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Activity-based proteomic profiling: application of releasable linker in photoaffinity probes
Drug Discovery Today Volume 00, Number 00 November 2019
Activity-based proteomic profiling: application of releasable linker in photoaffinity probes Q9
Jin Wang1,y, Qinhua Chen2,y, Yuanyuan Shan3, Xiaoyan Pan1 and Jie Zhang1
1 Q10 2 School of Pharmacy, Health Science Center, Xi’an Jiaotong University, Xi’an, 710061, China Q11 3 Affiliated Dongfeng Hospital, Hubei University of Medicine, Shiyan, 442008, China Department of Pharmacy, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, 710061, China
Combining releasable chemical crosslinkers with photoaffinity probes represents a valuable tool for identifying protein–protein interactions (PPIs). The biomacromolecule photoaffinity probe prepared by using releasable photoaffinity linkers can be used to exploring PPIs by triggering release of the releasable group. More importantly, it can overcome the shortcomings of macromolecular photoaffinity probes without label transfer functionality to accurately confirm defects in specific structural sites. It shows particular promise for research exploring the interaction of unknown proteins and transient–weak PPIs Q12 in living organisms to discover new drug targets. In this review, we highlight recent progress in the development and application of chemical releasable linkers in photoaffinity probes. Several comparative studies are described in which the efficiency of various photoaffinity probes are compared.
Introduction Q13 In pharmaceutics, releasable linkers have an important role in drug delivery [1–8]. Researchers always choose a releasable linker to link targeting agents with drugs. When the prodrug arrives at the target site, it can trigger the releasable linker to release drugs into the target site. At the same time, the releasable linker can also be used to explore PPIs [9,10]. Proteins are an essential part of cellular activity, participating in cell regulation and signaling [11–13]. Therefore, the exploration of PPIs is vital. Traditional methods of exploring PPIs focused on known PPIs, and included western blot, immunofluorescence, and surface plasmon resonance. ignoring potentially unknown PPIs. However, unknown PPIs have an important role in cell signals and help us understand the complex environment of the cell. Currently, photoaffinity labeling (PAL) can be used in drug target discovery to explore such PPIs, although challenges remain. For example, the bait protein might interfere with the study of prey–protein interactions, especially because it exists in low levels within the cell [14]. Therefore, some researchers
Corresponding author: Zhang, J. ([email protected])
y Both authors contributed equally to this work and should be regarded as joint first authors.
1359-6446/ã 2019 Elsevier Ltd. All rights reserved. https://doi.org/10.1016/j.drudis.2019.10.016
have introduced releasable linkers in bait-protein probes. When the bait-protein captures prey proteins, the releasable linker releases the bait protein by triggering certain conditions. The prey protein is then analyzed and identified, despite its low abundance. For example, He et al. selected selenium (Se) as a releasable linker to explore PPIs [9], whereas Horne et al. used S–S bonds as releasable linkers to modify the protein. Therefore, further exploration of releasable linkers is becoming increasingly essential [15].
Photoaffinity labeling PAL is the use of a chemical probe that can covalently bind to its target in response to activation by light [16,17]. Upon irradiation with a specific wavelength of light, the photo group forms a reactive intermediate that rapidly reacts with, and binds to, the nearest molecule, which ideally will be the target protein. Therefore, we can use related analysis and identification technology to explore the target protein. At the same time, researchers found that this technology can also be used to explore PPIs by designing and synthesizing a bait-protein photoaffinity probe. When a specific wavelength of light irradiates the probe, it can rapidly bind to the prey protein. However, such strategies suffer from the following limitations, especially in living systems: (i) the biomacromolecule probe has
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FIGURE 1
Q1 Exploring protein–protein interactions (PPIs) by photoaffinity labeling (PAL). Photoaffinity linker covalently modified protein molecules are released to construct bait-protein photoaffinity probes. Specific PPIs are then used to generate protein complexes, which can be transformed a covalently complex prey-bait protein under specific-wavelength ultraviolet (UV) irradiation. Then, the complex can release other unrelated groups by triggering the releasable group. Finally, the related biochemical analysis technology can be combined with the bio-orthogonal reaction to trace and identify the prey protein.
poor stability, and the structural characteristics of the probe cannot be clarified by chemical modification; (ii) the use of genetically encoded tools to construct probes that require the introduction of key groups at specific active sites is not universally applicable; (iii) complicated crosslinking results often include both intermolecular and intramolecular complexes, which require intensive software development to decipher [18–21]; and (iv) large molecular volume probes interfere with subsequent identification analysis. Therefore, researchers have introduced releasable linkers to construct probes to improve the outcomes, enabling them to create certain conditions where the releasable linker can be activated and the bait protein released. Therefore, it is possible to understand the characteristics of the prey protein while avoiding the influence of the bait protein Q14 (Fig. 1). He et al. were one of the first to adopt this technology and explored the client protein DegP in the periplasm of Escherichia coli [9].
Releasable linkers Releasable linkers are used in drug delivery so that when the prodrug arrives at the target site, the drug is released. Combining this technology and PAL, it is possible to analyze and identify PPIs. By only using the releasable linker, the protein probe can be specifically enriched with low-abundance interacting proteins in the cell. Here, we provide a summary of releasable linkers and release conditions used in recent years.
Chemical activation Se Se is an essential element in the human body with potential antioxidant properties. Given its electronegativity and atomic radius, Se-containing compounds exhibit unique bond energies (C–Se bond: 244 kJ mol–1; Se–Se bond: 172 kJ mol–1) [22]. These values give C–Se or Se–Se covalent bonds a dynamic character and make them responsive to mild stimuli. Therefore, Se-containing polymers can disassemble in response to changes under physiologically relevant conditions. This property makes these polymers 2
a promising biomaterial for the controlled release of drugs or as synthetic mimics for enzymes. Given the unique redox properties of elemental Se, Se-containing polymers have proven to be ideal candidates for responsive disassembly under mild conditions, which could be more applicable for clinical use [23]. C–Se bonds can be broken by H2O2. Yang et al. utilized C–Se bonds to synthesize a photocrosslinker that can undergo oxidative cleavage, allowing subsequent release of the captured client pools from their respective chaperones for unbiased 2D-PAGE analysis [24]. The authors used the method to capture the low-abundance substrate protein DegP in the periplasm of E. coli (Fig. 2). Q15 Selenide is a promising candidate for a dual redox response because of its good activity in the presence of either oxidants or reductants (Fig. 3). Normally, selenide bonds can be oxidized to selenic acid in the presence of oxidants to Se in a reducing environment. Ma et al. successfully synthesized a dual redoxresponsive block copolymer with selenide bonds located at the polymer main chains that could be used as a drug delivery vehicle in a controlled manner [25]. Meanwhile, the selenide bond can also be released in the presence of the redox agent glutathione (1–10 mM). Han et al. synthesized Se-DSA nanoparticles (NPs), which could co-assemble with an antitumor prodrug, selenidecontaining paclitaxel (Se-PTX), which could be obtained by precipitation, to form SeDSA-SePTX Co-NPs (Co-NPs).These selenidecontaining NPs can release PTX in the presence of the redox agent glutathione. thus, the Co-NPs combine the advantages of Se-DSA and Se-PTX for cell imaging and antineoplastic activity and exhibit selective cytotoxicity between neoplasia cells and normal cells [26].
