SMaSh: A Streptavidin Mass Shift Assay for Rapidly Quantifying Target Occupancy by Irreversible Inhibitors.

The streptavidin mass shift (SMaSh) assay is a robust and fast approach for quantifying target protein occupancy by a covalent inhibitor or ligand. It exploits the biotin-streptavidin bond using the Simple Western platform. One measurement on a single sample determines both total and occupied target protein simultaneously and is, therefore, self-normalizing. The approach works in diverse and complex biological matrices and, with no need for matched vehicle-treated controls, readily applies to tissues from animal pharmacology models. Assessing occupancy is critical in the development of targeted covalent drugs. We demonstrate its use by characterizing and validating a variety of chemical probes for Bruton's tyrosine kinase (BTK, UniprotKB Q10607) and mitogen-activated protein kinase (ERK1/2/MAPK1/2, UniprotKB P28482 and P27361) and determining target engagement of covalent inhibitors for both targets and off-target engagement for ERK. We demonstrated that it works in cell lysates, tissues, and human peripheral blood mononuclear cells. The SMaSh assay is superior to traditional methods and broadly useful as a tool in assessing covalent biological probes or targeted covalent inhibitors.

Profiling CELMoD-Mediated Degradation of Cereblon Neosubstrates.

Targeted protein degradation is garnering increased attention as a therapeutic modality due in part to its promise of modulating targets previously considered undruggable. Cereblon E3 Ligase Modulating Drugs (CELMoDs) are one of the most well-characterized therapeutics employing this modality. CELMoDs hijack Cereblon E3 ligase activity causing neosubstrates to be ubiquitinated and degraded in the proteasome. Here, we describe a suite of assays-cellular substrate degradation, confirmation of CELMoD mechanism of action, in vitro ubiquitination, and Cereblon binding-that can be used to characterize CELMoD-mediated degradation of Cereblon neosubstrates. While the assays presented herein can be run independently, when combined they provide a strong platform to support the discovery and optimization of CELMoDs and fuel validation of targets degraded by this drug modality.

Ablation of PM20D1 reveals N-acyl amino acid control of metabolism and nociception.

N-acyl amino acids (NAAs) are a structurally diverse class of bioactive signaling lipids whose endogenous functions have largely remained uncharacterized. To clarify the physiologic roles of NAAs, we generated mice deficient in the circulating enzyme peptidase M20 domain-containing 1 (PM20D1). Global PM20D1-KO mice have dramatically reduced NAA hydrolase/synthase activities in tissues and blood with concomitant bidirectional dysregulation of endogenous NAAs. Compared with control animals, PM20D1-KO mice exhibit a variety of metabolic and pain phenotypes, including insulin resistance, altered body temperature in cold, and antinociceptive behaviors. Guided by these phenotypes, we identify N-oleoyl-glutamine (C18:1-Gln) as a key PM20D1-regulated NAA. In addition to its mitochondrial uncoupling bioactivity, C18:1-Gln also antagonizes certain members of the transient receptor potential (TRP) calcium channels including TRPV1. Direct administration of C18:1-Gln to mice is sufficient to recapitulate a subset of phenotypes observed in PM20D1-KO animals. These data demonstrate that PM20D1 is a dominant enzymatic regulator of NAA levels in vivo and elucidate physiologic functions for NAA signaling in metabolism and nociception.

Novel K-Ras G12C Switch-II Covalent Binders Destabilize Ras and Accelerate Nucleotide Exchange.

The success of targeted covalent inhibitors in the global pharmaceutical industry has led to a resurgence of covalent drug discovery. However, covalent inhibitor design for flexible binding sites remains a difficult task due to a lack of methodological development. Here, we compared covalent docking to empirical electrophile screening against the highly dynamic target K-RasG12C. While the overall hit rate of both methods was comparable, we were able to rapidly progress a docking hit to a potent irreversible covalent binder that modifies the inactive, GDP-bound state of K-RasG12C. Hydrogen-deuterium exchange mass spectrometry was used to probe the protein dynamics of compound binding to the switch-II pocket and subsequent destabilization of the nucleotide-binding region. SOS-mediated nucleotide exchange assays showed that, contrary to prior switch-II pocket inhibitors, these new compounds appear to accelerate nucleotide exchange. This study highlights the efficiency of covalent docking as a tool for the discovery of chemically novel hits against challenging targets.

