ActivityBased Protein Profiling: Recent ... - Wiley Online Library

DOI: 10.1002/cbic.201402582

Reviews

Activity-Based Protein Profiling: Recent Advances in Probe Development and Applications Pengyu Yang[b] and Kai Liu*[a]

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Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Reviews mentary for gene expression analysis and an ideal utensil in decoding this overflow of genomic information. This approach makes use of synthetic small molecules that covalently modify a set of related proteins and subsequently facilitates identification of the target protein, enabling rapid biochemical analysis and inhibitor discovery. This tutorial review introduces recent advances in the field of ABPP and its applications.

The completion of the human genome sequencing project has provided a wealth of new information regarding the genomic blueprint of the cell. Although, to date, there are roughly 20 000 genes in the human genome, the functions of only a handful of proteins are clear. The major challenge lies in translating genomic information into an understanding of their cellular functions. The recently developed activity-based protein profiling (ABPP) is an unconventional approach that is comple-

Introduction

lar biochemical activity when combined with mass spectrometry, accelerating the functional annotation of proteins. With an ABPP probe derived from a bioactive small molecule, direct investigation of the interaction between small molecules and proteins in the native proteome provides valuable insights about the role that these target proteins play in disease states. In addition, such methods also offer a more accurate depiction of the compound’s mechanism of action in vivo than traditional biochemical assays with recombinant proteins, and thus, provide a better prediction of its suitability as a candidate drug. Owing to the great impact of ABPP technology on protein functional annotation and target validation in both basic biology and drug discovery, in this review, we briefly introduce the basics of this technology, the recent development of ABPP probes for different classes of proteins, innovative improvements and new concepts in ABPP probe design and development, and their biological applications.

The complete sequence of the human genome, in addition to the larger framework of other model organisms, has established a firm foundation for modern biological investigations to unveil the blueprint of life.[1] Rapid functional assignment and global characterization of those poorly annotated gene and proteins are the next major step for the human genome project.[2] Proteomics aims to accelerate such functional assignment by developing analytical methods for large numbers of proteins.[3] However, unlike static genomic sequences, proteins inside the cell are perpetually being created, modified, and discarded. As highly diverse entities inside the cells, proteins’ abundance, modification states, sub-cellular locations, and most importantly, their functions, change dynamically.[4] Therefore, comparative proteome profiling that compares the dynamic change in multiple proteomes will provide more insights for our comprehensive understanding of protein function.[5] This multidisciplinary field utilizes a collection of various technologies, namely, liquid chromatography-mass spectrometry (LC-MS) analysis of protein expression pattern and modification states,[6] protein microarray for proteome wide analysis of protein activities[7] and yeast two-hybrid assays for global mapping of protein–protein interactions.[8] These state-of-theart technologies have provided major breakthroughs in our understanding of protein expression patterns, modification states, protein–protein interactions, etc. Despite huge successes, these technologies provide limited insights into the functional states of proteins in their native proteome. To address these limitations, activity-based protein profiling (ABPP) technologies, which make use of synthetic small molecules that are directed to the active site of proteins in complex proteomes, have been developed. An ABPP probe detects a defined set of proteins within a proteome, depending on the protein’s enzymatic or binding activity under certain conditions. Therefore, it can be applied to discover proteins that possess such particu-

Activity- and Affinity-Based Probes for Proteomic Profiling ABPP, pioneered by Cravatt and Bogyo, has emerged as a powerful chemical proteomic method towards the analysis of functional states of proteins in complex proteomes.[9] ABPP probes, which include activity-based probes (ABPs) and affinity-based probes (AfBPs), usually consist of three fundamental building blocks with distinct functions (Scheme 1): 1) an active warhead (WH) that contains reactive groups or affinity binding groups that target the conserved mechanistic or structural feature of

[a] Dr. K. Liu Gladstone Institute of Cardiovascular Disease The J. David Gladstone Institute 1650 Owens St, San Francisco, CA 94158 (USA) E-mail: [email protected]

Scheme 1. Activity-based protein profiling (ABPP). Proteomes are treated with activity-based probes (ABPs) or photo-reactive affinity-based probes (AfBPs) that label proteins depending on their activities. Probe-labeled proteomes can be analyzed through several platforms, including fluorescent gel or LC-MS/MS.

