Kinases are important key regulators in cellular signaling and are often involved in the development and progression of disease. Small molecule kinase inhibitors often target the conserved ATP-binding kinase domain and are quite successful molecularly targeted therapies. Over 30 drugs are approved for various indications and over 250 inhibitors are currently evaluated in clinical trials. Due to their chemical nature, these inhibitors can be rather unselective and not only act on their intended target but also inhibit other proteins. This polypharmacology does not necessarily lead to toxic side effects but can also open up new therapeutic opportunities where an inhibitor can be administered. So far, the target landscape of these drugs has not been evaluated in a systematic manner.
Chemoproteomic approaches, like Kinobeads, offer valuable insights into drug-protein interactions in close to endogenous settings and can evaluate the effect of one drug on multiple proteins in one experiment. Therefore, this method should be applied to profile the selectivity of clinical kinase inhibitors systematically. The resulting target spectra can then be used to deduce insights into inhibitor mode of action, propose repositioning possibilities and identify off-targets leading to side effects.
This thesis presents an improved workflow to apply Kinobeads in a higher throughput setting enabling the screening of over 200 molecules (3.1). With this, 242 small molecule inhibitors were profiled for their selectivity resulting in the druggable kinome (3.2). The use of this resource is then demonstrated in the following paragraphs. Selectivity could be evaluated across drugs, binding modes or targets (3.3). Selected targets and inhibitors were then characterized concerning their mode of action or new uses for the respective drug (3.4). Finally, the discovery of a non-kinase off-target was characterized further (3.5).
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2 Experimental procedures
Cell culture, reagents and affinity matrices
The cell lysate mixture (cell line mix) used to profile all kinase inhibitors in this study was generated from K-562, COLO 205 and MV4-11 cells grown in RPMI1640 medium (Biochrom GmbH), SK-N-BE(2) cultured in DMEM/HAM’s F-12 medium (Biochrom GmbH). All were supplemented with 10% FBS (Biochrom GmbH) and 1% antibiotic solution (Sigma). For MET-inhibitor profiling, Caki-1 cells were cultured in IMDM (Biorad) with 10% FBS. For EGFR-inhibitor profiling, BT-474 cells were grown in DMEM/HAM’s F-12 supplemented with 15% FBS (Biochrom). KM-12 and HEK-293 cells were grown in IMDM-medium (Biochom GmbH) in the presence of 10% FBS. For phosphorylated SK-N-BE(2) cell lysate, cells were treated with 100 µM pervanadate for 15 min.
Affinity matrices were produced in house as published previously92. PPIX was coupled to beads by first reversing NHS-beads with aminoethanol (4:1 mixture), ethylendiamine and triethylamine in DMSO for 20 h on an end-over-end shaker at r.t. in the dark. PPIX was coupled to the beads at a coupling density of 1 µmol/mL in the presence of Hünig’s base, triethylamine and PyBrOP in DMF for 20 h on an end-over-end shaker at r.t. in the dark. Remaining free residues on the beads were blocked with NHS-activated acetic acid92. Small molecule inhibitors were purchased from Selleckchem, MedChemExpress, Active Biochem, Abmole, Merck or LC Labs (a complete list of inhibitors can be found in the Appendix I). Hemin and Protoporphyrin IX (PPIX) were obtained from Sigma Aldrich (H9039 and 25385). The stable and soluble recombinant FECH R115L mutant was a gift from Vipul Gupta (Tokyo Institute of Technology, Tokyo). It was expressed and purified as described previously207.
Binding profiling using affinity matrices
Kinobeads selectivity profiling, as well as profiling with PPIX affinity matrices, was performed as described previously92.
For affinity pulldowns, cells were lysed in so called 1x CP buffer (50 mM Tris/HCl pH7.5, 5% Glycerol, 1.5 mM MgCl2, 150 mM NaCl, 1 mM Na3VO4, 25 mM NaF, 1 mM DTT) with 0.8% NP40 ( Igepal-CA630, Sigma Aldrich) supplemented with Protease inhibitor (SigmaFast (S8820-20TAB) and Phosphatase inhibitor I (2.5 mM (-)-p-Bromotetramisole oxalate (190047, Sigma Aldrich), 500 µM Cantharidin (C7632, Sigma Aldrich), 500 nM Mirocystin LR (ALX-350-012-C050, Enzo)), II (200 mM Imidazole, 100 mM NaF, 115 mM NaMoO4, 100 mM Na3VO4, 400 mM Sodium tartrate dehydrate), III (20 µM Calyculin A (C-3987, LC Laboratories)).
