Mapping proteome-wide interactions of reactive chemicals using chemoproteomic platforms

Abstract

A large number of pharmaceuticals, endogenous metabolites, and environmental chemicals act through covalent mechanisms with protein targets. Yet, their specific interactions with the proteome still remain poorly defined for most of these reactive chemicals. Deciphering direct protein targets of reactive small-molecules is critical in understanding their biological action, off-target effects, potential toxicological liabilities, and development of safer and more selective agents. Chemoproteomic technologies have arisen as a powerful strategy that enable the assessment of proteome-wide interactions of these irreversible agents directly in complex biological systems. We review here several chemoproteomic strategies that have facilitated our understanding of specific protein interactions of irreversibly-acting pharmaceuticals, endogenous metabolites, and environmental electrophiles to reveal novel pharmacological, biological, and toxicological mechanisms.

Figure 1

Examples of irreversibly-acting drugs, tool compounds, environmental chemicals, and endogenous electrophiles.

Figure 2. Chemoproteomic platforms for assessing proteome-wide targets of irreversibly-acting chemicals

(A) SILAC-ABPP uses active-site directed chemical probes to assess the functional state of large numbers of enzymes directly in complex proteomes. Small-molecule inhibitors can be competed against the binding of activity-based probes to enzymes to assess enzyme class-wide selectivity. Cells can be labeled with light or heavy isotopic amino acids for quantitative proteomic analysis. (B) Analogs of these inhibitors bearing a bioorthogonal handle (e.g. alkyne) can be used to assess proteome-wide selectivity of small-molecule inhibitors using chemoproteomic approaches. (C) Isotopic Tandem Orthogonal Proteolysis-ABPP (isoTOP-ABPP) can be used to map hyper-reactive and functional sites across the proteome using reactivity-based chemical probes bearing bioorthogonal handles (e.g. alkyne). Reactive electrophiles can be competed against probe binding to hyper-reactive sites to map protein targets of these reactive agents. Probe-labeled peptides can be identified through subsequent appending of a biotin-azide analytical handle bearing a TEV protease recognition sequence and heavy or light isotopic valine tag using copper-catalyzed click chemistry. Upon mixing control and treated proteomes, probe-labeled proteins can be avidin-enriched, tryptically digested, and probe-labeled peptides can be subsequently enriched and released by TEV protease for subsequent quantitative proteomic analysis.

Figure 3. Biological insights gained from using chemoproteomic platforms

(A) ABPP has been successfully used to develop selective small-molecule inhibitors against enzymes involved the synthesis and degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG). Selective DAGL inhibitors KT109 and KT172 and selective MAGL inhibitors JZL184, KML29, and MJN110 have been used to not only identify that DAGL and MAGL regulate endocannabioid metabolism and signaling, but also to show that this pathway controls arachidonic metabolism that fuels pro-inflammatory prostaglandin synthesis. (B) ABPP was used to develop the selective DDHD2 inhibitor KLH45, which was used to show that the previously uncharacterized enzyme DDHD2 was the primary triacylglycerol (TAG) hydrolase in the brain, and that inhibiting this enzyme led to accumulation in brain triacylglycerol levels and accumulation of lipid droplets. (C) ABPP was used to develop the selective ABHD16A inhibitor KC01 to determine that ABHD16A was the primary phosphatidylserine (PS) hydrolase that generates the pro-inflammatory signaling lipid lyso-PS, which is in-turn hydrolyzed by ABHD12. Previous studies had shown that ABHD12 inactivation caused a neurodegenerative disease known as PHARC. ABHD16A inhibition protected mice from the neurotoxicological markers associated with PHARC. (D) Lipid aldehydes such as HNE were competed against the cysteine-reactive iodoacetamide-alkyne (IAyne) probe and coupled to the isoTOP-ABPP platform to map the direct protein targets of HNE. HNE showed selective interactions with certain sites such as the active-site proximal cysteine of ZAK, leading to ZAK inhibition and JAK inactivation. (E) Reactive environmental chemicals, such as the fungicide chlorothalonil (CTN) were competed against IAyne to map direct protein targets of these chemicals, leading to the discovery that CTN binds to and inhibits multiple enzymes involved in fatty acid oxidation.