Improved Deconvolution of Protein Targets for Bioactive Compounds

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Improved Deconvolution of Protein Targets for Bioactive Compounds Using a Palladium Cleavable Chloroalkane Capture Tag Rachel Friedman Ohana, Sergiy Levin, Monika G. Wood, Kris Zimmerman, Melanie L. Dart, Marie K. Schwinn, Thomas A. Kirkland, Robin Hurst, H. Tetsuo Uyeda, Lance P Encell, and Keith V. Wood ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00408 • Publication Date (Web): 14 Jul 2016 Downloaded from http://pubs.acs.org on July 18, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Biology

Improved Deconvolution of Protein Targets for Bioactive Compounds Using a Palladium Cleavable Chloroalkane Capture Tag

*○†Rachel Friedman Ohana, ○‡Sergiy Levin, †Monika G. Wood, †Kris Zimmerman, †Melanie L. Dart, †Marie K. Schwinn, ‡Thomas A. Kirkland, †Robin Hurst, ‡H. Tetsuo Uyeda, †Lance P. Encell and †Keith V. Wood

†

Promega Corporation, Madison, WI, USA

‡

Promega Biosciences LLC, San Luis Obispo, CA, USA

*Corresponding Author: [email protected]

○

These authors contributed equally to this work.

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT The benefits provided by phenotypic screening of compound libraries are often countered by difficulties in identifying the underlying cellular targets. We recently described a new approach utilizing a chloroalkane capture tag, which can be chemically attached to bioactive compounds to facilitate the isolation of their respective targets for subsequent identification by mass spectrometry. The tag minimally affects compound potency and membrane permeability, enabling target engagement inside cells. Effective enrichment of these targets is achieved through selectivity in both their rapid capture onto immobilized HaloTag and their subsequent release by competitive elution. Here, we describe a significant improvement to this method where selective elution was achieved through palladium-catalyzed cleavage of an allyl-carbamate linkage incorporated into the chloroalkane capture tag. Selective tag cleavage provided robust release of captured targets exhibiting different modes of binding to the bioactive compound, including prolonged residence time and covalent interactions. Using the kinase inhibitors ibrutinib and BIRB796 as model compounds, we demonstrated the capability of this new method to identify both expected targets and “off-targets” exhibiting a range of binding affinities, cellular abundances and binding characteristics.


Page 2 of 40

Page 3 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION The benefits of phenotypic screening for discovering bioactive compounds able to induce a desired cellular response have been well recognized.1, 2 Yet, identifying the underlying cellular targets of these compounds remains a critical challenge for understanding their mode of action, side effects involving “off-targets”, and overall therapeutic potential. 1-4 We recently described a new method facilitating target enrichment from cultured cells, including targets exhibiting low affinity, low abundance or fast dissociation rates.5 The method utilizes a chloroalkane capture tag that can be chemically attached to bioactive compounds, enabling the isolation of their respective cellular targets for subsequent identification by mass spectrometry. The tag was shown to minimally alter compound potency in cultured cells, allowing binding interactions to occur under conditions relevant to the desired cellular phenotype. Maintained potency ensures that compounds modified by the appended tag retain their capacity to engage with relevant pharmacological targets and that they can therefore be used for target isolation. Because bioactive compounds generally interact reversibly with their cellular targets, effective enrichment requires rapid isolation of tagged compounds with their bound targets from cultured cells. This is achieved through capture of the chloroalkane tagged compounds onto immobilized HaloTag6, which was shown to be significantly faster than a similarly configured system utilizing a biotin-tagged compound.5 Selective release of the bound targets is then accomplished by competitive elution using excess unmodified compound. Competitive elution reduces release of nonspecifically bound proteins. This can facilitate target identification by improving the sensitivity of mass spectrometry, particularly for targets with low abundance or low affinity.3, 4, 7 However, the efficiency of this approach relies on the aqueous solubility of the bioactive compound4, 7 and its dissociation rates from bound targets. This may present a challenge for early-stage research compounds, which often exhibit low solubility. Furthermore, dissociation rates can vary widely among diverse cellular targets. Finally, elution is difficult for compounds with prolonged residence times, and for covalent interactions the use of competitive elution is not possible. A cleavable tag would address the shortcomings of competitive elution and still provide selective release of captured targets. Ideally, the cleavable tag would retain previously established features of the chloroalkane capture tag, such as minimal influence on compound potency and cell permeability, as well as efficient capture of cellular targets. This could be achieved by incorporating into the chloroalkane tag a small chemically-cleavable linkage, which would minimally affect the tag’s physical properties. This linkage should be stable in cellular environments and common buffers, but susceptible to quantitative and selective cleavage upon treatment with a mild reagent. 3 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A variety of bioorthogonal cleavage chemistries exist, yet most lack one or more of the aforementioned desired features.8, 9 For example, disulfide linkages are small and require mild cleavage conditions, but they are unstable in cellular environments. Unfortunately, stability of many other linkages coincides with harsh cleavage conditions such as low pH, intense ultraviolet light or oxidative reagents, all which can damage proteins and increase nonspecific background released from the affinity surface. In contrast, stable linkages susceptible to mild cleavage conditions (e.g., azobenzene derivatives) generally require bulky groups, which can limit cell permeability, alter binding interactions with cellular targets, and cause nonspecific protein binding to the capture tag. We opted to use a small allyl-carbamate linkage that can be specifically cleaved by a water-soluble palladium catalyst.10 We hypothesized that a minor structural change to the chloroalkane tag, i.e., replacing an alkyl-carbamate with an allyl-carbamate, would introduce a bond susceptible to mild cleavage conditions while minimally influencing other tag properties (Figure 1, panel B). Briefly, a phosphine-derived palladium catalyst causes breakage of a carbonoxygen bond in the allyl-carbamate linkage. The released carbamic acid spontaneously decomposes to yield an amine and CO2, while an exogenous amine nucleophile displaces palladium from the allyl-palladium intermediate10 (Figure 1, panel A). Despite widespread use of palladium catalyzed allylic substitution reactions in organic chemistry, their utility has only recently been demonstrated in the context of complex biological environments.11-13 This is likely due to potential decrease in catalyst reactivity caused by competing interactions with protein functional groups (e.g., amines, amides and thiols).14 By screening a panel of water-soluble phosphines in combination with various nucleophiles, we identified a pair that supported highly efficient cleavage of an allyl-carbamate linkage in a proteinaceous environment. We then utilized the cleavable chloroalkane capture tag to test the capability of this new approach to enrich cellular targets of two model compounds, ibrutinib15 and BIRB796,16, 17 which bind to their primary targets covalently15 or with prolonged residence time,16, 17 respectively. We identified the expected kinase targets for both compounds, demonstrating the applicability of this method to different compound interaction modes. We also identified and verified multiple kinase and non-kinase “off-targets” of BIRB796, some of which have not been previously described. Three of these “off-targets”, cyclin C, the cyclin-dependent kinase (CDK) CDK8, and CDK19 belong to the CDK mediator module.18 This suggests that in addition to its effects on inflammation,16, 17 BIRB796 may impact the regulation of transcription.


