Rapid covalent-probe discovery by electrophile fragment screening

Efrat Resnick ¹ Anthony Bradley ^2,3,4 Jinrui Gan ¹ Alice Douangamath ⁴ Tobias Krojer ² Ritika Sethi ^2,5,6 Anthony Aimon ^3,4 Gabriel Amitai ⁷ Dom Belini ⁸ James Bennett ^2,9 Michael Fairhead ² Oleg Fedorov ^2,9 Paul P. Geurink ¹⁰ Jingxu Guo ¹¹ Alexander Plotnikov ⁷ Nava Reznik ¹ Gian Filippo Ruda ^2,9 Laura Diaz Saez ^2,9 Verena M. Straub ^2,9 Tamas Szommer ^2,9 Srikannathasan Velupillai ^2,9 Daniel Zaidman ¹ Alun R. Coker ¹¹ Christopher G. Dowson ⁸ Haim Barr ⁷ Killian V.M. Huber ^2,9 Paul E. Brennan ^2,9 Huib Ovaa ¹⁰ Frank von Delft ^2,4,12 Nir London ¹

¹Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel
²Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford, Oxford, UK
³Department of Chemistry, University of Oxford, Oxford, UK
⁴Diamond Light Source Ltd., Harwell Science and Innovation Campus, Didcot, UK
⁵Structural Biology Research Center, VIB, Brussels, Belgium
⁶Department of Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium
⁷The Wohl Institute for Drug Discovery and the Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot, Israel
⁸School of Life Sciences, University of Warwick, Coventry, UK
⁹Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK
¹⁰Oncode Institute and Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, Netherlands
¹¹Division of Medicine, University College London, London, UK
¹²Department of Biochemistry, University of Johannesburg, Johannesburg, 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. Such electrophilic fragments were considered non-selective 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 ten cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. By contrast, we found selective hits for most targets. Combination 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.