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Detection of ubiquitin–proteasome enzymatic activities in cells: Application of activity-based probes to inhibitor development☆

Background: Synthetic probes that mimic natural substrates can enable the detection of enzymatic activities in a cellular environment. One area where such activity-based probes have been applied is the ubiquitin–proteasome pathway, which is emerging as an important therapeutic target. A family of reagents has been developed that specifically label deubiquitylating enzymes (DUBs) and facilitate characterization of their inhibitors. Scope of review: Here we focus on the application of probes for intracellular DUBs, a group of specific proteases involved in the ubiquitin proteasome system. In particular, the functional characterization of the active subunits of this family of proteases that specifically recognize ubiquitin and ubiquitin-like proteins will be discussed. In addition we present the potential and design of activity-based probes targeting kinases and phosphatases to study phosphorylation. Major conclusions: Synthetic molecular probes have increased our understanding of the functional role of DUBs in living cells. In addition to the detection of enzymatic activities of known members, activity-based probes have contributed to a number of functional assignments of previously uncharacterized enzymes. This method enables cellular validation of the specificity of small molecule DUB inhibitors. General significance: Molecular probes combined with mass spectrometry-based proteomics and cellular assays represent a powerful approach for discovery and functional validation, a concept that can be expanded to other enzyme classes. This addresses a need for more informative cell-based assays that are required to accelerate the drug development process. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.


Introduction
The last two decades have witnessed the development and application of activity based probes for the detection of enzymatic activities related to a wide range of protein post-translational modifications (PTMs). These tools have allowed for the detection of many enzymatic activities in relation to conjugation and deconjugation of PTMs. The greatest success and widest application of this approach have probably been found within the ubiquitin proteasome pathway [1]. The ubiquitin proteasome system (UPS) consists of three different elements with partially opposing, but complementary roles; the ubiquitin conjugating cascade, the deubiquitylating enzymes and the proteasome complex (Fig. 1). The ubiquitin conjugating cascade of enzymes (E1, E2, E3 and E4) is responsible for conjugating ubiquitin (Ub)/ubiquitin-like proteins (Ubls) to other proteins as signaling molecules in several biological processes [2,3]. The deubiquitylating enzymes (DUBs) are responsible for removing, trimming or editing the Ub/Ubl modifications from proteins [2]. The proteasome complex, composed of at least three different proteolytically active subunits β1, β2 and β5, degrades the majority of soluble proteins within the cell following polyubiquitylation [4]. The UPS not only enables the targeted destruction of proteins no longer required by the cell, but also enables the selective deactivation of signaling molecules, removal of pathogenic proteins and production of peptides for presentation to the immune system. The entire pathway comprises close to 1000 genes, is vital for many normal cellular functions and is frequently disrupted in cellular pathologies [5,6].
A likely explanation for the remarkable success of development of activity-based probes in the UPS when compared to small molecule probes related to other PTMs, is the ability to target cysteine and threonine proteases successfully by covalent inhibition. These represent the major enzymatic activities of DUBs and the proteasome respectively. Improved recognition of the protein-based ubiquitin and peptide-derived proteasome probes as compared with small molecule based probes may also contribute. The generation of activity based probes for the ubiquitin pathway as important tools to obtain insights into the ubiquitin proteasome system has been reviewed previously [7][8][9][10]. The focus of this review is the application of activity-based probes in the context of inhibitor development and characterization. This process represents a critical step in arriving at a better understanding of fundamental cellular processes and enables the development of novel targeted therapeutic agents.

Activity-based proteomics probes
Activity-based probes (ABPs) are defined by their ability to target subsets of a complex proteome based on labeling of specific enzyme classes in a covalent fashion. The incorporation of specific tags for detection and enrichment allows for visualization and further analysis for example by tandem mass spectrometry. The ABP approach is highly complementary to mass spectrometry-based proteomics due to the ability to enrich for specifically captured substrates with the aid of an affinity tag. This also distinguishes the method from the use of radiolabeled covalent inhibitors that support detection but not affinity enrichment. A critical parameter is also the choice of reactive group for covalent labeling of the target enzyme at or close to the enzyme active site. Depending on the mechanism of enzymatic catalysis different reactive groups can be chosen for covalent capture. The alkylation of nucleophilic residues (Cys, Ser, Thr) present in protease active sites by reactive electrophiles has been a very successful realization of this method. The utility of ABPs is particularly evident in the study of enzyme systems involved in the attachment and removal of PTMs. Due to the enzyme active site chemistries involved ABP development has been generally more fruitful for the targeting of deconjugating enzymes that usually contain strong nucleophiles at their active sites when compared to the respective ligases/transferases.

