Sulfonyl Fluoride Analogues as Activity‐Based Probes for Serine Proteases

Abstract

Enriched with fluoride: To expand on the available tools to interrogate proteases, we explored sulfonyl fluorides as activity-based probes. An alkyne-tagged sulfonyl fluoride covalently modifies members of the S1 family of serine proteases. By applying click chemistry, avidin enrichment and mass spectrometry, we can enrich and identify active endogenous serine proteases from a complex proteome.

Covalent protein modification: the current landscape of residue-specific electrophiles

“…Sulfonyl-fluoride derivatives were shown to covalently label multiple serine protease sub-classes, demonstrating utility as a serine-reactive electrophile [18]. However, proteome-wide evaluation of protein labeling showed additional targeting of activated tyrosine residues, particularly within glutathione S-transferases (GSTs) [19 ].…”

SuFEx-enabled, agnostic discovery of covalent inhibitors of human neutrophil elastase

“…This protease is therefore a key target for the development of antiinflammatory agents to combat these diseases. Alkyl and aryl sulfonyl fluorides have a long history as rather promiscuous covalent inhibitors of serine proteases (78)(79)(80)(81)(82)(83)(84)(85)(86)(87). In the late 1970s, the Powers group demonstrated the intrinsic reactivity differences across several serine proteases, including elastase, with 2-amido(peptido) benzenesulfonyl fluoride inhibitors (88,89).…”

Unbiased Mass Spectrometry Elucidation of the Targets and Mechanisms of Activity-Based Probes: A Case Study Involving Sulfonyl Fluorides

“…Sulfonyl fluorides (SF) in particular have become very popular warheads for ABPs, as demonstrated in a number of studies. [5][6][7][8] Their wide-use has prompted a recent foray exploring the factors influencing their reactivity as well as the stability of the resulting adducts, using protected amino acids as model nucleophiles. 9 The primary endpoint assay coupled to ABPP workflows is liquid chromatography tandem mass spectrometry (LC-MS/MS) since it provides unbiased identification of the protein targets and can, at least in principle, identify the specific residue associated with the ABP interaction based upon the mass shift of the probe.…”

“…Using a published sulfonyl fluoride probe (SFABP) as a case study, 5 we demonstrate how unbiased mass spectrometry combined with unconstrained sequencing can be used to characterise unexpected probe-protein interactions to an unprecedented level. The fundamental tenet behind this approach is that mass spectrometry data contains all the necessary information, but that the target residue and elemental composition of the probe must be treated as variables for it to be fully exploited.…”