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J Am Chem Soc. 2019 Jun 5;141(22):8951-8968. doi: 10.1021/jacs.9b02822. Epub 2019 May 22.

Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening.

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Department of Chemistry , Chemistry Research Laboratory , 12 Mansfield Road , Oxford OX1 3TA , U.K.
Diamond Light Source Ltd., Harwell Science and Innovation Campus , Didcot OX11 0QX , U.K.
Structural Biology Research Center , VIB , Brussels , Belgium.
Structural Biology Brussels , Vrije Universiteit Brussel , Brussels , Belgium.
Oncode Institute and Department of Cell and Chemical Biology , Leiden University Medical Center , Einthovenweg 20 , 2333 ZC Leiden , The Netherlands.
School of Life Sciences , University of Warwick , Coventry , U.K.
Division of Medicine , University College London , Gower Street , London WC1E 6BT , U.K.
Alzheimer's Research UK Oxford Drug Discovery Institute , University of Oxford , NDMRB, Roosevelt Drive , Oxford OX3 7FZ , U.K.
Department of Biochemistry , University of Johannesburg , Auckland Park 2006 , South Africa.


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.

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