Proteasome active site probes
The discovery and development of proteasome inhibitors have been previously covered by comprehensive reviews [10,11]. Here, we will therefore only give a brief introduction to the proteasome complex and proteasome active site probes.
The proteolytically active sites of the proteasome complex are located on distinct subunits (for example β1, β2 and β5 in the constitutive proteasome) and this is reflected by the labeling pattern obtained by active site-directed proteasome probes [12]. The three subunits responsible for proteolysis also correspond to different activity profiles with respect to substrate specificity which were named after prototypic proteases: chymotrypsin-like activity for β5 (CT-L), trypsinlike activity for β2 (T-L) and petidylglutamyl peptide hydrolyzinglike (PGPH-L) activity (also referred to as caspase-like) for β1 [13]. Exchange of the active subunits with close homologues can be induced in immune cells [14] and the thymus [15]. This allows for the tuning of the peptide repertoire produced by proteasomal proteolysis, which underscores the importance of presentation of these peptides (after further processing steps) in immune surveillance.
Proteolytically active subunits of the proteasome have been targeted by probes based on peptide scaffolds bearing a C-terminal electrophile (Fig. 2). The most popular designs have been based on the trileucine vinylsulfone (L 3 VS) sub-structure and on functionalized epoxomicin derivatives. Profiling with these probes is enabled by detection with dansyl [16] and biotin [17,18] tags and with strong fluorophores [19,20] as well as by radiolabels [12,21]. Alternatively, a two step protocol can be applied whereby the tag is attached in a bioorthogonal ligation step following labeling of active proteasome subunits in a cell lysate [22]. This selective ligation is possible through application of the Staudinger ligation [23] or 1,4-triazole formation also known as "click" chemistry [24,25]. A more recent addition to the arsenal of proteasome-directed probes has been the Belacostin A derived dansyl-KF33955 probe [26]. This β-lactone containing probe was used successfully in the labeling of β1, β2 and β5 subunits of the 26S proteasome in HeLa cells as well as the 20S proteasome from erythrocytes.

Ubiquitin-derived probes
Ubiquitin derived ABPs [27,28] have been used for the profiling of the active DUB population in cell lines, primary cells and tissues [29]. Subsequently this concept has been extended to the study of proteases specific for ubiquitin-like proteins (Ubls) [30]. A complex proteome such as a cell or tissue lysate is labeled by incubation with ubiquitin probe, followed by SDS-PAGE separation and immunoblotting against the epitope tag genetically encoded in the probe construct. Thereby a read-out, which is dependent on DUB abundance and activity, is obtained. This approach was also extended to the investigation of cancer cell lines [29,31] and patient biopsies [31]. Particularly fruitful was the study of in vitro infection models of viral [32][33][34][35], parasitic [36,37] and bacterial [38,39] pathogens. In this way previously unknown DUBs encoded by pathogens were identified and other interactions of pathogen encoded factors with the host ubiquitin proteasome system were elucidated. DUB related pathogen-host interactions have been reviewed in detail elsewhere [40][41][42][43]. Ubiquitin probes have further enabled structural studies on DUB-ubiquitin complexes [44]. In these investigations the crystallization and X-ray diffraction of covalent adducts formed between enzyme and the ubiquitin probe allowed insights into some of the molecular determinants of interactions of DUBs with ubiquitylated substrates.
In addition to the utility of ubiquitin ABPs targeting Cys protease DUBs, MS-based proteomics experiments of ubiquitin probes have also been shown to retrieve other regulators of ubiquitylation, for example Metalloprotease DUBs were retrieved under native pull-down conditions [51]. This finding might be explained by the formation of non-covalent complexes which can be enriched in immunoprecipitations (IPs) under non-denaturing conditions. The targeting and retrieval of ubiquitin ligases by ubiquitin probes have also been observed [52]. Usually the reduced nucleophilicity of Cys residues in this enzyme class (when compared to Cys as part of the catalytic triad in DUB proteases) results in strongly reduced capture. This can apparently be compensated for to a certain extend by strong C-terminal electrophiles conjugated to the Ub probes [52].

