Competitive Activity-based Protein Profiling Research Articles

Molecular targets of cisplatin in HeLa cells explored through competitive activity-based protein profiling strategy

Cisplatin is widely used as anticancer drugs, and DNA is considered as the main target. Considering its high affinity towards cysteines and the important role of cystine containing proteins, we applied a competitive activity-based protein profiling strategy to identify protein cysteines that bind with cisplatin in HeLa cells. Living cells were treated with cisplatin at cytotoxic concentrations, then the protein was extracted. After labeling with desthiobiotin iodoacetamide (DBIA) probe, protein was precipitated, digested and isotopically labeled, subsequently the peptides were combined, and the biotinylated cysteine-containing peptides were enriched and quantified by LC-MS/MS. A total of 3571 peptides which originated from 1871 proteins were identified using the DBIA probe. Among them, 46 proteins were screened as targets, including proteins that have been identified as binding proteins by previous study. A novel cisplatin target, calpain-1 (CAPN1), was identified and validated as binding with cisplatin in vitro.

Probing the Interactions of Thiazole Abietane Inhibitors with the Human Serine Hydrolases ABHD16A and ABHD12.

12-Thiazole abietanes are highly selective reversible inhibitors of hABHD16A that could potentially alleviate neuroinflammation. In this study, we used synthetic chemistry, competitive activity-based protein profiling, and computational methodologies to try to establish relevant structural determinants of activity and selectivity of this class of compounds for inhibiting ABHD16A over ABHD12. Five compounds significantly inhibited hABHD16A but also very efficiently discriminated between inhibition of hABHD16A and hABHD12, with compound 35 being the most effective, at 100 μM (55.1 ± 8.7%; p < 0.0001). However, an outstanding switch in the selectivity toward ABHD12 was observed in the presence of a ring A ester, if the C2' position of the thiazole ring possessed a 1-hydroxyethyl group, as in compound 28. Although our data were inconclusive as to whether the observed enzyme inhibition is allosteric or not, we anticipate that the structure-activity relationships presented herein will inspire future drug discovery efforts in this field.

Cell-active, irreversible covalent inhibitors that selectively target the catalytic lysine of EGFR by using fluorosulfate-based SuFEx chemistry

EGFR signaling is involved in multiple cellular processes including cell proliferation, differentiation and development, making this protein kinase one of the most valuable drug targets for the treatment of non–small cell lung carcinomas (NSCLC). Herein, we describe the design and synthesis of a series of potential covalent inhibitors targeting the catalytically conserved lysine (K745) of EGFR on the basis of Erlotinib, an FDA-approved first-generation EGFR drug. Different amine-reactive electrophiles were introduced at positions on the Erlotinib scaffold proximal to K745 in EGFR. The optimized compound 26 (as well as its close analog 30), possessing a novel arylfluorosulfate group (ArOSO2F), showed excellent in vitro potency (as low as 0.19 nM in independent IC50 determination) and selectivity against EGFR and many of its drug-resistant mutants. Both intact protein mass spectrometry (MS) and site-mapping analysis revealed that compound 26 covalently bound to EGFR at K745 through the formation of a sulfamate. In addition, compound 26 displayed good anti-proliferative potency against EGFR-overexpressing HCC827 cells by inhibiting endogenous EGFR autophosphorylation. The pharmacokinetic studies of compound 26 demonstrated the druggable potential of other ArOSO2F-containing compounds. Finally, competitive activity-based protein profiling (ABPP), cellular thermal shift assay (CETSA), as well as cellular wash-out experiments, all showed compound 26 to be the first cell-active, fluorosulfate-based targeted covalent inhibitor (TCI) of protein kinases capable of covalently engaging the catalytically conserved lysine of its target in live mammalian cells.

Cellular Exposure to Chloroacetanilide Herbicides Induces Distinct Protein Destabilization Profiles.