[b] Dr. P. Yang Department of Chemistry and the Skaggs Institute for Chemical Biology The Scripps Research Institute 10550 North Torrey Pines Road, La Jolla, California 92037 (USA)

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Reviews a set of proteins; 2) a reporter tag for identification and purification of modified proteins; and 3) a linker region that can modulate the reactivity and specificity of the reactive group and binding group, while providing enough space between the reporter and the reactive or binding group. For ABPs, the reactive groups of many successful examples have been developed based on covalent, mechanism-based inhibitors (i.e., suicide inhibitors) of various enzyme families (Scheme 2). The selective targeting of these inhibitors depends largely on the conserved mechanistic differences of each protein class. Therefore, many of the best examples of ABPs have been designed to target hydrolases, such as fluorophosphonate probes (1) for serine hydrolases[10] and epoxide and AOMK probes for cysteine proteases (2, 3),[11] which have a reactive nucleophilic residue in the active site and possess a distinct catalytic mechanism.[12] Other such directed ABPs includes probes targeting cathepsins, legumains,[13] caspases,[14] protein arginine methyltransferases (4),[15] proteasomal proteases (5),[16] kinases (6),[17] tyrosine phosphatase,[18] serine/threonine phosphatase,[19] and glycosidases.[20] It may be expected that the design of ABPs for enzyme classes with known covalent inhibitors is, at least in concept, straightforward. However, unlike these classes of en-

zymes, a majority of proteins do not yet have a known covalent inhibitor attacking a conserved residue. Therefore, AfBPs adopt several strategies by utilizing: 1) a photo-reactive group (e.g., benzophenone (BP), diazirine, aryl azide) conjugated to the affinity binding groups that target the active site, resulting in modification of potentially non-catalytic residues upon irradiation with UV light (e.g., glutarate oxygenase (7)[21] aspartic proteases (8),[22] metalloproteases (9);[23] 2) a masked electrophile (also referred as to a latent electrophilic probe) that becomes activated upon cleavage by the enzyme, which means that reagents are recognized by the catalytic site only become reactive probes after enzymatic turnover (e.g., tyrosine phosphatase, glycosidase, and protease quinone methide probes (10));[24] and 3) a novel ligand-directed tosyl (11; LDT) chemistry that allows site-specific introduction of synthetic probes to the surface of proteins, accompanying release of the affinity ligands.[25]

Bioorthogonal Chemical Reactions Facilitating Tandem Labeling in ABPP A drawback encountered in first generation ABPP, however, is the finding that the presence of the reporter entity generally diminishes or even precludes cellular uptake of the probes, necessitating cell lysis prior to the labeling experiment. However, certain interactions between small molecules and proteins might require conditions in the living systems that are not preserved in cell lysates.[26] To overcome this shortcoming, several small latent ligation groups (mainly alkyne or azide groups) for a reporter tag have been introduced (Scheme 3), with the idea that such constructs would likely to be cell-permeable. After the sample is labeled in living cells, attachment of the reporter tag may then be performed ex vivo through highly specific bioorthogonal ligation chemistry that shows low reactivity towards other biomolecules. In this section, we discuss such tandem ABPP labeling, facilitated by bioorthogonal reactions. Most bioorthogonal reactions employed in ABPP include the Staudinger ligation between azides and methyl ester-modified triphenylphosphines,[27] click chemistry (CC) between terminal alkynes and azides, or [3+ +2] cycloaddition with strained cyclooctynes.[28] However, the Staudinger ligation suffers from slow reaction kinetics and background phosphine oxidation. The copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) is a rapid reaction, but the CuI catalyst is toxic to live cells, limiting its applications.[29] Strain-promoted cycloaddition with fluorinated cyclooctyne reagents is an alternative bioorthogonal reaction for the azide; however, the inherent difficulty in synthesizing cyclooctynes and the one order of magnitude faster reactivity than the Staudinger ligation are still sub-optimal. Compared to CuAAC, strained alkynes are reactive toward the thiol group of reduced cysteine on proteins under certain conditions. This reactivity causes non-specific labeling and considerable background.[30] These limitations have therefore promoted the recent expansion of the arsenal of bioorthogonal chemical reactions used to label proteins in living systems, such as inverse-electron demand Diels–Alder reactions between strained alkenes and tetrazines.[31] Due to its faster reaction rates under