Page 4 of 40

Page 5 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RESULTS AND DISCUSSION

Optimization of palladium-catalyzed cleavage reaction Efficient release of captured cellular targets requires effective palladium-catalyzed cleavage of the chloroalkane tag when it is covalently bound to HaloTag.8, 9 We tested this by replacing the alkyl-carbamate in the chloroalkane tag CA-T15 with an allyl-carbamate, generating CA-T2Z and CA-T2E wherein the allyl group double bond is in cis or trans configuration, respectively (Figure 1, panel B). By appending each tag with a tetramethylrhodamine (TAMRA) fluorophore, we covalently labeled HaloTag with TAMRA and monitored cleavage of the bound tags by loss of fluorescence. Cleavage was performed using a catalyst generated from sodium tetrachloropalladate (Na2PdCl4) and a commonly used phosphine, triarylphosphine tris-(sulfonate) (TPPTS),19 in HEPES buffer, which also served as the amine nucleophile. We found that, when bound to HaloTag, tags bearing an allyl-carbamate linkage were efficiently and selectively cleaved in a manner dependent on both catalyst concentration and time (Figure 1, panel C). We further tested cleavage efficiency in the presence of cell lysate because competing interactions of proteins with the catalyst may decrease its reactivity.14 Not surprisingly, cleavage was significantly reduced in a manner dependent on lysate concentration (Figure 1, panel D). This negative influence of a proteinaceous environment was even more pronounced when cleavage of 6-TAMRA-CA-T2Z bound to HaloTag coated beads was carried out in the presence of increasing amounts of beads (Supporting Figure 1, panel A). To optimize the reaction, we chose challenging conditions relevant to our desired application (i.e., cleavage of 6-TAMRA-CA-T2Z bound to beads containing 400 µg immobilized HaloTag), which yielded cleavage efficiencies of 20% or less. The choice of phosphine ligand and nucleophile can significantly influence catalyst reactivity and cleavage efficiency.10 However, given the complex kinetics of palladium-catalyzed allylic substitutions, 20-22 it was difficult to predict the optimal combination that would enhance cleavage efficiency within a proteinaceous environment. Therefore, we screened a panel of commercially available water-soluble phosphines for enhanced cleavage efficiency relative to TPPTS. We found that electron-deficient sulfonated phosphines of the DANPHOS23 family, particularly DANPHOS and o-DANPHOS, significantly enhanced cleavage efficiency (Figure 1, panel E and Supporting Figure 2). Notably, the cleavage efficiency rank order (oDANPHOS>DANPHOS>p-DANPHOS>TPPTS) correlates well with the relative electronegativity of these phosphines.23 It is not clear why catalysts derived from these electron-deficient phosphines are more reactive than those derived from the more electron-rich TPPTS.20-22 It is known that electron-deficient phosphines reduce palladium to its catalytically active zero 5 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

oxidation state faster than electron-rich phosphines,24 but this is probably not the cause for their higher reactivity. Amatore et. al19 showed that reduction of palladium (II) by TPPTS takes approximately one hour. Since we ensured palladium was completely reduced by waiting at least 24 h before using the catalyst solutions, we would not expect reduction rate to be a factor. Having selected the optimal phosphines (DANPHOS and o-DANPHOS) for this reaction, we screened a panel of nucleophiles for their ability to further enhance cleavage efficiency. We included common amine-based buffers and frequently used allyl-palladium scavengers such as carbon-based and sulfur-based nucleophiles (Figure 1, panel F and Supporting Figure 3). oDANPHOS outperformed DANPHOS with all tested nucleophiles. In combination with the amine-based buffer, MOPS, 0.22—2 mM o-DANPHOS-derived palladium catalyst provided nearly complete cleavage within 30 min. It is likely that under these conditions, the nucleophilic morpholine associated with MOPS is most effective at displacing palladium from the allyl group. The molar ratio of phosphine to palladium was also investigated. Excess phosphine is required to stabilize the catalyst, but when this excess is too high it can lead to inhibition of the catalytic process.24 By testing a range of o-DANPHOS-to-palladium ratios we confirmed that an 8-fold molar excess of phosphine over palladium was optimal (Supporting Figure 1, panel B). In conclusion, we identified a combination of phosphine and nucleophile (i.e., o-DANPHOS and MOPS) that provided highly efficient palladium-catalyzed cleavage of an allyl-carbamate linkage in a protein-rich environment relevant to our enrichment process.