Herbicides in the widely used chloroacetanilide class harbor a potent electrophilic moiety, which can damage proteins through nucleophilic substitution. In general, damaged proteins are subject to misfolding. Accumulation of misfolded proteins compromises cellular integrity by disrupting cellular proteostasis networks, which can further destabilize the cellular proteome. While direct conjugation targets can be discovered through affinity-based protein profiling, there are few approaches to probe how cellular exposure to toxicants impacts the stability of the proteome. We apply a quantitative proteomics methodology to identify chloroacetanilide-destabilized proteins in HEK293T cells based on their binding to the H31Q mutant of the human Hsp40 chaperone DNAJB8. We find that a brief cellular exposure to the chloroacetanilides acetochlor, alachlor, and propachlor induces misfolding of dozens of cellular proteins. These herbicides feature distinct but overlapping profiles of protein destabilization, highly concentrated in proteins with reactive cysteine residues. Consistent with the recent literature from the pharmacology field, reactivity is driven by neither inherent nucleophilic nor electrophilic reactivity but is idiosyncratic. We discover that propachlor induces a general increase in protein aggregation and selectively targets GAPDH and PARK7, leading to a decrease in their cellular activities. Hsp40 affinity profiling identifies a majority of propachlor targets identified by competitive activity-based protein profiling (ABPP), but ABPP can only identify about 10% of protein targets identified by Hsp40 affinity profiling. GAPDH is primarily modified by the direct conjugation of propachlor at a catalytic cysteine residue, leading to global destabilization of the protein. The Hsp40 affinity strategy is an effective technique to profile cellular proteins that are destabilized by cellular toxin exposure. Raw proteomics data is available through the PRIDE Archive at PXD030635.

N-Acryloylindole-alkyne (NAIA) enables imaging and profiling new ligandable cysteines and oxidized thiols by chemoproteomics

Cysteine has been exploited as the binding site of covalent drugs. Its high sensitivity to oxidation is also important for regulating cellular processes. To identify new ligandable cysteines which can be hotspots for therapy and to better study cysteine oxidations, we develop cysteine-reactive probes, N-acryloylindole-alkynes (NAIAs), which have superior cysteine reactivity owing to delocalization of π electrons of the acrylamide warhead over the whole indole scaffold. This allows NAIAs to probe functional cysteines more effectively than conventional iodoacetamide-alkyne, and to image oxidized thiols by confocal fluorescence microscopy. In mass spectrometry experiments, NAIAs successfully capture new oxidized cysteines, as well as a new pool of ligandable cysteines and proteins. Competitive activity-based protein profiling experiments further demonstrate the ability of NAIA to discover lead compounds targeting these cysteines and proteins. We show the development of NAIAs with activated acrylamide for advancing proteome-wide profiling and imaging ligandable cysteines and oxidized thiols.

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

AbstractSince 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.

Activity‐Based Protein Profiling – Celebrating the Groundbreaking Contributions of Benjamin Cravatt

Activity‐Based Protein Profiling – Celebrating the Groundbreaking Contributions of Benjamin Cravatt

Discovery of cisplatin-binding proteins by competitive cysteinome profiling.

Cisplatin is a widely used cancer metallodrug that induces cytotoxicity by targeting DNA and chelating cysteines in proteins. Here we applied a competitive activity-based protein profiling strategy to identify cisplatin-binding cysteines in cancer proteomes. A novel cisplatin target, MetAP1, was identified and functionally validated to contribute to cisplatin's cytotoxicity.

Quantitative Chemoproteomic Profiling of Targets of Au(I) Complexes by Competitive Activity-Based Protein Profiling.

Owing to the encouraging pharmacological action and acceptable toxicity profile, Au(I) complexes have attracted growing interest in the application of disease treatment. In order to investigate their potential target proteins and related bioinformation, herein, we screened four Au(I) complexes and explored the binding proteins utilizing a competitive activity-based protein profiling (ABPP) strategy, including identification experiments and reactivity classification experiments, which offers a simple and robust method to identify the target proteins of Au(I) complexes. We quantified the target proteins of the four Au(I) complexes and found that most of proteins were associated with cancer. In addition, the newly Au(I)-binding proteins and biological gold-protein interaction pathways were exhibited. Furthermore, we estimated the correlation between target proteins of Au(I) complexes and various cancers, which will promote the development of the gold anticancer drugs.