Kai Liu received his BSc from Huazhong University of Science and Technology in 2005 and his PhD from the National University of Singapore in 2011, under the supervision of Prof. Shao Q. Yao. Since 2011, he has been a postdoctoral fellow at the J. David Gladstone Institute at the University of California, San Francisco, under the supervision of Prof. Sheng Ding, focused on discovering and characterizing novel small molecules that control various cell fates and functions, as well as stem cell safety and sequencing-based gene expression analysis to discover small molecules that improve cell function in various disease models. Peng-yu Yang received his MSc from Chinese Academy of Sciences in 2004 and his PhD from the National University of Singapore in 2011, under the supervision of Prof. Shao Q. Yao. Since 2011, he has been a postdoctoral fellow at the Scripps Research Institute, under the supervision of Prof. Peter G. Schultz. In his postdoctoral work, he has focused on developing techniques for half-life extension of peptide and protein drugs, genetically encoding unnatural amino acids to probe protein structure, and high-throughput screening for small-molecule activators of the Hippo-YAP pathway, as well as development of novel homogeneous antibody–drug conjugates.

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Scheme 2. Structures of activity-based probes (upper panel) containing mechanism-based reactive groups (shaded rectangles) and affinity-based probes (lower panel) containing affinity binding groups (shaded rectangles) and photo-reactive groups, quinine methide, or tosyl groups (shaded ovals).

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Reviews of endogenous RSK1 and RSK2 in mammalian cells.[35] Later, Yao and co-workers reported clickable probe 15, derived from a reversible inhibitor for Abelson tyrosine kinases (Abl).[36] Abl plays an essential role in the development of chronic myelogenous leukemia. A clickable probe was synthesized based on the FDAapproved drug Imatinib, containing photo-reactive group BP and an alkyne handle for CC tagging. Compared to a probe containing a bulky Rhodamine tag, this clickable probe achieved much better selectivity in proteome labeling. However, identifying the cellular targets of endogenous kinases in living cells remains a challenge. In a proof of concept study, a cellpermeable design was achieved by converting Staurosporine, a highly potent and broad-spectrum kinase inhibitor, into AfBP for in situ labeling.[37] In 2012, the first cell-permeable probe for Src kinase family was developed by Taunton’s group.[38] Later, a cell-permeable design of a Dasatinib probe was synScheme 3. Tandem ABPP labeling, assisted by bioorthogonal reactions. a) Staudinger ligation; b) “Click” chemistry thesized to enable the identifi+2] cycloaddition with “strained” cyclooctynes; c) Inverse-electron demand via terminal alkynes and azides or [3+ cation of Dasatinib targets in Diels–Alder reactions between strained alkenes and tetrazines. situ. This in situ capturing of interactions between Dasatinib and its cellular targets was shown to be more effective than a limited concentration of reagents that cross cell membrane, the previous approach performed in cell lysates.[39] In order to this reaction is currently a more optimal reaction inside living cells. The activatable “turn-on” probe developed by Dr. Weisminimize interference upon binding to the target proteins, the sleder’s group best represents the usefulness of live-cell fluosame group developed Dasatinib and Staurosporine probes rescent labeling between trans-cyclooctenes and tetrazines.[32] with “minimalist” linkers, in which both the photo-reactive and However, the steric impact of trans-cyclooctenes limits the apreporter groups are made as small as possible.[40] This series of ABPP probes, derived from reversible kinase inhibitors, showplication of tetrazine reactions. A recent development emcased how the efficiency and specificity of probes could be imployed the reaction between tetrazines and cyclopropenes, proved by minimizing the photo-reactive linker on non-mechawhich offers more optimal size, reactivity, and stability.[33] With nism-based probes. the successful development of the above-mentioned bioorAaron and Cravatt exploited this “label-free” design to study thogonal reactions, “label-free” probes carrying small surrogate cytochrome P450s, a large and diverse group of monooxygegroups have been developed for various classes of enzymes nases that catalyze the oxidization of a large number of me(Scheme 4). Using small surrogate groups on chemical probes tabolites and signaling intermediates but lack functional charusually alters and improves the specificity compared to probes acterization.[41] The Cravatt group synthesized a suite of probes directly modified by fluorescent groups, which is already apparent for serine protease probe 12 and cysteine protease (16) containing chemical scaffolds that were validated as probe 13.[34] mechanism-based inhibitors of the P450 family and an alkyne functionality for subsequent CC conjugation with reporter In 2007, Taunton and co-workers developed clickable probe tags. Screening with this set of probes led to the discovery of 14 for kinase RSK, based on the selective irreversible inhibitor optimal probes having a broad coverage of this enzyme family. fmk. Click reaction tagging of an fmk-derived probe reveals Profiling of small-molecule inhibitor effects with the optimized that this probe achieves selective and saturable modification ChemBioChem 2015, 16, 712 – 724

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Scheme 4. Structures of clickable activity-based probes (left) containing mechanism based reactive groups (shaded, rounded rectangles) and clickable affinitybased probes (right) containing affinity binding groups (shaded, rounded rectangles) and photo-reactive groups (shaded ovals) for tandem labeling.

