Activity-based protein profiling in vivo using a copper(i)-catalyzed azide-alkyne [3 + 2] cycloaddition

Toward the goal of assigning function to the tens of thousands of protein products encoded by eukaryotic and prokaryotic genomes, the field of proteomics requires new technologies that can functionally characterize proteins within the dynamic environment of the cell, where these biomolecules are subject to myriad posttranslational modifications and the actions of endogenous activators and inhibitors. Here, we report an advanced strategy for activity-based protein profiling (ABPP) that addresses this important need. We show that several enzymes can be labeled in an activity-based manner both in vitro and in vivo by an azido-sulfonate ester probe and that these labeling events can be detected in whole proteomes by copper-catalyzed ligation with a rhodamine-alkyne reagent. This click chemistry-based strategy for ABPP represents a unique and versatile method for functional proteome analysis.     

Activity-based probes: discovering new biology and new drug targets

The development and application of chemical technologies enabling direct analysis of enzyme activity in living systems has undergone explosive growth in recent years. Activity-based protein profiling (ABPP) is a key constituent of this broad field, and is among the most powerful and mature chemical proteomic technologies. This tutorial review introduces the essential features of ABPP and the design and application of activity-based probes (ABPs) from drug target elucidation and in vivo visualisation of enzyme activity to comprehensive profiling of the catalytic content of living systems, and the discovery of new biological pathways.     

Confirming target engagement for reversible inhibitors in vivo by kinetically tuned activity-based probes

The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPLA1 and LYPLA2, two enzymes for which selective, in vivo active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo.     

Optimization of activity-based probes for proteomic profiling of histone deacetylase complexes

Histone deacetylases (HDACs) are key enzymatic regulators of the epigenome and serve as promising targets for anticancer therapeutics. Recently, we developed a photoreactive "clickable" probe, SAHA-BPyne, to report on HDAC activity and complex formation in native biological systems. Here, we investigate the selectivity, sensitivity, and inhibitory properties of SAHA-BPyne and related potential activity-based probes for HDACs. While we identified several probes that are potent HDAC inhibitors and label HDAC complex components in native proteomic preparations, SAHA-BPyne was markedly superior for profiling HDAC activities in live cells. Interestingly, the enhanced performance of SAHA-BPyne as an in situ activity-based probe could not be solely ascribed to potency in HDAC binding, implying that other features of the molecule were key to efficient active site-directed labeling in living systems. Finally, we demonstrate the value of in situ profiling of HDACs by comparing the activity and expression of HDAC1 in cancer cells treated with the cytotoxic agent parthenolide. These results underscore the utility of activity-based protein profiling for studying HDAC function and may provide insight for the future development of click chemistry-based photoreactive probes for the in situ analysis of additional enzyme activities.     

An activity-based protein profiling probe for the nicotinic acetylcholine receptor

Activity-based protein profiling (ABPP) has been used extensively to characterize the physiological functions of enzymes but has not yet been extended to ion channels. We have synthesized a state-dependent photoaffinity probe for the nicotinic acetylcholine receptor (nAChR) as a proof of concept for the development of ion channel directed ABPP probes. The candidate probe BPyneTEA comprises an nAChR binding moiety, a benzophenone moiety for photolabeling, and an alkyne moiety for biotinylation via "click chemistry". Single-molecule current measurements show that BPyneTEA blocks both the closed and open (i.e., nondesensitized) conformations of the nAChR with similar kinetics. In living cells, BPyneTEA photolabels the closed state selectively over the inactive desensitized state. BPyneTEA thus shows promise as a probe for nondesensitized nAChRs and may be useful in studying the molecular physiology of desensitization. The structure and reactivity of ion channel pores in general suggest that they will be a broadly useful target for ABPP probes.     

