Characterization of the Novel Broad-Spectrum Kinase Inhibitor CTx

Article pubs.acs.org/jpr

Characterization of the Novel Broad-Spectrum Kinase Inhibitor CTx0294885 As an Affinity Reagent for Mass Spectrometry-Based Kinome Profiling Luxi Zhang,† Ian P. Holmes,‡ Falko Hochgraf̈ e,† Scott R. Walker,‡,§ Naveid A. Ali,† Emily S. Humphrey,† Jianmin Wu,† Melanie de Silva,‡,⊥,¶ Wilhelmus J. A. Kersten,‡,⊥,¶ Theresa Connor,‡,⊥,¶ Hendrik Falk,‡,⊥,¶ Lynda Allan,‡,⊥,¶ Ian P. Street,‡,⊥,¶ John D. Bentley,∥ Patricia A. Pilling,∥ Brendon J. Monahan,∥ Thomas S. Peat,∥ and Roger J. Daly*,†,■ †

Cancer Research Program, Garvan Institute of Medical Research, 384 Victoria Street, Sydney, New South Wales 2010, Australia Cancer Therapeutics CRC, 4 Research Avenue, Bundoora, Victoria 3083, Australia § Monash Institute of Pharmaceutical Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia ⊥ Division of Chemical Biology, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia ¶ Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia ∥ Biosciences Program, CSIRO Materials Science and Engineering, 343 Royal Parade, Parkville, Victoria 3052, Australia ‡

S Supporting Information *

ABSTRACT: Kinase enrichment utilizing broad-spectrum kinase inhibitors enables the identification of large proportions of the expressed kinome by mass spectrometry. However, the existing inhibitors are still inadequate in covering the entire kinome. Here, we identified a novel bisanilino pyrimidine, CTx-0294885, exhibiting inhibitory activity against a broad range of kinases in vitro, and further developed it into a Sepharose-supported kinase capture reagent. Use of a quantitative proteomics approach confirmed the selectivity of CTx-0294885-bound beads for kinase enrichment. Large-scale CTx-0294885-based affinity purification followed by LC−MS/MS led to the identification of 235 protein kinases from MDA-MB-231 cells, including all members of the AKT family that had not been previously detected by other broad-spectrum kinase inhibitors. Addition of CTx-0294885 to a mixture of three kinase inhibitors commonly used for kinase-enrichment increased the number of kinase identifications to 261, representing the largest kinome coverage from a single cell line reported to date. Coupling phosphopeptide enrichment with affinity purification using the four inhibitors enabled the identification of 799 highconfidence phosphosites on 183 kinases, ∼10% of which were localized to the activation loop, and included previously unreported phosphosites on BMP2K, MELK, HIPK2, and PRKDC. Therefore, CTx-0294885 represents a powerful new reagent for analysis of kinome signaling networks that may facilitate development of targeted therapeutic strategies. Proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the data set identifier PXD000239. KEYWORDS: cell signaling, chemical proteomics, kinase, mass spectrometry, protein phosphorylation

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genesis.4 In several cases, disease progression can be reversed by inhibition of the aberrant kinase activity, as exemplified by targeting the BCR-ABL fusion protein by imatinib in treatment of chronic myeloid leukemia.5 The clinical success of imatinib has fueled the development of a large number of smallmolecule inhibitors as well as antisense oligonucleotides and

INTRODUCTION

Protein kinases are key components of cell signaling networks and thereby regulate fundamental biological processes such as cellular proliferation, metabolism, and survival.1 The human kinome currently contains 518 members, nearly half of which can be mapped to disease loci, and perturbation in kinasemediated signaling cascades is a common cause of diseases such as inflammation, diabetes, and cancer.2,3 For example, up to one-quarter of all protein kinases may contribute to onco© 2013 American Chemical Society

Received: September 6, 2012 Published: May 21, 2013 3104

dx.doi.org/10.1021/pr3008495 | J. Proteome Res. 2013, 12, 3104−3116

Journal of Proteome Research

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monoclonal antibodies as anticancer drugs for specific kinase inhibition.6−8 Despite the human kinome being recognized as a potentially rich source of drug targets, much research effort has been focused on a rather small fraction of the human kinome, and only 10% of all protein kinases are being evaluated in the clinical setting.9−11 As a result, a large proportion of the human kinome still remains poorly characterized. Several approaches have been developed for unraveling kinase-mediated signaling networks. Antibody arrays, for instance, allow determination of the status of over 100 phosphorylation sites from kinases and their substrates in a given biological sample.12,13 Peptide arrays, on the other hand, contain up to thousands of kinase substrate sequences that can be phosphorylated in vitro by kinases present in the applied protein lysates. 14 The pattern and degree of peptide phosphorylation provide information regarding upstream kinase activities; however, since the majority of the peptide sequences can be recognized by more than one kinase, pinpointing the upstream kinase is challenging. In addition, array-based techniques do not reveal any novel phosphorylation events and permit the investigation of only kinases with a corresponding antibody or known substrate; thus, these methods do not shed any light on the poorly characterized kinome. In contrast, mass spectrometry-based phosphoproteomics has no such restrictions, and large-scale studies have demonstrated that tens of thousands of phosphorylated peptides can be identified when an appropriate enrichment strategy is employed, such as TiO2 or an antiphosphotyrosine antibody.15−19 However, because of the low cellular abundance of many protein kinases, most kinase-derived peptides are undetectable in a complex protein sample; therefore, an additional kinase enrichment step prior to phosphopeptide enrichment is necessary for comprehensive analysis of the kinome at a global level.20 Chemical proteomics utilizing immobilized small-molecule kinase inhibitors as protein kinase-capturing agents represents the most efficient kinase enrichment strategy to date.20−22 As all protein kinases possess a highly conserved ATP binding domain, ATP competitive kinase inhibitors can be designed to recognize a large number of kinases and act as kinase-capturing tools. Several studies have demonstrated that by using a mixture of broad-spectrum kinase inhibitors with distinct but overlapping binding profiles, over 200 different protein kinases can be detected from cell extracts.23 In addition, this kinase enrichment technique in combination with quantitative mass spectrometry can be applied to a wide range of studies, including investigating the kinome response to a particular stimulus or mutation, kinomic changes between different phases of the cell cycle and, through competitive binding assays, identifying physiological targets of more-specific kinase inhibitors.20,24−26 These studies have significantly enhanced our knowledge of the kinome and highlight the potential of this approach for identification of candidate drug targets. Nevertheless, the power of this kinase enrichment technique is limited to the kinome coverage of the inhibitors used, and the existing kinase inhibitors are still inadequate in capturing the entire kinome; hence, there is a need for development of novel broad-spectrum kinase inhibitors to fill this void. In this study, we have evaluated the binding profile of a novel kinase capture reagent, CTx-0294885, using mass spectrometry. Combining CTx-0294885 with three other broad-specificity inhibitors for kinase enrichment has resulted in the largest kinome coverage from a single cell line reported to date.