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Reviews sequent reporter tagging by bioorthogonal CC reactions.[55] With this approach, the probe MJE3 was identified, with antiproliferation effects against breast cancer. The in situ target of MJE3 was identified as brain-type phosphoglycerate mutase 1 (PGAM1), a key enzyme in glycolysis that converts 3-phosphoglycerate to 2-phosphoglycerate. A similar approach was also applied to study hepatitis C virus (HCV) replication by Pezacki and co-workers.[56] In this study, a nondirected phenyl sulfonate ester probe library with alkyne handles was used to selectively target various enzyme families involved in HCV replication.

P450 probe set surprisingly discovered inhibitors that bear heterotypic cooperativity with P450 1A2 and potentially other P450s, accentuating the versatility of label-free design for monitoring both the increase and decrease of enzyme activities. In addition to successes with kinases and cytochrome P450s, increasingly more enzymes classes are profiled by newly developed “label-free” ABPP probes. In 2010, Tully et al. reported the design and synthesis of phosphatidylcholine (PC) probes (17, 18) containing reactive fluorophosphonate elements at the SN1 and SN2 positions of a PC scaffold targeting the serine phospholipase subclass of serine hydrolases.[42] These probes have been shown to accurately report the phospholipase A1 (PLA1)-selective catalytic activity from the phospholipase DDHD1 and have further demonstrated that DDHD2, which displays similar reactivity with the two probes, might exhibit both PLA1 and PLA2 activity. Proteomic profiling with these probes revealed labeling of several additional proteins, suggesting that the discovery of new serine phospholipases might be facilitated by these specific probes. Another class of enzymes starting to be profiled by the “label-free” ABPP strategy is dimethylarginine dimethylaminohydrolase (DDAH).[43] DDAH-1 and DDAH-2, two isoforms with different tissue distributions, control the physiological concentration of asymmetric Nw,Nw-dimethyl-l-arginine (ADMA), an endogenously produced inhibitor of human nitric oxide synthase. The design of probe 19 was based on 2-chloroacetamidine (CAA), which selectively modifies the active site cysteine of DDAH. A simple butynyl chain was appended to CAA to minimize its impact on selectivity and bioavailability, at the same time providing a handle for variable functionalization through a click reaction after cell lysis. This probe has been shown to label the active fraction of DDAH-1 in intact mammalian cells specifically, whereas the labeling can be impeded by the presence of competitive, reversible, and irreversible inhibitors. Other “label-free”, surrogate group-bearing probes reported so far include probes for histone deacetylases (HDACs, 20, 21),[44] methionine aminopeptidase (22),[45] transglutaminase (23),[46] protein phosphatase methylesterase (24),[47] fatty acid amide hydrolase (FAAH) (25),[48] the 14-3-3 family of phosphorbinding protein (26),[49] fatty acid associate proteins (27, 28),[50] flavin-dependent oxidases (29),[51] and protein arginine deiminase (30).[52] Although the aforementioned directed “label-free” probes are widely employed for targeting proteins with known inhibitors, many enzyme classes lack such cognate inhibitors that could inspire probe development. Fortunately, “label-free” probe design has also enabled the ABPP profiling of proteins lacking such known cognate inhibitors by facilitating the development of a nondirected approach. A nondirected approach usually employs screening of probe libraries consisting of large numbers of small molecule against complex proteomes for activity-dependent protein reactivity; for example, a nondirected library of sulfonate esters[53] and a-chloroacetamides.[54] This was first displayed by the Cravatt group in 2005. A library of small molecules with a reactive spiroepoxide towards distinct sets of protein in the proteome were transformed to alkyne handle-containing probes amenable for subChemBioChem 2015, 16, 712 – 724