For kinase or protein enrichment, a protein mixture of the four cell lines or a single cell line were ultra-centrifuged at 52,000 rpm (167,177 xg) at 4°C for 20 min and diluted with 1x CP buffer with Protease and Phosphatase inhibitors to a 0.4% NP40 concentration in the lysate. This was then equally distributed into wells of a 96 deep well plate and adjusted to 5 mg/mL. Kinase inhibitors of interest were spiked into 1 mL cell lysates in increasing concentrations(3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 µM, 3 µM, 30 µM and vehicle control) in suggested solvent (mostly DMSO) or as single compound dose (5 µM) and incubated for 45 min at 4°C. The preincubation step was followed by incubation with kinobeads or other affinity matrices (35 µl settled beads) for 30 min at 4 °C.
Unbound proteins were washed away by first applying 3 mL of 1x CP buffer with 0.4% NP40 to the filter plate, followed by 2 mL of 1x CP buffer with 0.2% NP40. Bound proteins were eluted with
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2xLDS sample buffer (NuPAGE, Invitrogen) containing 50 mM DTT by heating to 50°C for 30 min at 700 rpm.
For the calculation of a correction factor, the unbound fraction of the DMSO control was incubated with fresh beads for a second time (pull down of pull down).
For pulldowns using recombinant FECH protein, 1 µg FECH were spiked into 1 mL of 1x CP buffer with 0.4% NP40. (Competition) pulldowns were performed following the procedure described above.
Protein digestion
Reduced eluates were alkylated with chloroacetamide (55 mM) for 30 min in the dark and the proteins were concentrated by a short electrophoresis on a 4-12% Bis-Tris NuPAGE gel (Invitrogen) at 200 V for 5 min. Gels were stained with Coomassie or silver nitrate following standard procedures to visualize protein bands. In-gel digestion was performed according to standard procedures by technical assistants at the Chair of Proteomics, Technical University of Munich.
TMT labeling
Dried peptides were dissolved in 20 µl 50 mM TEAB (pH 8.5) and incubated for 10 min at 20 °C and 400 rpm. Up to 12 mM TMT labeling reagent (60 mM in anhydrous ACN) was added to each sample and incubated for 1 h at 20 °C and 400 rpm according to manufacturer’s instructions. The labeling reaction was stopped by adding 2 µl 5% hydroxylamine for 14 min at 20 °C and 400 rpm. 75 µl of differentially labeled peptides were then combined in equal amounts by adding 50 µl 0.1% FA in H2O to each sample.
StageTip desalting
After TMT labeling, peptides were desalted using StageTips123, 208. Five disks of EmporeTM C18 47 mm SPE extraction disks (Supelco) were packed into the end of 200 µl pipet tip. Disks were equilibrated by first soaking the material with up to 100 µl of ACN and subsequent washing with 2x 100 µl 0.1% FA in H2O and centrifugation of the tip at 2,000 rpm. Acidified samples (pH 2) were loaded onto the micro-column and washed with 2x 100 µl 0.1% FA in H2O. Peptides were eluted in 100 µL 60% ACN/0.1% FA in H2O and dried in a vacuum concentrator centrifuge.
High pH reverse phase fractionation
For high pH reverse phase fractionation, five disks of EmporeTM C18 47 mm SPE extraction disks (Supelco) were packed into the end of 200 µl pipet tip, soaked with ACN and equilibrated with 25 mM NH4FA. Samples were dissolved in 100 µl 25 mM NH4FA and sonicated three times for 1 min with 1 min on ice after each step. They were then loaded onto the micro-column with reloading the first flowthrough. The second flowthrough was collected and desalted. Peptides on the micro-column were then washed with 2 x 40 µl 25 mM NH4FA and sequentially eluted with 40 µl of elution buffer with increasing ACN concentrations (5%, 12.5%, 15%, 50%). Peptides in the flowthrough were pooled with the 50% fraction and dried in a vacuum concentrator centrifuge.
LC MS/MS analysis
Peptides generated by in-gel trypsin digestion were analyzed via LC-MS/MS on a nanoLC-Ultra 1D+
(Eksigent) coupled to a LTQ-Orbitrap Elite mass spectrometer (Thermo Scientific). Peptides were delivered to a trap column (75 µm × 2 cm, self-packed with Reprosil-Pur C18 ODS-3 5 µm resin, Dr.
