Strategies for Competitive Activity-Based Protein Profiling in Small Molecule Inhibitor Discovery and Characterization

Abstract

Since its introduction by Cravatt and colleagues in 1999, activity-based protein profiling (ABPP) has become widely utilized throughout academia, government, and industry laboratories to study enzymes spanning numerous gene families in a multitude of biological systems. As a variation of ABPP, competitive ABPP provides a powerful approach to characterize the binding behavior of small molecule probes and clinical drugs throughout the functional proteome. The power and flexibility of competitive ABPP are exemplified by a wide range of creative adaptions which increase assay throughput, enable diverse detection schemes, and support the implementation of this approach within a hybrid target-based screening platform. We review major developments in competitive ABPP through a compare-contrast format to provide a useful introduction to this enabling technology for scientists in chemical biology and drug discovery.

Figure 1.

(A) Representative structure of the fluorophosphonate (FP) activity probe (ABP). In this example, the FP moiety serves as both enzyme recognition motif and covalent warhead. Integrated biotin tag provides a handle for biochemical pulldown as well as detection substrate. (B) General workflow for activity-based protein profiling (ABPP) of endogenous enzymes/proteins. The flexible ABPP platform supports multiple detection schemes.

Figure 2.

(left) General set-up for competitive ABPP comprising co-incubation of test compounds and activity-based probe (ABP). (right) Fluorescent based detection schemes to quantify test compound binding to target.

Figure 3.

General workflow for LC–MS/MS-based competitive ABPP (LC–MS ABPP) as used to screen the binding behavior of small molecules against a target enzyme class. As reviewed (Ref. [50]), a variety of mass spectrometry data acquisition strategies, including data-dependent (DDA), data-independent (DIA) and parallel reaction monitoring (PRM), have been used for identification of ABP-bound enzymes and quantification of small molecule binding.

Figure 4.

The concept of DNA-barcoding as used in high-throughput screening. (A) DNA-encoded small molecules are incubated with a purified target tethered to a bead or other solid support. qPCR is used to quantify small molecules bound to the target. (B) DNA-encoded kinases are incubated with test compounds and then co-incubated with ABPs tethered to beads. qPCR quantifies the relative enrichment of kinases on the beads as an indirect measure of small molecule binding.

Figure 5.

Workflow for in silico, structure-guided virtual screening

Figure 6.

Workflow for DUB ABPP using a focused covalent library paired with LC–MS/MS. Reproducible detection of 56 endogenous DUBs across multiple LC–MS/MS experiments enabled target-class SAR and development of first-in-class covalent inhibitor of the understudied DUB VCPIP1.

Figure 7.

Radar plots illustrate the relative strengths of different strategies used in competitive ABPP (cABPP). The estimation of performance metrics is based on single assay.