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High-Throughput-Amenable Chemistry Enabling Efficient Assembly and Evaluation of Focused Probe Libraries Despite the merits of the aforementioned development of probes for well-studied protein families, it is still a daunting task to design, synthesize, and search for high quality ABPP probes that selectively label each member of a large homologous protein family (e.g., protein kinases) or protein isoforms. In addition, for structurally and functionally poorly understood proteins, the development of ABPP probes is highly valuable for understanding their functional roles but is often met with enormous difficulties, especially in design and synthesis. These tasks thus necessitate a complementary high-throughput screening approach that utilizes a focused probe-candidate library, while at the same time, managing to make the synthesis and evaluation of a large number of structurally diverse probe candidates feasible and convenient. Evaluations of a library of candidate are usually less complicated and performed by screening these probe candidates against purified proteins or complex proteomes to identify “specific” protein-labeling events, which are defined as those that occurred with the native protein or proteome but not with heat-denatured protein or proteome. The underlying rationale is that heat-sensitive probe–protein reactions are more likely to occur in a defined, small-molecule binding site of well-folded proteins. These binding sites define the functional role of proteins, for example, the catalytic site of an enzyme or the ligand binding pocket of a receptor. In contrast, labeling reactions between probes and proteins occurring in a heatinsensitive manner are considered to be non-specific reactions. The fact that these reactions could occur with either native or heat-denatured proteins indicates that these reactions are not determined by the correct folding or structure of proteins. Such heat-sensitive assays are essential for the evaluation of probes based on structures without known structure–activity relationships (SARs) or well-studied selectivity for a given class of enzymes. In contrast, the synthesis of a library of candidate probes demands special synthetic considerations.[57] The primary consideration and challenge is to minimize synthetic difficulties by utilizing chemoselective reactions. The secondary consideration is to produce compounds that are sufficiently pure so that the quality of activity of the final product probe is not compromised.[58] Given these considerations, combinatorial strategies 718


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Scheme 5. Probe design and development strategies utilizing high-throughput-amenable chemistry (top) and successful examples of warhead structures (in box at bottom) bearing affinity binding groups (orange) and high-throughput-amenable connecting groups (blue).

using high-throughput-amenable chemistry, including CC,[59] amide-forming reactions, and solid-phase synthesis (SPS),[59a, 60] have been frequently employed to build large libraries of diverse molecules (Scheme 5). A library of WHs can be conveniently assembled by using appropriate diversity building blocks bearing carefully selected connecting groups, then evaluated by high-throughput-amenable technologies (e.g., small-molecule microarray), and finally converted to a focused library of ABPP candidate probes when they are conjugated with distinct functional molecules for distinct purposes, while minimizing the synthetic effort. Specifically, the WH that selectively targets the active site and bears an azide can be reacted with a trifunctional molecule bearing 1) an alkyne, 2) a photo-reactive group, and 3) a biotin or fluorophore tag to assemble an ABPP probe intended for target validation or gel profiling. Among these suitable chemistry approaches, CC CuI-catalyzed 1,3-dipolar cycloaddition reactions between azides and terminal alkynes are both efficient and specific and most widely adopted for ABPP probe synthesis. As early as 2004, the Cravatt group designed and synthesized a photo-reactive probe for metalloproteases by clicking a succinyl hydroxamate-based photo-reactive WH-containing alkyne with a Rhodamine azide. Later, Yao’s group employed CC to achieve a highly modular and facile synthesis of a library of twelve hydroxamate based probes for metalloprotease. This was achieved by utilizing CC reactions between alkyne-derivatized succinyl hydroxamate 31 and a trifunctional molecule bearing an azide, a photo-reactive BP, and Rhodamine. Protein profiling with these probes enabled generation of activity-based fingerprints of a variety of metalloproteases.[61] The same success was achieved for click synthesis of probes for dipeptidic g-secretase by using benzodiazepine and dibenzoazepine-derived WHs.[62] To develop probes that could selectively distinguish highly similar homologous enzymes in the protein tyrosine phosphatase (PTP) protein family, Yao and co-workers synthesized a novel unnatural amino acid (2-FMPT, 32).[18a] 2-FMPT can be conveniently incorporated into peptide sequences through SPS for the efficient synthesis of peptide-based probes. Using this strategy, eleven peptide probes designed against five different PTPs were synChemBioChem 2015, 16, 712 – 724