Identification of cell wall synthesis inhibitors active against Mycobacterium tuberculosis by competitive activity-based protein profiling

SummaryThe identification and validation of a small molecule’s targets is a major bottleneck in the discovery process for tuberculosis antibiotics. Activity-based protein profiling (ABPP) is an efficient tool for determining a small molecule’s targets within complex proteomes. However, how target inhibition relates to biological activity is often left unexplored. Here, we study the effects of 1,2,3-triazole ureas on Mycobacterium tuberculosis (Mtb). After screening ∼200 compounds, we focus on 4 compounds that form a structure-activity series. The compound with negligible activity reveals targets, the inhibition of which is functionally less relevant for Mtb growth and viability, an aspect not addressed in other ABPP studies. Biochemistry, computational docking, and morphological analysis confirms that active compounds preferentially inhibit serine hydrolases with cell wall and lipid metabolism functions and that disruption of the cell wall underlies biological activity. Our findings show that ABPP identifies the targets most likely relevant to a compound's antibacterial activity.

Identification of covalent inhibitors that disrupt M. tuberculosis growth by targeting multiple serine hydrolases involved in lipid metabolism

The increasing incidence of antibiotic-resistant Mycobacterium tuberculosis infections is a global health threat necessitating the development of new antibiotics. Serine hydrolases (SHs) are a promising class of targets because of their importance for the synthesis of the mycobacterial cell envelope. We screen a library of small molecules containing serine-reactive electrophiles and identify narrow-spectrum inhibitors of M.tuberculosis growth. Using these lead molecules, we perform competitive activity-based protein profiling and identify multiple SH targets, including enzymes with uncharacterized functions. Lipidomic analyses of compound-treated cultures reveal an accumulation of free lipids and a substantial decrease in lipooligosaccharides, linking SH inhibition to defects in cell envelope biogenesis. Mutant analysis reveals a path to resistance via the synthesis of mycocerates, but not through mutations to SH targets. Our results suggest that simultaneous inhibition of multiple SH enzymes is likely to be an effective therapeutic strategy for the treatment of M.tuberculosis infections.

Application of a Highly Selective Cathepsin S Two-step Activity-Based Probe in Multicolor Bio-Orthogonal Correlative Light-Electron Microscopy.

Cathepsin S is a lysosomal cysteine protease highly expressed in immune cells such as dendritic cells, B cells and macrophages. Its functions include extracellular matrix breakdown and cleavage of cell adhesion molecules to facilitate immune cell motility, as well as cleavage of the invariant chain during maturation of major histocompatibility complex II. The identification of these diverse specific functions has brought the challenge of delineating cathepsin S activity with great spatial precision, relative to related enzymes and substrates. Here, the development of a potent and highly selective two-step activity-based probe for cathepsin S and the application in multicolor bio-orthogonal correlative light-electron microscopy is presented. LHVS, which has been reported as a selective inhibitor of cathepsin S with nanomolar potency, formed the basis for our probe design. However, in competitive activity-based protein profiling experiments LHVS showed significant cross-reactivity toward Cat L. Introduction of an azide group in the P2 position expanded the selectivity window for cathepsin S, but rendered the probe undetectable, as demonstrated in bio-orthogonal competitive activity-based protein profiling. Incorporation of an additional azide handle for click chemistry on the solvent-exposed P1 position allowed for selective labeling of cathepsin S. This highlights the influence of click handle positioning on probe efficacy. This probe was utilized in multicolor bio-orthogonal confocal and correlative light-electron microscopy to investigate the localization of cathepsin S activity at an ultrastructural level in bone marrow-derived dendritic cells. The tools developed in this study will aid the characterization of the variety of functions of cathepsin S throughout biology.

