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.     

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.     

Qualitative analysis of the fluorophosphonate-based chemical probes using the serine hydrolases from mouse liver and poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis

The serine hydrolase family consists of more than 200 members and is one of the largest enzyme families in the human genome. Although up to 50?% of this family remains unannotated, there are increasing evidences that activities of certain serine hydrolases are associated with diseases like cancer neoplasia, invasiveness, etc. By now, several activity-based chemical probes have been developed and are applied to profile the global activity of serine hydrolases in diverse proteomes. In this study, two fluorophosphonate (FP)-based chemical probes were synthesized. Further examination of their abilities to label and pull down serine hydrolases was conducted. In addition, the poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis was demonstrated as an appropriate standard serine hydrolase, which can be applied to measure the labeling ability and pull-down efficiency of FP-based probes. Furthermore, mass spectrometry (MS) was used to identify the serine residue that covalently bonded to the active probes. Finally, these FP-based probes were shown capable of establishing the serine hydrolase profiles in diverse mouse tissues; the serine hydrolases pulled down from mouse liver organ were further identified by MS. In summary, our study provides an adequate method to evaluate the reactivity of FP-based probes targeting serine hydrolases.

The putative endocannabinoid transport blocker LY2183240 is a potent inhibitor of FAAH and several other brain serine hydrolases

How lipid transmitters move within and between cells to communicate signals remains an important and largely unanswered question. Integral membrane transporters, soluble lipid-binding proteins, and metabolic enzymes have all been proposed to collaboratively regulate lipid signaling dynamics in vivo. Assignment of the relative contributions made by each of these classes of proteins requires selective pharmacological agents to perturb their individual functions. Recently, LY2183240, a heterocyclic urea inhibitor of the putative endocannabinoid (EC) transporter, was shown to disrupt the cellular uptake of the lipid EC anandamide and promote analgesia in vivo. Here, we show that LY2183240 is a potent, covalent inhibitor of the EC-degrading enzyme fatty acid amide hydrolase (FAAH). LY2183240 inactivates FAAH by carbamylation of the enzyme's serine nucleophile. More global screens using activity-based proteomic probes identified several additional serine hydrolases that are also inhibited by LY2183240. These results indicate that the blockade of anandamide uptake observed with LY2183240 may be due primarily to the inactivation of FAAH, providing further evidence that this enzyme serves as a metabolic driving force that promotes the diffusion of anandamide into cells. More generally, the proteome-wide target promiscuity of LY2183240 designates the heterocyclic urea as a chemotype with potentially excessive protein reactivity for drug design.     

Engineering Ecotin for Identifying Proteins with a Trypsin Fold

Ecotin is a bidentate, fold-specific inhibitor of mammalian serine-proteases produced by Escherichia coli. This molecule may be engineered to increase and/or change its affinity and specificity providing significant biotechnological potential. Since ecotin binds tightly to serine proteases of the trypsin fold, it may help to identify the role of these enzymes in different biological processes. In this work, we tested ecotin variants as an affinity purification reagent for identifying enzymes in samples of tumor progression and mammary gland involution. Initially, we used a commercial source of urokinase-type plasminogen activator (u-PA) that remained fully active after elution from an affinity column of the ecotin variant (M84R, M85R). We then successfully identified u-PA from more complex mixtures including lysates from a prostate cancer cell line and involuting mouse mammary glands. Interestingly, a membrane-type serine protease 1 was isolated from the Triton X-100-solubilized PC-3 cell lysates, and surprisingly, haptoglobin, a serine-protease homolog protein, was also identified in mammary gland lysates and in blood. Haptoglobin does not prevent ecotin inhibition of u-PA, but it may act as a carrier within blood when ecotin is used in vivo. Finally, this affinity purification matrix was also able to identify a thrombin-like enzyme from snake venom using an ecotin variant directed against thrombin. Overall, the ecotin variants acted as robust tools for the isolation and characterization of proteins with a trypsin fold. Thus, they may assist in the understanding of the role of these serine proteases and homologous proteins in different biological processes.     