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thesized, and distinctive labeling profiles of individual PTPs were observed, corresponding to a large extent with their known substrate preferences. In addition, by using CC, the specificity information obtained from the discovery of specific ABPP probes readily facilitates inhibitor library design and minimizes the synthetic effort in developing an inhibitor library against the same protein targets.[21b] In an elegant study aiming to develop AfBPs as well as inhibitors against malaria parasite aspartic proteases, plasmepsins (PMs), this approach starts with the assembly of AfBPs by using hydroxyethyl-containing WHs (33) with varying R1 and R2 groups. These AfBPs were subsequently used to label PMs in the cell extracts of highly synchronized malaria parasites obtained at different stages of their life cycle. A 37 kDa protein band, which corresponds to the molecular weight of the four known PMs, was highly visible across various probes, with different labeling intensities indicating different levels of potency and specificity. Click assembly between the same azido WHs utilized in these specific AfBPs and 19 different aromatic alkynes readily generated a 152-member hydroxyethyl inhibitor library. Using AfBP competitive labeling intensity as readout, researchers performed an in situ inhibitor screen of this 152-member library against the four PMs in the parasite proteome and identified a cell-permeable compound with decent inhibition against all four PMs and even parasite growth in infected red blood cells. The throughput of ABPP probe discovery could be further boosted by combining high-throughput-amenable chemical reactions with small-molecule microarray (SMM) technology.[63] Yao and co-workers created a 198-member WH library through combinatorial synthesis of hydroxyethylene-based inhibitors of aspartic proteases by using an SPS method. All 198 members coupled with a biotin tag were directly immobilized onto avidin-functionalized slides to generate the corresponding small-molecule microarrays, which were subsequently screened with fluorescently labeled membrane fractions of mammalian cell lysates overexpressing g-secretase. Identified hits were conveniently converted to AfBPs by click assembly of their azido intermediates with a Rhodamine–BP alkyne for in-gel, ac719


Reviews tivity-based profiling and a biotin–BP alkyne for target identification experiments. Proteomic labeling and validation studies were carried out with these AfBPs to confirm the utility of SMM-assisted screening and CC-assisted conversion of smallmolecule inhibitors to specific AfBPs. This work was further extended with an expanded library of hydroxylethylamine-derivatized inhibitors that non-exclusively target aspartic proteases.[64] A similar approach was employed to assemble 86 new inhibitors diversified across the P2, P1, P1’, and P2’ positions, generating a total 284-member library of biotinylated compounds, arrayed onto avidin slides. Hits arising from mammalian lysate screening with SMM were conveniently converted to AfBPs. These AfBPs were then used individually or in a cocktail in pull-down/mass spectrometric analysis for putative protein target identification, leading to well-known aspartic proteases such as cathepsin D. Together, these studies exemplify the usefulness of high-throughput-amenable chemistry in the development of novel ABPP probes and innovative strategies of probe evaluation.

ases, the Rosenblum group reported for the first time that AX7503 potently inhibits Polo-like kinase 1 (PLK1). An extension of this study identified PLK3 as another target of wortmannin.[68] These findings collectively discovered and validated PLK1 and PLK3 as novel targets of wortmannin. Using a similar approach of appending a reporter tag to a natural product structure, Adam et al. synthesized ABP (35) by modifying cyclostreptin (¢)-FR182877. This work identified carboxylesterase-1 as the main target of the natural product in the proteome, derived from a variety of mouse tissues.[69] Similarly, a microcystin-derived fluorescent probe, AX7574 (36), was synthesized to target serine/threonine phosphatases in cell extracts.[19] Most recently, Sieber and other groups further advanced a “label-free” design by adding a short alkyne/azide group rather than a bulky fluorescent reporter to the natural product. As discussed before, such a design eliminated the adverse effect of the bulky fluorescent group on binding specificity and bioavailability of the natural product derivatives. In 2008, Sieber’s group reported the synthesis of a b-lactone-derived ABP library, comprising ten compounds with aliphatic or aromatic varying in length and branches.[70] Twenty specific targets of this b-lactone probe library were identified as different enzymes belonging to four major families including ligases, oxidoreductases, hydrolases, and transferases.[71] Later, the same group carried out an elegant study by extending this “labelfree” design to another group of natural products: b-lactams, including natural b-lactam probes based on the antibiotics penicillin (37), aztreonam (38), and cephalosporin (39).[72] The synthesized b-lactam probe library (40) was applied to label and identify b-lactam-binding enzymes under in vivo conditions. By tuning the core scaffold b-lactam ring, not only penicillin-binding proteins (PBPs), but also other PBP-unrelated enzymes, were identified to be b-lactam targets. In order to profile this lactam probe library in antibiotic-sensitive and -resistant Staphylococcus aureus strains (MRSA), these probes were further used to identify b-lactam targets that are associated with the resistance of MRSA.[73] Natural product analogue Tetrahydrolipstatin (THL) is an FDA-approved drug (generic name: orlistat) designed for the treatment of obesity by targeting lipase. Recently, THL was found to inhibit the thioesterase domain of fatty acid synthase (FAS) and cancer cell line proliferation, exhibiting its potential in cancer treatment. By converting THL to an ABP (41), Yao and co-workers reported an elegant chemical proteomic strategy aiming to identify THL targets other than FAS in cancer cell lines.[74] With this approach, eight new targets other than FAS were identified, including Hsp90 and three ribosomal proteins, which were further validated by alternative methods. An expanded list of THL analogues, incorporating various degrees of molecular complexities, was further synthesized.[75] Evaluation of a total of 22 of these compounds for their anti-proliferative activities in cancer cell lines revealed a noteworthy SAR between the tested compounds and their anti-proliferative activities. Large-scale affinity pull-down/LCMS identifications of 60 putative protein hits were enabled by these THL probes. These findings revealed that subtle structural modifications of