Structure Optimization of Cleavable Chloroalkane Tag Given the putative size of the o-DANPHOS-derived palladium catalyst, we suspected its access to the cleavage site could be restricted when the captured tagged compound is also bound to a protein target. Partial occlusion of the cleavage site is suggested by a space filling model of HaloTag and MAPK14 (the primary target of BIRB796 16, 17) bound simultaneously to BIRB796 conjugated to CA-T2Z (BIRB796-CA-T2Z) (Figure 2, panel A). To address this, we synthesized a series of incrementally longer cleavable tags (Figure 2, panel B). During evaluation of these cleavable tags we were mindful that, in addition to efficient cleavage, previously established capabilities for engagement with intracellular targets and efficient capture onto HaloTag coated beads must be retained.5 To minimize compound related bias, we compared these tags for the aforementioned parameters using three kinase inhibitors. We chose inhibitors that are not suitable for competitive elution because they interact with their primary targets either covalently (ibrutinib15) or with prolonged residence time (ponatinib25 and BIRB79616, 17). 6 ACS Paragon Plus Environment

Page 6 of 40

Page 7 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


We interrogated the influence of these tags on compound potency using both biochemical and cellular assays. Inhibition of mitogen-activated protein kinases (MAPK) by BIRB796 reduces production of proinflammatory cytokines such as TNFα.17 Therefore, we compared the BIRB796 conjugates for inhibition of both purified MAPK14 and TNFα secretion from LPS-stimulated THP-1 cells (Figure 3, panel A and Supporting Figure 4, panels A and B). In both assays, we observed an inverse correlation between the length of the cleavable tag and its influence on BIRB796 potency. Interestingly, only the longer tags, CA-T4Z and CA-T4E, exhibited potency similar to CA-T1. Ponatinib inhibits the BCR-ABL oncogenic signaling pathway, thereby reducing the downstream STAT5 activity.25 We compared the ponatinib conjugates for inhibition of both purified ABL1 and STAT5 reporter expression in K-562 cells (Figure 3, panel B and Supporting Figure 4, panels C and D). In both assays only the shorter tags, CA-T2Z and CA-T2E, reduced ponatinib potency relative to CA-T1. Finally, ibrutinib targets BTK and thereby inhibits BCR signaling and its downstream NFκ B activated survival pathway.15 We compared ibrutinib conjugates for inhibition of both purified BTK and NFκB reporter expression in anti-IgM stimulated Ramos cells. Unlike the other models, we did not observe any reduction in potency relative to CA-T1 (Figure 3, panel C and Supporting Figure 4, panels E and F). These results indicate that for these model compounds the longest tags, CA-T4Z and CAT4E, minimally influenced compound potency (reducing it at most by 6-fold). The larger effect on potency displayed by shorter tags suggests potential influence of the allyl group on binding interactions, which is ameliorated by increased distance between the allyl and the compound. The tags displayed similar relative influence on compounds potency in biochemical and cellular assays (Figure 3, panels A, B, and C), suggesting cell permeability is not a limiting factor for the binding of these conjugates to their respective targets. Since cellular assays were performed over 24 h, a shorter incubation time was required to verify minimal influence of the tags on cell permeability. For this, we compared the binding kinetics of the tagged compounds to HaloTag in lysates and intact cells (Supporting Figure 5). The tags displayed similar relative influence on binding kinetics in both assays, indicating the tagged compounds maintained cell permeability. Notably, cleavable tags with a trans double bond (i.e., CA-T2E, CA-T3E and CA-T4E) displayed faster binding to HaloTag than respective tags with a cis double bond (Supporting Figure 5). The binding kinetics of CA-T4E was most similar to CA-T1. We also compared tags for their ability to capture cellular targets by genetically fusing them to NanoLuc luciferase (NLuc).26 Following incubation of cells expressing NLuc fusions with the tagged compounds, cells were lysed and tagged compounds together with their bound 7 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

targets were captured onto HaloTag beads. Capture efficiency was determined based on decreased luminescence in the unbound fractions relative to untreated cellular lysates (Figure 3, panel D). We found the length of the cleavable tag had a significant impact on capture efficiency. Compared to CA-T1, tags of equal or shorter length exhibited reduced capture, while longer tags exhibited similar or greater capture. In particular, CA-T4Z and CA-T4E were most effective at capturing targets. Because effective target enrichment depends on both capture and elution, we also compared the tags for efficiency of palladium-catalyzed elution. Briefly, we engaged tagged compounds with targets fused to NLuc in a cellular lysate, and subsequently captured them onto HaloTag beads. We then compared palladium-catalyzed elution by tag cleavage to other elution methods (Figure 4). As BIRB796 exhibits slow dissociation from its primary targets,16, 17 palladium-catalyzed elution of a NLuc:MAPK9 fusion was compared to non-selective elution by SDS (Figure 4, panel A). Unlike SDS, the efficiency of palladium-catalyzed elution correlated with tag length. Only the longest tags (CA-T4Z and CA-T4E) exhibited similar enrichment by both elution methods, suggesting the increased length facilitated catalyst accessibility to the cleavage site. Notably, palladium treatment also led to minor release of NLuc:MAPK9 captured by BIRB796-CA-T1, which does not contain an allyl group. We suspect this may be caused by palladium interaction with functional groups found in proteins. We then used the covalent interaction between ibrutinib and BTK to evaluate palladium-catalyzed elution of a covalently bound target. As SDS elution would be ineffective in this scenario, we inserted a TEV protease recognition site between NLuc and BTK in the BTK:NLuc fusion to compare palladium-catalyzed elution of BTK:NLuc with proteolytic elution of NLuc (Figure 4, panel B). For each tag we observed similar enrichment by both elution methods, indicating catalyst accessibility to the cleavage site is not a limiting factor for this model. Notably, longer tags provided the best enrichment, presumably due to their ability to promote higher capture efficiency. Data presented so far indicate that the longest cleavable tags tested, CA-T4Z and CAT4E, provide a significant advantage over CA-T1. They exhibit minimal influence on compound potency and cell permeability, promote better capture of interacting targets, and enable efficient palladium-catalyzed elution of these targets. We favored CA-T4E due to its faster binding kinetics to HaloTag. Because endogenous proteins are often expressed at levels lower than recombinant targets, effective enrichment can require a larger mass of cultured cells.5 Anticipating the increased cellular content may reduce cleavage efficiency, we tested enrichment from higher cell numbers (1—5 x 107 cells) and found that efficient enrichment required a similar increase in the amount of catalyst (Supporting Figure 6).