Covalent Ligand Discovery against Druggable Hotspots Targeted by Anti-cancer Natural Products.

Many natural products that show therapeutic activities are often difficult to synthesize or isolate and have unknown targets, hindering their development as drugs. Identifying druggable hotspots targeted by covalently acting anti-cancer natural products can enable pharmacological interrogation of these sites with more synthetically tractable compounds. Here, we used chemoproteomic platforms to discover that the anti-cancer natural product withaferin A targets C377 on the regulatory subunit PPP2R1A of the tumor-suppressor protein phosphatase 2A (PP2A) complex leading to activation of PP2A activity, inactivation of AKT, and impaired breast cancer cell proliferation. We developed a more synthetically tractable cysteine-reactive covalent ligand, JNS 1-40, that selectively targets C377 of PPP2R1A to impair breast cancer signaling, proliferation, and in vivo tumor growth. Our study highlights the utility of using chemoproteomics to map druggable hotspots targeted by complex natural products and subsequently interrogating these sites with more synthetically tractable covalent ligands for cancer therapy.

Mapping Novel Metabolic Nodes Targeted by Anti-Cancer Drugs that Impair Triple-Negative Breast Cancer Pathogenicity.

Triple-negative breast cancers (TNBCs) are estrogen receptor, progesterone receptor, and HER2 receptor-negative subtypes of breast cancers that show the worst prognoses and lack targeted therapies. Here, we have coupled the screening of ∼400 anticancer agents that are under development or in the clinic with chemoproteomic and metabolomic profiling to identify novel metabolic mechanisms for agents that impair TNBC pathogenicity. We identify 20 anticancer compounds that significantly impaired cell survival across multiple types of TNBC cells. Among these 20 leads, the phytoestrogenic natural product licochalcone A was of interest, since TNBCs are unresponsive to estrogenic therapies, indicating that licochalcone A was likely acting through another target. Using chemoproteomic profiling approaches, we reveal that licochalcone A impairs TNBC pathogenicity, not through modulating estrogen receptor activity but rather through inhibiting prostaglandin reductase 1, a metabolic enzyme involved in leukotriene B4 inactivation. We also more broadly performed metabolomic profiling to map additional metabolic mechanisms of compounds that impair TNBC pathogenicity. Overlaying lipidomic profiling with drug responses, we find that deubiquitinase inhibitors cause dramatic elevations in acyl carnitine levels, which impair mitochondrial respiration and contribute to TNBC pathogenic impairments. We thus put forth two unique metabolic nodes that are targeted by drugs or drug candidates that impair TNBC pathogenicity. Our results also showcase the utility of coupling drug screens with chemoproteomic and metabolomic profiling to uncover unique metabolic drivers of TNBC pathogenicity.

Argininosuccinate Synthase 1 is a Metabolic Regulator of Colorectal Cancer Pathogenicity.

Like many cancer types, colorectal cancers have dysregulated metabolism that promotes their pathogenic features. In this study, we used the activity-based protein profiling chemoproteomic platform to profile cysteine-reactive metabolic enzymes that are upregulated in primary human colorectal tumors. We identified argininosuccinate synthase 1 (ASS1) as an upregulated target in primary human colorectal tumors and show that pharmacological inhibition or genetic ablation of ASS1 impairs colorectal cancer pathogenicity. Using metabolomic profiling, we show that ASS1 inhibition leads to reductions in the levels of oncogenic metabolite fumarate, leading to impairments in glycolytic metabolism that supports colorectal cancer cell pathogenicity. We show here that ASS1 inhibitors may represent a novel therapeutic approach for attenuating colorectal cancer through compromising critical metabolic and metabolite signaling pathways and demonstrate the utility of coupling chemoproteomic and metabolomic strategies to map novel metabolic regulators of cancer.

Chemoproteomic Screening of Covalent Ligands Reveals UBA5 As a Novel Pancreatic Cancer Target.

