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

Abstract

The elucidation of protein/drug interactions remains a major challenge in drug discovery. Liquid chromatography-tandem mass spectrometry has emerged as a tremendously powerful technology for this endeavor, but its full potential has yet to be realized owing in part to unresolved challenges in data analysis. Herein, we demonstrate how tandem mass spectrometry can comprehensively map small molecule/peptide adducts when combined with unconstrained sequencing. Using a published sulfonyl fluoride activity-based probe as a model system, this method enabled the discovery of several unreported sites of interaction with its target proteins. Crucially, this probe was found to undergo quantitative displacement and hydrolysis from the target protein's active site. Isotopic labeling experiments provided a mechanistic rationale for the observed hydrolysis that involves neighboring-group participation. A chemical biology tagging strategy that leverages the probe's observed lability was developed and shown to be compatible with the original small molecule inhibitor in discovery profiling experiments.

Bioinspired Nitroalkylation for Selective Protein Modification and Peptide Stapling

“…Theb romo-derivative of the nitroalkane 1e was synthesized for easy characterization of am odified protein since ab romine atom leads to au nique signature by MS analysis given the 1:1a bundance of the two bromine isotopes ( 79 Br: 81 Br,F igure 6). [24] To confirm our hypothesis,n itro-bromomodified myoglobin was analyzed by MS and the two expected adducts of modified myoglobin bearing 79 Br and 81 Br were identified (see Figure S10). To make nitro-reagents as ag eneral approach to prepare diverse protein conjugates, we synthesized the alkyne-nitro-reagent 1h and carried out reaction with 5 to generate the alkyne-nitro protein conjugate with high conversion (99 %, Figure 6; see Figures S7 and S8).…”

Sulfur(vi) fluorides as tools in biomolecular and medicinal chemistry

“…Moreover, utilizing the DAS1 probe, additional labeling of Tyr and Lys residues was also observed. 61,62…”

“…Using this method, an alkyne-tagged AEBSF was demonstrated to covalently label the serine proteases, elastase, chymotrypsin, and trypsin through both in-gel fluorescence analysis and tandem mass spectrometry. 60,61 However, DiMaggio Jr, and coworkers revealed a challenge using sulfonyl fluoride-based probes for evaluating serine modification, where they demonstrated that the corresponding sulfonyl ester adduct formed on trypsin is hydrolyzed in the workflow and therefore can be challenging to monitor. The authors also developed an isotopic signature strategy that relies on the displacement of the serine-modified sulfonate adduct by 3-bromothiophenol to provide a stable isotopic signature to further aid in understanding probe-modified peptides.…”

“…Moreover, utilizing the DAS1 probe, additional labeling of Tyr and Lys residues was also observed. 61,62 While the sulfonyl fluoride headgroup is necessary for covalent linkage, it must first be appropriately positioned in the proximity of a nucleophilic residue. 61,62 Structure-based drug design (SBDD) guided modifications to sulfonyl fluoride structures enable the tuning of the reactivity toward specific binding-site residues and the selective modification of particular protein classes.…”

3-Oxo-β-sultam as a Sulfonylating Chemotype for Inhibition of Serine Hydrolases and Activity-Based Protein Profiling

“…The landscape of covalent inhibitors and activity-based probes (ABPs) has been dominated by a wide range of electrophilic reactive groups that enable the formation of carbon–heteroatom or phosphorus–oxygen bonds with a nucleophile in the active site of the target enzyme. − In contrast, covalent modification of proteins using sulfur–oxygen and sulfur–nitrogen bond formation has been focused on sulfur­(VI) fluoride exchange chemistry . Sulfonyl fluorides and aryl fluorosulfates have provided the chemical biology and medicinal chemistry communities with sulfonylating tools to assess the functionality of lysine, tyrosine, cysteine, and threonine residues, with a reactivity profile that can be modulated by adjusting the electronic properties of the reactive group. ,− However, the potential of other sulfonylation chemistries in target and drug discovery remains largely underexplored. For example, the β-sultam is the only sulfur­(VI)-nitrogen-based reactive group used to successfully develop selective enzyme inhibitors and ABPs. − …”