Chemoproteomics Demonstrates Target Engagement and Exquisite

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Chemoproteomics Demonstrates Target Engagement and Exquisite Selectivity of the Clinical PDE10 Inhibitor MP10 in Its Native Environment Jan-Philip Schülke, Laura A. McAllister, Kieran F Geoghegan, Vinod Parikh, Thomas A. Chappie, Patrick R. Verhoest, Christopher J Schmidt, Douglas S. Johnson, and Nicholas J Brandon ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/cb500671j • Publication Date (Web): 08 Oct 2014 Downloaded from http://pubs.acs.org on October 14, 2014

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ACS Chemical Biology

Chemoproteomics Demonstrates Target Engagement and Exquisite Selectivity of the Clinical PDE10 Inhibitor MP-10 in Its Native Environment Jan-Philip Schülke1, Laura A McAllister2, Kieran F Geoghegan3, Vinod Parikh3, Thomas A Chappie2, Patrick R Verhoest2, Christopher J Schmidt1, Douglas S Johnson2, Nicholas J Brandon1,4. 1-3

Pfizer Worldwide Research and Development:

1

Neuroscience Research Unit, Cambridge, MA 02139 (USA).

2

Neuroscience Medicinal Chemistry and Chemical Biology, Cambridge, MA 02139 (USA).

3

Center of Chemistry, Groton, CT 06340 (USA).

4

AstraZeneca Neuroscience iMED, Cambridge, MA 02139 (USA) (present address).

ABSTRACT: Phosphodiesterases (PDEs) regulate the levels of the second messengers cAMP and cGMP and are important drug targets. PDE10A is highly enriched in medium spiny neurons of the striatum and is an attractive drug target for the treatment of basal ganglia diseases like schizophrenia, Parkinson’s or Huntington’s disease. Here we describe the design, synthesis and application of a variety of chemical biology probes, based on the first clinically tested PDE10A inhibitor MP-10, which were used to characterize the chemoproteomic profile of the clinical candidate in its native environment. A clickable photoaffinity probe was used to measure target-engagement of MP-10, and revealed differences between whole cell and membrane preparations. Moreover, our results illustrate the importance of the linker design in the creation of functional probes. Biotinylated affinity probes allowed identification of drug-interaction partners in rodent and human tissue and quantitative mass spectrometry analysis revealed highly specific binding of MP-10 to PDE10A with virtually no offtarget binding. The profiling of PDE10A chemical biology probes described herein illustrates a strategy by which high affinity inhibitors can be converted into probes for determining selectivity and target engagement of drug candidates in complex biological matrices from native sources.

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Phosphodiesterases (PDEs) are a class of enzymes that hydrolyze cyclic nucleotides and contribute to cell signaling by controlling levels of the second messengers cAMP and cGMP in the cell. PDEs are believed to be excellent drug targets based on their role in regulating key intracellular processes and the demonstrated ability to develop small molecule inhibitors of these enzymes.1–3 This very diverse class of proteins consists of 11 different families (PDE1 to PDE11), classified based on sequence homology, substrate specificity and domain architecture, with multiple different isoforms derived from separate genes or splice variants.4 PDE10A is highly enriched in medium spiny neurons of the striatum and is therefore well-positioned to regulate basal ganglia circuitry which is central to neurological diseases like Parkinson’s disease, Huntington’s disease, Tourette syndrome, addiction and schizophrenia.5–7 PDE10A inhibition has been shown to regulate cyclic nucleotide signaling pathways in the striatum and be efficacious in preclinical models for assessing antipsychotic drug action and reversing behavioral cellular phenotypes in Huntington’s disease models.8–10 Besides its impact on brain pathways, PDE10A has also been shown to be involved in the regulation of proliferative states in pulmonary hypertension and colon cancer.11,12 Therefore, PDE10A inhibitors are promising pharmaceuticals and demonstration of their efficacy to ameliorate disease symptoms is actively being pursued by several drug companies.2 Pfizer’s PDE10A inhibitor MP-10 (PF-2545920) was found to be ineffective in a 4-week monotherapy trial investigating the effect of the drug on the acute exacerbation of psychosis in patients suffering from schizophrenia13, but is still in clinical testing for adjunctive therapy in schizophrenia and Huntington’s disease (www.ClinicalTrials.gov: NCT01939548, NCT01806896). Given the considerable clinical investment in MP-10, it is important to have a detailed understanding of the selectivity and target engagement of MP-10 in native biological systems. Chemical proteomic technologies allow the characterization of the spectrum of protein-drug interactions and the measurement of target engagement of a particular drug in its native environment.14–16 Furthermore, being able to identify and quantify the protein binding partners of biologically active compounds can not only provide information about potential off-target activity, but can also increase our mechanistic understanding of the pharmacology of a given compound. Chemical biology probes have been designed for various enzyme classes17,18 including kinases19, histone deactylases (HDACs)20, serine hydrolases21, metalloproteases22, cysteine proteases23, γ-secretase24, cytochrome P45025 and adenylating enzymes26. In this study, we report the development and application of various chemical biology probes for the chemoproteomic [2] ACS Paragon Plus Environment

