Peptidyl Acyloxymethyl Ketones as Activity-Based Probes for the Main Protease of SARS-CoV-2

Abstract

<div>The global pandemic caused by SARS-CoV-2 calls for a fast development of antiviral drugs </div><div>against this particular coronavirus. Chemical tools to facilitate inhibitor discovery as well as </div><div>detection of target engagement by hit or lead compounds from high throughput screens are </div><div>therefore in urgent need. We here report novel, selective activity-based probes that enable </div><div>detection of the SARS-CoV-2 main protease. The probes are based on acyloxymethyl ketone </div><div>reactive electrophiles combined with a peptide sequence including non-natural amino acids </div><div>that targets the non-primed site of the main protease substrate binding cleft. They are the first </div><div>activity-based probes for the main protease of coronaviruses and display target labeling within </div><div>in a human proteome without background. We expect that these reagents will be useful in the </div><div>drug development pipeline, not only for the current SARS-CoV-2, but also for other </div><div>coronaviruses. </div>

Detection of Active SARS-CoV-2 3CL Protease in Infected Cells Using Activity-Based Probes with a 2,6-Dichlorobenzoyloxymethyl Ketone Reactive Warhead

“…10 Moreover, other inhibitor-based ABPs with different warhead groups have been described, which have only been examined against recombinant 3CLpro. 25,26 Hence, visualizing active 3CLpro at a subcellular level using ABPs remains challenging.…”

Phosphinate Esters as Novel Warheads for Quenched Activity-Based Probes Targeting Serine Proteases

“…ABPs usually rely on binding elements that match the optimal substrate specificity of the target protease − either incorporating natural or non-natural amino acids − in combination with a reactive electrophile and a detection tag (Figure 1C). Highly selective ABPs have been reported for different protease targets including members of the cathepsin family, 9,10 several caspases, 11,12 MALT1, 13−15 neutrophil serine proteases (NSPs), 16−21 the SARS-CoV-2 main protease, 22,23 as well as different kallikreins 24,25 and granzymes. 26,27 The read-out possibilities for ABPs heavily depend on the nature of the detection tag.…”