Abstract
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.
Highlights
The most widespread approach to designing such inhibitors has relied on the incorporation of an electrophile into an already optimized reversible recognition element,[4,9−11] most notably in kinase inhibitors.[4,12−16] More recently, large-scale covalent virtual screens have emerged as a method for the discovery of covalent binders.[17−23] While successful, in silico docking still has its limitations: it is limited to targets for which a crystal structure is available, it cannot efficiently address protein flexibility, and it cannot predict the intrinsic reactivity of electrophiles and may result in highly reactive compounds
The molecular-weight distribution of the reversible recognition elements is shifted to even lower masses
Discovering selective covalent acting compounds is challenging. We approach this problem by significantly increasing the chemical space of recognition elements, through the use of mild electrophiles, while carefully accounting for reactivity and promiscuity
Summary
The most widespread approach to designing such inhibitors has relied on the incorporation of an electrophile into an already optimized reversible recognition element,[4,9−11] most notably in kinase inhibitors.[4,12−16] More recently, large-scale covalent virtual screens have emerged as a method for the discovery of covalent binders.[17−23] While successful, in silico docking still has its limitations: it is limited to targets for which a crystal structure (or a high-quality model) is available, it cannot efficiently address protein flexibility, and it cannot predict the intrinsic reactivity of electrophiles and may result in highly reactive compounds. Empirical highthroughput screening (HTS) for covalent binders is typically avoided,[24] owing to concerns about promiscuous activity.[25−27] A major risk in screening large covalent libraries is that hits will be dominated by overly reactive compounds rather than by specific recognition.[28]
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