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characterization of the clinical PDE10A inhibitor MP-10.27 Using a probe containing a photo activatable crosslinker and an alkyne moiety for click chemistry we determined the PDE10A target engagement in membrane preparations and primary striatal cultures.24 By performing a comprehensive analysis of the probe binding partners using a label free liquid chromatography–mass spectrometry (LC-MS) approach, we identified that MP-10 binds to PDE10A with exquisite selectivity. This information is critical as it suggests little off-target activity in the striatum and reinforces the use of MP-10 to evaluate the therapeutic potential of selective PDE10 inhibition. RESULTS AND DISCUSSION Design and Synthesis of a Clickable Photoaffinity Probe for Phosphodiesterase 10A. The biopharmaceutical industry’s first PDE10A inhibitor to reach the clinic was MP-10 (Figure 1A).27 By being the first PDE10A inhibitor to advance so far, it has had a large influence on the design of subsequent inhibitors. Specifically, the publication of MP-10 and similar compounds was the first to describe the PDE10A “selectivity pocket”, occupation of which resulted in in vitro selectivities vs. other PDEs of >1000 fold. We designed chemical probes with a linker attached to the PDE10A recognition element (MP-10) such that a reporter group could be introduced without detrimental effects on activity. Based on the analysis of the PDE10A/MP-10 co-crystal structure (Figure 1A), it was deemed best to attach a linker to the pyrazole nitrogen which is unsubstituted in MP-10.27 It was proposed that this attachment point on MP-10 should not significantly interfere with binding to the enzyme and provides a relatively easy synthetically accessible point of modification. Additionally, the SAR taken from the original disclosure of MP-10 shows just a fourfold decrease in potency by putting a methyl group on this nitrogen as compared to MP-10.27 Additionally, we used the X-ray co-crystal structure of MP-10 and PDE10A to identify a suitable linker length such that a photoreactive group and/or reporter group could be appended without disrupting the binding of the probe to the enzyme. The first target probe that we designed was PF-942 (Figure 1B, Figure S2). At this early stage of experimentation, not knowing the true affinity of this MP-10/linker combination for PDE10A, we chose to include a benzophenone group within the linker in order to covalently attach the probe to the enzyme upon UV irradiation. This would alleviate the possibility of the probe dissociating from the enzyme during experimental manipulations. The ethylene glycol linker would extend the benzophenone group far enough from the ligand binding pocket while also not extending it too far into solvent limiting the ability to [3] ACS Paragon Plus Environment


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covalently attach to PDE10 (Figure 1B). An alkyne functional group was attached to the benzophenone moiety to allow for click chemistry-mediated conjugation of biotin-azide for affinity capture or a fluorophore-azide for visualization by in-gel fluorescence.24 This resulted in a probe that inhibits PDE10A with an IC50 of 29 nM and PDE2 with an IC50 of >1 µM and therefore has a potency comparable to the parent inhibitor MP-10 (PDE10A IC50: 1.26 nM) when measured in phosphodiesterase assays using recombinant enzyme.9 Therefore, PF-942 was well suited to bind and label PDE10A such that the target engagement of MP-10 and other PDE10A inhibitors could be measured as described below. Photoaffinity Probe Labels PDE10A and Can Be Used to Measure Target Engagement. Target validation of pharmacological compounds often relies on assays using recombinant proteins or artificial systems which might not reflect the protein-drug interactions in its native environment accurately. In this study we aim to characterize the native chemoproteomic interactions and potencies of the PDE10A inhibitor MP-10 by utilizing chemical biology probes. We established our protocol in membrane preparations, since PDE10A is abundant in these fractions.28,29 To test whether the PDE10A photoaffinity probe PF-942 can be used to label native PDE10A, we isolated membrane fractions from rat striatal lysates by ultracentrifugation and verified that PDE10A is enriched in our membrane preparations (Figure S3). To determine the optimal conditions for labeling, we incubated the membranes with increasing amounts of PF-942. After UV irradiation to cross-link the photoprobe to its binding partner(s) and click chemistry with TAMRA-azide, we separated the membranes by SDS-PAGE and analyzed labeled proteins by in gel fluorescence (Figure 2A). We observed a fluorescent band at about 90 kDa, similar to the size of endogenous rat PDE10A (Uniprot Q9QYJ6: 90.2 kDa), at low concentrations of the probe which saturated at about 1 µM. At high probe concentrations multiple other bands were present in the gel, which likely represent nonspecific labeling of abundant proteins since Coomassie staining showed bands at similar molecular weights (Figure 2A; compare left and right gel). Using 30 nM of the photoprobe which gave an optimal combination of labeling intensity and specificity for PDE10A (Figure 2A), we varied the UV-irradiation time and found that 20 min provided optimal labeling intensity without eroding the specificity (Figure 2B). Therefore, we used 30 nM PF-942 and a UV-irradiation time of 20-min for subsequent experiments. To verify the 90 kDa band as PDE10A, we incubated striatal membranes with PF-942 (30 nM) and after UV crosslinking, conjugated a biotinylated TAMRA fluorophore (Biotin-TAMRA-N3) to the probe-labeled protein(s). This approach allowed us to [4] ACS Paragon Plus Environment

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enrich for proteins which were labeled with the photoprobe using streptavidin (SA) magnetic beads. After SA pulldown and separation of the eluates by SDS-PAGE, we observed a single 90 kDa band by in gel fluorescence (Figure 2C; left-hand side). Importantly, after transferring the gel to nitrocellulose for immunoblot analysis, this band was positively identified as PDE10A (Figure 2C; right-hand side) showing that we have developed a photoprobe which specifically labels PDE10A from native sources The band at 200 kDa is likely due to aggregation of PDE10A resulting from the harsh elution conditions of the SA pulldown which is reliably detected (although with varying intensities) using our PDE10A antibody (Figure S3) and by mass spectrometry of the excised band (data not shown). To test if the PF-942 probe can be used to measure target engagement of a PDE10A inhibitor, we incubated the striatal membranes with increasing concentrations of the PDE10A inhibitor MP-10 before photolabeling with PF-942, followed by click chemistry conjugation of TAMRA-azide. Quantification of the fluorescent PDE10A band (Figure 2D, left gel, arrowhead) revealed that MP-10 decreased labeling of the PF-942 photoprobe in a concentration dependent manner with an IC50 of 4.2 nM which is slightly higher than the potency determined in previous studies using recombinant enzyme (1.26 nM).9 This result shows that MP-10 is directly competing for PF-942 probe labeling and that that MP-10 engages its endogenous target PDE10A in striatal membranes at low nanomolar concentrations. Importantly, we showed that the PDE10A inhibitor Papaverine engaged PDE10A with an IC50 of 186.2 nM (IC50 using recombinant enzyme is 92.3 nM)9, showing that the photoprobe PF-942 can also measure target engagement of a lower potency and structurally distinct active-site directed inhibitor (Figure 2D, right gel). However, when applying these probes to live striatal cultures, which are expressing PDE10A in levels comparable to striatal tissue (data not shown), we observed that labeling and target engagement was different between membranes and the cultures (Figure 2A+C, Figure 3A+B). As for the membranes we established the optimal probe concentration for labeling of primary striatal neurons. After incubation of rat primary striatal cultures with increasing amounts of PF-942 followed by UVcrosslinking, we lysed the cells and performed click chemistry to conjugate TAMRA-azide to the alkyne handles. We again observed a fluorescent band at about 90 kDa (Figure 3A). In contrast to the experiment using membranes, higher concentrations of the probe were needed to reach saturation of the label (Figure 3A). Additionally, in the striatal cultures the 90 kDa label appeared as a double band rather than a single band as observed in the striatal membranes. The intensity of that double band was greatly reduced by preincubation with the parent inhibitor MP-10 before photolabeling with PF-942 (Figure 3B) demonstrating the specificity of the probe for PDE10A in [5] ACS Paragon Plus Environment