Activity-based probes allow enhanced inhibitor screening
In addition to the above-mentioned applications activity-based proteomics probes have also enabled the development of pharmacologically active enzyme inhibitors. In one format, competition assays between an inhibitor candidate compound and the activitybased probe can be performed in cell lysates. In this type of assay format the competition between the small molecule inhibitor and the probe leads to a reduced labeling profile for the activity-based probe. The loss of signal for labeling of specific target enzymes allows assessment of the specificity of inhibition, while titration of concentration ranges provides a measure of IC 50 values for the respective inhibitor. Several features make this type of assay format particularly attractive: The approach represents a cell-based assay in which treatment by the inhibitor candidate can be performed on intact cells. In contrast to the majority of in vitro enzyme assays, a range of active cellular enzymes can be assayed simultaneously. This is only limited by the number of enzymes successfully labeled by the activity-based probe and the representation of active enzymes in the cellular proteome. Quantifiable results can be obtained using various readout formats including immunoblotting, in-gel fluorescence detection or IP followed by quantitative MSbased proteomics.
Such an approach has been employed successfully for a number of ABPs including in the identification and characterization of selective inhibitors of the proteasome [53,54], of cysteine proteases in the DUB enzyme family [51] and serine hydrolases in the fatty acid amide hydrolase class [55]. Promiscuous inhibitors can be tested in this way in order to demonstrate their broad inhibition profile [56] and competition assays can be applied to the characterization of the specificity of known drugs.

Characterization of the subunit specificity of proteasome inhibitors
The development of novel cancer therapeutics is a prime example where the success of bortezomib [16,57] for treatment of multiple myeloma [58] has provided proof of concept for the value of interventions targeting the ubiquitin proteasome system. The effects of Bortezomib (PS-341) on the activity and composition of proteasomes in multiple myeloma cells were investigated by labeling with proteasome probe Ada-[ 125 I]-Y-Ahx 3 L 3 VS demonstrating that the inhibitor selectively targets β5 and β1 subunits (as well as immunoproteasome subunits β5i and β1i) [57]. The corresponding experiments were carried out in the presence or absence of cytokines, which induce the formation of immunoproteasomes under conditions that may be encountered in the bone marrow.
A similar characterization of the subunit specificity of proteasome inhibition was also carried out for the orally active inhibitor NPI-0052 (Salinosporamide A) in multiple myeloma cells [59]. The results of competitive labeling with the proteasome probe Dansyl-Ahx 3 L 3 VS indicated that the inhibitor targets the β1, β2 and β5 subunits. This result was consistent with the finding that cleavage of fluorogenic substrates showed inhibition of the CT-L, T-L and PGPH-L activities of the proteasome. In a comparable study the specificity of the three proteasome inhibitors BzLLLCOCHO, Bortezomib (PS-341) and MG132 was assessed by labeling with the cell permeable Dansyl-Ahx 3 L 3 VS probe following treatment with individual inhibitors for 24 h [60]. This demonstrated clearly distinct labeling pattern of the proteolytically active proteasome subunits β1, β2 and β5 for the three inhibitors. The observed labeling patterns were positively correlated with the inhibition of the CT-L, T-L and PGPH-L activities of the proteasome by hydrolysis of suitable fluorogenic substrates in the presence of inhibitors.
The selective inhibition of the immunoproteasome subunit β1i (LMP2) by synthetic Dihydroeponemycin analogs was demonstrated by competitive labeling experiments in EL4 cells using biotinepoxomicin and biotin-eponemycin as a proteasome active site probes [61].
These examples highlight the value of utilizing active site-directed probes in the development and characterization of proteasome inhibitors. This is enabled by their ability to provide a rapid assay of the corresponding proteolytically active subunits in different cell types and in the presence or absence of added competitive inhibitors the specificities of which can thereby be assessed. This approach has been informative to basic science as well as to the development of lead compounds for novel therapeutics.