A Screen of Covalent Inhibitors In&amp;nbsp; &lt;i&gt;Mycobacterium Tuberculosis&lt;/i&gt;&amp;nbsp;Identifies Serine Hydrolases Involved in Lipid Metabolism as Potential Therapeutic Targets

The increasing incidence of antibiotic-resistant Mycobacterium tuberculosis infections is a global health threat necessitating the development of new antibiotics. Serine hydrolases (SHs) are a promising class of targets because of their importance for the synthesis of the mycobacterial cell envelope. We screened a library of small molecules containing serine-reactive electrophiles and identified narrow spectrum inhibitors of M. tuberculous growth. Using these lead molecules, we performed competitive activity-based protein profiling and identified multiple SH targets, including enzymes with uncharacterized functions. Lipidomic analyses of compound-treated cultures revealed an accumulation of free lipids and a substantial decrease in lipooligosaccharides, linking SH inhibition to defects in cell envelope biogenesis. Mutant analysis revealed a path to resistance via the synthesis of mycocerates, but not through mutations to target enzymes. Our results suggest that simultaneous inhibition of multiple SH enzymes is likely to be an effective therapeutic strategy for the treatment of M. tuberculosis infections.

Design, synthesis and biological evaluation of second-generation benzoylpiperidine derivatives as reversible monoacylglycerol lipase (MAGL) inhibitors.

An interesting enzyme of the endocannabinoid system is monoacylglycerol lipase (MAGL). This enzyme, which metabolizes the endocannabinoid 2-arachidonoylglycerol (2-AG), has attracted great interest due to its involvement in several physiological and pathological processes, such as cancer progression. Experimental evidences highlighted some drawbacks associated with the use of irreversible MAGL inhibitors invivo, therefore the research field concerning reversible inhibitors is rapidly growing. In the present manuscript, the class of benzoylpiperidine-based MAGL inhibitors was further expanded and optimized. Enzymatic assays identified some compounds in the low nanomolar range and steered molecular dynamics simulations predicted the dissociation itinerary of one of the best compounds from the enzyme, confirming the observed structure-activity relationship. Biological evaluation, including assays in intact U937cells and competitive activity-based protein profiling experiments in mouse brain membranes, confirmed the selectivity of the selected compounds for MAGL versus other components of the endocannabinoid system. An antiproliferative ability in a panel of cancer cell lines highlighted their potential as potential anticancer agents. Future studies on the potential use of these compounds in the clinical setting are also supported by the inhibition of cell growth observed both in cancer organoids derived from high grade serous ovarian cancer patients and in pancreatic ductal adenocarcinoma primary cells, which showed genetic and histological features very similar to the primary tumors.

Small-Molecule Activity-Based Probe for Monitoring Ubiquitin C-Terminal Hydrolase L1 (UCHL1) Activity in Live Cells and Zebrafish Embryos.

Many reagents have emerged to study the function of specific enzymes in vitro. On the other hand, target specific reagents are scarce or need improvement, allowing investigations of the function of individual enzymes in their native cellular context. Here we report the development of a target-selective fluorescent small-molecule activity-based DUB probe that is active in live cells and an in vivo animal model. The probe labels active ubiquitin carboxy-terminal hydrolase L1 (UCHL1), also known as neuron-specific protein PGP9.5 (PGP9.5) and Parkinson disease 5 (PARK5), a DUB active in neurons that constitutes 1 to 2% of the total brain protein. UCHL1 variants have been linked with neurodegenerative disorders Parkinson’s and Alzheimer’s diseases. In addition, high levels of UCHL1 also correlate often with cancer and especially metastasis. The function of UCHL1 activity or its role in cancer and neurodegenerative disease is poorly understood and few UCHL1-specific activity tools exist. We show that the reagents reported here are specific to UCHL1 over all other DUBs detectable by competitive activity-based protein profiling and by mass spectrometry. Our cell-penetrable probe, which contains a cyanimide reactive moiety, binds to the active-site cysteine residue of UCHL1 in an activity-dependent manner. Its use is demonstrated by the fluorescent labeling of active UCHL1 both in vitro and in live cells. We furthermore show that this probe can selectively and spatiotemporally report UCHL1 activity during the development of zebrafish embryos. Our results indicate that our probe has potential applications as a diagnostic tool for diseases with perturbed UCHL1 activity.