Chemical genetic screening of small-molecule libraries has been a promising strategy for discovering unique and novel therapeutic compounds. However, identifying the targets of lead molecules that arise from these screens has remained a major bottleneck in understanding the mechanism of action of these compounds. Here, we have coupled the screening of a cysteine-reactive fragment-based covalent ligand library with an isotopic tandem orthogonal proteolysis-enabled activity-based protein profiling (isoTOP-ABPP) chemoproteomic platform to rapidly couple the discovery of lead small molecules that impair pancreatic cancer pathogenicity with the identification of druggable hotspots for potential cancer therapy. Through this coupled approach, we have discovered a covalent ligand DKM 2-93 that impairs pancreatic cancer cell survival and in vivo tumor growth through covalently modifying the catalytic cysteine of the ubiquitin-like modifier activating enzyme 5 (UBA5), thereby inhibiting its activity as a protein that activates the ubiquitin-like protein UFM1 to UFMylate proteins. We show that UBA5 is a novel pancreatic cancer therapeutic target and show DKM 2-93 as a relatively selective lead inhibitor of UBA5. Our results underscore the utility of coupling the screening of covalent ligand libraries with isoTOP-ABPP platforms for mining the proteome for druggable hotspots for cancer therapy.

Chemoproteomic Profiling of Acetanilide Herbicides Reveals Their Role in Inhibiting Fatty Acid Oxidation.

Acetanilide herbicides are among the most widely used pesticides in the United States, but their toxicological potential and mechanisms remain poorly understood. Here, we have used chemoproteomic platforms to map proteome-wide cysteine reactivity of acetochlor (AC), the most widely used acetanilide herbicide, in vivo in mice. We show that AC directly reacts with >20 protein targets in vivo in mouse liver, including the catalytic cysteines of several thiolase enzymes involved in mitochondrial and peroxisomal fatty acid oxidation. We show that the fatty acids that are not oxidized, due to impaired fatty acid oxidation, are instead diverted into other lipid pathways, resulting in heightened free fatty acids, triglycerides, cholesteryl esters, and other lipid species in the liver. Our findings show the utility of chemoproteomic approaches for identifying novel mechanisms of toxicity associated with environmental chemicals like acetanilide herbicides.

Mapping Proteome-wide Targets of Glyphosate in Mice.

Glyphosate, the active ingredient in the herbicide Roundup, is one of the most widely used pesticides in agriculture and home garden use. Whether glyphosate causes any mammalian toxicity remains highly controversial. While many studies have associated glyphosate with numerous adverse health effects, the mechanisms underlying glyphosate toxicity in mammals remain poorly understood. Here, we used activity-based protein profiling to map glyphosate targets in mice. We show that glyphosate at high doses can be metabolized in vivo to reactive metabolites such as glyoxylate and react with cysteines across many proteins in mouse liver. We show that glyoxylate inhibits liver fatty acid oxidation enzymes and glyphosate treatment in mice increases the levels of triglycerides and cholesteryl esters, likely resulting from diversion of fatty acids away from oxidation and toward other lipid pathways. Our study highlights the utility of using chemoproteomics to identify novel toxicological mechanisms of environmental chemicals such as glyphosate.

The Secreted Enzyme PM20D1 Regulates Lipidated Amino Acid Uncouplers of Mitochondria.

Brown and beige adipocytes are specialized cells that express uncoupling protein 1 (UCP1) and dissipate chemical energy as heat. These cells likely possess alternative UCP1-independent thermogenic mechanisms. Here, we identify a secreted enzyme, peptidase M20 domain containing 1 (PM20D1), that is enriched in UCP1(+) versus UCP1(-) adipocytes. We demonstrate that PM20D1 is a bidirectional enzyme in vitro, catalyzing both the condensation of fatty acids and amino acids to generate N-acyl amino acids and also the reverse hydrolytic reaction. N-acyl amino acids directly bind mitochondria and function as endogenous uncouplers of UCP1-independent respiration. Mice with increased circulating PM20D1 have augmented respiration and increased N-acyl amino acids in blood. Lastly, administration of N-acyl amino acids to mice improves glucose homeostasis and increases energy expenditure. These data identify an enzymatic node and a family of metabolites that regulate energy homeostasis. This pathway might be useful for treating obesity and associated disorders.

Mapping Proteome-Wide Targets of Environmental Chemicals Using Reactivity-Based Chemoproteomic Platforms.

