , Karim Rafie , David E Blair , Vladimir S Borodkin , Alexander W Schüttelkopf , Osama Albarbarawi , Daan M F Van Aalten Nature Structural & Molecular Biology 22 (9), 744 - 750 DOI: 10.1038/nsmb.3063 PMID: 26237509 Show Abstract ▼ Abstract: O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoans. Although this enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, we describe the application of a high-throughput OGT assay to a library of peptides. We mapped sites of O-GlcNAc modification by electron transfer dissociation MS and found that they correlate with previously detected O-GlcNAc sites. Crystal structures of four acceptor peptides in complex with Homo sapiens OGT suggest that a combination of size and conformational restriction defines sequence specificity in the −3 to +2 subsites. This work reveals that although the N-terminal TPR repeats of OGT may have roles in substrate recognition, the sequence restriction imposed by the peptide-binding site makes a substantial contribution to O-GlcNAc site specificity.	Oct 2015
I03-Macromolecular Crystallography	Bisubstrate UDPpeptide conjugates as human O-GlcNAc transferase inhibitors Vladimir s. Borodkin , Marianne Schimpl , Mehmet Gundogdu , Karim Rafie , Helge Dorfmueller , David Robinson , Daniel Van Aalten Biochemical Journal 457, 497 - 502 DOI: 10.1042/BJ20131272 Diamond Proposal Number(s): [8268] Open Access Show Abstract ▼ Abstract: Inhibitors of OGT (O-GlcNAc transferase) are valuable tools to study the cell biology of protein O-GlcNAcylation. We report OGT bisubstrate-linked inhibitors (goblins) in which the acceptor serine in the peptide VTPVSTA is covalently linked to UDP, eliminating the GlcNAc pyranoside ring. Goblin1 co-crystallizes with OGT, revealing an ordered C3 linker and retained substrate-binding modes, and binds the enzyme with micromolar affinity, inhibiting glycosyltransfer on to protein and peptide substrates.	Feb 2014
I03-Macromolecular Crystallography	Synergy of Peptide and Sugar in O-GlcNAcase Substrate Recognition Marianne Schimpl , Vladimir s. Borodkin , Lindsey j. Gray , Daan m.f. Van aalten Chemistry & Biology 19 (2), 173 - 178 DOI: 10.1016/j.chembiol.2012.01.011 PMID: 22365600 Diamond Proposal Number(s): [1223] Open Access Show Abstract ▼ Abstract: Protein O-GlcNAcylation is an essential reversible posttranslational modification in higher eukaryotes. O-GlcNAc addition and removal is catalyzed by O-GlcNAc transferase and O-GlcNAcase, respectively. We report the molecular details of the interaction of a bacterial O-GlcNAcase homolog with three different synthetic glycopeptides derived from characterized O-GlcNAc sites in the human proteome. Strikingly, the peptides bind a conserved O-GlcNAcase substrate binding groove with similar orientation and conformation. In addition to extensive contacts with the sugar, O-GlcNAcase recognizes the peptide backbone through hydrophobic interactions and intramolecular hydrogen bonds, while avoiding interactions with the glycopeptide side chains. These findings elucidate the molecular basis of O-GlcNAcase substrate specificity, explaining how a single enzyme achieves cycling of the complete O-GlcNAc proteome. In addition, this work will aid development of O-GlcNAcase inhibitors that target the peptide binding site.	Feb 2012

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