ABPP Probes Derived from and Inspired by Natural Products for Protein Target Identification Natural products are small molecules produced by living organisms found in nature, and many of these compounds are pharmacologically or biologically active, offering high selectivity and potency as a result of evolutionary selection.[65] Natural products, and their derivatives and mimics, have made an enormous contribution to the treatment of diseases such as infections and cancers over the past decades. Notable examples include antibiotics like b-lactam penicillin G, the tetracyclines (e.g., tetracycline, chlortetracycline), the glycopeptide vancomycin, the lipopetide daptomycin, and chemotherapeutic anticancer agents such as Taxol (paclitaxel), vinca alkaloids, and the plant-derived etoposide. Natural products are biosynthesized or modified by proteins, and as such, are bound by natural products. However, the interactions between many bioactive natural products and their protein targets are often unknown. In this section, we discuss recent advances and development of ABPP probes derived from and inspired by natural products and their applications in elucidating their potential targets and mechanism of action (Scheme 6). Many of the natural products consist of highly reactive functional groups which are highly specific and selective, due to optimization resulting from millions of years of evolution. Conversion of these natural products into ABPP probes merely requires modification of the structure to append a reporter or handle.[66] Rosenblum’s group reported the conversion of wortmannin to ABP.[67] Wortmannin is a metabolite natural product produced by the fungus P. wortmannin and has long been recognized as highly specific inhibitor of the phosphoinositide-3kinase (PI3K) enzyme superfamily. By attachment of a tetramethylrhodamine to wortmannin, the conjugate AX7503 (34) was synthesized, permitting both visualization and purification of proteins modified by AX7503 in a complex proteome. Other than the previously reported target PI3K and PI3K-related kinChemBioChem 2015, 16, 712 – 724

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Scheme 6. Structures of natural product-derived ABPP probes bearing mechanism-based reactive groups (upper panel; shaded, rounded rectangles) or photo-reactive groups (lower panel; shaded ovals) for protein target identification.

THL led to noticeable changes in both the cellular potency and target profiles of the drug. An interesting combination of this ABPP strategy with well-established AGT/SNAP-tag technology utilizing synthesized, cell-permeable, benzylguaninecontaining THL derivative probes realized effective drug delivery and retention in different sub-cellular organelles of living cells.[76] Extended application of this drug delivery approach in ChemBioChem 2015, 16, 712 – 724

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the Trypanosoma brucei parasite enabled accurate visualization of the cellular uptake and subsequent organelle-specific localization of these probes. Such specific localization information, together with the associated morphological changes in T. brucei, will potentially aid in elucidating the mechanism of action of the drug and the most efficient stage in the T. brucei life cycle at which to treat.[77] 721