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striatal cultures and target engagement of MP-10. However, in contrast to the competition experiments in membranes, which revealed that MP-10 was able to engage its target with an IC50 of 4.2 nM, much higher levels of MP-10 were needed to engage PDE10A in the striatal cultures (Figure 3B; IC50 = 1.7 µM). This difference in target engagement could be a result of limited accessibility of the compound to its target in the context of the primary striatal cultures. Indeed, poor cell permeability can be an issue with some chemical biology probes.30 We addressed this potential issue by using a clickable probe containing an alkyne handle in which the reporter group is attached after covalently labeling the target with our probe using a photo-activatable benzophenone group.24 Indeed PF-942 exhibited good cell permeability of 56 x 10-6cm/sec, as tested in a RRCK cell transwell assay.31 To further ensure cell penetration of our photoprobe, we tested if PF-942 could also be used to label PDE10A from intact rat striatal tissue. We incubated rat striatal tissue pieces of ~1 mm3 with PF-942 and looked for PDE10A labeling by in gel fluorescence after UVcrosslinking and click chemistry. PF-942 labeled a 90 kDa band which was competed by preincubation with MP-10 showing efficient and specific labeling of PDE10A using the probe in situ (Figure S5) and supporting that the probe reaches sufficient concentrations at the site of action. The higher IC50s observed in this study could result from slower cross-linking in cells, but we were not able to cross-link longer than 20 min due to cell toxicity concerns. The differences in target engagement between striatal membranes and dissociated striatal cultures could also be a result of a particular environment present in the cellular system, which is for instance reflected by altered posttranslational modifications. Indeed, we observed in the cultures, that the photoaffinity probe PF-942 labels a double band at the molecular weight of PDE10A. Thus in the striatal cultures, the compound may be binding to particular pools of PDE10A that could potentially harbor a posttranslational modification causing the mass shift in the SDS-PAGE (Figure 3A+B and Figure S4). Moreover, the double band could reflect labeling of the two major PDE10A splice variants, PDE10A1 and PDE10A2, which differ in their N-terminal sequence, phosphorylation status and subcellular localization.28 Alternatively, this double band around the molecular weight of PDE10A could reflect a potential off-target. The potential PDE10A double bands observed in the striatal cultures are unlikely the result of non-specific labeling, since we do not observe highly abundant proteins in Coomassie stained gels at that particular molecular weight range (Figure S4). However, high concentrations of the probe resulted in labeling of several other bands. Since these bands had corresponding bands in the Coomassie stained gel and were competed to a lesser extent by the parent compound (Figure 2A+3B and Figure S4), it suggests that these high abundant [6] ACS Paragon Plus Environment

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proteins are labeled non-specifically at higher probe concentrations. Nevertheless, these findings show that even for a selective photoaffinity probe like PF-942, it is important to optimize labeling conditions such that a sub-saturating concentration of photoprobe is used to avoid excessive nonspecific labeling. In summary, we have developed a PDE10A photoaffinity probe which labels endogenously expressed PDE10A and can be used to measure target engagement of PDE10A inhibitors such as MP-10 from native sources. Biotinylated Affinity Probes Enrich PDE10A from Striatal Tissue. We synthesized another class of PDE10A chemical biology probes to further understand the spectrum of protein-drug interactions and identify possible protein binding partners in the striatum to increase our mechanistic understanding of the molecular pharmacology of MP-10. Based on the structural information that the compound is bound deep in the ligand binding pocket, we designed affinity probes in which the target recognition structure was coupled to a biotin moiety through a PEG linker that should allow isolation of protein complexes from tissue lysates (Figure 4). We attached the linker and biotin moieties to MP-10 using the same amide motif as for the photoaffinity probe PF-942 to afford biotinylated probe PF-621, a 51 nM PDE10 inhibitor as tested in the recombinant enzyme assay.27 We directly compared the ability of the biotinylated probe and the photoaffinity probe to precipitate PDE10A from striatal lysates. 15 nmol Biotin-TAMRA-N3 was conjugated to excess PF-942 by click chemistry and then bound to 250 µl slurry of SA-agarose (capable of binding 30-300 nmol free biotin). Excess PF-942 probe was washed away. In parallel, 15 nmol of the PF-621 was bound to SA-agarose. We found that both probes precipitated similar amounts of PDE10A from rat striatal lysates (Figure 5A). We also synthesized a biotinylated probe that contained an ether linker in place of the amide (PF-267, PDE10A IC50 = 335 nM). In contrast to PF-621, much less PDE10A was precipitated using PF-267 (Figure 5B+C). The reduced efficiency might be due to the less rigid linker in PF-267, which may fold and interact with the outside of the protein rather than project out of the active site such that the biotin is able to interact with streptavidin. Therefore, the acetamide linker attached to the pyrazole appears to be playing a critical role that enables pull down of PDE10A. As can be seen from the Coomassie stained gels, many proteins are released together with PDE10A when eluting the SA-beads under harsh conditions using sample buffer or ammonium hydroxide (Figure 5A+C). These are background proteins bound to the resin material since they are also released in the negative control reactions and they might potentially interfere with downstream identification. To reduce the amount of [7] ACS Paragon Plus Environment