Page 8 of 40

Page 9 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Although the extended length of CA-T4E would be expected to reduce steric crowding, it is counterintuitive that a longer tag would also have minimal influence on cell permeability. We suspected the four carbamate groups in CA-T4E may be important for retention of cell permeability and speculated that replacing them with amides, which are structurally comparable but more polar, would lead to reduced permeability. Since the carbamate proximal to the chloride is necessary for rapid binding to HaloTag,5 we synthesized an analogous tag replacing the three remaining carbamates with amides. Appending ponatinib to both tags, we compared their influence on binding to HaloTag and ABL1 in biochemical and cellular assays. Both tags exhibited similar binding kinetics to HaloTag in lysate and comparable inhibition of purified ABL1, indicating the amides had no influence on binding to either protein (Supporting Figure 7, panels B and D). In cellular assays amides reduced binding kinetics to intracellular HaloTag and decreased ponatinib potency by 20-fold, implying substantial impact on cell permeability (Supporting Figure 7, panels C and E). These results suggest carbamates contribute to the minimal influence of CA-T4E on cell permeability.

Enrichment of Endogenous BIRB796 Targets To evaluate the capability to enrich endogenous targets, we compared the new method utilizing palladium-catalyzed release to our earlier method employing competitive elution.5 BIRB796 was an appropriate model because it presents several challenges. The MAPK targets differ in cellular abundance and exhibit a range of affinities for BIRB796 with generally slow dissociation rates.16, 17, 27 Using CA-T1 and CA-T4E appended to BIRB796, we compared the enrichment of relevant MAPK targets from THP-1 cells using competitive elution with 400 µM BIRB796 and treatment with 6 mM palladium catalyst, respectively (Figure 5, panel A). Western analysis revealed significantly higher enrichment by the new method, which was likely due to both enhanced capture afforded by CA-T4E and a more efficient elution. We then investigated the influence of binding affinity and dissociation rates of BIRB796 for these MAPKs on enrichment. We previously demonstrated that bioluminescence resonance energy transfer (BRET) can be used to interrogate binding of a compound with its protein targets within cell lysates or inside live cells.5, 28 Exchanging the chloroalkane with a nonchloroTOM (NCT) fluorophore, we synthesized BIRB796-NCT and confirmed by BRET that it binds specifically to MAPK targets fused to NLuc. Binding affinities determined through competitive displacement of BIRB796-NCT by unmodified BIRB796 were generally consistent with reported values,16, 27 demonstrating a range of binding strength to BIRB796 (nM—low µM) (Figure 5, panel B). Although BIRB796 has been shown to have prolonged residence time for MAPK targets,16, 17 relative dissociation rates were unknown. We used BRET to monitor real-time 9 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 40

competitive displacement of unmodified BIRB796 by BIRB796-NCT in cells expressing MAPK:NLuc fusions. BIRB796 displayed a range of apparent dissociation kinetics for these MAPKs, affecting the ability to enrich them by competitive elution (Figure 5, panel C and Supporting Figure 8). The extremely slow dissociation observed for the high affinity targets, MAPK14 and MAPK11, is likely the cause for their poor or undetectable enrichment by competitive elution. Further, the efficient enrichment of low affinity MAPK12 by the same method is probably due to fast dissociation. These results demonstrate the dependence of competitive elution on residence time. Furthermore, they emphasize the advantage of the cleavable chloroalkane capture tag as a more robust approach for elution of captured targets. Mass Spectrometry analysis (LC-MS/MS) of proteins captured by BIRB796-CA-T4E and released by palladium treatment revealed significant enrichment of these MAPKs (Table 1). In addition to the expected MAPKs, we identified other kinases as well as non-kinase proteins. Using fusions of NLuc to these putative targets we confirmed by BRET that there was a direct binding relationship between BIRB796 and all but PARK7 and YEATS4 (Table 1 and Supporting Figure 9). Most of the kinase “off-targets” were previously described as BIRB796 targets, and their binding affinities were in agreement with reported values.27 However, none of the nonkinase “off-targets” have been reported before. One of these novel non-kinase “off-targets” was cyclin C (CCNC), which exhibited moderate affinity to BIRB796 (Ki = ~300 nM). Cyclin C associates with two kinase “off-targets”, CDK8 and its paralog, CDK19, and is known to regulate their activity.18, 29 Furthermore, cyclin C/CDK8 or cyclin C/CDK19 together with MED12 and MED13 forms the CDK mediator module, which is involved in transcription regulation.18, 29 We therefore tested the binding of BIRB796 to cyclin C in the absence of CDK8 and CDK19 by expressing a cyclin C:NLuc fusion in a bacterial cell-free lysate. Using high dilutions of the lysate to suppress possible influences of non-specific interactions, we were able to further confirm by BRET specific binding of BIRB796NCT to cyclin C (Figure 6, panel A). BIRB796 is a type II kinase inhibitor that binds to its targets at two sites, an ATP binding site and an adjacent allosteric site. The allosteric site is accessible only when the kinase adopts a catalytically inactive conformation, where an Asp-Phe-Gly (DFG) motif of the kinase activation loop is flipped “out” relative to its conformation in the active state.17, 30 To further characterize the interaction between BIRB796 with cyclin C, we tested the ability of other kinase inhibitors to bind this non-kinase target. We specifically used dasatinib and staurosporine, which bind to the ATP binding pocket, and truncated BIRB796 (i.e., BIRB*30 ), which binds only to the allosteric site. BRET experiments confirmed specific binding only to BIRB* (Figure 6, panel B), suggesting cyclin C may have a binding pocket similar to the allosteric site.


