Development of a diverse activity-based probe library for studying serine proteases

Abstract

Serine proteases are ubiquitous enzymes that are involved in a variety of physiological processes ranging from digestion to immune system responses. While the functions of several serine proteases are well-characterized, many of these enzymes in the human body still lack functional annotation. Activity-based protein profiling (ABPP) technology has provided a strategy for studying these enzymes, particularly those with no known substrates. The use of a small-molecule probe that binds selectively to the active, but not the inactive, protein can enable enzyme detection, functional elucidation, and inhibitor discovery. While several activity-based probes, most notably fluorophosphonates, have been developed for serine proteases, we have found that a subset of serine proteases, including several rhomboid intramembrane proteases (RIPs), are not readily engaged by these probes. We therefore designed and synthesized a library of alkyne-functionalized structures, including phosphonates, β-lactams, and succinimides, to improve coverage of this enzyme class. We studied the ability of these structures to engage the well-studied serine protease trypsin as well as the bacterial RIP GlpG. Probe labeling was visualized by performing the azide-alkyne Huisgen cycloaddition reaction on probe-treated protein using an azide-functionalized rhodamine. These experiments have demonstrated that several of these structures react with the serine protease targets in an activity-dependent manner and have also revealed differential patterns in activity-dependent labeling between the two proteases. The chemical structures generated as part of these efforts hold promise as alternative activity-based probes for studying serine proteases that have so far eluded characterization.

Publisher

American Society for Biochemistry and Molecular Biology

Publication Date

3-25-2024

Publication Title

Journal of Biological Chemistry

Department

Chemistry and Biochemistry

Document Type

Abstract

DOI

https://doi.org/10.1016/j.jbc.2024.106000

Keywords

Activity-based protein profiling, Serine proteases

Language

English

Format

text

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