Activity-based probes for studying the activity of flavin-dependent oxidases and for the protein target profiling of monoamine oxidase inhibitors

High profile: new activity-based protein profiling (ABPP) probes have been designed that target exclusively monoamine oxidases A and B within living cells (see picture; FAD=flavin adenine dinucleotide, FMN=flavin monodinucleotide). With these probes it could be shown that the MAO inhibitor deprenyl, which is in clinical use against Parkinson's disease, shows unique protein specificity despite its covalent mechanism of action.     

A tandem orthogonal proteolysis strategy for high-content chemical proteomics

The field of proteomics aims to assign functions to the numerous protein products encoded by eukaryotic and prokaryotic genomes. Toward this end, chemical strategies have emerged as a powerful means to enrich specific classes of proteins based on shared functional properties, such as catalytic activity [activity-based protein profiling (ABPP)], and post-translational modification state. The theoretical information content in chemical proteomic experiments greatly exceeds the actual data procured, due in large part to limitations in existing analytical technologies. Here, we present a tandem orthogonal proteolysis (TOP) strategy for high-content chemical proteomics that enables the parallel characterization of probe-labeled proteins and sites of probe modification. The TOP approach exploits "click chemistry" to introduce a multifunctional tag onto probe-labeled proteins that contains both a biotin group for protein enrichment and a tobacco etch virus (TEV) protease cleavage site for selective release of probe-modified peptides. Following capture on streptavidin beads, protein targets of probes and their sites of labeling are sequentially identified by a two-step proteolysis strategy (trypsin and TEV, respectively). We apply the TOP method to characterize targets of sulfonate ester ABPP probes in tissue proteomes, resulting in the discovery of numerous active site-labeled enzymes. Enzymes modified on regulatory sites and proteins of unknown function were also identified. These findings indicate that a wide range of functional residues are targeted by sulfonate ester probes and highlight the value of TOP-based chemical proteomics for the characterization of proteins and the residues that regulate their activity.     

Beta-lactams as selective chemical probes for the in vivo labeling of bacterial enzymes involved in cell wall biosynthesis, antibiotic resistance, and virulence

With the development of antibiotic-resistant bacterial strains, infectious diseases have become again a life-threatening problem. One of the reasons for this dilemma is the limited number and breadth of current therapeutic targets for which several resistance strategies have evolved over time. To expand the number of addressable enzyme targets and to understand their function, activity, and regulation, we utilized a chemical proteomic strategy, called activity-based protein profiling (ABPP) pioneered by Cravatt, for the identification of beta-lactam-binding enzymes under in vivo conditions. In this two-tiered strategy, we first prepared a selection of conventional antibiotics for labeling diverse penicillin binding proteins (PBPs) and second introduced a new synthetic generation of beta-lactam probes, which labeled and inhibited a selection of additional PBP unrelated bacterial targets. Among these, the virulence-associated enzyme ClpP and a resistance-associated beta-lactamase were labeled and inhibited by selected probes, indicating that the specificity of beta-lactams can be adjusted to versatile enzyme families with important cellular functions.     

Recent advances in activity-based probes (ABPs) and affinity-based probes (AfBPs) for profiling of enzymes

Activity-based protein profiling (ABPP) is a technique that uses highly selective active-site targeted chemical probes to label and monitor the state of proteins. ABPP integrates the strengths of both chemical and biological disciplines. By utilizing chemically synthesized or modified bioactive molecules, ABPP is able to reveal complex physiological and pathological enzyme–substrate interactions at molecular and cellular levels. It is also able to provide critical information of the catalytic activity changes of enzymes, annotate new functions of enzymes, discover new substrates of enzymes, and allow real-time monitoring of the cellular location of enzymes. Based on the mechanism of probe-enzyme interaction, two types of probes that have been used in ABPP are activity-based probes (ABPs) and affinity-based probes (AfBPs). This review highlights the recent advances in the use of ABPs and AfBPs, and summarizes their design strategies (based on inhibitors and substrates) and detection approaches.

This review highlights the recent advances in the use of activity-based probes (ABPs) and affinity-based probes (AfBPs), and summarizes their design strategies (based on inhibitors and substrates) and detection approaches.     