We are exposed to a growing number of chemicals in our environment, most of which have not been characterized in terms of their toxicological potential or mechanisms. Here, we employ a chemoproteomic platform to map the cysteine reactivity of environmental chemicals using reactivity-based probes to mine for hyper-reactive hotspots across the proteome. We show that environmental contaminants such as monomethylarsonous acid and widely used pesticides such as chlorothalonil and chloropicrin possess common reactivity with a distinct set of proteins. Many of these proteins are involved in key metabolic processes, suggesting that these targets may be particularly sensitive to environmental electrophiles. We show that the widely used fungicide chlorothalonil specifically inhibits several metabolic enzymes involved in fatty acid metabolism and energetics, leading to dysregulated lipid metabolism in mice. Our results underscore the utility of using reactivity-based chemoproteomic platforms to uncover novel mechanistic insights into the toxicity of environmental chemicals.

Chemical genetics screening reveals KIAA1363 as a cytokine-lowering target.

Inflammation is a hallmark of many human diseases, including pain, arthritis, atherosclerosis, obesity and diabetes, cancer, and neurodegenerative diseases. Although there are several successfully marketed small molecules anti-inflammatory drugs such as cyclooxygenase inhibitors and glucocorticoids, many of these compounds are also associated with various adverse cardiovascular or immunosuppressive side effects. Thus, identifying novel anti-inflammatory small molecules and their targets is critical for developing safer and more effective next-generation treatment strategies for inflammatory diseases. Here, we have conducted a chemical genetics screen to identify small molecules that suppress the release of the inflammatory cytokine TNFα from stimulated macrophages. We have used an enzyme class-directed chemical library for our screening efforts to facilitate subsequent target identification using activity-based protein profiling (ABPP). Using this strategy, we have found that KIAA1363 is a novel target for lowering key pro-inflammatory cytokines through affecting key ether lipid metabolism pathways. Our study highlights the application of combining chemical genetics with chemoproteomic and metabolomic approaches toward identifying and characterizing anti-inflammatory smal molecules and their targets.

An alkyne-aspirin chemical reporter for the detection of aspirin-dependent protein modification in living cells.

Aspirin (acetylsalicylic acid) is widely used for the acute treatment of inflammation and the management of cardiovascular disease. More recently, it has also been shown to reduce the risk of a variety of cancers. The anti-inflammatory properties of aspirin in pain-relief, cardio-protection, and chemoprevention are well-known to result from the covalent inhibition of cyclooxygenase enzymes through nonenzymatic acetylation of key serine residues. However, any additional molecular mechanisms that may contribute to the beneficial effects of aspirin remain poorly defined. Interestingly, studies over the past 50 years using radiolabeled aspirin demonstrated that other proteins are acetylated by aspirin and enrichment with antiacetyl-lysine antibodies identified 33 potential targets of aspirin-dependent acetylation. Herein we describe the development of an alkyne-modified aspirin analogue (AspAlk) as a chemical reporters of aspirin-dependent acetylation in living cells. When combined with the Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition, this chemical reporter allowed for the robust in-gel fluorescent detection of acetylation and the subsequent enrichment and identification of 120 proteins, 112 of which have not been previously reported to be acetylated by aspirin in cellular or in vivo contexts. Finally, AspAlk was shown to modify the core histone proteins, implicating aspirin as a potential chemical-regulator of transcription.

N-Propargyloxycarbamate monosaccharides as metabolic chemical reporters of carbohydrate salvage pathways and protein glycosylation.

Metabolic chemical reporters of glycosylation allow for the visualization and identification of a variety of glycoconjugates by taking advantage of the promiscuity of carbohydrate metabolism. Here we describe the synthesis and characterization of metabolic chemical reporters bearing an N-propargyloxycarbamate (Poc) group that creates discrimination between glycosylation pathways.

Robust in-gel fluorescence detection of mucin-type O-linked glycosylation.

Mucin-type O-linked glycosylation is a common post translational modification of cell-surface and secretory pathway proteins and is implicated in vital biological processes as well as human disease. We report here the use of the metabolic chemical reporter GalNAz along with Cu(I)-catalyzed [3+2] azide-alkyne cycloaddition conditions for the robust, in-gel fluorescent visualization of mucin-type O-linked glycoproteins.