Activity-based protein profiling for mapping and pharmacologically interrogating proteome-wide ligandable hotspots

Abstract

Despite the completion of human genome sequencing efforts nearly 15 years ago that brought with it the promise of genome-based discoveries that would cure human diseases, most protein targets that control human diseases have remained largely untranslated, in-part because they represent difficult protein targets to drug. In addition, many of these protein targets lack screening assays or accessible binding pockets, making the development of small-molecule modulators very challenging. Here, we discuss modern methods for activity-based protein profiling-based chemoproteomic strategies to map 'ligandable' hotspots in proteomes using activity and reactivity-based chemical probes to allow for pharmacological interrogation of these previously difficult targets. We will showcase several recent examples of how these technologies have been used to develop highly selective small-molecule inhibitors against disease-related protein targets.

Figure 1. ABPP platforms and their applications

A) Design of activity-based and reactivity-based biorthogonal probes. B) Using isoTOP-ABPP platforms for identifying disease-relevant targets. Normal and diseased proteomes can be labeled with activity or reactivity-based probes, followed by appendage of isotopically light or heavy analytical biotin handles bearing a TEV protease cleavage sequence, followed by mixing of proteomes in a 1:1 ratio, avidin enrichment of probe-labeled proteins, TEV digestion to release probe-modified tryptic peptides, and quantitative proteomic analysis of probe-modified peptides. C) Using isoTOP-ABPP for mapping hyper-reactive and functional hotspots in the proteome. The procedures would mirror those described in B), except that proteomes would be labeled with varying concentrations of probes (e.g. 10 × vs 1 × probe concentration) to quantitatively map hyper-reactive sites. D) Using isoTOP-ABPP for pharmacological interrogation of ligandable hotspots. The procedures would mirror those described in B), except that proteomes would be treated with vehicle or inhibitor to map not only the sites of probe labeling, but also sites where the inhibitor displaced probe labeling, facilitating both inhibitor discovery for targets of interest as well as an assessment of its proteome-wide selectivity.

Figure 2. Examples of activity and reactivity-based probes

“TAG” refers to an analytical handle for analysis of probe-labeled proteins such as a biotin or fluorophore handle.

Figure 3

Examples of small-molecule inhibitors developed using competitive ABPP platforms.