Competitive activity-based protein profiling identifies aza-β-lactams as a versatile chemotype for serine hydrolase inhibition

Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Most serine hydrolases lack selective inhibitors, which are valuable probes for assigning functions to these enzymes. We recently discovered a set of aza-β-lactams (ABLs) that act as potent and selective inhibitors of the mammalian serine hydrolase protein-phosphatase methylesterase-1 (PME-1). The ABLs inactivate PME-1 by covalent acylation of the enzyme's serine nucleophile, suggesting that they could offer a general scaffold for serine hydrolase inhibitor discovery. Here, we have tested this hypothesis by screening ABLs more broadly against cell and tissue proteomes by competitive activity-based protein profiling (ABPP), leading to the discovery of lead inhibitors for several serine hydrolases, including the uncharacterized enzyme α,β-hydrolase domain-containing 10 (ABHD10). ABPP-guided medicinal chemistry yielded a compound ABL303 that potently (IC(50) ≈ 30 nM) and selectively inactivated ABHD10 in vitro and in living cells. A comparison of optimized inhibitors for PME-1 and ABHD10 indicates that modest structural changes that alter steric bulk can tailor the ABL to selectively react with distinct, distantly related serine hydrolases. Our findings, taken together, designate the ABL as a versatile reactive group for creating first-in-class serine hydrolase inhibitors.     

Synthesis and use of mechanism-based protein-profiling probes for retaining beta-D-glucosaminidases facilitate identification of Pseudomonas aeruginosa NagZ

The NagZ class of retaining exo-glucosaminidases play a critical role in peptidoglycan recycling in Gram-negative bacteria and the induction of resistance to beta-lactams. Here we describe the concise synthesis of 2-azidoacetyl-2-deoxy-5-fluoro-beta-d-glucopyranosyl fluoride as an activity-based proteomics probe for profiling these exo-glycosidases. This active-site directed reagent covalently inactivates this class of retaining N-acetylglucosaminidases with exquisite selectivity by stabilizing the glycosyl-enzyme intermediate. Inactivated Vibrio cholerae NagZ can be elaborated with biotin or a FLAG-peptide epitope using the Staudinger ligation or the Sharpless-Meldal click reaction and detected at nanogram levels. This ABPP enabled the profiling of the Pseudomonas aeruginosa proteome and identification at endogenous levels of a tagged protein with properties consistent with those of PA3005. Cloning of the gene encoding this hypothetical protein and biochemical characterization enabled unambiguous assignment of this hypothetical protein as a NagZ. The identification and cloning of this NagZ may facilitate the development of strategies to circumvent resistance to beta-lactams in this human pathogen. As well, this general strategy, involving such 5-fluoro inactivators, may prove to be of general use for profiling proteomes and identifying glycoside hydrolases of medical importance or having desirable properties for biotechnology.     

Biochemical reactions in metabolite-protein interaction

An update on the recently developed chemical proteomics called activity-based protein profiling (ABPP) has been reviewed. ABPP is able to identify proteins interacted either covalently or non-covalently with metabolites significantly, which will facilitate the characterization of specific metabolite regulating proteins in human disease progression.     

Protein-reactive natural products

Researchers in the post-genome era are confronted with the daunting task of assigning structure and function to tens of thousands of encoded proteins. To realize this goal, new technologies are emerging for the analysis of protein function on a global scale, such as activity-based protein profiling (ABPP), which aims to develop active site-directed chemical probes for enzyme analysis in whole proteomes. For the pursuit of such chemical proteomic technologies, it is helpful to derive inspiration from protein-reactive natural products. Natural products use a remarkably diverse set of mechanisms to covalently modify enzymes from distinct mechanistic classes, thus providing a wellspring of chemical concepts that can be exploited for the design of active-site-directed proteomic probes. Herein, we highlight several examples of protein-reactive natural products and illustrate how their mechanisms of action have influenced and continue to shape the progression of chemical proteomic technologies like ABPP.     