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MATERIALS AND METHODS

Cell Culture and Lysis

MDA-MB-231 cells were cultured in RPMI1640 (Invitrogen) supplemented with insulin at 0.25 IU/mL and 10% fetal bovine serum (Invitrogen). For SILAC, cells were grown for 10 days in RPMI 1640 (Thermo Scientific) containing either normal Larginine (Arg0) at 15 mg/L and L-lysine (Lys0) at 40 mg/L or equimolar amounts of L-[13C6,15N4]-arginine (Arg10) and L[13C6,15N2]-lysine (Lys8) (Silantes). Subconfluent cells were lysed with (I) urea lysis buffer containing 8 M urea and 50 mM HEPES-NaOH for total and phosphoproteome analyses; (II) modified RIPA buffer containing 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, and 50 mM Tris (pH 7.5) for SILAC-MS; or (III) kinome profiling buffer containing 50 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, and 1 mM EGTA for large-scale kinome profiling. All lysis buffers were supplemented freshly with 10 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mM PMSF, 10 mM NaF, 50 ng/mL calyculin A, 1% phosphatase inhibitor mixture 3 (Sigma), and 2.5 mM Na3VO4 prior to cell lysis. Cell debris was removed by centrifugation at 16500g at 4 °C for 30 min, and the supernatant was subsequently filtered through a 0.45 μm PVDF membrane (Millipore). Synthesis of Chemical Compounds

CTx-0152960, CTx-0152857, CTx-0294885, and CTx-0294857 were synthesized by initial regioselective nucleophilic displacement of the 4-Cl group of 2,4,5-trichloropyrimidine with 2amino-N-methylbenzamide. Subsequent displacement of the 2Cl group with an appropriately decorated aniline under acidic conditions gave the target compounds. A more detailed description of the synthesis strategy will be published elsewhere. Kinase Inhibition Assays

A panel of 10 kinases consisting of FAK, Flt3, JAK2, JAK3, CSF-1R, SRC, VEGFR3, Aurora A, LIMK1, and ROCK2 was initially used to evaluate kinase inhibition profiles. For the tyrosine kinase biochemical assays, compounds were tested for inhibitory activity using the P-Tyr-100 Alphascreen kit (PerkinElmer) according to the manufacturer’s recommendations. Briefly, 15 μL assay reactions were performed in Greiner brand white 384-well, low-volume plates. All reactions contained assay buffer (10 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% (v/v) Tween-20, 50 μM Na3VO4, 0.01% (w/v) albumin from chicken egg white, 1 mM dithiothreitol), a Nterminally biotinylated peptide substrate (concentration 111 nM), and ATP at a concentration equaling the KM (ATP) of the kinase. The compounds were added in a volume of 100 nL from an 11-point-dilution series prepared in DMSO, positive and negative control reactions receiving the same volume of DMSO without compound. The kinases were added at predetermined concentrations, generally ranging between 0.2 and 8 nM, with the enzyme being omitted from negative control reactions. The reactions were incubated for 90 min at 30 °C and stopped by adding 5 μL of Stop buffer (10 mM HEPES pH 7.4, 25 mM NaCl, 100 mM EDTA, 0.01% (v/v) Tween-20) containing streptavidin-coated donor and antiphosphotyrosine (P-Tyr-100) acceptor beads. Plates were incubated for 4−6 h before being read on a PerkinElmer EnVision plate reader in HTS Alphascreen mode. IC50 values were obtained by calculating percent inhibition for each 3105



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reaction relative to controls on the same plate and fitting the dose−response curves to a four-parameter logistic equation. For serine/threonine kinase biochemical assays, inhibition of serine-threonine kinases was tested using the Transcreener ADP FP kit (Bellbrook Laboratories) according to the manufacturer’s recommendations. Briefly, 10 μL reactions were performed in Greiner brand black 384-well, low-volume plates. All reactions contained assay buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 10 mM MgCl2, 0.25 mM EGTA, 0.01% (v/ v) Triton X100, 0.01% (w/v) albumin from chicken egg white, 1 mM dithiothreitol), ATP at a concentration equaling the Km (ATP) of the kinase, and a peptide substrate at the same concentration as ATP. The compounds were added in a volume of 100 nL from an 11-point-dilution series prepared in DMSO, positive and negative control reactions receiving the same volume of DMSO without compound. The kinases were added at predetermined concentrations resulting in about 10% of the ATP turned over, with the enzyme being omitted from negative control reactions. The reactions were incubated for 60 min at 30 °C and stopped by adding 10 μL of Transcreener Stop & Detect buffer containing Tracer (3 nM final) and ADPantibody as appropriate for the ATP starting concentration. The plates were incubated for at least 90 min before being read on a PerkinElmer EnVision plate reader in fluorescence polarization mode. IC50 values were obtained by calculating percent inhibition for each reaction relative to controls on the same plate and fitting the dose−response curves to a fourparameter logistic model.