S–S bonds Disulfide bonds are basic chemical bonds in the secondary structure of protein molecules that link the thiol groups of different peptide chains or two different cysteine residues in the same peptide chain. In a protein molecule, disulfide bonds have an important role in stabilizing the spatial structure of the peptide
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Q2 Using selenium (Se) to investigate protein–protein interactions (PPIs) by photoaffinity labeling (PAL). Se is selected as releasable group to construct an amino acid photoaffinity linker, which can be used to encode a prey-protein photoaffinity probe. Once specific recognition of the bait proteins has been achieved, the identified prey protein can covalently bind the bait protein by photocrosslinking. The release of the Se group releases the prey-protein molecule via H2O2, resulting in the efficient analysis of PPIs in living organisms.
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Release conditions for selenium (Se)–Se bonds. Se–Se bonds can be released under redox conditions in 0.01% H2O2 and 0.01% glutathione (GSH).
chain [27]. In general, the number of disulfide bonds is correlated with the stability of the protein. Disulfide bonds are also a type of releasable group that can be released under reducing conditions. In recent years, this technology has been mainly used for the preparation of prodrugs for the study of drug delivery [28]. Xi et al. used the bonds as releasable linkers to construct the novel prodrug, meso-tetra (m-hydroxy phenyl) chlorin [29]. The authors combined the drug with the target molecule in the novel prodrug that showed significant targeting effects on HeLa cells. Borrelli et al. utilized disulfide bonds to link squalene with active drug to construct a conjugation that can self-assemble in water and release the parent drug in vitro (Fig. 4) [30]. This research also showed that the introduction of disulfide bonds and squalene did not affect the activity of the parent drugs. In 2009, Apparat et al. adopted the strategy to deliver nitric oxide (NO) and nonsteroidal anti-inflammatory drugs (NO-NSAIDs), which showed promising pharmacokinetic, anti-inflammatory, and NO-releasing properties and Q16 protected rats from NSAID-induced gastric damage [XX]. Other than applications in drug delivery using the prodrug strategy, the releasable disulfide bonds have also been used to deliver genes. In
2014, Feng et al. adopted releasable disulfide bonds to prepare disulfide-containing crosslinked polyethyleneimines (PEI-SS-CLs) for gene delivery [31]. Their results indicated that incorporating thiol-specific cleavable disulfide bonds into crosslinked PEIs to implement the regulated release of DNA is an effective strategy for designing safe and effective gene vectors. In 2009, Sun et al. introduced S–S bonds to DNA [32]. These authors found statistically significant differences between the expression levels from immobilized and releasable DNA, and discussed these differences in relation to the distinct accessibility and mode of action of glutathione, an intracellular reducing agent responsible for releasing the bound double-stranded (ds)DNA. In 2004, Sough Ayer et al. found that free substrate peptide concentrations of 10–20–10–18 M were attainable in a cell when substrates were delivered utilizing these conjugates [33]. In addition, disulfide bonds can also be used to modify active peptides and proteins, which can prevent biology activity loss. During the 1990s, Repeal et al. modified the active-site cysteine residue of papain with a 4-pyridyl disulfide [34]. Recently, Bontemps et al. reported the synthesis of thiol-reactive polymers, which were synthesized by atom transfer radical polymerization, and their reversible conjugation to bovine serum albumin [35]. The conjugation was achieved in methanol/phosphate buffer solution at basic pH, and the release of the protein could be achieved with dithiothreitol. In addition to the above applications, Townsend et al. combined the one-bead-one-compound (OBOC) combinatorial library method with the releasable linker strategy via in situ ultrahigh-throughput releasable solution phase screening of OBOC libraries [36]. The authors screened three positive beads from a total of 20 000 beads and found that these compounds had a strong consensus motif and cytotoxicity (IC50 50–150 mM) against two T lymphoma cell lines but less toxicity
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Q3 The pathway for drug release of disulfide-containing squalene conjugates. Disulfide bonds are used to link squalene with active drug to construct a conjugate that can self-assemble in water and release the parent drug following exposure to glutathione (GSH) in vitro, resulting in the delivery of drug in vivo.
against the MDA-MB 231 breast cancer cell line. This novel ultrahigh-throughput OBOC releasable method could be adapted to many existing 96 or 384-well solution-phase cell-based or biochemical assays. In recent years, cell-penetrating peptides have been used in molecular delivery in both biology and medicine. Given the ever-increasing number of reported cell-penetrating Q17 peptides (CPPs), reliable assays are urgently needed for evaluating their comparative internalization efficacy and further characterization as cellular delivery agents. Eiriksdottir et al. developed a novel releasable luminescence linker that can conjugate CPPs to assess delivery efficacy and advantageously provide a strong signal and low background [37].