Competitive ubiquitin probe labeling for DUB inhibitor development
Due to the large number of cellular DUBs there is a need for innovative assay designs that allow assessment of DUB activity in a cellular context. Competitive labeling with Ub probes provides characterization of lead compounds in a cell culture model thereby providing valuable data for assessment of both compound potency and selectivity of different DUBs relevant to a given cell type. A number of DUB inhibitors have been characterized and developed into lead compounds for medicinal chemistry programs. Generally the initial hit has been obtained by high throughput screening (HTS) of compound libraries followed by further optimization using structureactivity relationships. It is noteworthy that many of the identified lead compounds and inhibitors have the ability to covalently label the active site cysteine residue present in the majority of DUBs (Fig. 3). This may occur by conjugate addition, nucleophilic aromatic substitution or disulfide bond formation depending on the chemistry of the small molecule inhibitor.
One example of Ub probe labeling in the presence of a small molecule inhibitor has been the characterization of WP1130 as a partially selective DUB inhibitor which induces aggresome formation and tumor cell apoptosis by Kapuria et al. [62]. The selectivity of inhibition for different members of the DUB family of enzymes was also investigated by in vitro inhibition assays using UbAMC as a substrate in combination with recombinant purified DUBs. The total cellular DUB activity in lysates treated with inhibitors was tested by UbAMC hydrolysis. Labeling of treated versus untreated cell lysates with the activity-based probe HAUbVS was performed in order to assess the DUB inhibitory profile of WP1130. In this assay format reduced labeling by the HAUbVS probe as detected in anti-HA immunoblotting of lysates from cells treated with WP1130 was used as readout of selectivity of DUB inhibition. A further focus of this work was the observation of accumulation of polyubiquitylated protein material upon treatment with WP1130 despite a lack of inhibition of proteasome activity. Altun et al. described a more extensive application of activitybased probes to the characterization and development of novel DUB inhibitors [51]. In this study two DUB inhibitors, PR-619 and P22077, were characterized by in vitro enzyme inhibition assays and in cell culture models. Labeling of HEK293T cell lysates treated with inhibitors at varying concentrations was performed with the ubiquitin probes HAUbVME and HAUbBr2 to investigate DUB inhibition profiles. As a read-out of these assays immunoblotting against HA-tag or specific DUBs of interest was carried out enabling a rapid assessment of inhibitor specificity. In order to obtain an alternative quantitative read-out format, anti-HA immunoprecipitation (IP) was combined with identification and label-free quantification by mass spectrometry based proteomics (Fig. 4). Using this approach, quantitative data illustrating the degree of inhibition of 25 cellular DUBs was obtained. These data were consistent with the in vitro enzyme characterization data, demonstrating that PR-619 exhibits a broad inhibition profile whereas P22077 is a selective inhibitor of USP7 and USP47. Both USP7 and USP47 are considered oncology targets and P22077 and related analogs are the subject of further development as anti-cancer therapeutics [63,64].
Another application of Ub probe labeling for the development of USP7 inhibitors was recently demonstrated by Reverdy et al. [65]. The screening hit HBX 19,818, which had been identified by high throughput screening (HTS) of a compound library employing the UbAMC assay, was characterized by HAUbVS labeling of HCT116 cells treated with increasing concentrations of the inhibitor. The readout of the assay was performed by anti-HA immunoblotting and showed reduced probe labeling of USP7 while leaving the competitive labeling of other cellular DUBs unaffected. This result was consistent with the in vitro enzyme inhibition data that also indicated a lack of inhibition of other tested cysteine proteases, DUBs and Ubl specific proteases, but the activity of HBX 19,818 towards the closely related DUB USP47 was not discussed. The mechanism of inhibition was investigated by the authors and covalent labeling of the active site Cys223 in USP7 was demonstrated, indicating that alkylation of this residue leads to irreversible inactivation of the DUB. Furthermore the cellular effects of the inhibitor were characterized as reduction in cell proliferation, induction of apoptosis and G1 arrest in a dosedependent fashion. A novel DUB inhibitor, b-AP15 was recently described by D'Arcy et al. [66]. DUB profiling was carried out in the presence of ubiquitin probe HAUbVS in HCT-116 cells after 3 h treatment with 1.0 μM inhibitor; demonstrating that USP14 and to a lesser extend UCHL5 were inhibited at this concentration. Treatment of cells with b-AP15 led to tumor cell apoptosis, which was independent of TP53 status. Additionally an accumulation of high molecular weight Ub conjugates and reduced levels of free ubiquitin were observed and ascribed to the inhibition of the 19S associated DUBs USP14 and UCHL5. The authors concluded that the compound does not act as a broad spectrum DUB inhibitor at the concentration employed, rather it inhibits the proteasome associated DUBs USP14 and UCH-L5. Thus, b-AP15 effectively inhibits proteasomal activity by inhibiting the DUBs that process polyubiquitylated proteins prior to their engagement by the proteasome.
This approach has utility for viral DUBs as well. The ubiquitin probe HAUbVS has been successfully applied to demonstrate the selectivity of inhibitor GRL0617 for the SARS Coronavirus protease and isopeptidase Papain-like protease (Plpro) in Vero E6 cell lysate [67]. No change in labeling profile was observed for cellular DUBs at inhibitor concentrations of up to 40 μM. In contrast, the labeling of added Plpro was almost completely abolished at the same inhibitor concentration.