Sepharose beads and ethanolamine-capped control Sepharose beads, respectively, for 16 h at 4 °C and protected from light. A replicate experiment was performed in parallel with the labeled lysates switched for each bead type for inverse labeling. After incubation, the beads were washed in modified RIPA buffer supplemented with a cocktail of protease and phosphatase inhibitors, as described earlier. Following washing, proteinbound CTx-0294885 beads and control beads were combined in both replicate experiments. Bound proteins from the combined beads were reduced and alkylated with direct addition of 1 mM DTT and 5 mM IAA and subsequently eluted with Laemmli sample buffer. Eluted proteins were resolved and separated using 8% SDS−PAGE, followed by an in-gel digest procedure as described elsewhere.27 Large-Scale Kinase Enrichment for Kinome Profiling

The protein concentration was ∼4 mg/mL, and the final NaCl concentration of protein lysates was adjusted to 1 M. The kinase inhibitor-bound resins were washed with 10 mL of H2O, followed by 10 mL of washing buffer A (lysis buffer with 1 M NaCl plus 10 mM NaF and 0.1 mM Na3VO4) prior to kinase enrichment. For single kinase inhibitors, 1 mL of inhibitorbound resin was incubated with 50 mg of protein lysate. For multiinhibitor-bound resins, a cocktail comprising 1 mL of each inhibitor-bound resin was incubated with 100 mg of protein lysate. All incubations were performed for 2 h at 4 °C while protected from light on a rotating wheel. Following incubation, protein-bound inhibitor resins were washed twice with washing buffer A, once with washing buffer B (same as washing buffer A except the NaCl concentration is 150 mM instead of 1 M), and once with washing buffer C (50 mM HEPES, 10 mM NaF, and 0.1 mM Na3VO4). Resin-bound proteins were eluted by five consecutive rounds of incubation with 4 mL of 5 mM DTT and 0.5% SDS for 3 min at 60 °C. Eluted fractions were pooled and lyophilized by freeze-drying.

Generation of Kinase Affinity Resin

The kinase inhibitors Purvalanol B (Tocris) and SU6668 (Biochempartner Chemical) were immobilized to EAHSepharose 4B (GE Healthcare), and VI16832 (Evotec) was coupled to ECH-Sepharose 4B (GE Healthcare) beads as previously described.20,23 For CTx-0294885 immobilization, 1.78 g of activated CH-Sepharose 4B (GE Healthcare) was swelled with 50 mL of 1 mM aqueous HCl, collected by filtration (porosity 4 glass frit), and then washed 9 times with 50 mL of 1 mM aqueous HCl. CTx-0294885 (14 mg) was dissolved in 5.3 mL of DMF then diluted 1:1 with 100 mM sodium bicarbonate. The swelled activated resin was added, and the resulting mixture was gently shaken for 18 h. After filtration, the resin was washed twice with 15 mL of 50% aqueous DMF. The resin was then resuspended in 10 mL of 1 M ethanolamine in 50% aqueous DMF and agitated for 1 h. The mixture was filtered, and the resin was washed sequentially 10 times with 10 mL of 50% aqueous DMF, 20 times with 25 mL of 0.1 M pH 4 sodium acetate buffer, 20 times with 25 mL of 0.1 M pH 8 sodium bicarbonate buffer, and 10 times with 20 mL of 20% aqueous ethanol. For ethanolamine-capped control beads, the preparation is essentially the same as previously described for the CTx0294885 beads, except the ligand-coupling step and the immediate washing steps were omitted from the procedure. The washed resin was collected, resuspended in 20% aqueous ethanol, and stored at 4 °C.

MS Sample Preparation

Lyophilized proteins from large-scale kinome profiling experiments were resuspended in H2O, followed by acetone precipitation and collection of proteins.28 Thirty percent of the precipitated proteins were solublized in a 20 mM HEPES and 8 M urea buffer (pH 7.5), followed by reduction and alkylation using 10 mM DTT and 55 mM iodoacetamide, respectively (30 min at RT). Tryptic digestion of the reduced and alkylated proteins was performed at 37 °C overnight using sequencing grade modified porcine trypsin (Promega) at a protein-to-enzyme ratio of 1:100.27 Following digestion, peptides were desalted using Sep-Pak C18 columns (Waters).29 The remaining 70% of the precipitated proteins were dissolved in 2× Laemmli sample buffer and separated by 10% SDS− PAGE. An in-gel digest was performed as previously described.27 One-tenth of the in-gel extracted peptides was desalted using C18 StageTips prior to MS analysis for total protein expression, and the remainder was prepared for phosphopeptide enrichment. For proteome analysis of total cell extracts, 50 μg of MDAMB-231 urea lysate was mixed with Laemmli sample buffer prior to protein separation by 10% SDS-PAGE and followed by protein in-gel reduction, alkylation, and digestion the same way as described for large-scale kinome enrichment samples. For phosphoproteome analysis, 8 mg of protein was reduced, alkylated, digested with trypsin, desalted using SepPak C18 cartridges, and lyophilized. Desalted peptides were resuspended in 1.2 mL of 30% ACN and 0.2% TFA and separated by strong

Determination of the Specificity of CTx-0294885 Binding by Quantitative Proteomics

Experiments were undertaken essentially as previously described.24 Briefly, MDA-MB-231 cells were SILAC-labeled and lysed as described in the Cell Culture and Lysis section. Two milligrams of light lysate and 2 mg of heavy lysate were incubated with 25 μL of a 50% slurry of CTx-0294885-bound 3106



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Figure 1. Chemical structures of the FAK inhibitor TAE-226 and its analogues. A. Structural comparison of TAE-226 with its analogues CTx0152960 and CTx-0152857 lacking the methyl ether group. B. Structural comparison of CTx-0294885 with its immobilized form and CTx-0294857 mimicking the immobilized CTx-0294885.

