Bioorthogonal Reactions in Activity-Based Protein Profiling

molecules Review Bioorthogonal Reactions in Activity-Based Protein Profiling Steven H. L. Verhelst 1,2,* , Kimberly M. Bonger 3,* and Lianne I. Willems 4,* 1 Laboratory of Chemical Biology, Department of Cellular and Molecular Medicine, KU Leuven, Herestr. 49, Box 802, 3000 Leuven, Belgium 2 AG Chemical Proteomics, Leibniz Institute for Analytical Sciences ISAS, e.V., Otto-Hahn-Str. 6b, 44227 Dortmund, Germany 3 Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands 4 York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, UK * Correspondence: [email protected] (S.H.L.V.); [email protected] (K.M.B.); [email protected] (L.I.W.) Academic Editor: Derek J. McPhee Received: 30 November 2020; Accepted: 17 December 2020; Published: 18 December 2020 Abstract: Activity-based protein profiling (ABPP) is a powerful technique to label and detect active enzyme species within cell lysates, cells, or whole animals. In the last two decades, a wide variety of applications and experimental read-out techniques have been pursued in order to increase our understanding of physiological and pathological processes, to identify novel drug targets, to evaluate selectivity of drugs, and to image probe targets in cells. Bioorthogonal chemistry has substantially contributed to the field of ABPP, as it allows the introduction of tags, which may be bulky or have unfavorable physicochemical properties, at a late stage in the experiment. In this review, we give an overview of the bioorthogonal reactions that have been implemented in ABPP, provide examples of applications of bioorthogonal chemistry in ABPP, and share some thoughts on future directions. Keywords: activity-based probes; activity-based protein profiling; bioorthogonal chemistry; chemoselective ligation; click chemistry; covalent inhibitors; enzyme probes; target identification 1. Introduction Approximately two decades ago, several seminal papers from the groups of Cravatt and Bogyo started the field of activity-based protein profiling (ABPP) [1–4]. ABPP was initially reported as a technique to detect active enzymes in a complex proteome, such as a cell lysate or a whole cell, with in-gel or immunoblot detection after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In the past two decades, the application areas have vastly expanded, accelerated by the implementation of bioorthogonal chemistry. This review is dedicated to discussing the topic of bioorthogonal chemistry in ABPP and highlighting recent advances in this field. ABPP makes use of chemical probes that covalently bind to an active target enzyme or enzyme family but not to inactive counterparts. They do so by making use of a mechanism-based reaction with active site residues. These activity-based probes (ABPs) (see Figure1A) usually consist of three elements. Molecules 2020, 25, 5994; doi:10.3390/molecules25245994 www.mdpi.com/journal/molecules Molecules 2020, 25, 5994 2 of 21 Molecules 2020, 25, x FOR PEER REVIEW 2 of 21 Figure 1.1. TheThe concept concept of ABPP and examplesexamples ofof probesprobes andand reactivereactive groups.groups. (A) SchematicSchematic representationrepresentation of one-one- andand two-steptwo-step ABPsABPs withwith theirtheir threethree structuralstructural elementselements (tag(tag oror bioorthogonalbioorthogonal handle, recognition element, and reactive group) and binding of a target protein byby thethe recognitionrecognition element. ( B) Example of the fluorophosphonate fluorophosphonate probe FP-RhFP-Rh (reactive group in red) and the covalent reactionreaction mechanismmechanism withwith thethe activeactive sitesite ofof aa serineserine hydrolase.hydrolase. (C) ExampleExample ofof epoxysuccinateepoxysuccinate probesprobes (reactive(reactive groupgroup inin red)red) andand the covalent reaction mechanismmechanism with the active site of a a cysteine cysteine protease. protease. ((DD)) StructureStructure of of probes probes based based on on the the 4-chloro-isocoumarin 4-chloro-isocoumar scain scaffoldffold (in red)(in red) for serinefor serine proteases, proteases, with with tags andtags recognitionand recognition elements elements at di ffaterent different positions. positions. (E) Two (E) examplesTwo examples of cyanimide of cyanimide probes probes for the for DUB the UCHL1DUB UCHL1 (reactive (reactive cyanimide cyanimide group group in red). in red). (1)(1) AA reactive groupgroup thatthat forms forms a a covalent covalent bond bond with with the the target target protein. protein. In theIn the case case of enzymes of enzymes that that use usea nucleophilic a nucleophilic amino amino acid sideacid chainside chain for attack for attack onto their onto substrates, their substrates, the reactive the reactive group is group