Utility of activity based proteomics in inhibitor characterization outside the UPS
The usefulness of activity based probes (ABPs) in the development and characterization of enzyme inhibitors has also been recognized in fields outside the ubiquitin proteasome system. Below we describe the advances made in the application of this concept to kinases and phosphatases involved in protein phosphorylation. ABPs for the targeting of kinases and phosphatases have received considerable attention due to the central role of protein phosphorylation in cellular signaling, protein translocation and ultimately its aberrations in disease.

Kinase targeted activity-based probes
Initial kinase probes have utilized ATP and ADP analogs as molecular scaffolds thereby targeting the essential kinase co-factor in probe design. Later approaches were built on modification of known kinase inhibitors of natural product origin or related to licensed drugs. Kinase probes based on acyl phosphate ATP and ADP analogs which lead to the transfer of a biotin to amino acid residues on the interacting protein resulted in the labeling and identification of a range of kinases, ATPases, nucleotide biosynthesis-related and other proteins [68]. The lack of selectivity for kinases in a cellular environment is understandable given the wide range of proteins that utilize ATP as a cofactor.
A tetramethylrhodamine-labeled Wortmannin analog AX7503 allowed labeling in mammalian cell lysates and readout of the assay by in gel fluorescence [69]. This fluorescent kinase probe labeled DNA dependent protein kinase (DNA-PK), phosphoinositide 3 kinase and mammalian Polo-like kinase 1 (PLK1). Previously it had not been appreciated that PLK1 is inhibited by Wortmannin at relevant concentrations. Further Wortmannin derivatives labeled with borondipyrromethene (BODIPY), tetramethylrhodamine or biotin were prepared successfully [70]. It was shown that the BODIPY derivative is cell-permeable thereby enabled labeling of intact cells while the biotin derivative allows isolation of labeled protein material from cell lysates. In a follow-up study to this work Liu et al. also demonstrated that Polo-like kinase 3 (PLK3) is a further molecular target of Wortmannin as shown by competitive labeling between AX7503 and Wortmannin [71]. The labeling sites of AX7503 for both PLK1 and PLK3 were determined by tandem mass spectrometry and were found to represent conserved lysine residues in the ATP binding site of each of the kinases.
A bisindolylmaleimide motif was chosen as the scaffold for the design of a further kinase probe AX4697 [72]. This recognition motif with a high affinity for protein kinase C (PKC) was combined with a chloroacetamide electrophile and a tetramethylrhodamine fluorophore. Successful labeling of α and β PKC in Jurkat cells was demonstrated as well as loss of signal in staurosporine-pretreated cells.
A recent design of a probe for the Abelson Tyr kinase (Abl) was based on the anti-cancer drug imatinib [73]. The reversible mode of enzyme inhibition necessitated the incorporation of a photoreactive benzophenone moiety or a dialdehyde motif for covalent labeling. Detection was enabled by the incorporation of a terminal alkyne into the structure allowing attachment of a rhodamine azide dye by Cu(I) catalyzed triazole formation (click chemistry) [24,25]. Targeting and detection of Abl kinase in the presence of added cell lysate occurred, but the labeling efficiency diminished with increasing amounts of lysate and the approach did not extend to labeling and detection at endogenous levels.
The reversible and non-selective kinase inhibitor staurosporine has also been utilized as the recognition motif in probe design by several groups. In these probe designs covalent labeling is enabled by incorporation of photoreactive phenylazide [74] or diazirine [75] groups and irradiation by UV light. Enrichment of the trapped proteins is performed by a further incorporated biotin moiety [74] or a terminal alkyne for attachment of either a biotin group or a fluorophore for detection following the covalent capture by click chemistry [75].