Maisch) at a flow rate of 5 µL/min in solvent A0 (0.1% formic acid in water). Peptides were separated
27 on an analytical column (75 µm × 40 cm, self-packed with Reprosil-Gold C18, 3 µm resin, Dr. Maisch) using a 100 min linear gradient from 4-32% solvent B (0.1% formic acid, 5% DMSO in acetonitrile) and solvent A1 (0.1% formic acid, 5% DMSO in water)153 at a flow rate of 300 nL/min. The mass spectrometer was operated in data dependent mode, automatically switching between MS and MS2 spectra. MS1 spectra were acquired over a mass-to-charge (m/z) range of 360-1300 m/z at a resolution of 30,000 (at m/z 400) in the Orbitrap using an automatic gain control (AGC) target value of 1e6 or maximum injection time of 100 ms. Up to 15 peptide precursors were selected for fragmentation by higher energy collision-induced dissociation (HCD; isolation width of 2 Th, maximum injection time of 100 ms, AGC value of 2e5) using 30% normalized collision energy (NCE) and analyzed in the Orbitrap (7,500 resolution). A previous-experimentally obtained inclusion list containing approximately 1,000 kinase peptide m/z and retention time values was enabled in the data acquisition regime. Dynamic exclusion was 20 s and singly charged precursors were excluded.
Profiles of EGFR inhibitors in BT-474 cells were obtained using a Dionex Ultimate 3000 nano HPLC coupled to a Q Exactive HF mass spectrometer. Peptides were delivered to a trap column as described above and separated on an analytical column using a 60 min gradient from 4-32% solvent B in solvent A1. MS1 spectra were acquired at a resolution of 60,000 (at m/z 200) using a maximum injection time of 10 ms and an AGC target value of 3e6. Up to 12 peptide precursors were isolated (isolation width of 1.7 Th, maximum injection time of 75 ms, AGC value of 2e5), fragmented by HCD using 25% NCE and analyzed at a resolution of 15,000. Precursor ions that were singly-charged, unassigned or with charge states >6+ were excluded. The dynamic exclusion duration of fragmented precursor ions was 30 s.
Peptide and protein identification and quantification
For label-free quantification of Kinobeads selectivity profiles, peptide and protein identification and quantification was performed using MaxQuant (version 1.5.3.30)188 by searching the tandem MS data against a human Swissprot reference database (human proteins only, 20,193 entries, downloaded 22.03.16, internally annotated with PFAM domains) using the embedded search engine Andromeda181. Carbamidomethylated cysteine was specified as fixed modification;
phosphorylation of serine, threonine, and tyrosine, oxidation of methionine, and N-terminal protein acetylation were variable modifications. Trypsin/P was specified as the proteolytic enzyme and up to two missed cleavage sites were allowed. Precursor tolerance was set to 10 ppm and fragment ion tolerance to 20 ppm. Label-free quantification206 and match between runs (alignment window 1 min) were enabled within MaxQuant. Search results were filtered for a minimum peptide length of seven amino acids, 1% peptide and protein FDR as well as common contaminants and reverse identifications. For consistent peptide identification and protein grouping, the MS data for each compound was supplemented with 15 standard controls. Each compound was analyzed separately.
TMT labeled samples were searched with MaxQuant (version 1.5.2.8) against a human UniProt reference database (88,354 entries, downloaded 22.07.13, internally annotated with PFAM domains). TMT10 reporter ions were specified for quantification, other parameters were set the same as for the label-free analysis. Furthermore, TMT data was also analyzed using the phyton package isobarQuant in combination with the Mascot search engine. Therefore, relative TMT intensities were extracted directly from the raw files, whereas protein identification was performed by Mascot with the same search parameters as for MaxQuant.
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Data analysis
For competition binding assays, protein intensities were normalized to the respective DMSO control. IC50 and EC50 values were deduced by a four-parameter, log-logistic regression using an internal pipeline that utilizes the ‘drc’ package209 in R. A Kdappwas then calculated by multiplying the estimated EC50 with a protein-dependent correction factor (depletion factor) that was limited to a maximum value of 1. The correction factor cf for a protein is defined as the ratio of the amount of protein captured from two consecutive pull downs of the same DMSO control lysate92, 97. In this study, protein-dependent correction factors were set to the median of correction factors across all experiments using the same lysate and beads. Representative dose dependent inhibition curves were analyzed using GraphPadPrism (version 5.03).
Target annotation
Targets were manually annotated. A protein was considered a high-confidence target if the binding curve showed a sigmoidal shape with a dose-dependent decrease in binding to the Kinobeads.
Proteins that only showed an effect at the highest inhibitor dose where not annotated as targets.