Affinity purification and enzymatic cleavage of inter-alpha inhibitor proteins using antibody and elastase immobilized on CIM monolithic disks

Epoxy-activated monolithic CIM disks seem to be excellent supports for immobilization of protein ligands. The potential use of enzymes, immobilized on monolithic disks for rapid preparative cleavage proteins in solution was investigated. Digestion of complex plasma proteins was demonstrated by using inter-alpha inhibitors with elastase, immobilized on epoxy-activated CIM disks. Recently, a monoclonal antibody against human inter-alpha inhibitor proteins (MAb 69.31) was developed. MAb 69.31 blocks the inhibitory activity of inter-alpha inhibitor proteins to serine proteases. These results suggest that the epitope defined by this antibody is located within or proximal to the active site of the inhibitor molecule. This antibody, immobilized on monolithic disk, was used for very rapid isolation of inter-alpha proteins. The isolated complex protein was used for enzymatic digestion and isolation of cleavage products, especially from inter-alpha inhibitor light chain to elucidate precisely the target sequence for MAb 69.31 by N-terminal amino acid sequencing. Bovine pancreatic elastase immobilized on monolithic disk cleaves inter-alpha inhibitor protein complex into small fragments which are still reactive with MAb 69.31. One of these proteolytic fragments was isolated and partially sequenced. It could be shown that this sequence is located at the beginning of two proteinase inhibitor domains of the inter-alpha inhibitor light chain (bikunin). Elastase immobilized on monolithic disk offers a simple and rapid method for preparative isolation of protease cleavage fragments. The immobilized enzyme is stable and still active after repeated runs. A partial or complete digestion can be achieved by varying the flow rate.     

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...     

Development of Ubiquitin‐Based Probe for Metalloprotease Deubiquitinases

Deubiquitinases (DUBs) are a family of enzymes that regulate the ubiquitin signaling cascade by removing ubiquitin from specific proteins in response to distinct signals. DUBs that belong to the metalloprotease family (metalloDUBs) contain Zn²⁺ in their active sites and are an integral part of distinct cellular protein complexes. Little is known about these enzymes because of the lack of specific probes. Described here is a Ub‐based probe that contains a ubiquitin moiety modified at its C‐terminus with a Zn²⁺ chelating group based on 8‐mercaptoquinoline, and a modification at the N‐terminus with either a fluorescent tag or a pull‐down tag. The probe is validated using Rpn11, a metalloDUB found in the 26S proteasome complex. This probe binds to metalloDUBs and efficiently pulled down overexpressed metalloDUBs from a HeLa cell lysate. Such probes may be used to study the mechanism of metalloDUBs in detail and allow better understanding of their biochemical processes.     

Discovering disease-associated enzymes by proteome reactivity profiling

Proteomics aims to identify new markers and targets for the diagnosis and treatment of human disease. To realize this goal, methods and reagents are needed to profile proteins based on their functional properties, rather than mere abundance. Here, we describe a general strategy for synthesizing and evaluating structurally diverse libraries of activity-based proteomic probes. Quantitative screening of probe-proteome reactions coupled with bioinformatic analysis enabled the selection of a suite of probes that exhibit complementary protein reactivity profiles. This optimal probe set was applied to discover several enzyme activities differentially expressed in lean and obese (ob/ob) mice. Interestingly, one of these enzymes, hydroxypyruvate reductase, which was 6-fold upregulated in ob/ob livers, participates in the conversion of serine to glucose, suggesting that this unusual metabolic pathway may contribute to gluconeogenesis selectively in states of obesity.     

Fluorescent Triazole Urea Activity‐Based Probes for the Single‐Cell Phenotypic Characterization of Staphylococcus aureus

Phenotypically distinct cellular (sub)populations are clinically relevant for the virulence and antibiotic resistance of a bacterial pathogen, but functionally different cells are usually indistinguishable from each other. Herein, we introduce fluorescent activity‐based probes as chemical tools for the single‐cell phenotypic characterization of enzyme activity levels in Staphylococcus aureus. We screened a 1,2,3‐triazole urea library to identify selective inhibitors of fluorophosphonate‐binding serine hydrolases and lipases in S. aureus and synthesized target‐selective activity‐based probes. Molecular imaging and activity‐based protein profiling studies with these probes revealed a dynamic network within this enzyme family involving compensatory regulation of specific family members and exposed single‐cell phenotypic heterogeneity. We propose the labeling of enzymatic activities by chemical probes as a generalizable method for the phenotyping of bacterial cells at the population and single‐cell level.     

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.     

Chemical force microscopy: applications in surface characterisation of natural hydroxyapatite

Detailed mapping of surface chemistry with nanometer resolution has application throughout the physical and life sciences. The atomic force microscope (AFM) has provided a tool that, when using functionalised probes, is capable of providing chemical information with this level of spatial resolution. Here, we describe the technique of chemical force microscopy (CFM) and demonstrate the sensitivity of the technique using chemical force titrations against pH. We describe in detail the specific application of mapping the surface charge on natural hydroxyapatite from skeletal tissue and show that this new information leads to a better understanding of the binding of matrix proteins to the mineral surface.     