Reviews Another important natural product that has been converted to ABP is the nucleoside analogue showdomycin.[78] It is produced by Streptomyces showdoensis and is active against many Gram-positive strains. Being structurally analogous to uridine and pseudouridine, showdomycin is potentially recognized by uridine- or pseudouridine- binding enzymes. The selective inhibition of such enzymes by showdomycin is related to its reactive maleimide structure, which is known to specifically react with nucleophilic cysteine thiols in the active site or binding pocket of these enzymes. Conversion of showdomycin to ABP was achieved by introducing an alkyne handle through attachment of a hexanoyl moiety on its free primary 5’-OH group (42). This alkyne handle, therefore, enables showdomycin’s target identification in living Gram-positive and Gram-negative pathogenic bacteria. A target identification study with a showdomycin probe discovered enzymes belonging to different classes in both pathogenic and non-pathogenic strains. Among these enzymes, UDP-N-acetylglucosamine 1-carboxyvinyl transferase (MurA transferase), an essential enzyme in bacteria cell wall biosynthesis, was found to be most likely accountable for showdomycin’s antibiotic activity. In contrast to the aforementioned natural products, which form covalent bonds with their protein targets, the cyclic heptadepsipeptide HUN-7293 binds reversibly and inhibits the expression of cell adhesion molecule.[79] To identify its cellular target, Taunton and co-workers incorporated photo-leucine, a diazirine-based photo-reactive analogue of leucine, into the scaffold of the cyclodepsipeptide (43). After incubation of this probe with an endoplasmic reticulum microsome fraction, followed by UV irradiation, a target of this HUN-7293-based probe was identified as Sec61a, the largest subunit of the Sec61 complex, which forms the translocation channel through which all proteins transit. Vancomycin is another example that only reversibly interacts with its protein targets. Vancomycinbased photoaffinity probe 44 was synthesized by Sieber’s group to identify its cellular targets in living S. aureus and Enterococcus faecalis.[80] This study identified two previously unknown targets on the bacterial membrane and revealed an unexplored mode of action. With these exciting developments of natural product-inspired ABPP probes, functional profiling of various proteomes has led to discovery and identification of novel protein activities and potential biomarkers in diseases states including, but not limited to, cancer and microbial infections. Furthermore, conversion of known drugs and other natural products of therapeutic value to ABPP probes has made a significant contribution in elucidating their cellular targets that are waiting to be characterized. This also provides a platform for effective evaluation and assessment of these molecules’ pharmaceutical effects, enhancing our comprehension of their SAR, mechanism of action, and exact function.

integration of ABPP with the latest bioorthogonal chemistry has enabled “label-free” design to facilitate probe labeling in living cells to capture the interactions between small molecules and proteins in their native environment. Furthermore, high-throughput-amenable synthesis and highly modular probe design have minimized the synthetic efforts and simplified the efficient assembly and evaluation of probe candidate libraries. Such efficient combinatorial syntheses of focused libraries and high-throughput screening approaches paved new paths to distinguish homologous proteins and to systematically characterize protein functions in complex proteomes, especially under circumstances where very limited biological information is known priori. Finally, natural-product-derived and -inspired probes have gradually become prevalent, due to their rich stereogenic centers and broad coverage of chemical space. Owing to these advantages, natural products will always be an essential source of inspiration for ABPP probe development.[81] However, due to limited space, we have not been able to cover all aspects of ABPP technology. For example, the fundamental development of in vivo imaging fluorescent ABPs by the Bogyo group was purposely not included for the reason that it has been extensively reviewed elsewhere.[82] More recently, kinase active site profiling by a novel active sitedirected probe has provided profound insights into the regulatory mechanism of multi-domain kinases.[83] Similarly, Overkleeft and colleagues recently reported a triple bioorthogonal strategy for simultaneous labeling of multiple enzymatic activities.[84] It is of enormous interest to see how these recent ABPP innovations will evolve and expand to provide discerning insights into our understanding of protein function. By now, the merits from these innovative developments are already evident. Nonetheless, advances in organic chemistry approaches, systems-level genetics, proteomics, and metabolomics will inspire future innovations and applications of ABPP technology that will further expand our comprehension of protein cellular functions and regulatory mechanisms. A systems-level understanding of protein activity network in living systems is the key to advancing drug discovery, pharmacology, and medicine in the post-genomic era.

Acknowledgements Dr. Kai Liu is kindly supported by Prof. Sheng Ding at the J. David Gladstone Institute, University of California, San Francisco. Dr. Peng-yu Yang is kindly supported by Prof. Peter G. Schultz at the Scripps Research Institute. Also, we thank Prof. Shao Q. Yao for his invaluable guidance for this review. We apologize to all scientists whose work could not be properly discussed and cited here due to limited space. Keywords: activity-based protein profiling · bioorthogonal · chemical proteomics · click chemistry · tandem labeling

Summary and Outlook

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