The number of unique peptides and MSMS spectra were also included as target selection criteria.
Peptide intensity in DMSO controls and MS/MS data quality was also taken into account. Proteins with low peptide counts, MS/MS spectral counts or MS1 intensity that nonetheless showed a reasonable dose response curve fit were considered as potential targets. In addition, if an inhibitor also interacted with similar kinases (e.g. CDK family) it was also considered as a potential target.
Low-confidence targets were excluded from further analysis. Note that for some targets, curve fitting with our data processing pipeline was not possible resulting in no or very high Kdapp values.
Targets were considered as direct Kinobeads binders if annotated in UniProt.org as a protein or lipid kinase. Furthermore, nucleotide binders, helicases, ATPases and GTPases, FAD (e.g., NQO2) and heme (e.g., FECH) containing proteins were also considered as potential direct binders. Most other target proteins are interaction partners/adaptor proteins of the kinases and are termed indirect Kinobeads binders.
All high confidence targets will from now on be referred to as targets, used for analysis and shown in figures if not stated otherwise.
Comparison of kinase inhibitor profiles/targets to other published data
To compare kinase inhibitor profiles to published screening data and major online databases, only targets annotated as either direct Kinobeads binders or kinases were considered. Datasets obtained from CHEMBL210, LINCS211, Anastassiadis et al.72 and Metz et al.212 were filtered for compounds used in the Kinobeads drug screen and protein names were annotated according to gene names as used in UniProt. Dose response data was filtered for half-maximal response at 30 μM or lower if the assay threshold concentration was lower. Single concentration data was filtered for a minimum of 25%
inhibition of binding or activity.
Kinase activity assays
Kinases activity assays where performed at Reaction Biology Corporation. IC50s were determined using 10 concentrations in semi-log steps. Kinases of interest were measured at an ATP concentration corresponding to the apparent Km for ATP of the corresponding kinase.
29 Cell viability assays
Cell viability was measured using the alamarBlue cell viability assay (DAL1100, Invitrogen). For KM12 viability, 1,000 cells were seeded per well in IMDM Medium with 10% FBS (Biochrom). Cells were allowed to attach overnight. On the following day, the cells were exposed to increasing concentrations of a specific inhibitor (final inhibitor concentration: 10 μM, 3 μM, 1 μM, 300 nM, 100 nM, 30 nM, 10 nM, 3 nM) to the cells, as well as a DMSO control. The cells were incubated for 72 h at 37 °C and 5% CO2. Cell viability assays were performed by adding 10% alamarBlue reagent to each well. The reduction from resazurin to resorufin was measured after 4 h using a fluorescence spectrophotometer (BMG Labtech) at 544 nm (excitation) and 584 nm (emission). Each compound was measured in a technical triplicate.
MV4-11 viability was measured using the Cell Proliferation Kit II (XTT, Cat. No. 11465 015 001, Roche) according to manufacturer’s procedure. Briefly, 104 cells were seeded per well and allowed to attach overnight. Cells were exposed to increasing concentrations of each inhibitor (1 μM, 100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 300 pM, 100 pM). The cells were incubated for 72 h at 37 °C and 5% CO2. Cell viability assays were performed by adding XTT reagent to each well. The metabolite formazan 4 h using an absorbance spectrophotometer (Tecan) at 492 nm. Each compound was measured in a technical triplicate.
Viability was then assessed by calculating the reduction in fluorescence of each inhibitor dose compared to the DMSO control. Graphs were generated in GraphPadPrism (version 5.03).
Immunodetection for NTRK1 signaling
After separation of up to 40 µg of previously treated KM12 lysate, immunoblots were performed using the Xcell II Blot Modul (Invitrogen) and PVDF Membranes (Biorad) according to manufacturer’s instructions. Downstream targets of NTRK1 signaling were detected with the respective antibodies: phospho-AKT (S473, (D9E) XPTM, Cell Signaling Technology), pan AKT (C67E7, Cell Signaling Technology), phospho-p44/41 Erk1/2 (Thr202/Tyr204, (D13.14.4E) XPTM, Cell Signaling Technology) and p44/41 Erk1/2 (137F5, Cell Signaling Technology). ß-actin (C4, sc-47778, Santa Cruz Biotechnology) was used as loading control. Secondary antibodies used were IRDye 800CW Goat-anti-Rabbit Antibody and IRDye 680LT Donkey-anti-Mouse antibody (LICOR Biosciences). Detected protein was readout with the Odyssey infrared imaging system (LI-COR Biosciences).