Thioester bonds Thioester bonds can also be released under certain conditions, Chen et al. prepared a thioester derivative of MPEG containing an activated acid for conjugation to amino groups on proteins [38]. When the conjugates were delivered to the target site, they utilized the thiol–thioester exchange reaction to release protein. One of the most investigated r-PEGylation strategies relies on the 1,6benzyl elimination of a linker molecule to release fully unmodified amino groups on proteins. This process is initiated by activation of a trigger group (Fig. 3).
pH sensitivity The chemical environment in living organisms is complicated. In normal organisms, cells maintain normal pH because of their internal environmental regulation [39,40]; by contrast, the internal environment of tumor cells is often destroyed and becomes either acidic or alkaline. For example, in gastric cancer cells, the internal environment is acidic [41]. Therefore, in terms of drug delivery, such pH conditions and acid-base releasable linkers can be used to construct prodrug molecules to achieve drug delivery. Hirschberg et al. developed a flexible and efficient method for the conjugation of Taxol to various arginine-based molecular transporters via the Taxol C2’ O-chloroacetic derivative [42]. These authors showed that the resultant Taxol-transporter conjugates were highly water soluble and released free Taxol with half-lives of minutes to hours. In addition, some of the key organelles in some organisms, such as lysosomes, are widely found in eukaryotic organisms [43–45], and contain a variety of hydrolases that break down substances that 4
enter the cell from the outside and digest cells. When a cell ages, the lysosome ruptures, releasing hydrolase, which digests the entire cell and causes it to die. However, because of excessive hydrolysis, some drugs targeting the nucleus cannot reach the lysosome and lose activity. Based on this, Zhou et al. conjugated a drug carrier to the nucleus of the target all-trans retinoic acid and then encapsulated the conjugate in the micelle [46]. The linker was cleaved in the lysosome, releasing the HA2 peptide and destroying the lysosomal membrane, so that the drug-loaded micelle can effectively escaped the lysosome and targeted MCF-7 breast cancer cells. Follow-up activity studies showed that micelle modification to escape lysosomes and target the nucleus can improve the efficiency of anticancer drugs. In recent years, chemical bonds have also been released under certain pH conditions, and some researchers found that hydrazine bonds could be fractured at certain pH levels. For example, Goodwin et al. utilized hydrazine bond-linked doxorubicin (Dox) with oxidized pullulan, the latter of which was exploited to generate a targeted drug delivery system by the conjugation of Dox to the polymer backbone; this was able to deliver Dox to target sites and showed a twofold increase in anticancer activity compared with the control [47]. In addition to the controlled release of chemical bonds under specific acid-base conditions, scientists have used specialized chemical reactions, such as b-elimination reactions [48,49]. For example, Hennies et al. developed a system for drug delivery using a multifunctional linker that can be self-cleaved by b-elimination to release natural drugs [50]. The results showed that the drug release occurred in a highly predictable manner, in which half-life of cleavage ranged from hours to months. In this approach (Fig. 6), a macromolecular carrier is connected to a linker that is attached to a drug or prodrug via a carbamate group. At the same time, Santi et al. also adopted this method for the half-life extension of peptides, proteins, and small-molecule drugs [51]. The linkers undergo b-elimination reactions with predictable cleavage rates to release the native drug. The authors utilized this technology for half-life extension of the 38-amino acid HIV-1 fusion inhibitor TRI-1144. Conjugation of TRI-1144 to 40-kDa PEG by an appropriate b-eliminative linker and administration of the conjugate increased the in vivo half-life of the released peptide from 4 to 34 h in rats, and the PK parameters were in excellent accordance with a one-compartment model [52].
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Enzyme activation In organisms, enzymatic reactions are widespread and support the normal activities of organisms, such as energy production in mitochondria. Given their specificity, enzymes can be used to achieve controlled chemical modification of certain structures, including chemical bonds [53–55]. Therefore, many researchers have used the selective cleavage of specific enzymes to achieve the release of biological linkers. Recently, research has focused on the modification of anticancer drugs to obtain self-assembling drug conjugates that spontaneously form NPs in aqueous media. Bornean et al. focused on the controlled release induced by the self-immolating ability of a phydroxybenzylalcohol-based linker [56]. The enzymatic cleavage of a scissile ester bond (acetate) affords a strongly electron-donating phenoxide that facilitates the formation of a quinone methide intermediate. The authors reported new self-assembling conjugates that obtained a self-immolating linker that secures controlled release induced by lipase cleavage and an appropriate appendage for anchoring the self-assembling inducer. The release of the thiocolchicine derivative, which they used as the drug prototype, was demonstrated in vitro in the presence of porcine pancreases lipase (PPL) and Elite-supported lipase (PS). The antiproliferative activity of the NPs obtained was shown on two human cancer cell lines (HeLa and MCF-7). Significant issues are associated with the development of antibiotic drugs [57,58], such as drug resistance and low cell permeability, which have prompted the development of new antibiotics. In recent years, the development of transferrin has been a major solution for the penetration of bacteria by drugs. The use of iron carriers to support antibiotics is an appropriate strategy to bypass the bacterial wall and enter the bacteria to exert efficacy. However, the design of the synthetic transfer carrier-antibiotic complex by this strategy can cause antibiotic cleavage when transporting antibiotics through the bacterial inner membrane. Therefore, researchers have considered incorporating a controllable releasable linker in the complex. When the complex enters the bacteria, it is further lysed by the action of a specific enzyme to complete
antibiotic transportation. Kelly et al. utilized the synthesis of a siderophore-cephalosporin compound and demonstrated that b-lactams (Fig. 4), such as cephalosporins, can serve as b-lactamase-triggered releasable linkers to allow intracellular delivery of Gram-positive antibiotics to Gram-negative bacteria [59]. In 2016, Chen et al. combined a prodrug strategy with a specific enzyme reaction to design a novel prodrug [60]. The authors linked Dox and a polymer to the prodrug molecule via a protease B labyrinth. The activity results indicated that the polymer prodrug combination had an additional antitumor effect that can be used to enhance AMP efficacy.
The application of releasable linkers in a photoaffinity label In recent years, PAL has been used not only for analyzing, identifying, and finding the target protein, but also for exploring PPIs [9,10,26]. When this technology is used to directly study the interaction between biological macromolecules, there are often defects, such as large probe molecules, poor biopermeability, and damage to normal cellular activities. Therefore, many scientists use this technology to study biomacromolecular interactions, often introducing releasable linkers to construct biomacromolecular photoaffinity probes during the probe design. In 2019, He et al. utilized C–Se bonds as releasable linkers to construct a probe [9], introducing an alkyne bio-orthogonal handle, a photocrosslinking group (bis-aziridine), and a releasable C–Se bond group in the amino acid structure as a basic unit to design a synthetic protein probe molecule by gene coding technology. The authors demonstrated covalent capture of the less abundant guest protein with which the probe was able to interact for subsequent purification analysis and identification. The authors utilized this technique under heat shock conditions for 14 proteolytic substrates of the essential E. coli PQC factor Dig. These included six newly identified proteins with lower abundance. The releasable nature of Disuse allows the transfer of an alkyne to the captured prey on Q18 the protein when the bait is separated, ensuring the efficiency and fidelity of subsequent bio-orthogonal labeling. Before this, the
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Q4 r-PEGylation achieved by thiol–thioester exchange. The conjugation of a methoxy poly(ethylene glycol) (mPEG) thioester derivative and proteins results in drug delivery to the target sites. At the same time, the thioester bonds can be released via thiol–thioester exchange to deliver the protein drugs in vivo.