Page 11 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Interestingly the crystal structure of cyclin C in complex with CDK829 revealed a candidate Glu-Phe-Gly (EFG) motif, located in the cyclin C interface with CDK8 (Supporting Figure 10). Examining all 12 published CDK8/cyclin C structures, we found that the DFG-like motifs of these two binding partners are consistently at opposite “in/out” conformations, suggesting they may influence each other’s conformational state (Supporting Figure 10). Docking analysis of BIRB796 to the cyclin C structure revealed a potential binding cavity near the interface with CDK8, which seems to be created by the EFG “out” conformation (Supporting Figure 11). We postulate that binding in this cavity may influence the association of cyclin C with CDK8 or the binding of other compounds to CDK8. Finally, the identification of three members of the CDK mediator module as targets for BIRB796 suggests it may also influence transcription.

Enrichment of Endogenous Ibrutinib Targets We also tested the capability of this new method to enrich endogenous targets that interact covalently with bioactive compounds. For this we chose ibrutinib, which binds covalently to a cysteine in the ATP binding pocket of its primary target, BTK.15 In addition, ibrutinib has been reported to interact either covalently or reversibly with other tyrosine kinases, depending on the presence of a corresponding cysteine in the ATP binding pocket.15 Western and mass spectrometry analyses (LC-MS/MS) of protein enriched from Ramos cells revealed significant enrichment of expected tyrosine kinases (Figure 7 and Supporting Figure 12). These included BTK and BLK, which were predicted to interact covalently with ibrutinib, and CSK, LYN, LCK and RIPK2, which were expected to interact reversibly.15 We also identified non-kinase putative targets consisting mostly of purine binding proteins. Kinase inhibitors designed as ATP mimetics are often promiscuous and can bind other purine binding proteins.3 These results demonstrate the applicability of palladium-catalyzed elution to different types of interaction with the bioactive compounds, including covalent interactions that cannot be addressed by other elution methods.

Summary By developing a chloroalkane capture tag that can be efficiently cleaved upon treatment with an optimized palladium catalyst, we have significantly improved our previous published method for target discovery.5 This cleavable capture tag promotes effective enrichment of cellular targets for bioactive compounds by enabling their engagement inside cells as well as their efficient capture and elution. We previously demonstrated that rapid capture of chloroalkane tagged compounds with their bound targets onto immobilized HaloTag is key for their enrichment. Here, we successfully exploited a palladium-catalyzed cleavage chemistry to 11 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 40

efficiently elute the captured targets. This was achieved by inserting a cleavable allyl-carbamate linkage into the tag and developing an optimized palladium catalyzed cleavage reaction that can be carried out in complex biological mixtures. In contrast to our prior use of competitive displacement for target elution, cleavage of the capture tag allows selective release that is unaffected by the nature of the interaction between the target and the compound. This eliminates bias associated with interactions offrates, which could be exacerbated by poor solubility of the competing compound. The effectiveness of this new approach for target discovery was demonstrated by the identification of relevant cellular targets of two model compounds, including targets exhibiting low affinity, low abundance and various compound interaction modes (including covalent and prolonged residence time). Targets identified by mass spectrometry must be verified for their ability to bind the compound by an independent method. We previously coupled our target identification approach with a BRET-based verification method, which also provides an estimate of binding affinities.5 Here, we expanded the utility of the BRET approach to interrogate the dynamics of target engagement and were able to provide mechanistic interpretation to the nature of the interactions between BIRB796 and its cellular targets. Further, the discovery and verification of novel targets of BIRB796 advocates for this target identification and validation workflow as a valuable tool for drug discovery, revealing “off target” interactions and novel pharmacological pathways. Finally, the relatively inert cleavable chloroalkane tag, which is stable in cellular environments (Supporting Figure 13) but susceptible to mild cleavage conditions, should be broadly useful for other proteomic applications that rely on target isolation (i.e, activity probes, photo crosslinking probes, or affinity surfaces). This is because it should help reduce background and enhance enrichment of relevant targets, thus shortening the time needed for target validation.

METHODS See Supporting Information for details about chemical synthesis of compound conjugates, analysis of palladium-catalyzed cleavage efficiency, structural models, biochemical and phenotypic assays, and mass spectrometry analysis. See prior publications for information related to analysis of binding kinetics to HaloTag5, and BRET-based analyses of compound binding affinity within cell lysates5 and dissociation kinetics. 28

Preparation of catalyst solution 12 ACS Paragon Plus Environment

Page 13 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


All operations were performed under argon atmosphere (Schlenk technique). Water was degassed by 3 freeze-pump-thaw cycles. Example for 12 mM Pd-o-DANPHOS 1:8 solution: A solution of Na2PdCl4 (72 mg , 0.24 mmol) in H2O (2 mL) was added to a solution of oDANPHOS dihydrate (1128 mg , 1.924 mmol, 97% pure) in H2O (18 mL) and mixed for 30 min. Pd-o-DANPHOS complex (clear yellow solution) was transferred into sealed vials (1 mL each) which were incubated under argon for 24 h at 22 °C protected from light and then frozen and stored at -80 °C. Prior to use, catalyst was diluted 2-folds into 2 x pull-down buffer (see below).