Bioresources) with 2% ACN in 0.05% TFA at 15 μL/min for 4 min. The precolumn was then switched online with a nano-C18 column (75 μ × ∼10 cm, 5 μ, 200 Å Magic, Michrom), and the reverse phase nanoeluent was subjected to nanoflow electrospray analysis in a data-dependent acquisition mode (250 nL/ min over 30 min). The survey scans (350−1750) were acquired with lockmass enabled in the orbitrap with a resolution of 60 000 at m/z 400. Up to 15 ions (ion selection threshold > 5000 counts) with charge states ≥2 were sequentially isolated and further subjected to MS/MS fragmentation within the linear ion trap using collision-induced dissociation (30% relative collision energy). MS/MS spectra were accumulated with an activation time of 30 ms at a target value of 30 000 ions, and ions were excluded from further selection for 30 s.

cation chromatography using a Resource S column (GE Healthcare) on an AKTA Purifier FPLC (GE Healthcare). Peptides were fractionated into 14 fractions (including the flowthrough) with a gradient of 100% buffer A (5 mM KH2PO4, 30% ACN, 0.01% TFA) to 30% buffer B (5 mM KH2PO4, 30% ACN, 0.01% TFA, 350 mM KCl). Phosphopeptide Enrichment Using TiO2

The remaining 90% of the gel extracts (with adjacent gel slices combined to give a total of six peptide samples) and peptides resulting from in-solution digestion prepared for large-scale kinome profiling, and the final nine peptide fractions from SCX for phosphoproteome analysis were individually subjected to TiO2 enrichment for phosphopeptides. TiO2 beads (GL Sciences) were suspended in 100% ACN and packed into an in-house C8 StageTip by centrifugation at 2000g for 2 min. Peptide samples were dissolved in loading buffer (25% lactic acid, 73% ACN, and 2% formic acid) and were loaded onto the packed TiO2 columns (0.5 mg of beads used for each of the kinome profiling peptide samples and 3 mg of beads were used for each fraction from the phosphoproteome analysis), followed by centrifugation of the column at 1000g for 10 min. The flowthrough of the in-solution digests from TiO2 column was reapplied to a new TiO2 column for two further consecutive rounds of phosphopeptide enrichment. The peptide-bound columns were washed four times with 50 μL of washing buffer (1% TFA, 80% ACN), and phosphopeptides were eluted using 50 μL of 5% ammonia solution in Milli-Q H2O, followed by a second elution with 50 μL of 30% ACN into the same tube. Phosphopeptide-enriched samples were vacuum-centrifugedried and desalted using C18 StageTips prior to MS analysis.

Protein Identification and Data Analysis

Raw files were processed with MaxQuant (version 1.1.1.25) for feature detection, protein identification, and quantification using the Andromeda search engine integrated into the MaxQuant environment for database searching.30 Extracted peak lists were searched against the UniProtKB/Swiss-Prot Homo sapiens database (Uniprot_human_2010_10) containing 35 052 entries and a separate reverse decoy database for controlling the false discovery rate (FDR). The following search parameters were selected: fixed cysteine carbamidomethylation modification; variable methionine oxidation modification; variable protein N-acetylation; variable phosphorylation of serine, threonine, and tyrosine. A minimum peptide length of 6 amino acids and up to two missed cleavages were allowed. In addition, for SILAC experiments, the SILAC labels Arg10 and Lys8 were selected as modifications, and the minimum peptide count for protein quantification was set to 1. The initial first search mass tolerance was 20 ppm for precursor ions and 0.5 Da for fragment ions, with individualized peptide mass tolerances used for the subsequent searches. The “match between runs” option in MaxQuant was used to transfer identifications between runs on the basis of matching of precursors with high mass accuracy.31 The FDR was limited to

LC−MS/MS Data Acquisition

Peptides were separated by nano-LC using an Ultimate 3000 HPLC and autosampler system (Dionex), and mass spectra were acquired on the Orbitrap Velos mass spectrometer (Thermo Electron). Samples were concentrated and desalted onto a micro C18 precolumn (500 μm × 2 mm, Michrom 3107



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Table 1. List of Kinases Showing >50% Inhibition When CTx-0294857 Was Used at Concentration of 1 μM kinase

% activity

SD

kinase

% activity

SD

kinase

% activity

SD

kinase

% activity

SD

ABL AMPK (Rat) AMPK Aurora A Aurora B BRK BRSK1 BRSK2 BTK CAMKKb CDK2 CHK1 CHK2 CK1δ CK2 CLK2 DAPK1 DDR2 DYRK1A DYRK2 DYRK3 EIF2AK3 EPHA2 EPHA4 EPHB1

5 3 1 1 1 4 14 10 6 10 29 18 0 21 23 1 25 5 12 4 6 18 19 34 22

0 1 1 0 1 2 2 2 2 1 2 3 9 4 0 0 9 3 2 1 2 0 4 23 0

EPHB2 EPHB4 ERK2 ERK8 FGFR1 GCK GSK3b HER4 HIPK1 HIPK2 HIPK3 IGF1R IKKe IR IRAK1 IRAK4 IRR JAK2 JNK1 JNK2 JNK3 Lck LKB1 MAP4K3 MAP4K5

30 30 50 8 3 8 27 7 5 9 14 2 20 3 4 10 18 1 3 11 28 24 30 7 9

2 3 8 7 3 3 1 1 4 1 1 0 0 0 2 1 3 0 0 4 6 12 0 0 1

MARK1 MARK2 MARK4 MELK MINK1 MKK1 MKK2 MLK3 MSK1 MST2 MST4 NEK2a NUAK1 PAK2 PAK4 PDGFRA PDK1 PHK PIM3 PKA AKT1 AKT2 PKD1 PLK1 PRK2