Chemical Proteomic Discovery of Isotype-Selective Covalent Inhibitors of the RNA Methyltransferase NSUN2

5-Methylcytosine (m⁵C) is an RNA modification prevalent on tRNAs, where it can protect tRNAs from endonucleolytic cleavage to maintain protein synthesis. The NSUN family (NSUN1-7 in humans) of RNA methyltransferases are capable of installing the methyl group onto the C⁵ position of cytosines in RNA. NSUNs are implicated in a wide range of (patho)physiological processes, but selective and cell-active inhibitors of these enzymes are lacking. Here, we use cysteine-directed activity-based protein profiling (ABPP) to discover azetidine acrylamides that act as stereoselective covalent inhibitors of human NSUN2. Despite targeting a conserved catalytic cysteine in the NSUN family, the NSUN2 inhibitors show negligible cross-reactivity with other human NSUNs and exhibit good proteome-wide selectivity. We verify that the azetidine acrylamides inhibit the catalytic activity of recombinant NSUN2, but not NSUN6, and demonstrate that these compounds stereoselectively disrupt NSUN2-tRNA interactions in cancer cells, leading to a global reduction in tRNA m⁵C content. Our findings thus highlight the potential to create isotype-selective and cell-active inhibitors of NSUN2 with covalent chemistry targeting a conserved catalytic cysteine.     

Microarray platform for profiling enzyme activities in complex proteomes

Activity-based protein profiling (ABPP) is a chemical method that utilizes active-site-directed probes to determine the functional state of enzymes in complex proteomes. Probe-labeled enzymes are typically detected by in-gel fluorescence scanning, a robust technique that nonetheless exhibits some key deficiencies, including limited sensitivity and resolution, as well as ambiguity regarding the molecular identity of the enzymes under investigation. Here, we report a microarray platform for ABPP that addresses these limitations. In this platform, proteomes are treated with ABPP probes in solution, after which labeled enzymes are captured and visualized on glass slides displaying an array of anti-enzyme antibodies. We show that ABPP microarrays exhibit superior sensitivity and resolution compared to gel-based methods, permitting the parallel analysis of several enzyme activities in proteomes, including cancer-associated proteases such as urokinase, matrix metalloproteinase-9, and prostate-specific antigen.     

Chemoproteomics identifies Ykt6 as the direct target of schisandrin A for neuroprotection

Schisandrin A is a natural dibenzocyclooctene lignan with potent neuroprotective activity. However, the specific mechanisms or direct target proteins have not been clarified up to now. In this study, we designed and synthesized the probes of schisandrin A with photoreactive diazirine and clickable alkyne to identify its direct target in SH-SY5Y cells by employing activity-based protein profiling (ABPP) technique. Ykt6 was prominent among the 13 proteins obtained with high confidence and we confirmed Ykt6 as the direct target of schisandrin A by CETSA, IF, SPR and knockdown assay. Functionally, schisandrin A protected the cells against the injury induced by glutamate by regulating autophagy via Ykt6. This discovery may provide a novel therapeutic option for various neuronal cell damage-mediated diseases.     

A Chemical Proteomic Analysis of Illudin-Interacting Proteins

The illudin natural product family are fungal secondary metabolites with a characteristic spirocyclopropyl-substituted fused 6,5-bicyclic ring system. They have been extensively studied for their cytotoxicity in various tumor cell types, and semisynthetic derivatives with improved therapeutic characteristics have progressed to clinical trials. Although it is believed that this potent alkylating compound class acts mainly through DNA modification, little is known about its binding to protein sites in a cellular context. To reveal putative protein targets of the illudin family in live cancer cells, we employed a semisynthetic strategy to access a series of illudin-based probes for activity-based protein profiling (ABPP). While the probes largely retained potent cytotoxicity, proteomic profiling studies unraveled multiple protein hits, suggesting that illudins exert their mode of action not from addressing a specific protein target but rather from DNA modification and unselective protein binding.     