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Q5 Drug delivery via b-elimination. This novel system for drug delivery uses a multifunctional linker that can be self-cleaved by b-elimination to release natural drugs. A macromolecular carrier is connected to a linker that is attached to a drug or prodrug via a carbamate group, which can release drugs via b-elimination. www.drugdiscoverytoday.com 5 Please cite this article in press as: Wang, J. et al. Activity-based proteomic profiling: application of releasable linker in photoaffinity probes, Drug Discov Today (2019), https://doi.org/ 10.1016/j.drudis.2019.10.016
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Q6 Siderophore–cephalosporin–oxazolidinone conjugates capable of delivering the Gram-positive antibiotic oxazolidinone into Gram-negative bacteria. When the antibiotic complex molecule, which contains transferrin and a releasable group, penetrates a bacterium, the target drugs are released under specific conditions, thus improving their poor permeability and enabling them to exert an improved therapeutic effect.
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Q7 Tag-transfer via disulfide (S–S) bonds. A releasable photoaffinity linker can specifically covalently modify the thiol site of a protein to construct a bait-protein probe. A combination of photoaffinity labeling and tag-transfer can be used to transfer the thiol group to the prey protein. Thus, it is only necessary to analyze and identify the thiol-containing protein using the relevant analytical techniques.
research group first proposed joint gene coding, PAL, and labeltransferring strategies for studying PPIs. In 2015, Yang et al. developed a genetically encoded photoaffinity unnatural amino acid that introduced a mass spectrometry-identifiable label (MS-label) to the captured prey proteins after photocrosslinking and prey– bait separation [28], which enabled the direct identification of photocaptured substrate peptides that are difficult to uncover by conventional genetically encoded photocrosslinkers. In 2018, the strategy of combining PAL and tag-transfer was used to detect protein complexes. For example, Horne et al. used tag transfer and PAL to introduce a releasable disulfide bond and a photoaffinity tag key group into a protein molecule to construct a protein photoaffinity probe molecule [15], which was useful for 6
identifying rare, transient, dynamic, and complex biomacromolecule interactions in cells (Fig. 5).
Concluding remarks and future perspective Coupling releasable chemical crosslinking or photocrosslinking reagents with mass spectrometry has become a valuable tool for identifying PPIs, particularly in living organisms. Chemical releasable crosslinking is a well-developed strategy for the identification of interaction partners and of interaction interfaces, especially in unbiased global proteomics studies, without the need for protein engineering [61–64] (Figs. 7 and 8). Q19 In the future, we will be able to design and synthesize different types of releasable photoaffinity biomacromolecule probe, including
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ronment, the internal environment of different tissues, organs, and cells is more complex and more unknown, which is a problem Q21 for exploring the releasability of the releasable probes. However, although there are many difficulties, we believe that, with the development of molecular biology research, these problems will be solved. At the same time, the new strategy of tag-transfer and PAL will become more efficient and accurate for studying the interactions between biological macromolecules. Q22
Acknowledgments This work was supported by the National Natural Science Foundation of China (NSFC, Grant No. 81573285 and 81602965), the Natural Science Basic Research Plan in Shaanxi Province of China (Program No. 2018JM7071), and the Fundamental Research Funds for the Central Universities (2015qngz13).
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nucleic acid probe molecules, to study the interaction between biomacromolecules along with related analytical identification techniques, such as mass spectrometry and fluorescence. Imaging and other tools for the exploration of the interaction between macromolecules, which are used to decipher the complex interactions between biological macromolecules in organisms, provide theoretical support for the further study of other disciplines. The key to exploring the interactions of biomacromolecules by using photoaffinity techniques and label transfer strategies is choosing a suitable releasing group, which is essential for the subsequent efficient release of unrelated groups in living organQ20 ism. Thus, to achieve the efficient release of unrelated groups once photocaptured by the bait biomacromolecules, we need to select the appropriate releasable linker according to differences in the biology of the organism. However, in a natural biological envi-
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41 Kim, E. et al. (2013) Hyaluronic acid receptor-targetable imidazolinenano vectors for induction of gastric cancer cell death by RNA interference. Biomaterials 34, 4327–4338 42 Hirschberg, T.A. et al. (2003) Arginine-based molecular transporters: the synthesis and chemical evaluation of releasable Taxol-transporter conjugates. Org. Lett. 5, 3459–3462 43 Feng, X. et al. (2018) Lysosomal potassium channels: potential roles in lysosomal function and neurodegenerative diseases. CNS Neurol. Disord. Drug Targets 17, 261–266 44 Salvatore, M. et al. (2018) The Sub Cons webserver: a user friendly web interface for state-of-the-art subcellular localization prediction. Protein Sci. 27, 195–201 45 Till, A. et al. (2015) Evolutionary trends and functional anatomy of the human expanded autophagy network. Autophagy 11, 1652–1667 46 Zhou, Z. et al. (2017) Enhanced nuclear delivery of anti-cancer drugs using micelles containing releasable membrane fusion peptide and nuclear-targeting retinoic acid. J. Mater. Chem. B 5, 7175–7185 Q23 47 Goodwin, A. (2018) Identifying covalent bond breakage in materials under strain: Mechanochemistry without mechanophores. ACS 4, 19–23 48 Santi, D.V. et al. (2012) Predictable and tunable half-life extension of therapeutic agents by controlled chemical release from macromolecular conjugates. Proc. Natl. Acad. Sci. U. S. A. 109, 6211–6216 49 Ashley, G.W. et al. (2013) Hydrogel drug delivery system with predictable and tunable drug release and degradation rates. Proc. Natl. Acad. Sci. U. S. A. 110, 2318– 2323 50 Hennies, J. et al. (2015) Biodegradable tetra-PEG hydrogels as carriers for a releasable drug delivery system. Bioconjug. Chem. 26, 270–278 Q24 51 Santi, D.V. et al. (2012) Predictable and tunable half-life extension of therapeutic agents by controlled chemical release from macromolecular conjugates. Proc. Natl. Acad. Sci. U. S. A. 109, 6211–6216