Release of Enzymatically Active Deubiquitinating Enzymes upon Reversible Capture by Disulfide Ubiquitin Reagents

Deubiquitinating enzymes (DUBs) catalyze the cleavage of ubiquitin from target proteins. Ubiquitin is post‐translationally attached to proteins and serves as an important regulatory signal for key cellular processes. In this study, novel activity‐based probes to study DUBs were synthesized that comprise a ubiquitin moiety and a novel disulfide warhead at the C‐terminus. These reagents can bind DUBs covalently by forming a disulfide bridge between the active‐site cysteine residue and the ubiquitin‐based probe. As disulfide bridges can be broken by the addition of a reducing agent, these novel ubiquitin reagents can be used to capture and subsequently release catalytically active DUBs, whereas existing capturing agents bind irreversibly. These novel reagents allow for the study of these enzymes in their active state under various conditions.     

Identification of Histone deacetylase (HDAC)‐Associated Proteins with DNA‐Programmed Affinity Labeling

Histone deacetylase (HDAC) is a major class of deacetylation enzymes. Many HDACs exist in large protein complexes in cells and their functions strongly depend on the complex composition. The identification of HDAC‐associated proteins is highly important in understanding their molecular mechanisms. Although affinity probes have been developed to study HDACs, they were mostly targeting the direct binder HDAC, while other proteins in the complex remain underexplored. We report a DNA‐based affinity labeling method capable of presenting different probe configurations without the need for preparing multiple probes. Using one binding probe, 9 probe configurations were created to profile HDAC complexes. Notably, this method identified indirect HDAC binders that may be inaccessible to traditional affinity probes, and it also revealed new biological implications for HDAC‐associated proteins. This study provided a simple and broadly applicable method for characterizing protein‐protein interactions.     

GlcNAcstatin: a picomolar, selective O-GlcNAcase inhibitor that modulates intracellular O-glcNAcylation levels

Many phosphorylation signal transduction pathways in the eukaryotic cell are modulated by posttranslational modification of specific serines/threonines with N-acetylglucosamine (O-GlcNAc). Levels of O-GlcNAc on key proteins regulate biological processes as diverse as the cell cycle, insulin signaling, and protein degradation. The two enzymes involved in this dynamic and abundant modification are the O-GlcNAc transferase and O-GlcNAcase. Structural data have recently revealed that the O-GlcNAcase possesses an active site with significant structural similarity to that of the human lysosomal hexosaminidases HexA/HexB. PUGNAc, an O-GlcNAcase inhibitor widely used to raise levels of O-GlcNAc in human cell lines, also inhibits these hexosaminidases. Here, we have exploited recent structural information of an O-GlcNAcase-PUGNAc complex to design and synthesize a glucoimidazole-based inhibitor, GlcNAcstatin, which is a 5 pM competitive inhibitor of enzymes of the O-GlcNAcase family, shows 100000-fold selectivity over HexA/B, and binds to the O-GlcNAcase active site by mimicking the transition state as revealed by X-ray crystallography. This compound is able to raise O-GlcNAc levels in human HEK 293 and SH-SY5Y neuroblastoma cell lines and thus provides a novel, potent tool for the study of the role of O-GlcNAc in intracellular signal transduction pathways.     

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.     

Identification of Histone deacetylase (HDAC)-Associated Proteins with DNA-Programmed Affinity Labeling

Histone deacetylase (HDAC) is a major class of deacetylation enzymes. Many HDACs exist in large protein complexes in cells and their functions strongly depend on the complex composition. The identification of HDAC-associated proteins is highly important in understanding their molecular mechanisms. Although affinity probes have been developed to study HDACs, they were mostly targeting the direct binder HDAC, while other proteins in the complex remain underexplored. We report a DNA-based affinity labeling method capable of presenting different probe configurations without the need for preparing multiple probes. Using one binding probe, 9 probe configurations were created to profile HDAC complexes. Notably, this method identified indirect HDAC binders that may be inaccessible to traditional affinity probes, and it also revealed new biological implications for HDAC-associated proteins. This study provided a simple and broadly applicable method for characterizing protein-protein interactions.     

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.     

Profiling enzyme activities in vivo using click chemistry methods

Methods for profiling the activity of enzymes in vivo are needed to understand the role that these proteins and their endogenous regulators play in physiological and pathological processes. Recently, we introduced a tag-free strategy for activity-based protein profiling (ABPP) that utilizes the copper(I)-catalyzed azide-alkyne cycloaddition reaction ("click chemistry") to analyze the functional state of enzymes in living cells and organisms. Here, we report a detailed characterization of the reaction parameters that affect click chemistry-based ABPP and identify conditions that maximize the speed, sensitivity, and bioorthogonality of this approach. Using these optimized conditions, we compare the enzyme activity profiles of living and homogenized breast cancer cells, resulting in the identification of several enzymes that are labeled by activity-based probes in situ but not in vitro.     

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.