Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening

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.

Structural and Evolutionary Analysis Indicate that the SARS-CoV-2 Mpro is an Inconvenient Target for Small-Molecule Inhibitors Design

“…However, our in-depth analysis indicates a very different situation taking place, with major shape and size differences emerging due to the binding site flexibility. The continuous effort of Diamond Light Source group [44] performing massive XChem crystallographic fragment screen against Mpro supports our finding on large binding site flexibility (Supplementary Figure S4). Therefore, repurposing SARS drugs against COVID-19 may not be successful due to major shape and size differences, and despite docking methods, the enhanced sampling should be considered.…”

Structural and Evolutionary Analysis Indicate That the SARS-CoV-2 Mpro Is a Challenging Target for Small-Molecule Inhibitor Design

“…The continuous effort of Diamond Light Source group [46] performing massive XChem crystallographic fragment screen against Mpro has resulted in 22 non-covalent hits in the active site and 44 covalent hits in the active site (March 17th). Interestingly, two hits were identified on the dimer interface.…”

Covalent Small Molecules as Enabling Platforms for Drug Discovery

“…For example, small alterations to the binding mode may force a different orientation of the bound electrophile in relation to the nucleophilic amino acid, preventing efficient covalent bond formation with the POI. To circumvent these issues, research groups have developed cysteine‐targeted covalent fragment libraries already employing physiologically compatible electrophiles such as acrylamides, cyanoacrylamides, chloroacetamides, or vinyl sulfones (Figure C).…”

“…Overall, the use of covalent fragments in drug discovery has gained traction in the past few years with various groups describing detailed screening protocols and highlighting their benefits over more traditional methods. Additionally, covalent and reversible fragment libraries can be used synergistically to map out covalent and reversible binding hotspots throughout a binding pocket.…”

“…Additionally, covalent and reversible fragment libraries can be used synergistically to map out covalent and reversible binding hotspots throughout a binding pocket. Merging these fragments can lead to more advanced and potent inhibitors that can be developed using classical structure‐based drug design techniques …”