Evaluation of FASN inhibitors by a versatile toolkit reveals differences in pharmacology between human and rodent FASN preparations and in antiproliferative efficacy in vitro vs. in situ in human cancer cells

De novo synthesis of fatty acids is essential to maintain intensive proliferation of cancer cells. Unlike normal cells that utilize food-derived circulating lipids for their fuel, cancer cells rely on heightened lipogenesis irrespective of exogenous lipid availability. Overexpression and activity of the multidomain enzyme fatty acid synthase (FASN) is crucial in supplying palmitate for protumorigenic activity. Therefore, FASN has been proposed as an attractive target for drug development. As an effort to set up an effective toolkit to study FASN inhibitors in human and rodent tissues, we validated activity-based protein profiling (ABPP) as a viable approach to unveil inhibitors targeting FASN thioesterase domain (FASN-TE). ABPP was combined with multi-well plate-assays designed for classical substrate-based FASN activity analysis together with powerful monitoring of cancer cell proliferation using IncuCyte® Live Cell Analyzing System. FASN-TE inhibitors were identified by competitive ABPP using HEK293 cell lysates in a screen of in-house compounds (200+) designed to target serine hydrolase (SH) family. The identified compounds were tested for their inhibitor potencies in vitro using a substrate-based activity assay monitoring FASN-dependent NADPH consumption in LNCaP prostate cancer cell preparation, in parallel with selected reference inhibitors, including orlistat (THL), GSK2194069, GSK837149A, platensimycin and BI-99179. LNCaP lysate supernatant was validated as a reliable native preparation to monitor FASN-dependent NADPH consumption as opposed to human glioma GAMG cells, whereas FASN enrichment was a prerequisite for accurate assays. While inhibitor pharmacology was identical between human prostate and glioma cancer cell FASN preparations, notable differences were revealed between human and rodent FASN preparations, especially for inhibitors targeting FASN-TE. ABPP combined with substrate-based assays facilitated identification of pan thiol-reactive inhibitor scaffolds, exemplified by the 1,2,4-thiadiazole moiety. Finally, selected compounds were evaluated for their antiproliferative efficacy in situ using GAMG cells. These studies revealed that while the tested compounds acted as potent FASN inhibitors in vitro, only a few showed antiproliferative efficacy in situ. To conclude, we describe a versatile toolkit to study FASN inhibitors in vitro and in situ using human cancer cells and reveal dramatic pharmacological differences between human and rodent FASN preparations.

Chemoproteomic Identification of Serine Hydrolase RBBP9 as a Valacyclovir-Activating Enzyme.

Prodrug discovery and development in the pharmaceutical industry have been hampered by a lack of knowledge of prodrug activation pathways. Such knowledge would minimize the risks of prodrug failure by enabling proper selection of preclinical animal models, prediction of pharmacogenomic variability, and identification of drug-drug interactions. Technologies for annotation of activating enzymes have not kept pace with the growing need. Activity-based protein profiling (ABPP) has matured considerably in recent decades, leading to widespread use in the pharmaceutical industry. Here, we report the extension of competitive ABPP (cABPP) to prodrug-activating enzyme identification in stable isotope-labeled cell lysates using a modified fluorophosphonate probe. Focusing on the antiviral ester prodrug valacyclovir (VACV), we identified serine hydrolase RBBP9 as an activating enzyme in Caco-2 cells via shotgun proteomics, validating the activity via the selective inhibitor emetine (EME). Kinetic characterization of RBBP9 revealed a catalytic efficiency (kcat·KM-1 = 104 mM-1·s-1) comparable to that of BPHL, the only known VACV-activating enzyme prior to this work. EME incubation in wild-type and Bphl-knockout jejunum and liver lysates demonstrated the near-exclusivity of VACV activation by RBBP9 in the intestine. Additionally, these studies showed that RBBP9 and BPHL are the two major and coequal VACV-activating enzymes in the liver. Single-pass intestinal perfusions of VACV ± EME in mice showed EME coperfusion significantly inhibited the intestinal activation of VACV, implying the in vivo relevance of RBBP9-mediated VACV activation. We envision that others might use the cABPP approach in the future for global, rapid, and efficient discovery of prodrug-activating enzymes.