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52 Schneider, E.L. et al. (2015) Half-life extension of the HIV-fusion inhibitor peptide TRI-1144 using a novel linker technology. Eur. J. Pharm. Biopharm. 93, 254–259 53 Nyberg, E. et al. (2016) Growth factor-eluting technologies for bone tissue engineering. Drug Deliv. Transl. Res. 6, 184–194 54 Petersen, R.C. (2015) Triclosan computational conformational chemistry analysis for antimicrobial properties in polymers. J. Nat. Sci. 1, e54 55 Fumagillin, G. et al. (2019) Self-assembling releasable thiocolchicine diphenylbutenylaniline conjugates. ACS Med. Chem. Lett. 10, 611–614 56 Bornean, R. et al. (2019) [Antibiotic therapy in Bielefeld (AnTib)-a local project for the promotion of rational antibiotic prescribing in the outpatient pediatric sector]. Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 62, 952–959 57 Engin, A.B. et al. (2019) Nanoantibiotics: a novel rational approach to antibiotic resistant infections. Curr. Drug Metab. 11–14 58 Schalk, I.J. (2018) A Trojan-horse strategy including a bacterial suicide action for the efficient use of a specific Gram-positive antibiotic on Gram-negative bacteria. J. Med. Chem. 61, 3842–3844 59 Kelly, G.J. et al. (2016) Polymeric prodrug combination to exploit the therapeutic potential of antimicrobial peptides against cancer cells. Org. Biomol. Chem. 14, 9278–9286 60 Chen, L.P. et al. (2007) Trophoblast apoptosis, its regulators, and missed abortion. Jiangxi Yiyi 42, 173–176 61 Beggared, T. et al. (2007) Methods for the detection and analysis of protein–protein interactions. Proteomics 7, 2833–2842 62 Gingras, A.C. et al. (2007) Analysis of protein complexes using mass spectrometry. Nat. Rev. Mol. Cell Biol. 8, 645–654 63 Sinz, A. (2010) Investigation of protein–protein interactions in living cells by chemical crosslinking and mass spectrometry. Anal. Bioanal. Chem. 397, 3433–3440 64 Pham, N.D. et al. (2013) Photo crosslinking approaches to interactome mapping. Curr. Opin. Chem. Biol. 17, 90–101
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Activity-based proteomic profiling: application of releasable linker in photoaffinity probes Q9
Jin Wang1,y, Qinhua Chen2,y, Yuanyuan Shan3, Xiaoyan Pan1 and Jie Zhang1
1 Q10 2 School of Pharmacy, Health Science Center, Xi’an Jiaotong University, Xi’an, 710061, China Q11 3 Affiliated Dongfeng Hospital, Hubei University of Medicine, Shiyan, 442008, China Department of Pharmacy, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, 710061, China
Combining releasable chemical crosslinkers with photoaffinity probes represents a valuable tool for identifying protein–protein interactions (PPIs). The biomacromolecule photoaffinity probe prepared by using releasable photoaffinity linkers can be used to exploring PPIs by triggering release of the releasable group. More importantly, it can overcome the shortcomings of macromolecular photoaffinity probes without label transfer functionality to accurately confirm defects in specific structural sites. It shows particular promise for research exploring the interaction of unknown proteins and transient–weak PPIs Q12 in living organisms to discover new drug targets. In this review, we highlight recent progress in the development and application of chemical releasable linkers in photoaffinity probes. Several comparative studies are described in which the efficiency of various photoaffinity probes are compared.
Introduction Q13 In pharmaceutics, releasable linkers have an important role in drug delivery [1–8]. Researchers always choose a releasable linker to link targeting agents with drugs. When the prodrug arrives at the target site, it can trigger the releasable linker to release drugs into the target site. At the same time, the releasable linker can also be used to explore PPIs [9,10]. Proteins are an essential part of cellular activity, participating in cell regulation and signaling [11–13]. Therefore, the exploration of PPIs is vital. Traditional methods of exploring PPIs focused on known PPIs, and included western blot, immunofluorescence, and surface plasmon resonance. ignoring potentially unknown PPIs. However, unknown PPIs have an important role in cell signals and help us understand the complex environment of the cell. Currently, photoaffinity labeling (PAL) can be used in drug target discovery to explore such PPIs, although challenges remain. For example, the bait protein might interfere with the study of prey–protein interactions, especially because it exists in low levels within the cell [14]. Therefore, some researchers
Corresponding author: Zhang, J. ([email protected])
y Both authors contributed equally to this work and should be regarded as joint first authors.
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have introduced releasable linkers in bait-protein probes. When the bait-protein captures prey proteins, the releasable linker releases the bait protein by triggering certain conditions. The prey protein is then analyzed and identified, despite its low abundance. For example, He et al. selected selenium (Se) as a releasable linker to explore PPIs [9], whereas Horne et al. used S–S bonds as releasable linkers to modify the protein. Therefore, further exploration of releasable linkers is becoming increasingly essential [15].
Photoaffinity labeling PAL is the use of a chemical probe that can covalently bind to its target in response to activation by light [16,17]. Upon irradiation with a specific wavelength of light, the photo group forms a reactive intermediate that rapidly reacts with, and binds to, the nearest molecule, which ideally will be the target protein. Therefore, we can use related analysis and identification technology to explore the target protein. At the same time, researchers found that this technology can also be used to explore PPIs by designing and synthesizing a bait-protein photoaffinity probe. When a specific wavelength of light irradiates the probe, it can rapidly bind to the prey protein. However, such strategies suffer from the following limitations, especially in living systems: (i) the biomacromolecule probe has
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FIGURE 1
Q1 Exploring protein–protein interactions (PPIs) by photoaffinity labeling (PAL). Photoaffinity linker covalently modified protein molecules are released to construct bait-protein photoaffinity probes. Specific PPIs are then used to generate protein complexes, which can be transformed a covalently complex prey-bait protein under specific-wavelength ultraviolet (UV) irradiation. Then, the complex can release other unrelated groups by triggering the releasable group. Finally, the related biochemical analysis technology can be combined with the bio-orthogonal reaction to trace and identify the prey protein.
poor stability, and the structural characteristics of the probe cannot be clarified by chemical modification; (ii) the use of genetically encoded tools to construct probes that require the introduction of key groups at specific active sites is not universally applicable; (iii) complicated crosslinking results often include both intermolecular and intramolecular complexes, which require intensive software development to decipher [18–21]; and (iv) large molecular volume probes interfere with subsequent identification analysis. Therefore, researchers have introduced releasable linkers to construct probes to improve the outcomes, enabling them to create certain conditions where the releasable linker can be activated and the bait protein released. Therefore, it is possible to understand the characteristics of the prey protein while avoiding the influence of the bait protein Q14 (Fig. 1). He et al. were one of the first to adopt this technology and explored the client protein DegP in the periplasm of Escherichia coli [9].