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background proteins, we attempted to elute specifically bound proteins using excess MP-10. However, full length PDE10A isolated from striatal lysates could not be released in sufficient amounts by competitive elution (data not shown) which suggests that inhibitor binding renders full length PDE10A inaccessible for compound (and likely also cyclic nucleotide) exchange. In contrast, the recombinantly expressed catalytic domain of PDE10A could be released by competition with MP-10. Therefore, the binding characteristics of MP-10 toward native full length PDE10A and the recombinantly expressed catalytic domain are subtly different highlighting the importance of characterizing inhibitors against the native form of the enzyme. As an alternative approach to reduce background, we constructed an affinity probe with a cleavable linker in which the biotinylated PEG linker was coupled to the PDE10A recognition structure with a disulfide bond (Figure 4; PF-631, PDE10A IC50 = 7.5 nM). As seen in Figure 5D, SA-enrichment using PF-631 with the cleavable disulfide linker enabled specific isolation of PDE10A from striatal lysate with less background proteins being co-released when eluting with 10 mM DTT. After establishing the use of the probes in rodent tissue, we sought to test the ability of our probes to analyze PDE10A in human post-mortem brain tissue. In humans the striatum is divided into the caudate nucleus and the putamen. We tested binding of PDE10A to the probe PF-621 in tissue from this region of three individuals (#3322, #3397, #3406) and detected high levels of PDE10A in eluates of all three samples (Figure 5E). Of note, PDE10A levels in the pull-down did not directly correlate with input levels, suggesting that binding might be influenced by properties intrinsic to the individual. In sample #3322, PDE10A protein levels are significantly lower compared to samples #3397 and #3406 (Figure 5E, left graph). However, the amount of PDE10A in the eluates is similar to sample #3397 which results in a higher relative binding of #3322 (Figure 5E, right graph). Furthermore, input levels of PDE10A are similar between sample #3397 and #3406, but binding is significantly stronger in sample #3406 compared to #3397 (Figure 5E). MP-10 binds in the active site of PDE10A which is likely to be dependent on the catalytic activity of the enzyme. The differential labeling observed herein could reflect a differential activity of PDE10A and exploiting the potential of our molecules as activity-based probes will therefore be a future consideration.18 In conclusion, we demonstrated that we have developed highly effective affinity probes that are potent binders of PDE10A in both rodent and human tissue which should further allow isolation and characterization of the probe-bound proteome. Comprehensive Analysis of PDE10A Affinity Probe-Interacting Proteins in the Striatum. [8] ACS Paragon Plus Environment

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Using recombinant enzyme assays, MP-10 shows >1000 fold selectivities over other PDEs.9,27 However, these in vitro assays determine selectivity only for a predetermined set of targets selected based on enzyme family or sequence similarity and they generally do not provide information on compound-biomolecule interactions in the target environment. To further evaluate the proteomewide selectivity of MP-10, we utilized our PDE10A probes to search for endogenous interacting proteins in the context of striatal lysates using affinity chromatography.32 Our biotinylated PDE10A affinity probes were coupled to SA-beads, incubated with mouse striatal lysates, and bound proteins were eluted using ammonium hydroxide for PF-621, or DTT for PF-631 containing a cleavable disulfide linker. The eluates were trypsin digested in solution and subjected to LC-MS/MS for protein identification and quantification using a label free approach.33 Mass spectrometry based target identification using affinity purification is prone to false positives because of background proteins which will bind directly to the resin material used for the pull downs.34 Even using the PF-631 probe with a cleavable linker we still observed a significant number of nonspecific proteins during the elution. To overcome this issue we conducted pull down experiments both in the absence or presence of MP-10 to identify proteins that were competed by MP-10 and therefore must be considered as binding partners of the free inhibitor.35 Quantitative mass spectrometry allowed us to compare protein abundances in the pull-down reactions and to determine the average enrichment and its statistical significance over multiple experiments. The results are summarized in volcano plots to show the log2 of the average fold enrichment (difference of the log2 transformed data) of the probe pull-downs over the competitor control and the statistical significance in 3 independent biological experiments (Figure 6A+B). Proteins non-specifically bound to the resin material are not competed by free compound and should have a difference of 0. Since we only expect enrichment in our experiment, negative values in the volcano plot indicate the variability within the experiment. We therefore consider proteins deviating greater than the spread of proteins around zero, to be significantly enriched. Strikingly, PDE10A was significantly enriched in the pull-down experiments using both probes. PF-621 exhibited a difference in PDE10A signal over control of 7.39 [log2] (P = 2.9x10-4) and PF-631 a difference of 7.35 [log2] (P = 5.3x10-5). However, when analyzing the overlap of proteins which are enriched by both probes, we observed that PDE10A was the only protein identified above the significance threshold (Figure 6C). While we could nicely demonstrate the exquisite selectivity of MP-10 for PDE10A in its native tissue environment, we did not detect any PDE10A interacting proteins which could have potentially informed us about previously unrecognized pathways to increase our mechanistic understanding of the MP-10 pharmacology. [9] ACS Paragon Plus Environment