15 14 20 9 30 25 41 10 37 13 23 9 1 49 20 29 47 4 36 47 32 36 28 13 12

1 3 7 0 4 11 4 1 4 5 5 1 0 4 5 9 11 1 3 4 11 3 12 1 0

RIPK2 RSK1 RSK2 S6K1 SGK1 SIK2 SIK3 SmMLCK Src SRPK1 STK33 SYK TAK1 TAO1 TBK1 TESK1 TIE2 TrkA TSSK1 TTK ULK1 ULK2 VEGFR YES1

38 4 12 14 40 11 27 12 21 49 22 24 11 14 27 48 26 3 45 15 8 40 6 2

11 1 0 3 5 2 1 0 9 7 3 7 0 4 1 1 18 0 0 3 5 1 3 0

and inhibit kinases. An in vitro screen using CTx-0294885 and CTx-0294857 against a panel of 10 kinases showed that the two derivatives exhibited broadly similar properties, both potently inhibiting 7 kinases at low nanomolar concentrations (Supporting Information Table 1). To gain a better picture of the kinase selectivity and potency of the immobilized CTx0294885, the chemically related CTx-0294857 was subjected to cross-screen profiling against 131 kinases (http://www.ppu. mrc.ac.uk/). At a concentration of 1 μM, CTx-0294857 displayed high potency against 99 kinases, inhibiting >50% of their activity (Table 1).

1% for both protein and peptide identifications. Peptides with posterior error probability greater than 10% were removed, and protein identification required a minimum of 1 unique peptide. For phosphopeptides, those exhibiting a phosphosite localization probability (LP) of >0.75 were included in further analyses. For enrichment analysis of gene ontology categories, nonprotein kinases bound by CTx-0294885 from two SILAC replicate experiments were submitted to the DAVID Bioinformatic database online (http://david.abcc.ncifcrf.gov/, version 6.7) to compare with a reference data set comprising all UniProt entries and their respective GO identifiers.32,33

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Characterization of the Selectivity Profile of CTx-0294885 by MS

RESULTS

Having shown that CTx-0294857 targets a large number of kinases by in vitro kinase assays, we went on to determine the binding profile of the immobilized CTx-0294885 from cell lysates by quantitative MS. The binding selectivity of CTx-0294885 beads and control beads was compared in a small-scale affinity purification using SILAC-labeled cell lysates. SILAC ratios were used to determine the relative amounts of particular proteins that were specifically bound by CTx-0294885 beads versus control beads in duplicate inversed labeling experiments. In total, 172 protein kinases and 830 proteins of other classes were identified as captured by one or both matrices from two replicate experiments (Supporting Information Table 2). All proteins (including 143 protein kinases) with a SILAC ratio from both experiments are plotted in Figure 2A. Application of a log2 cutoff of 1.5 identified 131 nonredundant kinases and 510 other proteins specifically enriched by the CTx-0294885 beads versus the control beads in both replicate experiments. On average, protein kinases were identified with a peptide count 2.2-fold greater than nonprotein kinases. Approximately 49% of the total spectral intensity was contributed by kinase-

Development of CTx-0294885

TAE-226 has been characterized as a specific inhibitor of focal adhesion kinase (FAK).34,35 Surprisingly, we found that two compounds based on the structure of TAE-226 but lacking a particular methyl ether group, termed CTx-0152960 and CTx0152857 (Figure 1A), demonstrated high potency against a range of tyrosine and serine/threonine kinases when assayed in vitro (Supporting Information Table 1). This finding led us to prepare further analogues with the aim of developing a kinase capture reagent suitable for chemical proteomics. In addition to activity against a broad range of kinases, another essential feature for kinase-capture reagents is the presence of a functional group, not required for kinase affinity, suitable for immobilization of the compound on a resin such as Sepharose. Since CTx-0152960 and CTx-0152857 did not possess suitable functionality, CTx-0294885 was prepared with a piperazine ring to provide a point of attachment. A derivative mimicking the immobilized form of CTx-0294885, CTx0294857 was also synthesized (Figure 1B) to provide an initial evaluation of the ability of the resin-bound compound to bind 3108



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Figure 2. Use of SILAC-MS to identify proteins specifically enriched by CTx-0294885 beads. A. Quantitation of CTx-0294885 binding selectivity. Proteins identified and quantified with SILAC ratios from two replicate experiments were plotted, with the upper right-hand quadrant (SILAC ratio > log2 1.5) containing proteins with a high affinity for CTx-0294885 beads versus control beads. Red circles indicate protein kinases; black circles, other protein types. B. Distribution of CTx-0294885-bound protein kinases among the different kinase groups. C. Relative enrichment of different kinase families by CTx-0294885. The histograms indicate the representation of a given kinase family within the CTx-0294885-bound fraction (number of CTx-0294885-bound kinases within a given kinase family divided by the total number of kinases bound by CTx-0294885) and the representation of each kinase family within the total kinome; that is, the number of protein kinases in each family/total number of human protein kinases.

proteins copurified along with protein kinases are ATP-binding proteins. This analysis also highlights that tRNA-binding protein and NAD- or NADH-binding proteins are highly enriched by CTx-0294885. Purine-binding proteins have emerged as attractive targets in drug discovery in recent years.36,37 Although the level of enrichment for purine-binding proteins is relatively low, the number of identified proteins categorized under this term contributed to nearly 25% of all non-kinase proteins enriched by CTx-0294885.