Releasable linkers Releasable linkers are used in drug delivery so that when the prodrug arrives at the target site, the drug is released. Combining this technology and PAL, it is possible to analyze and identify PPIs. By only using the releasable linker, the protein probe can be specifically enriched with low-abundance interacting proteins in the cell. Here, we provide a summary of releasable linkers and release conditions used in recent years.
Chemical activation Se Se is an essential element in the human body with potential antioxidant properties. Given its electronegativity and atomic radius, Se-containing compounds exhibit unique bond energies (C–Se bond: 244 kJ mol–1; Se–Se bond: 172 kJ mol–1) [22]. These values give C–Se or Se–Se covalent bonds a dynamic character and make them responsive to mild stimuli. Therefore, Se-containing polymers can disassemble in response to changes under physiologically relevant conditions. This property makes these polymers 2
a promising biomaterial for the controlled release of drugs or as synthetic mimics for enzymes. Given the unique redox properties of elemental Se, Se-containing polymers have proven to be ideal candidates for responsive disassembly under mild conditions, which could be more applicable for clinical use [23]. C–Se bonds can be broken by H2O2. Yang et al. utilized C–Se bonds to synthesize a photocrosslinker that can undergo oxidative cleavage, allowing subsequent release of the captured client pools from their respective chaperones for unbiased 2D-PAGE analysis [24]. The authors used the method to capture the low-abundance substrate protein DegP in the periplasm of E. coli (Fig. 2). Q15 Selenide is a promising candidate for a dual redox response because of its good activity in the presence of either oxidants or reductants (Fig. 3). Normally, selenide bonds can be oxidized to selenic acid in the presence of oxidants to Se in a reducing environment. Ma et al. successfully synthesized a dual redoxresponsive block copolymer with selenide bonds located at the polymer main chains that could be used as a drug delivery vehicle in a controlled manner [25]. Meanwhile, the selenide bond can also be released in the presence of the redox agent glutathione (1–10 mM). Han et al. synthesized Se-DSA nanoparticles (NPs), which could co-assemble with an antitumor prodrug, selenidecontaining paclitaxel (Se-PTX), which could be obtained by precipitation, to form SeDSA-SePTX Co-NPs (Co-NPs).These selenidecontaining NPs can release PTX in the presence of the redox agent glutathione. thus, the Co-NPs combine the advantages of Se-DSA and Se-PTX for cell imaging and antineoplastic activity and exhibit selective cytotoxicity between neoplasia cells and normal cells [26].
S–S bonds Disulfide bonds are basic chemical bonds in the secondary structure of protein molecules that link the thiol groups of different peptide chains or two different cysteine residues in the same peptide chain. In a protein molecule, disulfide bonds have an important role in stabilizing the spatial structure of the peptide
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Q2 Using selenium (Se) to investigate protein–protein interactions (PPIs) by photoaffinity labeling (PAL). Se is selected as releasable group to construct an amino acid photoaffinity linker, which can be used to encode a prey-protein photoaffinity probe. Once specific recognition of the bait proteins has been achieved, the identified prey protein can covalently bind the bait protein by photocrosslinking. The release of the Se group releases the prey-protein molecule via H2O2, resulting in the efficient analysis of PPIs in living organisms.
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FIGURE 3
Release conditions for selenium (Se)–Se bonds. Se–Se bonds can be released under redox conditions in 0.01% H2O2 and 0.01% glutathione (GSH).
chain [27]. In general, the number of disulfide bonds is correlated with the stability of the protein. Disulfide bonds are also a type of releasable group that can be released under reducing conditions. In recent years, this technology has been mainly used for the preparation of prodrugs for the study of drug delivery [28]. Xi et al. used the bonds as releasable linkers to construct the novel prodrug, meso-tetra (m-hydroxy phenyl) chlorin [29]. The authors combined the drug with the target molecule in the novel prodrug that showed significant targeting effects on HeLa cells. Borrelli et al. utilized disulfide bonds to link squalene with active drug to construct a conjugation that can self-assemble in water and release the parent drug in vitro (Fig. 4) [30]. This research also showed that the introduction of disulfide bonds and squalene did not affect the activity of the parent drugs. In 2009, Apparat et al. adopted the strategy to deliver nitric oxide (NO) and nonsteroidal anti-inflammatory drugs (NO-NSAIDs), which showed promising pharmacokinetic, anti-inflammatory, and NO-releasing properties and Q16 protected rats from NSAID-induced gastric damage [XX]. Other than applications in drug delivery using the prodrug strategy, the releasable disulfide bonds have also been used to deliver genes. In
2014, Feng et al. adopted releasable disulfide bonds to prepare disulfide-containing crosslinked polyethyleneimines (PEI-SS-CLs) for gene delivery [31]. Their results indicated that incorporating thiol-specific cleavable disulfide bonds into crosslinked PEIs to implement the regulated release of DNA is an effective strategy for designing safe and effective gene vectors. In 2009, Sun et al. introduced S–S bonds to DNA [32]. These authors found statistically significant differences between the expression levels from immobilized and releasable DNA, and discussed these differences in relation to the distinct accessibility and mode of action of glutathione, an intracellular reducing agent responsible for releasing the bound double-stranded (ds)DNA. In 2004, Sough Ayer et al. found that free substrate peptide concentrations of 10–20–10–18 M were attainable in a cell when substrates were delivered utilizing these conjugates [33]. In addition, disulfide bonds can also be used to modify active peptides and proteins, which can prevent biology activity loss. During the 1990s, Repeal et al. modified the active-site cysteine residue of papain with a 4-pyridyl disulfide [34]. Recently, Bontemps et al. reported the synthesis of thiol-reactive polymers, which were synthesized by atom transfer radical polymerization, and their reversible conjugation to bovine serum albumin [35]. The conjugation was achieved in methanol/phosphate buffer solution at basic pH, and the release of the protein could be achieved with dithiothreitol. In addition to the above applications, Townsend et al. combined the one-bead-one-compound (OBOC) combinatorial library method with the releasable linker strategy via in situ ultrahigh-throughput releasable solution phase screening of OBOC libraries [36]. The authors screened three positive beads from a total of 20 000 beads and found that these compounds had a strong consensus motif and cytotoxicity (IC50 50–150 mM) against two T lymphoma cell lines but less toxicity
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Q3 The pathway for drug release of disulfide-containing squalene conjugates. Disulfide bonds are used to link squalene with active drug to construct a conjugate that can self-assemble in water and release the parent drug following exposure to glutathione (GSH) in vitro, resulting in the delivery of drug in vivo.
against the MDA-MB 231 breast cancer cell line. This novel ultrahigh-throughput OBOC releasable method could be adapted to many existing 96 or 384-well solution-phase cell-based or biochemical assays. In recent years, cell-penetrating peptides have been used in molecular delivery in both biology and medicine. Given the ever-increasing number of reported cell-penetrating Q17 peptides (CPPs), reliable assays are urgently needed for evaluating their comparative internalization efficacy and further characterization as cellular delivery agents. Eiriksdottir et al. developed a novel releasable luminescence linker that can conjugate CPPs to assess delivery efficacy and advantageously provide a strong signal and low background [37].
