Activity-Based Protein Profiling Methods to Study Bacteria: The Power of Small-Molecule Electrophiles

Abstract

ABPP methods have been utilized for the last two decades as a means to investigate complex proteomes in all three domains of life. Extensive use in eukaryotes has provided a more fundamental understanding of the biological processes involved in numerous diseases and has driven drug discovery and treatment campaigns. However, the use of ABPP in prokaryotes has been less common, although it has gained more attention over the last decade. The urgent need for understanding bacteriophysiology and bacterial pathogenicity at a foundational level has never been more apparent, as the rise in antibiotic resistance has resulted in the inadequate and ineffective treatment of infections. This is not only a result of resistance to clinically used antibiotics, but also a lack of new drugs and equally as important, new drug targets. ABPP provides a means for which new, clinically relevant drug targets may be identified through gaining insight into biological processes. In this chapter, we place particular focus on the discussion of ABPP strategies that have been applied to study different classes of bacterial enzymes.

From Differential Stains to Next Generation Physiology: Chemical Probes to Visualize Bacterial Cell Structure and Physiology

“…The ABPs can rapidly and irreversibly bind to catalytically active target enzymes by selectively and covalently modifying active site residues of the enzyme (Figure 1C). The ABPs basically comprises of three elements: (i) reactive group (also called 'warhead'), usually an electrophilic group that covalently bind with a conserved active site nucleophile; (ii) linker region or binding group that can modulate the reactivity and specificity of the probe profile [74,75]; (iii) reporter tag for the identification, enrichment and/or visualization of labeled (as reviewed in [74,[76][77][78][79]). The modular nature of ABPs in combination with their covalent interaction with probe targets, makes ABPs one of the most versatile group of chemical probes.…”

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

“…20 It relies on small molecules called activity-based probes (ABPs) that specifically form covalent bonds with active site residues of the target enzyme in a mechanism-based reaction. Although ABPP has been extensively applied for the characterization for the study of cancer-related enzymes 21 as well as enzymes from infectious bacteria, 22 application to viral infections has been much less investigated. 23 One of the underlying reasons may be the lack of ABPs specifically designed for viral targets, except for probes targeting the NS2B-NS3 protease of flaviviruses 24 25 and the smallpox virus protease K7L.…”

Harnessing β-Lactam Antibiotics for Illumination of the Activity of Penicillin-Binding Proteins inBacillus subtilis

“…Although none of the tested inhibitors only targeted PBP3, we pursed the development of a meropenem-based probe as this scaffold should label PBP3 and PBP5 and could be used in conjunction with a pbp5 deletion mutant (∆pbpC; DK695) to readily visualize PBP3 activity, as we have done in previous studies. (8,25) The secondary amino group located in the pyrrolidine ring of meropenem was used to attach a fluorophore to enable visualization of the TP activity (MEM-BODIPY; Scheme S1).…”

“…(22,23) Moreover, fluorescent penicillin-based probes have enabled monitoring of PBP transpeptidation activity in live cells. (21,24,25) Previously, we generated fluorescent Ceph C-based probes that demonstrated selectivity for a subset of PBPs and used them to label active PBPs in Gram-positive bacteria. subtilis.…”