Phosphine and nucleophile screen Following 30 min binding of 1 nmol 6-TAMRA-CA-T2Z (S89) to beads containing 400 µg (12 nmol) immobilized HaloTag, beads were washed 3 times, then treated for 30 min with 0.22—2 mM (13.3—120 nmol) palladium catalyst. To determine cleavage efficiencies, 1% of the released TAMRA-amine fractions were resolved on SDS-PAGE against a control of 10 pmol TAMR-CA-T2Z (pre-incubated with same amount of beads that do not contain HaloTag). Gel was scanned on a Typhoon 9400 fluorescent imager (GE Healthcare) and bands were quantitated using ImageQuant (GE Healthcare). Phosphine screen was conducted in 50 mM HEPES buffer containing 150 mM NaCl and 0.01% IGEPAL (Sigma). For nucleophile screen HEPES buffer was also replaced with either Tris, MOPS or PBS + 2 mM allyl-palladium scavengers. Targets enrichment Enrichment was performed on HSM 2.0 Heater Shaker Magnet instrument (Promega). Detergent lysis buffer: mammalian lysis buffer (Promega) containing 20 unit mL-1, RQ1 DNase (Promega) and 1x RQ1 DNase buffer. Pull-down buffer: 50 mM MOPS pH 7.5, 150 mM NaCl and 0.01% IGEPAL (Sigma). Enrichment of NLuc fusions: Each enrichment was performed from 1 x 107 HEK293 cells expressing the NLuc fusion. Cell were lysed in 2.5 mL detergent lysis buffer for 10 min, centrifuged at 3000 x g for 1 min and supernatants were diluted 1:2 with pull-down buffer. Lysates were either treated with the tagged compound at a final concentration of 1 µM or remained untreated (control). After 2.5 h binding, lysates were transferred to 50 mL tubes containing 75 µL settled HaloTag beads (34 nmol HaloTag). Following 15 min binding, unbound fractions were removed, beads were washed three times, 3 min each, then transferred to 1.5 mL tubes. Targets were eluted by 30 min treatment with 150 µL of either 1% SDS, buffer containing 20 units AcTEV (Thermo Fisher) or 0.66—2 mM (100—300 nmol) palladium catalyst. Enrichment of endogenous targets: targets were enriched from 1—5 x 107 cells, which were either treated with tagged compound at a final concentration of 20 µM or remained untreated (controls). Following 2.5 h incubation, medium was removed, cells were washed with PBS, then lysed in 2.5 mL detergent lysis buffer for 10 min. Cell lysates were centrifuged at 3000 x g for 1 13 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 40

min, supernatants were diluted 1:2 with pull-down buffer and transferred to 50 mL tubes containing 75 µL of settled HaloTag beads and pull down was performed as described above, except elution was also tested with 400 µM unmodified compound.

ASSOCIATED CONTENT Supporting Information Supporting figures 1—13, additional experimental procedures, compounds synthesis and characterization as well as raw mass spectrometry data are provided in Supporting Information. This material is available free of charge via the Internet.

REFERENCES [1] Lee, J., and Bogyo, M. (2013) Target deconvolution techniques in modern phenotypic profiling, Curr. Opin. Chem. Biol. 17, 118-126. [2] Sleno, L., and Emili, A. (2008) Proteomic methods for drug target discovery, Curr. Opin. Chem. Biol. 12, 46-54. [3] Bantscheff, M., Scholten, A., and Heck, A. J. (2009) Revealing promiscuous drug-target interactions by chemical proteomics, Drug Discov. Today 14, 1021-1029. [4] Rix, U., and Superti-Furga, G. (2009) Target profiling of small molecules by chemical proteomics, Nat. Chem. Biol. 5, 616-624. [5] Friedman Ohana, R., Kirkland, T. A., Woodroofe, C. C., Levin, S., Uyeda, H. T., Otto, P., Hurst, R., Robers, M. B., Zimmerman, K., Encell, L. P., and Wood, K. V. (2015) Deciphering the cellular targets of bioactive compounds using a chloroalkane capture tag, ACS Chem. Biol. 10, 23162324. [6] Encell, L. P., Friedman Ohana, R., Zimmerman, K., Otto, P., Vidugiris, G., Wood, M. G., Los, G. V., McDougall, M. G., Zimprich, C., Karassina, N., Learish, R. D., Hurst, R., Hartnett, J., Wheeler, S., Stecha, P., English, J., Zhao, K., Mendez, J., Benink, H. A., Murphy, N., Daniels, D. L., Slater, M. R., Urh, M., Darzins, A., Klaubert, D. H., Bulleit, R. F., and Wood, K. V. (2012) Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands, Curr. Chem. Genomics 6, 55-71. [7] Sato, S., Murata, A., Shirakawa, T., and Uesugi, M. (2010) Biochemical target isolation for novices: affinity-based strategies, Chem. Biol. 17, 616-623. [8] Leriche, G., Chisholm, L., and Wagner, A. (2012) Cleavable linkers in chemical biology, Bioorg. Med. Chem. 20, 571-582. [9] Rudolf, G. C., Heydenreuter, W., and Sieber, S. A. (2013) Chemical proteomics: ligation and cleavage of protein modifications, Curr. Opin. Chem. Biol. 17, 110-117. [10] Trost, B. M., and Lee, C. (2000) Asymmetric allylic alkylation reaction in catalytic asymmetric synthesis, 2 ed., Wiley-VCH, New York.