Thioester bonds Thioester bonds can also be released under certain conditions, Chen et al. prepared a thioester derivative of MPEG containing an activated acid for conjugation to amino groups on proteins [38]. When the conjugates were delivered to the target site, they utilized the thiol–thioester exchange reaction to release protein. One of the most investigated r-PEGylation strategies relies on the 1,6benzyl elimination of a linker molecule to release fully unmodified amino groups on proteins. This process is initiated by activation of a trigger group (Fig. 3).
pH sensitivity The chemical environment in living organisms is complicated. In normal organisms, cells maintain normal pH because of their internal environmental regulation [39,40]; by contrast, the internal environment of tumor cells is often destroyed and becomes either acidic or alkaline. For example, in gastric cancer cells, the internal environment is acidic [41]. Therefore, in terms of drug delivery, such pH conditions and acid-base releasable linkers can be used to construct prodrug molecules to achieve drug delivery. Hirschberg et al. developed a flexible and efficient method for the conjugation of Taxol to various arginine-based molecular transporters via the Taxol C2’ O-chloroacetic derivative [42]. These authors showed that the resultant Taxol-transporter conjugates were highly water soluble and released free Taxol with half-lives of minutes to hours. In addition, some of the key organelles in some organisms, such as lysosomes, are widely found in eukaryotic organisms [43–45], and contain a variety of hydrolases that break down substances that 4
enter the cell from the outside and digest cells. When a cell ages, the lysosome ruptures, releasing hydrolase, which digests the entire cell and causes it to die. However, because of excessive hydrolysis, some drugs targeting the nucleus cannot reach the lysosome and lose activity. Based on this, Zhou et al. conjugated a drug carrier to the nucleus of the target all-trans retinoic acid and then encapsulated the conjugate in the micelle [46]. The linker was cleaved in the lysosome, releasing the HA2 peptide and destroying the lysosomal membrane, so that the drug-loaded micelle can effectively escaped the lysosome and targeted MCF-7 breast cancer cells. Follow-up activity studies showed that micelle modification to escape lysosomes and target the nucleus can improve the efficiency of anticancer drugs. In recent years, chemical bonds have also been released under certain pH conditions, and some researchers found that hydrazine bonds could be fractured at certain pH levels. For example, Goodwin et al. utilized hydrazine bond-linked doxorubicin (Dox) with oxidized pullulan, the latter of which was exploited to generate a targeted drug delivery system by the conjugation of Dox to the polymer backbone; this was able to deliver Dox to target sites and showed a twofold increase in anticancer activity compared with the control [47]. In addition to the controlled release of chemical bonds under specific acid-base conditions, scientists have used specialized chemical reactions, such as b-elimination reactions [48,49]. For example, Hennies et al. developed a system for drug delivery using a multifunctional linker that can be self-cleaved by b-elimination to release natural drugs [50]. The results showed that the drug release occurred in a highly predictable manner, in which half-life of cleavage ranged from hours to months. In this approach (Fig. 6), a macromolecular carrier is connected to a linker that is attached to a drug or prodrug via a carbamate group. At the same time, Santi et al. also adopted this method for the half-life extension of peptides, proteins, and small-molecule drugs [51]. The linkers undergo b-elimination reactions with predictable cleavage rates to release the native drug. The authors utilized this technology for half-life extension of the 38-amino acid HIV-1 fusion inhibitor TRI-1144. Conjugation of TRI-1144 to 40-kDa PEG by an appropriate b-eliminative linker and administration of the conjugate increased the in vivo half-life of the released peptide from 4 to 34 h in rats, and the PK parameters were in excellent accordance with a one-compartment model [52].
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Enzyme activation In organisms, enzymatic reactions are widespread and support the normal activities of organisms, such as energy production in mitochondria. Given their specificity, enzymes can be used to achieve controlled chemical modification of certain structures, including chemical bonds [53–55]. Therefore, many researchers have used the selective cleavage of specific enzymes to achieve the release of biological linkers. Recently, research has focused on the modification of anticancer drugs to obtain self-assembling drug conjugates that spontaneously form NPs in aqueous media. Bornean et al. focused on the controlled release induced by the self-immolating ability of a phydroxybenzylalcohol-based linker [56]. The enzymatic cleavage of a scissile ester bond (acetate) affords a strongly electron-donating phenoxide that facilitates the formation of a quinone methide intermediate. The authors reported new self-assembling conjugates that obtained a self-immolating linker that secures controlled release induced by lipase cleavage and an appropriate appendage for anchoring the self-assembling inducer. The release of the thiocolchicine derivative, which they used as the drug prototype, was demonstrated in vitro in the presence of porcine pancreases lipase (PPL) and Elite-supported lipase (PS). The antiproliferative activity of the NPs obtained was shown on two human cancer cell lines (HeLa and MCF-7). Significant issues are associated with the development of antibiotic drugs [57,58], such as drug resistance and low cell permeability, which have prompted the development of new antibiotics. In recent years, the development of transferrin has been a major solution for the penetration of bacteria by drugs. The use of iron carriers to support antibiotics is an appropriate strategy to bypass the bacterial wall and enter the bacteria to exert efficacy. However, the design of the synthetic transfer carrier-antibiotic complex by this strategy can cause antibiotic cleavage when transporting antibiotics through the bacterial inner membrane. Therefore, researchers have considered incorporating a controllable releasable linker in the complex. When the complex enters the bacteria, it is further lysed by the action of a specific enzyme to complete
antibiotic transportation. Kelly et al. utilized the synthesis of a siderophore-cephalosporin compound and demonstrated that b-lactams (Fig. 4), such as cephalosporins, can serve as b-lactamase-triggered releasable linkers to allow intracellular delivery of Gram-positive antibiotics to Gram-negative bacteria [59]. In 2016, Chen et al. combined a prodrug strategy with a specific enzyme reaction to design a novel prodrug [60]. The authors linked Dox and a polymer to the prodrug molecule via a protease B labyrinth. The activity results indicated that the polymer prodrug combination had an additional antitumor effect that can be used to enhance AMP efficacy.