Functional annotation of serine hydrolases in the asexual erythrocytic stage of Plasmodium falciparum

Enzymes of the serine hydrolase superfamily are ubiquitous, highly versatile catalysts that mediate a wide variety of metabolic reactions in eukaryotic cells, while also being amenable to selective inhibition. We have employed a fluorophosphonate-based affinity capture probe and mass spectrometry to explore the expression profile and metabolic roles of the 56-member P. falciparum serine hydrolase superfamily in the asexual erythrocytic stage of P. falciparum. This approach provided a detailed census of active serine hydrolases in the asexual parasite, with identification of 21 active serine hydrolases from α/β hydrolase, patatin, and rhomboid protease families. To gain insight into their functional roles and substrates, the pan-lipase inhibitor isopropyl dodecylfluorophosphonate was employed for competitive activity-based protein profiling, leading to the identification of seven serine hydrolases with potential lipolytic activity. We demonstrated how a chemoproteomic approach can provide clues to the specificity of serine hydrolases by using a panel of neutral lipase inhibitors to identify an enzyme that reacts potently with a covalent monoacylglycerol lipase inhibitor. In combination with existing phenotypic data, our studies define a set of serine hydrolases that likely mediate critical metabolic reactions in asexual parasites and enable rational prioritization of future functional characterization and inhibitor development efforts.

ABHD2 Inhibitor Identified by Activity-Based Protein Profiling Reduces Acrosome Reaction.

ABHD2 is a serine hydrolase that belongs to the subgroup of the α,β-hydrolase fold-containing proteins, which is involved in virus propagation, immune response, and fertilization. Chemical tools to selectively modulate the activity of ABHD2 in an acute setting are highly desired to investigate its biological role, but are currently lacking. Here, we report a library-versus-library screening using activity-based protein profiling (ABPP) to evaluate in parallel the selectivity and activity of a focused lipase inhibitor library against ABHD2 and a panel of closely related ABHD proteins. This screen resulted in the rapid identification of novel inhibitors for ABHD2. The selectivity of the inhibitor was further investigated in native mouse testis proteome by competitive ABPP, revealing a highly restricted off-target profile. The progesterone-induced acrosome reaction was reduced in a dose-dependent manner by the newly identified inhibitor, which provides further support for the key-role of ABHD2 in the P4-stimulated acrosome reaction. On this basis, the ABHD2 inhibitor is an excellent starting point for further optimization of ABHD2 inhibitors that can modulate sperm fertility and may lead to novel contraceptives.

Piperidine and piperazine inhibitors of fatty acid amide hydrolase targeting excitotoxic pathology

FAAH inhibitors offer safety advantages by augmenting the anandamide levels “on demand” to promote neuroprotective mechanisms without the adverse psychotropic effects usually seen with direct and chronic activation of the CB1 receptor. FAAH is an enzyme implicated in the hydrolysis of the endocannabinoid N-arachidonoylethanolamine (AEA), which is a partial agonist of the CB1 receptor. Herein, we report the discovery of a new series of highly potent and selective carbamate FAAH inhibitors and their evaluation for neuroprotection. The new inhibitors showed potent nanomolar inhibitory activity against human recombinant and purified rat FAAH, were selective (>1000-fold) against serine hydrolases MGL and ABHD6 and lacked any affinity for the cannabinoid receptors CB1 and CB2. Evaluation of FAAH inhibitors 9 and 31 using the in vitro competitive activity-based protein profiling (ABPP) assay confirmed that both inhibitors were highly selective for FAAH in the brain, since none of the other FP-reactive serine hydrolases in this tissue were inhibited by these agents. Our design strategy followed a traditional SAR approach and was supported by molecular modeling studies based on known FAAH cocrystal structures. To rationally design new molecules that are irreversibly bound to FAAH, we have constructed “precovalent” FAAH-ligand complexes to identify good binding geometries of the ligands within the binding pocket of FAAH and then calculated covalent docking poses to select compounds for synthesis. FAAH inhibitors 9 and 31 were evaluated for neuroprotection in rat hippocampal slice cultures. In the brain tissue, both inhibitors displayed protection against synaptic deterioration produced by kainic acid-induced excitotoxicity. Thus, the resultant compounds produced through rational design are providing early leads for developing therapeutics against seizure-related damage associated with a variety of disorders.