Page 15 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


[11] Li, J., Yu, J., Zhao, J., Wang, J., Zheng, S., Lin, S., Chen, L., Yang, M., Jia, S., Zhang, X., and Chen, P. R. (2014) Palladium-triggered deprotection chemistry for protein activation in living cells, Nat. Chem. 6, 352-361. [12] Tilley, S. D., and Francis, M. B. (2006) Tyrosine-selective protein alkylation using pi-allylpalladium complexes, J. Am. Chem. Soc. 128, 1080-1081. [13] Volker, T., and Meggers, E. (2015) Transition-metal-mediated uncaging in living human cells-an emerging alternative to photolabile protecting groups, Curr. Opin. Chem. Biol. 25, 48-54. [14] Vinogradova, E. V., Zhang, C., Spokoyny, A. M., Pentelute, B. L., and Buchwald, S. L. (2015) Organometallic palladium reagents for cysteine bioconjugation, Nature 526, 687-691. [15] Berglof, A., Hamasy, A., Meinke, S., Palma, M., Krstic, A., Mansson, R., Kimby, E., Osterborg, A., and Smith, C. I. (2015) Targets for Ibrutinib beyond B cell malignancies, Scand. J. Immunol. 82, 208217. [16] Kuma, Y., Sabio, G., Bain, J., Shpiro, N., Marquez, R., and Cuenda, A. (2005) BIRB796 inhibits all p38 MAPK isoforms in vitro and in vivo, J. Biol. Chem. 280, 19472-19479. [17] Pargellis, C., Tong, L., Churchill, L., Cirillo, P. F., Gilmore, T., Graham, A. G., Grob, P. M., Hickey, E. R., Moss, N., Pav, S., and Regan, J. (2002) Inhibition of p38 MAP kinase by utilizing a novel allosteric binding site, Nat. Struct. Biol. 9, 268-272. [18] Daniels, D. L., Ford, M., Schwinn, M. K., Benink, H., Galbraith, M. D., Amunugama, R., Jones, R., Allen, D., Okazaki, N., Yamakawa, H., Miki, F., Nagase, T., Espinosa, J. M., and Urh, M. (2013) Mutual exclusivity of MED 12/12L, MED 13/13L and CDK8/19 paralogs revealed within the CDKMediator kinase module., J. Proteomics Bioinform. S2: 004. [19] Amatore, C., Blart, E., Genet, J. P., Jutand, A., Lemaire-Audoire, S., and Savignac, M. (1995) New synthetic applications of water-soluble acetate Pd/TPPTS catalyst generated in situ. Evidence for a true Pd(0) species intermediate, J. Org. Chem. 60, 6829-6839. [20] Evans, L. A., Fey, N., Harvey, J. N., Hose, D., Lloyd-Jones, G. C., Murray, P., Orpen, A. G., Osborne, R., Owen-Smith, G. J., and Purdie, M. (2008) Counterintuitive kinetics in Tsuji-Trost allylation: ionpair partitioning and implications for asymmetric catalysis, J. Am. Chem. Soc. 130, 14471-14473. [21] Kuhn, O., and Mayr, H. (1999) Kinetics and mechanisms of the reactions of π-allylpalladium complexes with nucleophiles., Angewandte Chemie. International 38, 343-346. [22] Ross, J., Chen, W., Xu, L., and Xiao, J. (2001) Ligand effects in palladium-catalyzed allylic alkylation in ionic liquids, Organometallics 20, 138-142. [23] Peral, D., Herrrera, D., Real, J., Flor, T., and Bayon, J. C. (2016) Strong π-acceptor sulfonated phosphines in biphasic rhodium-catalyzed hydroformylation of polar alkenes, Catal. Sci. Technol. 6, 800-808. [24] Beletskaya, I. P., and Cheprakov, A. V. (2000) The heck reaction as a sharpening stone of palladium catalysis, Chem. Rev. 100, 3009-3066. [25] O'Hare, T., Eide, C. A., Agarwal, A., Adrian, L. T., Zabriskie, M. S., Mackenzie, R. J., Latocha, D. H., Johnson, K. J., You, H., Luo, J., Riddle, S. M., Marks, B. D., Vogel, K. W., Koop, D. R., Apgar, J., Tyner, J. W., Deininger, M. W., and Druker, B. J. (2013) Threshold levels of ABL tyrosine kinase inhibitors retained in chronic myeloid leukemia cells determine their commitment to apoptosis, Cancer Res. 73, 3356-3370. [26] Hall, M. P., Unch, J., Binkowski, B. F., Valley, M. P., Butler, B. L., Wood, M. G., Otto, P., Zimmerman, K., Vidugiris, G., Machleidt, T., Robers, M. B., Benink, H. A., Eggers, C. T., Slater, M. R., Meisenheimer, P. L., Klaubert, D. H., Fan, F., Encell, L. P., and Wood, K. V. (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate, ACS Chem. Biol. 7, 1848-1857. 15 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 40

[27] Davis, M. I., Hunt, J. P., Herrgard, S., Ciceri, P., Wodicka, L. M., Pallares, G., Hocker, M., Treiber, D. K., and Zarrinkar, P. P. (2011) Comprehensive analysis of kinase inhibitor selectivity, Nat. Biotechnol. 29, 1046-1051. [28] Robers, M. B., Dart, M. L., Woodroofe, C. C., Zimprich, C. A., Kirkland, T. A., Machleidt, T., Kupcho, K. R., Levin, S., Hartnett, J. R., Zimmerman, K., Niles, A. L., Ohana, R. F., Daniels, D. L., Slater, M., Wood, M. G., Cong, M., Cheng, Y. Q., and Wood, K. V. (2015) Target engagement and drug residence time can be observed in living cells with BRET, Nat. Commun. 6, 10091. [29] Schneider, E. V., Bottcher, J., Huber, R., Maskos, K., and Neumann, L. (2013) Structure-kinetic relationship study of CDK8/CycC specific compounds, Proc. Natl. Acad. Sci. U.S.A. 110, 80818086. [30] Liu, H., Kuhn, C., Feru, F., Jacques, S. L., Deshmukh, G. D., Ye, P., Rennie, G. R., Johnson, T., Kazmirski, S., Low, S., Coli, R., Ding, Y. H., Cheng, A. C., Tecle, H., English, J. M., Stanton, R., and Wu, J. C. (2010) Enhanced selectivity profile of pyrazole-urea based DFG-out p38alpha inhibitors, Bioorg. Med. Chem. Lett. 20, 4885-4891.


Page 17 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1 Protein MW MAPK14 41 MAPK9 48 MAPK12 42 CDK19 57 RIPK2 61 RET 124 CCNC 33 CDK5 33 YEATS4 27 CDK8 53 PARK7 20 MAPK11 41 MAPKAPK2 46 MAPK13 42 EPHB3 110 PEX11B 28 RAF1 73 EPHB4 108 MKNK1 51 MAPKAPK3 43 CTSC 52 PLD3 55 MAP4K1 91 EPHB1 110

Mean SpC Ctrl 0.0 0.0 0.0 0.0 0.0 4.3 0.0 4.7 1.3 0.0 2.3 0.0 0.0 0.0 0.0 1.3 4.3 0.0 0.0 0.0 0.0 0.0 1.7 0.0

BIRB796-CA Ratio 290.0 NA 139.0 NA 68.0 NA 77.0 NA 58.0 NA 118.0 27.2 28.3 NA 19.3 4.1 15.3 11.5 27.3 NA 10.0 4.3 20.3 NA 23.3 NA 16.7 NA 42.0 NA 8.7 6.5 20.7 4.8 22.7 NA 9.3 NA 7.0 NA 6.3 NA 6.7 NA 7.0 4.2 7.3 NA