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However, another study aiming to identify protein binding partners of a PDE5 inhibitor also only identified direct probe binding targets.36 Moreover, since the pull down probe is based on an active site-directed inhibitor, protein binding partners would have to exist in the inhibitor-bound complex to be identified. This suggests that possible protein binding partners might be transient in nature or that certain phosphodiesterases might not be incorporated into a protein complex when the inhibitor is bound. This finding may have implications regarding the potential of the probes to characterize the chemoproteomic context of a phosphodiesterase in a certain activity state. Conclusions. To our knowledge, we are the first to present the design, synthesis and application of a clickable photoaffinity probe targeting a phosphodiesterase and our aim is to provide information which enables the further development of chemical biology probes for this important enzyme class. To achieve this goal, we used structural information of the catalytic domain of PDE10A to determine where to attach the prosthetic groups on the inhibitor MP-10 without disrupting its activity and selectivity for PDE10A. We were able to develop a small, cell-permeable photoprobe that labeled endogenous PDE10A in striatal cultures and membranes. The clickable nature of the probe not only helped with cell permeability but it also expanded the versatility since we could attach various azidelinked reporter groups for subsequent detection or enrichment of probe-labeled proteins. PDE10A inhibitors are promising drugs to treat basal ganglia diseases and several are being tested in clinical trials.2 Since MP-10 was found not to be effective in a monotherapy trial for the acute exacerbation of psychosis in patients suffering from schizophrenia, we were wondering if our data could provide information about protein-drug interactions that can be translated to humans. First, we demonstrated the exquisite selectivity of MP-10 for PDE10A in tissue lysates which makes any potential off-target activity in the striatum unlikely. Second, we showed that MP-10 engages its target with single digit nanomolar affinities. However using our probes, we could show that affinities differ between striatal membrane preparations and primary striatal cultures which suggest a different potency of MP-10 in primary cell assays. It has been shown previously that an inhibitor profile in native tissues can differ from the affinity and specificity determined using recombinant enzymes.37 Our results show that our chemical biology probes are useful to reveal those differences.


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METHODS Detailed information on the chemical synthesis and the experimental procedures is provided in the supplemental materials. Membrane Labeling Using PDE10A Photo Affinity Probes. Rat striatal membranes in (500 µg in 1 ml PBS +1 mM DTT) were incubated with photo affinity probe and competitor from 1000x DMSO stocks and incubated for 30 min on ice and then crosslinked by exposure to UV light for 20 min at 4°C. The membranes were pelleted by ultracentrifugation (100,000 G, 1 h, 4°C) and membranes at ~0.5 mg / ml were subjected to click chemistry based on an established protocol38: 0.1 mM TBTA from 1.7 mM stock in 1:4 DMSO:tBuOH, 1 mM TCEP, 1 mM CuSO4, 30 µM azide, 0.3-1% SDS, in PBS, 20-60 min at RT. For streptavidin pulldown, proteins were precipitated by Methanol / Chloroform isolation as described in

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and the probes were isolated in 5 ml modified

RIPA buffer (50 mM Tris pH 8.0, 0.5% Deoxycholate, 1% NP40, 0.1% SDS, 2 mM EDTA, 1 mM DTT, 500 mM NaCl, 1 mg/ml BSA, protease inhibitors) using 100 µl SA-magnetic beads (Thermo Scientific), extensively washed (2x with RIPA; 3x with 6 M Urea 1% SDS; 2x with PBS +0.1% SDS; 2x with RIPA (high salt: 500 mM NaCl); 2x with RIPA (low salt: 150 mM NaCl); 3x with dH2O +0.1% SDS) and eluted in 30 µl NuPage LDS sample buffer (Invitrogen) supplemented with 2.5% SDS, 1.2 mM Biotin, reducing agent (Invitrogen) or 100 mM DTT and 9% Glycerol for 10 min at 95°C. Labeling of Striatal Cultures Using Photo Affinity Probes. Competitor compounds and photo affinity probes were added to the rat striatal cultures (100,000 cells / cm2; DIV15 - DIV21) in conditioned media (0.1–0.2% final DMSO conc.) and incubated for ~30 min at 37°C. The cultures were placed on ice, washed twice with ice cold ACSF before adding the competitor and the probes in ACSF and incubation on ice for 10-20 min. Probes were crosslinked by exposure to UV light for 20 min at 4°C. The cultures were washed twice with ice cold ACSF to remove unbound probe and the cells were lysed in PBS +0.33% SDS. Lysates were subjected to click chemistry under conditions as described above by adding 5 µl of a 10x click mix to 45 µl of the lysate. Streptavidin Pulldown Using Biotinylated PDE10A Chemical Biology Probes. Tissues were lysed in IP-buffer (IPB) (50 mM Tris pH 8.0, 100 mM NaCl, 2 mM EDTA, 1% Triton X-100, supplemented with protease and phosphatase inhibitors) by sonication. Biotinylated probes were coupled to streptavidin magnetic beads (Thermo Scientific) in PBS and the coupled beads were [11] ACS Paragon Plus Environment