Cellular thermal shift (CETSA) and isothermal dose response assay (ITDR)
Cellular shift and isothermal dose response assays were performed as described before110. For CETSA, K562 cell lysate was incubated with up to 10 µM of drug for 30 min. Compound treated lysate was then distributed into PCR-tubes à 50 µl and heated to increasing temperatures from 40-70 °C for 3 min, followed by a 3 min cooling phase at 25 °C.
For ITDR, K562 cells were incubated with increasing concentrations of drug for 1h at 37 °C. Then, cells were washed with PBS and heated to 55 °C for 3 min followed by a 3 min cooling phase at 25 °C. After heating, cells were lysed by freeze thaw cycles in liquid N2.
After CETSA or ITDR, denatured proteins were precipitated by centrifugation at 20,000 xg for 30 min. Supernatants were reduced in LDS Sample buffer and proteins were separated in a 4-12%
NuPAGE gel (Invitrogen) at 200 V for 45 min. FECH protein was detected by immunoblotting with a mouse monoclonal antibody (sc-271434, Santa Cruz Biotechnology) in a 1:400 dilution in 0.2% BSA in 1x TBS-T for at least 16 h at 4 °C. For secondary detection IRDye 800CW conjugated goat anti-mouse (LI-COR Biosciences) was used (1:5000 in 0.2% BSA and 0.02% SDS in 1x TBS-T, 1 h, RT).
Detected protein was quantified with the Odyssey infrared imaging system (LI-COR Biosciences).
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The data are normalized with the quantity of soluble target at highest compound concentration, reflecting maximum thermal stabilization, set to 100%. Dose dependent stabilization curves were analyzed using GraphPadPrism (version 5.03) and EC50-values were calculated using nonlinear regression analysis.
FECH activity assay
For cell viability measurements, differentiated K562 cells were incubated with 1 µM of drug for up to 6 days and the amount of live cells was quantified using the alamarBlue assay (Thermo Scientific).
Experiments were performed in triplicates for each compound. The activity assay was modified based on Smith et al.213 Briefly, differentiated K562 cells were incubated with 1 µM drug for up to 6 days, washed with PBS and lysed using only ddH2O for 4 hours. Cell debris were pelleted by centrifugation at 20,000 xg at 4 °C for 10 min. Heme was measured from these cleared supernatants by LC-MS, using an Agilent 1100 HPLC system and a Triart C18 (10 x 1mm I.D., 3 µm resin, YMC Europe GmbH) column, applying a 60-95% gradient of solvent B (0.1% FA in acetonitrile) in solvent A0 (0.1% FA in water) coupled online to an Amazon ion trap (Bruker). For quantification of heme content, the area under the curve of the UV trace at 400 nm was determined and normalized to the protein content of the sample. The change in heme content was calculated with respect to the vehicle treated sample.
Cell-free FECH enzymatic assay
Inhibition of FECH enzymatic activity was performed as described previously.207 Recombinant FECH R115L mutant was used for the assay because it is more stable and better soluble than the wild type enzyme and this mutant has also been used in the literature including the reported crystal structure (see below).
Docking studies
Docking was performed by Bjoern Oliver Gohlke (Structural Bioinformatics Group, Charité-Universitätsmedizin, Berlin, Germany). The X-ray structure of Ferrochelatase (PDB: 3w1w) was selected for all docking studies. In this structure, salicylic acid as well as cholic acid were co-crystallized with the protein. For docking, hydrogens were added and ligands as well as possible side chain rotamers removed from the protein. Docking studies were carried out using GOLD 5.2214 by applying a radius of 10.0 Å around the respective coordinates, using standard parameters and the ChemScore scoring function for ranking the docking poses. This set of parameters was kept for every docking step to guarantee reproducibility. Separate docking experiments were performed for the dimerization region and protoporphyrin site. For the dimerization site, residues around salicylic acid were defined as active site and docking was performed by using standard parameters and applying the ChemScore scoring function to rank the results. For the protoporphyrin site, compounds were docked in an iterative process based on the known binding mode of cholic acid.
Cholic acid was removed from the complex, whereas the definition of the binding site for every of the three steps was based on the coordinates of the corresponding cholic acid molecule. To consider intermolecular interactions to the previously docked compounds, the best docking position for every compound was placed in the protoporphyrin site and kept for the next docking
Cholic acid was removed from the complex, whereas the definition of the binding site for every of the three steps was based on the coordinates of the corresponding cholic acid molecule. To consider intermolecular interactions to the previously docked compounds, the best docking position for every compound was placed in the protoporphyrin site and kept for the next docking