The application of releasable linkers in a photoaffinity label In recent years, PAL has been used not only for analyzing, identifying, and finding the target protein, but also for exploring PPIs [9,10,26]. When this technology is used to directly study the interaction between biological macromolecules, there are often defects, such as large probe molecules, poor biopermeability, and damage to normal cellular activities. Therefore, many scientists use this technology to study biomacromolecular interactions, often introducing releasable linkers to construct biomacromolecular photoaffinity probes during the probe design. In 2019, He et al. utilized C–Se bonds as releasable linkers to construct a probe [9], introducing an alkyne bio-orthogonal handle, a photocrosslinking group (bis-aziridine), and a releasable C–Se bond group in the amino acid structure as a basic unit to design a synthetic protein probe molecule by gene coding technology. The authors demonstrated covalent capture of the less abundant guest protein with which the probe was able to interact for subsequent purification analysis and identification. The authors utilized this technique under heat shock conditions for 14 proteolytic substrates of the essential E. coli PQC factor Dig. These included six newly identified proteins with lower abundance. The releasable nature of Disuse allows the transfer of an alkyne to the captured prey on Q18 the protein when the bait is separated, ensuring the efficiency and fidelity of subsequent bio-orthogonal labeling. Before this, the
1,6-benzyl elimination Protein
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FIGURE 5
Q4 r-PEGylation achieved by thiol–thioester exchange. The conjugation of a methoxy poly(ethylene glycol) (mPEG) thioester derivative and proteins results in drug delivery to the target sites. At the same time, the thioester bonds can be released via thiol–thioester exchange to deliver the protein drugs in vivo.
Mod
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Q5 Drug delivery via b-elimination. This novel system for drug delivery uses a multifunctional linker that can be self-cleaved by b-elimination to release natural drugs. A macromolecular carrier is connected to a linker that is attached to a drug or prodrug via a carbamate group, which can release drugs via b-elimination. www.drugdiscoverytoday.com 5 Please cite this article in press as: Wang, J. et al. Activity-based proteomic profiling: application of releasable linker in photoaffinity probes, Drug Discov Today (2019), https://doi.org/ 10.1016/j.drudis.2019.10.016
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DRUDIS 2577 1–8 Drug Discovery Today Volume 00, Number 00 November 2019
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Extracellular medium
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Oxazolidinone
Cephalosporin Siderophore β-Lactamase Inner membrane
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FIGURE 7
Q6 Siderophore–cephalosporin–oxazolidinone conjugates capable of delivering the Gram-positive antibiotic oxazolidinone into Gram-negative bacteria. When the antibiotic complex molecule, which contains transferrin and a releasable group, penetrates a bacterium, the target drugs are released under specific conditions, thus improving their poor permeability and enabling them to exert an improved therapeutic effect.
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Q7 Tag-transfer via disulfide (S–S) bonds. A releasable photoaffinity linker can specifically covalently modify the thiol site of a protein to construct a bait-protein probe. A combination of photoaffinity labeling and tag-transfer can be used to transfer the thiol group to the prey protein. Thus, it is only necessary to analyze and identify the thiol-containing protein using the relevant analytical techniques.
research group first proposed joint gene coding, PAL, and labeltransferring strategies for studying PPIs. In 2015, Yang et al. developed a genetically encoded photoaffinity unnatural amino acid that introduced a mass spectrometry-identifiable label (MS-label) to the captured prey proteins after photocrosslinking and prey– bait separation [28], which enabled the direct identification of photocaptured substrate peptides that are difficult to uncover by conventional genetically encoded photocrosslinkers. In 2018, the strategy of combining PAL and tag-transfer was used to detect protein complexes. For example, Horne et al. used tag transfer and PAL to introduce a releasable disulfide bond and a photoaffinity tag key group into a protein molecule to construct a protein photoaffinity probe molecule [15], which was useful for 6
identifying rare, transient, dynamic, and complex biomacromolecule interactions in cells (Fig. 5).
Concluding remarks and future perspective Coupling releasable chemical crosslinking or photocrosslinking reagents with mass spectrometry has become a valuable tool for identifying PPIs, particularly in living organisms. Chemical releasable crosslinking is a well-developed strategy for the identification of interaction partners and of interaction interfaces, especially in unbiased global proteomics studies, without the need for protein engineering [61–64] (Figs. 7 and 8). Q19 In the future, we will be able to design and synthesize different types of releasable photoaffinity biomacromolecule probe, including
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ronment, the internal environment of different tissues, organs, and cells is more complex and more unknown, which is a problem Q21 for exploring the releasability of the releasable probes. However, although there are many difficulties, we believe that, with the development of molecular biology research, these problems will be solved. At the same time, the new strategy of tag-transfer and PAL will become more efficient and accurate for studying the interactions between biological macromolecules. Q22
Acknowledgments This work was supported by the National Natural Science Foundation of China (NSFC, Grant No. 81573285 and 81602965), the Natural Science Basic Research Plan in Shaanxi Province of China (Program No. 2018JM7071), and the Fundamental Research Funds for the Central Universities (2015qngz13).
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www.drugdiscoverytoday.com 7 Please cite this article in press as: Wang, J. et al. Activity-based proteomic profiling: application of releasable linker in photoaffinity probes, Drug Discov Today (2019), https://doi.org/ 10.1016/j.drudis.2019.10.016
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nucleic acid probe molecules, to study the interaction between biomacromolecules along with related analytical identification techniques, such as mass spectrometry and fluorescence. Imaging and other tools for the exploration of the interaction between macromolecules, which are used to decipher the complex interactions between biological macromolecules in organisms, provide theoretical support for the further study of other disciplines. The key to exploring the interactions of biomacromolecules by using photoaffinity techniques and label transfer strategies is choosing a suitable releasing group, which is essential for the subsequent efficient release of unrelated groups in living organQ20 ism. Thus, to achieve the efficient release of unrelated groups once photocaptured by the bait biomacromolecules, we need to select the appropriate releasable linker according to differences in the biology of the organism. However, in a natural biological envi-
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