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blocked with BSA (10 mg / ml) in IPB. For the competition, lysates were incubated with 50 µM of the compound (1% DMSO). Biotinylated probes were precipitated by adding 20 µl of the coupled beads to 2-4 mg (5-10 mg / ml) of the crude lysate in IPB and incubation for 1.5 h at 4°C while rotating. The beads were washed 4x with IPB and eluted with 600 µl ammonium hydroxide (0.1 N NH4OH, 0.2 mM EDTA) for 20 min at RT, 30 µl NuPage LDS sample buffer (Invitrogen) supplemented with 1% SDS, reducing agent (Invitrogen) and 4.5% Glycerol for 5 min at 95°C, or with 120 ul 10 mM DTT, 10 mM NaPO4 pH 7.4, 0.5 mM EDTA for 30 min at RT in case probe PF-631 with a cleavable disulfide linker was used. For the mass spectrometry analysis, ammonium hydroxide or DTT eluates were evaporated using a Speedvac (Eppendorf) and stored at -20°C until further analysis. Mass Spectrometry Analysis and Quantification. For each immobilized probe, 3 independent biological replicate experiments were performed and each eluate was analyzed by liquid chromatography-mass spectrometry (LC-MS). Each dried eluate was redissolved in 25 µl of 8 M urea, 0.4 M NH4HCO3, 10 mM TCEP and incubated at 50 oC for 30 min, then allowed to cool to room temperature and treated with 55 mM iodoacetamide (Thermo Scientific) for 20 min. Digestion was initiated by adding 1 µg of trypsin (Promega) in 75 µl dH2O and the samples were incubated at 37°C overnight before being acidified by addition of 8 µl of formic acid and desalted and concentrated using Pierce 100 µl C18 Tips (Thermo Scientific). Elution was performed using 0.1 ml of 70% acetonitrile, 0.1% formic acid. The eluted samples were dried in a SpeedVac, redissolved in 40 µl of 0.1% formic acid, and then analyzed by nano-LC-MS (6 µl injections) LC-MS was conducted using Waters nanoAcquity HPLC equipment interfaced with either a Q Exactive or an LTQ Orbitrap Elite mass spectrometer (Thermo Scientific in each case). Raw files from the mass spectrometer have been analyzed using MaxQuant 1.3.0.5 and searched against the Uniprot mouse reference 43K proteome using the andromeda search engine. LFQ intensities were used for the analysis loaded into Perseus software (version 1.4.1.3).33 Missing values were replaced by a gaussian distribution (by column separately, width 0.2, downshift 1.8). The average fold enrichment of a protein was determined by calculating the difference between the protein intensities in pull-down reactions containing DMSO and reactions in the presence of 50 µM MP-10. Enrichment was calculated for each biological experiment first, to normalize for difference in absolute LFQ values between different MS-instruments, before averaging. P-values were calculated using a one-sample TTest. [12] ACS Paragon Plus Environment

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Figure Legends Figure 1. Design and synthesis of PDE10A photoaffinity probe. (A) Top: Structure of the Pfizer PDE10A clinical candidate PF-920 (MP-10). Below: X-ray co-crystal structure of MP-10 bound to the catalytic subunit of PDE10A. The red arrow highlights the attachment point for a linker group that should not significantly interfere with binding. (B) Top: Structure of the PDE10A photoaffinity probe PF-942. PDE10A recognition structure is shown in red, photoactivatable benzophenone crosslinker in blue and alkyne-handle for click-chemistry in green. Below: PF-942 modeled in the catalytic subunit of PDE10A. Figure 2. Photoaffinity labeling of PDE10A and target engagement of MP-10 in rat striatal membranes. (A) Incubation of membranes with PF-942 followed by UV-crosslinking, click chemistry with TAMRA-azide, SDS-PAGE and in-gel fluorescence (left) analysis resulted in labeling of a band at ~90 kDa (arrowhead). Middle: Coomassie staining of the gel after in-gel fluorescence shows equal loading. Right: Quantification of the 90 kDa band in the TAMRA gel (N=3). (B) Membranes were incubated with 30 nM PF-942 in presence of 0.1% DMSO (-) or 10 µM MP-10 (+) and UV-crosslinked for indicated times. Lysates were labeled with TAMRA-azide, analyzed by SDSPAGE and in-gel fluorescence (left) and the PDE10A band (arrowhead) quantified (right, N=2). (C) PDE10A is specifically enriched by a pulldown using streptavidin (SA) beads after labeling of membranes with 30 nM PF-942 followed by UV irradiation and click chemistry with BiotinTAMRA-azide. Eluates were analyzed by SDS-PAGE and TAMRA in gel fluorescence (left) and PDE10A immunoblot (right). (D) Target engagement of PDE10A by MP-10 was measured by competition of the PF-942-TAMRA label in striatal membranes by MP-10 (left; PDE10A band denoted by arrowhead) or the structurally distinct PDE10A inhibitor Papaverine (middle). Quantification of the PDE10A band in the TAMRA gels (right, N=3). Figure 3. Photoaffinity labeling of PDE10A and target engagement of MP-10 in rat striatal cultures. (A) Rat striatal cultures (100,000 cells / cm2; DIV15 - DIV21) were incubated with PF-942 followed by UV-crosslinking, lysis of the cells and click chemistry with TAMRA-azide. Left: SDSPAGE and in-gel fluorescence revealed labeling of a ~90 kDa band (arrowhead). Right: Quantification of the 90 kDa band in the TAMRA gel (N=4; N=3 for 1 µM PF-942). (B) PDE10A target engagement by MP-10 in rat striatal cultures (100,000 cells / cm2; DIV14 - DIV16) was measured by quantification of the 90 kDa band in the TAMRA gel (N=3; N=2 for 30 µM MP-10) [13] ACS Paragon Plus Environment


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after treating the cultures with MP-10 followed by photolabeling with with 0.3-1 µM PF-942, click chemistry with TAMRA-azide and analysis by in gel fluorescence as before. Figure 4. Design and synthesis of biotinylated PDE10A affinity probes. Chemical structures of the PDE10A biotinylated affinity probes PF-267, PF-621 and PF-631 which are derived from PF-920 (MP-10). PDE10A recognition structure is shown in red, linker sequence in black and biotin in green. PF-631 contains a cleavable disulfide in the linker. Figure 5. Enrichment of native PDE10A using biotinylated PDE10A probes. (A) PDE10A pulldown from striatal lysates. Equal molar quantities of biotinylated PF-621 probe and PF-942 conjugated to biotin-TAMRA-N3 by click chemistry were coupled to streptavidin beads. Coupled beads were incubated with striatal lysates and the eluates were analyzed by SDS-PAGE and Coomassie stain. (B+C) Comparison of PDE10A isolation by PF-267 and PF-621. Probe coupled beads were incubated with striatal lysates and the eluates were analyzed by SDS-PAGE, PDE10A immunoblot (B) and Coomassie stain (C). (D) PDE10A pulldown using PF-631 with cleavable disulfide linker. Probe coupled beads were incubated with striatal lysates and bound proteins were eluted using 10 mM DTT and analyzed by SDS-PAGE and Coomassie stain. (E) PDE10A pulldown from human post-mortem brain tissue from three individuals indicated by the LA-brain bank number. Left panel is showing expression of PDE10A in caudate / putamen lysates by immunoblot analysis. Blot was reprobed with GAPDH antibodies to show equal loading. Right panel is showing PDE10A levels in eluates from PF-621 pulldowns. Graphs are showing quantification of relative PDE10A levels (left) and relative PDE10A binding in (N=4) biological replicates. P-values are calculated using an unpaired T-test Figure 6. Mass spectrometry analysis of probe-interacting proteins. Eluates of SA-pulldowns using the indicated biotinylated affinity probes in striatal lysates were trypsin digested in solution, analyzed by LC-MS/MS and quantified using a label-free approach (MaxQuant). (A+B) Volcano plots showing proteins identified by the probe pull-downs. Difference is indicating the log2 of the average ratio (N=3) of probe pull-downs in absence or presence of the parent inhibitor MP-10. P-values are calculated using a one-sample T-Test and presented as negative log10. (C) Comparison of the overlap of enriched proteins between the non-cleavable probe (PF-621) and the probe containing the cleavable linker (PF-631) showing specificity of the compounds for PDE10A.