derived peptides. When this is compared with a proteome analysis of the same total cell lysates, in which 132 kinases were identified, the combined kinase-derived spectral intensity accounted for only 1.7% (Supporting Information Table 3). This marked difference in spectral intensity indicates a 29-fold enrichment of protein kinases through affinity purification using CTx-0294885 beads. CTx-0294885 displays affinity for kinases across all major kinase groups, and the relative distribution of these kinase groups is shown in Figure 2B. Comparison of the representation of each kinase group (among bound kinases) with the contribution of that kinase group to the entire kinome revealed that CTx-0294885 exhibits a relatively high affinity for kinases from the CMGC group, indicated by a strong trend toward statistical significance (p = 0.06) (Figure 2C). Of note, unlike other kinase-capture reagents, CTx-0294885 purified all members of the AKT family (Supporting Information Table 2). To further examine the binding profile of CTx-0294885, we performed a gene ontology analysis on the nonkinase proteins purified by CTx-0294885 to characterize other enriched proteins by their molecular functions (Figure 3 and Supporting Information Table 4). Consistent with the ATP competitive kinase inhibitor property of CTx-0294885, ∼1/5 of all other

CTx-0294885 As a Kinase Capture Tool in Large-Scale Kinome Profiling Experiments

Next, we characterized the properties of CTx-0294885 as a kinase capture tool in a large-scale purification using a commonly used kinome profiling protocol (Figure 4).20 Kinase enrichment using immobilized CTx-0294885 and lysates from MDA-MB-231 breast cancer cells was carried out under high salt conditions to minimize nonspecific binding to the affinity resin.38 A small aliquot from each elution of the resin-bound proteins was resolved by SDS−PAGE and visualized by silver staining. The enriched fractions produced a banding pattern distinctive to the total protein lysate and negative control, indicating that a subset of cellular proteins was captured by 3109



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Figure 3. Gene ontology classification of nonprotein kinases bound by CTx-0294885. Nonprotein kinases bound by CTx found in common in both replicate experiments were compared with the entire list of UniProtKB entries. Over-represented gene ontology (GO) molecular function terms with statistical significance of p < 0.001 were identified, and of these, only nonredundant terms from GOTERM_MF_FAT set from DAVID are shown here. Fold change for each GO term was calculated by dividing the CTx-bound ratio (ratio of CTx-bound proteins annotated to a particular GO term/total number of CTx-bound proteins) by the total ratio (total number of proteins annotated to that particular GO term/total number of proteins in the database).

for phosphopeptides from 8 mg of lysate, without any previous kinase enrichment (Supporting Information Table 7). Comparison of the results for phosphosite identification from the two approaches is shown in Table 2. In summary, kinome enrichment using CTx-0294885 resulted in identification of an additional 400 phosphosites, providing a more in-depth characterization of the kinome. In total, 235 kinases were identified from this large-scale kinome profiling workflow with 185 kinases identified as total protein, and 179 identified from TiO2 enrichment for phosphopeptides (Figure 5B). In addition, 5 kinases were identified by nonphosphorylated peptides from the latter analysis.

CTx-0294885 (Figure 5A). The remaining eluates were pooled and prepared for protein identification by MS through in-gel digestion. Ten percent of the gel extract was used for total protein identification, and 185 protein kinases were identified from two replicate experiments with high reproducibility (Supporting Information Table 5). To characterize phosphorylation events within the CTx0294885-bound fraction, we conducted a TiO2 enrichment for phosphopeptides using the remaining 90% of gel extracts. Following peptide analysis by MS, 3448 unique phosphosites were identified (Supporting Information Table 6). Nearly half of the total phosphopeptide intensity was contributed by peptides derived from protein kinases. The final high confidence list of phosphosites was compiled by filtering phosphopeptides on the basis of their MaxQuant determined LP scores. A total of 622 phosphopeptides with a LP > 0.75 were classified as class I. To demonstrate the level of kinase enrichment by CTx-0294885 beads, we also examined the phosphoproteome of the same cell line with TiO2 enrichment

Comparison of the Kinome Coverage of CTx-0294885 with Other Broad-Spectrum Kinase Inhibitors

Broad-spectrum kinase inhibitors exhibiting distinct kinase binding profiles have been commonly used in combination for maximal kinome coverage. To identify potential kinase inhibitors complementary to CTx-0294885, we compared the 3110



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Table 2. Comparison of Phosphosite (pSite) Identification from Large Scale Kinome Enrichment Using CTx-0294885 with Phosphoproteomics on Total Cell Lysates all pSites kinase pSites % of kinase pSites intensity kinase pSites (class I) kinase pS kinase pT kinase pY

CTx enrichment

phosphoproteomics

3448 891 46.4 622 483 112 27

6254 307 4.8 212 174 29 9

However, since the expressed kinome differs among cell lines, we first determined the binding profiles of each kinase inhibitor using lysates from MDA-MB-231 cells. Purvalanol B, SU6668, and VI16832 were immobilized on the appropriate Sepharose beads according to the published protocols, and the same kinomic enrichment and phosphopeptide enrichment as described for CTx-0294885 were carried out using each inhibitor resin individually. Subsequent MS analysis confirmed that Purvalanol B, SU6668, and VI16832 bind a distinct but overlapping set of protein kinases and collectively identified 197 kinases from three separate enrichment experiments (Figure 6). Taking these in combination with the 235 kinases identified by CTx-0294885, a total of 269 nonredundant protein kinases were identified through individual use of the four inhibitors (Figure 6). A comparison of the CTx-0294885bound kinases with all kinases identified by Purvalanol B, SU6668, or VI16832 revealed that 72 kinases were unique to CTx-0294885, and 34 kinases were unique to the other three inhibitors. The 106 protein kinases unique to either Purvalanol B/SU6668/VI16832 or CTx-0294885 enrichment accounted for 40% of total kinase identifications, providing us with a strong rationale for testing the kinome coverage achieved via combined use of the four inhibitors.

