1. Rapid covalent-probe discovery by electrophile-fragment screening
- Author
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Alexander N. Plotnikov, Haim Barr, S. Velupillai, T. Krojer, Jingxu Guo, Jinrui Gan, Huib Ovaa, Oleg Fedorov, Christopher G. Dowson, Gian Filippo Ruda, R. Sethi, Chu Wang, Michael Fairhead, Ronen Gabizon, Nava Reznik, Gabriel Amitai, Daniel Zaidman, T. Szommer, James Bennett, Dom Bellini, Nir London, Paul P. Geurink, Efrat Resnick, Anthony Aimon, Verena M. Straub, Anthony R. Bradley, Frank von Delft, Y. Zhang, Alun R. Coker, Alice Douangamath, Laura Diaz Saez, Paul Brennan, Kilian Huber, and Jin Gan
- Subjects
Models, Molecular ,Time Factors ,Protein Conformation ,Drug Evaluation, Preclinical ,Electrons ,Ligands ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Catalysis ,Deubiquitinating enzyme ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,Protein structure ,Humans ,QC ,chemistry.chemical_classification ,Pyrophosphatase ,biology ,Chemistry ,HEK 293 cells ,General Chemistry ,Combinatorial chemistry ,QP ,0104 chemical sciences ,3. Good health ,Molecular Weight ,HEK293 Cells ,Enzyme ,Covalent bond ,Electrophile ,biology.protein ,Selectivity - Abstract
[Image: see text] Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. In contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.
- Published
- 2019