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References (1) Francis, S. H., Houslay, M. D., and Conti, M. (2011) Phosphodiesterase Inhibitors: Factors That Influence Potency, Selectivity, and Action, in Phosphodiesterases as Drug Targets (Francis, S. H., Conti, M., and Houslay, M. D., Eds.), pp 47–84. Springer Berlin Heidelberg, Berlin, Heidelberg. (2) Maurice, D. H., Ke, H., Ahmad, F., Wang, Y., Chung, J., and Manganiello, V. C. (2014) Advances in targeting cyclic nucleotide phosphodiesterases. Nat. Rev. Drug Discov. 13, 290–314. (3) Menniti, F. S., Plath, N., Svenstrup, N., and Schmidt, C. J. (2014) Pharmacological Manipulation of Cyclic Nucleotide Phosphodiesterase Signaling for The Treatment of Neurological and Psychiatric Disorders In The Brain, in Cyclic-Nucleotide Phosphodiesterases In The Central Nervous System (Brandon, N. J., and West, A. R., Eds.), pp 77–114. John Wiley & Sons, Inc. (4) Bender, A. T., and Beavo, J. A. (2006) Cyclic Nucleotide Phosphodiesterases: Molecular Regulation to Clinical Use. Pharmacol. Rev. 58, 488–520. (5) Charych, E. I., and Brandon, N. J. (2014) Molecular And Cellular Understanding of PDE10A: A Dual-Substrate Phosphodiesterase with Therapeutic Potential to Modulate Basal Ganglia Function, in Cyclic-Nucleotide Phosphodiesterases In The Central Nervous System (Brandon, N. J., and West, A. R., Eds.), pp 247–268. John Wiley & Sons, Inc. (6) Coskran, T. M., Morton, D., Menniti, F. S., Adamowicz, W. O., Kleiman, R. J., Ryan, A. M., Strick, C. A., Schmidt, C. J., and Stephenson, D. T. (2006) Immunohistochemical localization of phosphodiesterase 10A in multiple mammalian species. J. Histochem. Cytochem. 54, 1205–1213. (7) DeLong, M. R., and Wichmann, T. (2007) Circuits and Circuit Disorders of the Basal Ganglia. Arch Neurol 64, 20–24. (8) Giampà, C., Laurenti, D., Anzilotti, S., Bernardi, G., Menniti, F. S., and Fusco, F. R. (2010) Inhibition of the Striatal Specific Phosphodiesterase PDE10A Ameliorates Striatal and Cortical Pathology in R6/2 Mouse Model of Huntington’s Disease. PLoS ONE 5, e13417. (9) Grauer, S. M., Pulito, V. L., Navarra, R. L., Kelly, M. P., Kelley, C., Graf, R., Langen, B., Logue, S., Brennan, J., Jiang, L., Charych, E., Egerland, U., Liu, F., Marquis, K. L., Malamas, M., Hage, T., Comery, T. A., and Brandon, N. J. (2009) Phosphodiesterase 10A Inhibitor Activity in Preclinical Models of the Positive, Cognitive, and Negative Symptoms of Schizophrenia. J. Pharmacol. Exp. Ther. 331, 574 –590. (10) Schmidt, C. J., Chapin, D. S., Cianfrogna, J., Corman, M. L., Hajos, M., Harms, J. F., Hoffman, W. E., Lebel, L. A., McCarthy, S. A., Nelson, F. R., Proulx-LaFrance, C., Majchrzak, M. J., Ramirez, A. D., Schmidt, K., Seymour, P. A., Siuciak, J. A., Tingley, F. D., Williams, R. D., Verhoest, P. R., and Menniti, F. S. (2008) Preclinical Characterization of Selective Phosphodiesterase 10A Inhibitors: A New Therapeutic Approach to the Treatment of Schizophrenia. J. Pharmacol. Exp. Ther. 325, 681– 690. (11) Li, N., Lee, K., Xi, Y., Zhu, B., Gary, B. D., Ramírez-Alcántara, V., Gurpinar, E., Canzoneri, J. C., Fajardo, A., Sigler, S., Piazza, J. T., Chen, X., Andrews, J., Thomas, M., Lu, W., Li, Y., Laan, D. J., Moyer, M. P., Russo, S., Eberhardt, B. T., Yet, L., Keeton, A. B., Grizzle, W. E., and Piazza, G. A. (2014) Phosphodiesterase 10A: a novel target for selective inhibition of colon tumor cell growth and β-catenin-dependent TCF transcriptional activity. Oncogene. [Epub ahead of print] DOI: 10.1038/onc.2014.94 (12) Tian, X., Vroom, C., Ghofrani, H. A., Weissmann, N., Bieniek, E., Grimminger, F., Seeger, W., Schermuly, R. T., and Pullamsetti, S. S. (2011) Phosphodiesterase 10A Upregulation Contributes to Pulmonary Vascular Remodeling. PLoS ONE 6, e18136. (13) DeMartinis, N., Banerjee, A., Kumar, V., Boyer, S., Schmidt, C. J., and Arrojo, S. (2012) Results of a Phase 2A Proof-of-Concept Trial with a PDE10A Inhibitor in the Treatment of Acute [15] ACS Paragon Plus Environment