Figure 4. Schematic diagram of the workflow used in this study. The kinome coverage of the CTx-0294885-coupled beads was examined by the outlined workflow. The schematic diagram is a representation of the general workflow commonly used in large-scale kinome profiling. CTx-0294885-, Purvalanol B-, SU6668-, and VI16832-coupled beads were used individually or in combination at different parts of the study. In-solution digestion was performed only in experiments in which inhibitors were used in combination.

Using CTx-0294885 in Combination with Other Kinase Capture Reagents

binding profile of CTx-0294885 to three other broad-spectrum kinase inhibitors. Purvalanol B, SU6668, and VI16832 were chosen on the basis of findings from previous studies using other cell lines23,39

To determine the kinome coverage achieved by using CTx0294885 in combination with other inhibitors, two kinomic enrichment experiments were conducted in parallel, with one

Figure 5. Examining the kinome coverage of CTx-0294885. A. Comparison of proteins enriched by CTx with total proteins in MDA-MB-231 cells. To visualize the proteins captured by CTx, 1% of each elution fraction from the CTx resin was compared with the total lysate by separation on a 10% SDS−PAGE gel and silver staining. B. Protein kinases detected in CTx-enriched fraction. The Venn diagram shows the total number of protein kinases identified as total protein and as phosphoproteins via TiO2 enrichment for phosphopeptides. 3111



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phosphokinases revealed a strong enrichment of the MAPK signaling pathway and ErbB signaling pathway, with an identification of 43 and 26 components from these pathways, respectively (Supporting Information Figure 2, Table 10). Comparison of all phosphokinases with the 234 kinases detected from the total kinome profiling identified 163 in common (Figure 8). Detection of both phosphorylated and nonphosphorylated peptides of the same kinase would enable quantitative MS to investigate whether phospho-specific events are regulatory or due to changes in protein abundance. Over 50 kinases were detected with phosphosites that reside within the activation loop, where the degree of phosphorylation is strongly correlated with catalytic activity (Supporting Information Table 11).1,40 Furthermore, manual validation of tandem MS/MS spectra for evidence of neutral loss (−98 Da) from serine and threonine phosphorylated peptides confirmed the identification of four novel phosphorylation sites (Table 3). Annotated spectra of novel phosphopeptides are shown in Supporting Information Figure 3.

Figure 6. Comparison of CTx-0294885 with other commonly used kinase capture reagents. Cell lysates from MDA-MB-231 breast cancer cells were subjected to affinity purification on columns containing purvalanol B (P), SU6668 (S), VI16832 (V), or CTx-0294885. The Venn diagram indicates the total number of kinases bound by P, S, and V affinity columns (P/S/V) and the CTx-0294885 column, as well as the overlap between the 2 groups.

■

DISCUSSION In this study, we modified the structure of the known FAK inhibitor TAE-226 to give rise to a novel kinase inhibitor CTx0294885 that displays high affinity toward an extensive range of kinases. The evaluation of the binding profile of CTx-0294885 in MDA-MB-231 cells by quantitative MS demonstrated its selective properties as a kinase enrichment reagent. In the largescale kinome profiling experiment, a total of 235 kinases representing ∼63% of the expressed kinome41 were identified using CTx-0294885 alone, with a notable enrichment of the CMGC group. In addition, CTx-0294885 captured all members of the AKT family, which to the best of our knowledge has never been achieved by similar studies using other broadspectrum kinase inhibitors.22,42−44 The AKT family consists of three functionally distinct members that have been implicated in a number of diseases.45,46 In addition, dysregulation of the AKT signaling pathway has been associated with anticancer drug resistance, which is often a result of cancer kinome remodeling.44,47,48 Recently, MEK inhibition in basal breast cancer models was shown to induce expression and activation of multiple kinases, ultimately leading to the reactivation of MEK2 and, hence, drug resistance; however, this study was unable to detect AKT from the kinome profiling because of the

using a mixture of Purvalanol B, SU6668, and VI16832 (Mix3) and another one including CTx-0294885 in addition to Mix3 (Mix4). Both total kinome profiling and phosphokinome profiling were performed for each type of inhibitor mix (Figure 4). Upon combining kinase identifications from both types of profiling, the use of Mix3 and Mix4 led to the detection of 199 and 261 nonredundant kinases, respectively (Figure 7A and Supporting Information Table 8). The 73 additional kinases identified by the inclusion of CTx-0294885 in Mix4 represent a 37% expansion in kinome coverage. A closer examination of the kinases uniquely bound to Mix4 revealed that these kinases consist of members from all major kinase groups, with a marked representation from AGC kinases (Figure 7B). Examining the Phosphokinome in MDA-MB-231 Cells Using Mix4

Given that Mix4 resulted in the most extensive kinome coverage in this study, we further interrogated the phosphokinome of MDA-MB-231 cells using Mix4. A combined search of all MS-generated raw files identified 799 phosphosites (class I) mapped to 183 protein kinases, with a phosphosite distribution of 77% pS, 17% pT, and 5.9% pY (Supporting Information Table 9). KEGG pathway analysis of the

Figure 7. Use of CTx-0294885 in combination with other commonly used kinase capture reagents. A. Combining CTx-0294885 with other kinase capture reagents greatly enhances kinome coverage. Cell lysates from MDA-MB-231 breast cancer cells were subject to affinity purification on columns containing a mixture of P, S, and V (Mix 3) or P, S, V, and CTx-0294885 (Mix 4). The Venn diagram indicates the total number of kinases bound by Mix 3 and Mix 4, as well as overlap between the 2 groups. B. Additional kinases in each family identified by Mix 4 compared with Mix 3. 3112



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Figure 8. Illustration of the kinome coverage of Mix4 on the human kinome tree. Protein kinases identified in MDA-MB-231 cells by Mix4 are plotted on the human kinome tree. Red dots indicate kinases that were identified with both nonphosphorylated and phosphorylated peptides, blue or orange indicate kinases identified with either nonphosphorylated or phosphorylated peptides, respectively. Kinase tree adapted with permission from Cell Signaling Inc. (http://www.cellsignal.com/).