Mean NSAF X1000

Validation

Ctrl BIRB796-CA Ratio 0.00 14.17 NA 0.00 5.80 NA 0.00 3.21 NA 0.00 2.76 NA 0.00 1.92 NA 0.07 1.89 28.8 0.00 1.75 NA 0.27 1.19 4.5 0.09 1.17 12.4 0.00 1.09 NA 0.22 1.03 4.7 0.00 1.02 NA 0.00 0.99 NA 0.00 0.82 NA 0.00 0.77 NA 0.09 0.63 6.8 0.11 0.56 5.0 0.00 0.41 NA 0.00 0.37 NA 0.00 0.32 NA 0.00 0.25 NA 0.00 0.25 NA 0.04 0.16 4.4 0.00 0.13 NA

Ki (nM) 0.60 4.7 2000 780 2100 2100 300 190 — 1000 — 1.6 + 100 320 2700 1700 360 3000 + 6100 6300 4000 830



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 40

Figure Legends Figure 1. Optimization of palladium-catalyzed cleavage reaction. (A) Schematic of palladiumcatalyzed allylic substitution where the leaving group is carbamate, allyl group is indicated in blue and cleaved bond in red. (B) Structure of chloroalkane tags conjugated to TAMRA fluorophore, cleavage sites are indicated in red. Analysis of cleavage efficiency in (C) HEPES buffer, (D) proteinaceous environment and in a screen for optimal (E) phosphine and (F) nucleophile. See Supporting Figures 2 and 3 for structures of phosphines and nucleophiles, respectively. Figure 2. Structure optimization of cleavable chloroalkane tag. (A) Crystal structures of HaloTag (PDB code 4KAJ, green) and human MAPK14 (PBD code 1KV2, purple), linked by modeled BIRB796-CA-T2Z (CA-T2Z in orange; cleavage site in red). Presumed catalyst structure (dark gray; Pd atom in yellow) shown to size. (B) Structure of chloroalkane tags, cleavable sites indicated in red. Figure 3. Influence of cleavable chloroalkane tags on compound potency and target capture. Influence on potency of (A) BIRB796, (B) ponatinib (C) ibrutinib tested in biochemical (red bars) and cellular assays (gray bars), double y-axis indicate different assays. Potency reduction relative to unaltered drug (X) is indicated in the graphs. (D) Specific capture of targets fused to NLuc fusion (n=6). Figure 4. Evaluation of cleavable chloroalkane tags for target enrichment by different elution methods. Lysates from cells expressing the appropriate NLuc fusion were either treated with 1 µM chloroalkane conjugates or remained untreated (Ctrl). Western analysis of eluted proteins using NLuc antibody for (A) enrichment of NLuc:MAPK9 by BIRB796 conjugates and (B) enrichment of BTK:NLuc by ibrutinib conjugates. Elution methods: (1) 1% SDS, (2) 2 mM catalyst, (3) 0.66 mM catalyst and (4) TEV protease. Figure 5. Enrichment of MAPK targets by CA-T1 and CA-T4E conjugated to BIRB796. (A) Western analysis of MAPKs enrichment in triplicates from THP-1 cells, which were either treated with 20 µM BIRB796 conjugates or remained untreated (Ctrl). Analysis by BRET of BIRB796 interaction with MAPK targets for (B) binding affinities and (C) apparent dissociation kinetics (i.e., rate of decreased BIRB796 occupancy).


Page 19 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Analysis of BIRB796 interaction with cyclin C. Analysis by BRET of cyclin C: NLuc fusion expressed in bacterial cell lysate for binding to (A) BIRB796-NCT and (B) fluorophoretagged kinase inhibitors. Figure 7. Enrichment of ibrutinib targets. Western analysis of tyrosine kinases enriched in triplicates from Ramos cells, which were either treated with 20 µM ibrutinib-CA-T4E or remained untreated (Ctrl). Table 1. BIRB796 targets identified by LC-MS/MS and verified by BRET. Enrichment in triplicates from THP-1 cells, which were either treated with 20 µM BIRB796-CA-T4E or remained untreated (Ctrl). Putative targets were determined based on the following criteria: protein had at least five spectral counts (SpC) in test sample and non in the control or at least 4-fold more SpC in the test sample over the control. Data from three different experiments are represented as the mean of SpC or the mean of normalized spectral abundance factors (NSAF). For results from individual runs and additional details on the LC-MS/MS analysis refer to Supporting Information. Putative targets were verified by BRET, estimated binding constant (Ki) are presented in the table, no binding (—) and binding only to BIRB796-NCT (+).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 20 of 40

Page 21 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC 80x39mm (300 x 300 DPI)

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig 1A 43x29mm (300 x 300 DPI)


Page 22 of 40

Page 23 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig 1B 31x15mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig 1C 38x23mm (300 x 300 DPI)


Page 24 of 40

Page 25 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig 1D 31x15mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig 1E 46x32mm (300 x 300 DPI)


Page 26 of 40

Page 27 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig 1F 53x43mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig 2A 65x54mm (300 x 300 DPI)


Page 28 of 40

Page 29 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig 2B 62x61mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig 3A 44x30mm (300 x 300 DPI)


Page 30 of 40

Page 31 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig 3B 50x38mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig 3C 48x36mm (300 x 300 DPI)


Page 32 of 40

Page 33 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig 3D 30x13mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig 4 65x80mm (300 x 300 DPI)


Page 34 of 40

Page 35 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig 5A 55x47mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig 5B 36x20mm (300 x 300 DPI)


Page 36 of 40

Page 37 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig 5C 42x27mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig 6A 35x19mm (300 x 300 DPI)


Page 38 of 40

Page 39 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig 6B 35x19mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig 7 65x78mm (300 x 300 DPI)


Page 40 of 40

Improved Deconvolution of Protein Targets for Bioactive Compounds

Recommend Documents