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ASSOCIATED CONTENT Supporting figures and a detailed description of the chemical synthesis and the materials and methods are in the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding author: Jan-Philip Schülke: [email protected]; +1-617-395-0654, or Douglas S Johnson: [email protected]; +1-617-395-0697 Notes: Conflict of Interest: JPS, LAM, KFG, VP, TAC, PRV, CJS, DSJ, are employees of Pfizer Inc; NJB is employee of AstraZeneca Ltd. ACKNOWLEDGEMENTS We thank Pfizer’s Comparative Medicine for help with the animal tissue dissection, K. Mou for the primary striatal cultures, T. Lanz for the human brain samples and H. (Simon) Xi for help establishing the label-free quantitation workflow.


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A

B

N

N N

O

N

N

N N

O

PF-2545920

N H N

O

PF-06481942

ACS Paragon Plus Environment

O

O

O O


250 150 100 75 50 37

250 150 100 75 50 37

25 20 15

25 20 15

0.0 -11 -10 -9 -8 -7 log10 PF-942 [M]

Coomassie

C

10

kDa 250 150 100 75 50 37

20 30 UV [min] MP-10

TAMRA In-Gel Fluorescence

kDa 250 150 100 75 50 37

250 150 100 75 50 37

0.5

0

10

20

Time [min]

MP-10 [nM]


TAMRA PDE10A

Papaverine [nM]

250 150 100 75 50 37

25 20 15

25

30

no probe 0 1 3 10 30 100 300 1,000 3,000

D

1.0

0.0

25 20

25 20 15

-6

Biotin-TAMRA-N3, SA-Pulldown

DMSO PF-942

3

MP-10 [IC50: 4.2 nM]

TAMRA Fluorescence

1

TAMRA Fluorescence

30 nM PF-942

0

EC50: 11.9 nM N=3

0.5


B

1.0

DMSO PF-942

kDa

DMSO 1 3 10 30 100 300 1,000 3,000 10,000

kDa

PF-942 [nM]

DMSO 1 3 10 30 100 300 1,000 3,000 10,000

PF-942 [nM]

TAMRA Fluorescence

A

no probe 0 1 3 10 30 100 300 1,000 3,000

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

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Papaverine [IC50: 186.8 nM]

1.0

0.5

0.0 TAMRA In-Gel Fluorescence


-10

-8 -6 log10 MP-10 [M]

-4

A

B

DMSO 1 3 10 30 100 300 1,000 3,000 10,000

PF-942 [nM]

MP-10 [nM]

kDa 250 150 100 75 50 37

25 20 15

1.0

TAMRA Fluorescence

TAMRA In-Gel Fluorescence TAMRA Fluorescence

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


EC50: 445 nM N=3

0.5

0.0

-10

-8 -6 log10 PF-942 [M]

-4

no probe 0 1 3 10 30 100 300 1,000 3,000 10,000 30,000

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1.0

IC50: 1.7 µM N=3

0.5

0.0

-10

-8 -6 log10 MP-10 [M]


-4


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

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N

N N

N

N H

H

H N

O

12

S O

N

N N

O

N

O

PF-06482621

HN

O

H N

O

O

O

N H

NH H

HN

O O

O

O

PF-06482267

H

H N

O

12

NH H S

O

N

O

PF-06646631 N N

O

N

O

H N

O O

O

N H

HN

O S

S

N H


O

H

H N 4

NH H S

O

2

kDa

kDa

200

200

116 97

116 97 66 55

66 55 36 31 Coomassie

IB: PDE10A PF-621 SA-Pulldown kDa 250 150 100 75

50 37

50 37

25 20 15

25 20 15

IB: PDE10A IB: GAPDH

25 20 15 Coomassie

Total PDE10A 1.5

2.0

ns

1.0 0.5

IB: PDE10A

PDE10A Binding

1.5 1.0 0.5

0.0

0.0 #3 32 #3 2 39 #3 7 40 6

kDa 250 150 100 75

#3322 #3397 #3406

Caudate / Putamen Input

50 37

#3 32 #3 2 39 #3 7 40 6

E

PF-631 SA-Pulldown DTT Elution MP-10 kDa 250 150 100 75

36 31 21 14 Coomassie

Relative Expression [normalized to GAPDH]

25 20 15 10

MP-10

D

SAPulldown

Relative Binding [normalized to input]

1

PF -9 4

PF -6 2 kDa 250 150 100 75 50 37

C

SAPulldown PF-267 PF-621 Input

B

SA-Pulldown

#3322 #3397 #3406

A

#3322 #3397 #3406

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


PF-267 PF-621

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A

PF-621 6 P-value [-log10]

5

PDE10A

4 3 2 1 0

-8 -6 -4 -2 0 2 4 6 8 Difference DMSO/MP-10 [log2]

B

PF-631 6 PDE10A

P-value [-log10]

5 4 3 2 1 0

C

-8 -6 -4 -2 0 2 4 6 8 Difference DMSO/MP-10 [log2] PF-621 vs. PF-631

10

PF-631 Difference DMSO/MP-10 [log2]

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 24 of 25

PDE10A

5 0

-5 -5

0 5 PF-621 Difference DMSO/MP-10 [log2]

10


Page 25 of 25

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


MP-10 N

Photo-Probe

N N

N

N N

O

Affinity-Probe

N

O

N

O

N H N

O

O

O

O

N N

O

N

O

H N

HN

O O

O

N H

O

H

H N 12

NH H S

O

O

In-Gel Fluorescence: Target Engagement

Affinity Chromatography / LC-MS: Chemoproteomic profile


Chemoproteomics Demonstrates Target Engagement and Exquisite

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