Table 3. Novel Phosphorylation Sites Identified on Protein Kinases

a

accession

kinase

group

peptide sequence

pSite

CTxa

Mix4a

Q9NSY1 Q9H2X6 Q14680 P78527

BMP2K HIPK2 MELK PRKDC

other CMGC CAMK atypical

GNDESEpSDFESDPPSPK STVpSLLDTYQK NKENVYpTPK LYpSLALHPNAFK

S742 S121 T428 S1065

x

x x x x

x x

x indicates identification of novel sites in the experiment mentioned in the column.

effective targeted treatments. Our previous phosphotyrosine profiling identified a prominent SRC family kinase (SFK) signaling network characteristic of this subtype.16 The current phosphokinome profiling using Mix4 provided an extensive coverage of this SFK signaling network, characterizing both expression and activity of many top-ranked kinases, such as EGFR, LYN, FAK, and EPHA2, and revealing the phosphorylation status of other potentially important serine/threonine residues. Taken together with the ability to monitor >200 kinases from other signaling networks, CTx-0294885 could serve as a useful tool in dissecting the aberrant signaling events associated with basal breast cancers, leading to the development

lack of an AKT-binding broad-spectrum kinase inhibitor. In contrast, our profiling from the same cell line using Mix4 captured the majority of players of the AKT/mTOR signaling pathway (Supporting Information Figure 4). The additional 70 kinases identified using CTx-0294885 also reveal a more complete picture of the expressed kinome. Therefore, the application of CTx-0294885 in kinome profiling may provide valuable information underpinning the rational design of combination therapies for cancer and create a system platform for studying AKT-associated signaling networks. The cell line MDA-MB-231 used in this study belongs to the aggressive basal breast cancer subtype that currently lacks 3113



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deposition. Proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral. proteomexchange.org) via the PRIDE partner repository with the data set identifier PXD000239.53 This work was supported by the National Health and Medical Research Council of Australia (Program Grant 535903 and Project Grant 1048550). ESH and LZ are recipients of Cancer Institute NSW and Sydney Catalyst Research Scholarships, respectively.

of novel therapeutic interventions for this cancer type. In addition, consistent with the activating KRAS and BRAF mutations carried by MDA-MB-231 cells,49 a highly phosphorylated RAF/MEK/ERK pathway was observed in our study. KEGG pathway analysis of all proteins bound by Mix4 showed a 4-fold enrichment of the RAF/MEK/ERK pathway (Supporting Information Figure 5), indicating that CTx0294885 could also be valuable for interrogating signaling in Ras-driven cancers, such as pancreatic and colorectal cancer. Interestingly, in concordance with findings from recent studies,20,23 our phosphokinome profiling identified multiple serine phosphorylations within the activation loop of various EPH receptor tyrosine kinases (RTKs). Complete activation of the EPH RTKs requires both tyrosine phosphorylations in the activation loop as well as the juxtamembrane domain that usually blocks the kinase domain when unphosphorylated.50 It is unclear which upstream kinases are responsible for the serine phosphorylations in the activation loop and how these may impact the 3D structure of the kinase domain or kinase activity. Although there is no direct evidence from our study suggesting the coexistence of serine phosphorylations and the tyrosine phosphorylation that is partly responsible for kinase activation, such possibilities cannot be ruled out because phosphopeptide enrichment by TiO2 has a bias toward monophosphorylated peptides.51 Alternative phosphopeptide enrichment strategies using immobilized metal affinity chromatography (IMAC) or sequential elution from IMAC (SIMAC) may be employed for detecting multiple phosphorylated peptides.52 Understanding the regulatory role of these serine phosphorylations may unveil novel mechanisms involved in the regulation and activation of the EPH RTKs. In conclusion, our study has shown that CTx-0294885 is capable of capturing more than half of the expressed kinome in MDA-MB-231 cells.41 More importantly, CTx-0294885 binds additional kinases that were not identified from previous studies using other kinase capture reagents. Therefore, using CTx0294885 in an inhibitor mixture can significantly expand kinome coverage, and hence, the characterization of this reagent opens new opportunities for detailed interrogation of signaling networks and development of novel therapeutic strategies.

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ABBREVIATIONS AGC, containing protein kinase A, G and C; CAMK, calcium/ calmodulin-dependent protein kinase; CMGC, containing cyclin-dependent kinase, mitogen-activated protein kinase, glycogen synthase kinase 3, and CDC2-like; TK, tyrosine kinase; TKL, tyrosine kinase-like; STE, homologues of yeast sterile 7, sterile 11, and sterile 20; CK1, casein kinase 1; pS, phosphoserine; pT, phosphothreonine; pY, phosphotyrosine; GO, gene ontology; DAVID, Database for Annotation, Visualization and Integrated Discovery; nanoLC−MS/MS, nanoliquid chromatography−tandem mass spectrometry; RTK, receptor tyrosine kinase; HIPK2, homeodomain-interacting protein kinase 2; BMP2K, BMP-2-inducible protein kinase; PRKDC, DNA-dependent protein kinase catalytic subunit; MELK, maternal embryonic leucine zipper kinase

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ASSOCIATED CONTENT

S Supporting Information *

Additional figures (.pdf; file 018) and tables (.xls, .xlsx; files 007−017). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +61-3-990-29301. E-mail: [email protected]. Present Address ■

(R. J. Daly) Dept. of Biochemistry and Molecular Biology, School of Biomedical Sciences, Level 1, Building 77, Monash University, VIC 3800, Australia.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors wish to thank the PRIDE Team at European Bioinformatics Institute for providing support for data 3114



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Characterization of the Novel Broad-Spectrum Kinase Inhibitor CTx

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