活性导向的化学探针在氨基酸反应活性表征中的应用进展

原创药物的研制得益于蛋白质新靶标的发现,而新靶标的发现依赖于高可信度、高通量的药物-蛋白质相互作用分析方法。蛋白质作为生命功能的执行者,其表达量、空间定位与结构差异直接影响药效的发挥。目前,超过85%的蛋白质尚被认为是无法成药的,主要原因是缺少药物分子靶向的空腔以及相应的反应活性位点。因此,基于蛋白质组学层次实现对氨基酸反应活性位点的表征成为原创共价靶向药物设计的关键,也是克服难以成药靶标蛋白问题的关键。近年来,质谱技术的飞速发展极大地推动了基于蛋白质组学技术的药物-靶蛋白相互作用研究。其中基于活性的蛋白质组分析(ABPP)策略是利用活性位点导向的化学探针分子在复杂样品中实现功能状态酶和药物靶标等蛋白质的检测。基于化学探针的开发和质谱定量技术的发展,ABPP技术在氨基酸反应活性表征研究中展现出重要的应用潜力,将助力于药物新靶标的发现和药物先导化合物的开发。ABPP策略主要基于蛋白质的活性特征进行富集,活性探针作为ABPP策略的核心,近年来取得了飞速进展。该文回顾了ABPP策略的发展历程,重点介绍基于广谱活性探针的ABPP技术在多种氨基酸反应活性筛选领域的研究进展,并对其在药物靶点发现中...     

An enzyme that regulates ether lipid signaling pathways in cancer annotated by multidimensional profiling

Hundreds, if not thousands, of uncharacterized enzymes currently populate the human proteome. Assembly of these proteins into the metabolic and signaling pathways that govern cell physiology and pathology constitutes a grand experimental challenge. Here, we address this problem by using a multidimensional profiling strategy that combines activity-based proteomics and metabolomics. This approach determined that KIAA1363, an uncharacterized enzyme highly elevated in aggressive cancer cells, serves as a central node in an ether lipid signaling network that bridges platelet-activating factor and lysophosphatidic acid. Biochemical studies confirmed that KIAA1363 regulates this pathway by hydrolyzing the metabolic intermediate 2-acetyl monoalkylglycerol. Inactivation of KIAA1363 disrupted ether lipid metabolism in cancer cells and impaired cell migration and tumor growth in vivo. The integrated molecular profiling method described herein should facilitate the functional annotation of metabolic enzymes in any living system.     

Mapping enzyme active sites in complex proteomes

Genome sequencing projects have uncovered many novel enzymes and enzyme classes for which knowledge of active site structure and mechanism is limited. To facilitate mechanistic investigations of the numerous enzymes encoded by prokaryotic and eukaryotic genomes, new methods are needed to analyze enzyme function in samples of high biocomplexity. Here, we describe a general strategy for profiling enzyme active sites in whole proteomes that utilizes activity-based chemical probes coupled with a gel-free analysis platform. We apply this gel-free strategy to identify the sites of labeling on enzymes targeted by sulfonate ester probes. For each enzyme examined, probe labeling was found to occur on a conserved active site residue, including catalytic nucleophiles (e.g., C32 in glutathione S-transferase omega) and bases/acids (e.g., E269 in aldehyde dehydrogenase-1; D204 in enoyl CoA hydratase-1), as well as residues of unknown function (e.g., D127 in 3 beta-hydroxysteroid dehydrogenase/isomerase-1). These results reveal that sulfonate ester probes are remarkably versatile activity-based profiling reagents capable of labeling a diversity of catalytic residues in a range of mechanistically distinct enzymes. More generally, the gel-free strategy described herein, by consolidating into a single step the identification of both protein targets of activity-based probes and the specific residues labeled by these reagents, provides a novel platform in which the proteomic comparison of enzymes can be accomplished in unison with a mechanistic analysis of their active sites.