usually is usuallyan electrophile. an electrophile. For example, For example, most serine most hydrolases serine hydrolases (SHs), a superfamily (SHs), a superfamily of enzymes thatof enzymes catalyze thatthe breakdowncatalyze the of breakdown amide and of ester amide bonds and [ester5], react bonds with [5], the react fluorophosphonate with the fluorophosphonate electrophile electrophilepresent in the present ABP FP-Rh in the (FigureABP FP-Rh1B) [ 6(Figure,7]. Because 1B) [6,7]. the nucleophilicBecause the nucleo attack ontophilic the attack electrophilic onto the electrophilictrap resembles trap the resembles first step the of thefirst catalytic step of the mechanism, catalytic mechanism, these probes these are consideredprobes are considered to be truly ‘activity-based’.to be truly ‘activity-based’. Consequently, Consequently, inactive enzyme inactive states,enzyme such states, as such zymogen as zymogen or inhibitor-bound or inhibitor- boundforms, forms, will not will react not react with with the ABPs. the ABPs. The The reactive reactive group group can can lead lead to to some some degree degree ofof probeprobe selectivity,selectivity, e.g., fluorophosphonates fluorophosphonates (Figure (Figure 11B)B) onlyonly reactreact withwith activatedactivated serineserine residuesresidues [[1],1], whereas epoxysuccinates (Figure 11C)C) selectivelyselectively reactreact withwith cysteinecysteine proteasesproteases [[3].3]. If the targettarget protein does not contain a nucleophilic residue in its active site, a photoaffinity photoaffinity labeling (PAL) strategystrategy can can be be used. used. Here, Here, covale covalentnt bond bond formation formation is ensured is ensured by a byphotoreactive a photoreactive group, group, such assuch a diazirine as a diazirine or a benzophenone. or a benzophenone. These probes These probes are generally are generally referred referred to as affinity-based to as affinity-based probes (AfBPs).probes (AfBPs). We will We not will discuss not discuss AfBPs AfBPs in detail in detail and andrefer refer for forthe the background background of of PAL PAL and and its applicationsapplications to a review that appeared elsewhere [[8].8]. (2)(2) AA spacer spacer or or recognition recognition element element that that induces induces a a high higherer degree degree of of selectivity selectivity to to the the target target protein. protein. Together withwith thethe reactivereactive group, group, it it will will determine determine which which target target proteins proteins are are covalently covalently modified modified [9]. [9]. Often, recognition elements are derivatives of the substrate, such as short stretches of amino acids (for proteases), modified mono- or polysaccharides (for glycosidases), or even whole Molecules 2020, 25, x FOR PEER REVIEW 3 of 21 proteins (for deubiquitinating enzymes; DUBs). The recognition element may also originate from a known inhibitor. For example, 4-chloro-isocoumarin (Figure 1D), a common heterocyclic scaffold used for covalent inhibition of serine proteases [10], has been equipped with biotin, alkyne, or fluorophores to target various soluble serine proteases [11,12], the intramembrane Molecules 2020, 25, 5994 3 of 21 protease family of rhomboids [13,14], as well as acyl protein thioesterase, a serine hydrolase that cleaves fatty acids from S-acylated cysteine residues [15]. Other examples include cyanimides 1 [16]Often, and recognition 2 [17], derived elements from areinhibitors derivatives of ubiquitin of the substrate,carboxy-terminal such as hydrolase short stretches L1 (UCHL1), of amino a DUBacids associated (for proteases), with modifiedvarious mono-human ordiseases polysaccharides, including (for neurodegeneration glycosidases), or and even cancer. whole Interestingly,proteins (for deubiquitinatingthese small-molecule enzymes; ABPs DUBs).are cell permeable, The recognition allowing element the study may alsoof DUBs originate in a cellularfrom a knownenvironment inhibitor. [16,17] For example,and even 4-chloro-isocoumarinin an in vivo zebrafish (Figure embryo1D), animal a common model heterocyclic [17]. (3) Asca tagffold that used enables for covalentdetection inhibition of the covalent of serine probe-protein proteases [complex10], has [18]. been Various equipped tags with have biotin, been usedalkyne, in ABPP, or fluorophores including tobiotin, target fluorophores, various soluble radioisotopes, serine proteases stable isotopes, [11,12], the and intramembrane nanoparticles. Theprotease introduced family oftag rhomboids determines [13 which,14], as possibilities well as acyl are protein available thioesterase, for read-out. a serine Many hydrolase studies thatuse thecleaves abovementioned fatty

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