Phosphatase targeted activity-based probes
Activity based probes for protein tyrosine phosphatases (PTPs) were developed on several scaffolds that allow mimicry of phosphotyrosine motifs while incorporating reactive electrophiles for the covalent capture of active phosphatases (Fig. 5). Several groups exploited the generation of quinone methides for the generation of reactive electrophiles for trapping of PTPs [76][77][78][79]. These are generated from substituted phenyl phosphates with benzylic fluoride substituents in ortho or para position. In a recent publication this concept was extended to the design of the unnatural amino acid 2fluoromethyl phosphotyrosine which was incorporated into peptides by solid phase peptide synthesis (SPPS) [80]. In this way peptidic activity based probes were produced based on known PTP target sequences.
In earlier work Kumar et al. combined a minimal motif incorporating a bromomethyl phosphonate-substituted phenyl derivative attached to either a fluorophore or biotin for detection [81]. Successful labeling of PTPs was demonstrated using both isolated enzymes in vitro and in cell lysates of different cancer cell lines.
A further alternative approach exploited conjugate addition to substituted aryl vinyl sulfonates and sulfones for the covalent labeling of active PTPs [82]. This methodology was then applied to a structural study of a covalent complex between the bacterial PTP YopH and its mechanism based inhibitor.
Recently a novel probe design was implemented that enables microscopy-based read out through the use of quenched activity based probes [83]. In this strategy the quencher is released upon quinone methide formation and reaction with the target enzyme. The methodology has also been applied to one-and two-photon imaging experiments.
In comparison, the targeting of Ser/Thr phosphatases by activitybased probes has been less frequently realized. A successful example is that of a microcystin derived probe which was prepared and applied to the labeling and isolation of endogenous levels of Ser/Thr protein phosphatases from Jurkat cell lysate [84].

Characterization of kinase inhibitors by competition assays and targeted proteomics
Acylphosphate kinase probes based on the ATP cofactor [68] were used recently in an extensive study evaluating the specificity profiles of a range of kinase inhibitors [85]. In this 'in situ native kinase profiling' (KiNativ) approach, labeling of mammalian cell lysates with the kinase probes was conducted in the presence or  absence of the respective inhibitor. The complex samples were then digested with trypsin and biotinylated peptides enriched with streptavidin resin. Following elution the captured peptides were analyzed by quantitative proteomics experiments using a targeted LC-MS 2 approach. This detection method significantly improved the signal to noise ratios and allows detection of lower amounts of analyte. Quantitative inhibition data for over 200 kinases with six kinase inhibitors was obtained by this method. This approach provides access to quantitative inhibition profiles that are not easily obtained by other experimental approaches and exploits the strengths of both activity-based probe profiling and targeted mass spectrometry-based proteomics.

Future perspectives
The introduction of activity-based probes into the development of enzyme inhibitors enables the assessment of compound selectivity in a cellular context. This ideally complements more conventional in vitro enzyme assays and allows implementation of this assay format at an early stage in drug development. Furthermore competitive labeling experiments have greatly aided the definition of specificities of inhibitors used in cellular research. New therapies in the UPS will employ the manipulation of DUB activity in a highly selective manner to achieve the desired outcomes. Therefore inhibition of selected intracellular DUB activities will receive increasing attention as a strategy for pharmacological intervention in disease. DUBs with a validated role in oncology are expected to be the first in line for future DUB inhibitors. Provided that challenges of developing specific inhibitors with desirable compound profiles can be overcome, it is expected that this will enable successful intervention in other pathologies such as inflammatory diseases.

Summary
Activity-based probe designs have provided insights into multiple cellular pathways and have proven extremely valuable as tools for the study of PTM conjugating and deconjugating enzymes. The investigation of the ubiquitin proteasome system has benefited from the development of probes for proteasomal proteolysis as well as deubiquitylating enzymes. Specificities and overlapping reactivities of ubiquitin-like proteases were investigated by analogous Ubl probes. Inhibitor development has been facilitated through competitive labeling assays enabling elucidation of inhibition profiles in a cellular environment. In addition, natural product derived kinase probes have demonstrated the protein targets of these covalent inhibitors, indicating previously unknown molecular targets and providing information in situations where specificities of the respective compounds are not always fully elucidated. The importance of access to selective inhibitors for basic cell biology and drug development makes this area highly suited for successful industry-academia collaborations.