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Chemical Proteomics Reveals Ferrochelatase as a Common Off-target of Kinase Inhibitors Susan Klaeger, Bjoern Gohlke, Jessica Perrin, Vipul Gupta, Stephanie Heinzlmeir, Dominic Helm, Huichao Qiao, Giovanna Bergamini, Hiroshi Handa, Mikhail M Savitski, Markus Bantscheff, Guillaume Médard, Robert Preissner, and Bernhard Kuster ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b01063 • Publication Date (Web): 10 Feb 2016 Downloaded from http://pubs.acs.org on February 12, 2016

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ACS Chemical Biology

Chemical Proteomics Reveals Ferrochelatase as a Common Off-target of Kinase Inhibitors

Susan Klaeger1,2,3, Bjoern Gohlke3,4,5, Jessica Perrin6, Vipul Gupta7, Stephanie Heinzlmeir1,2,3, Dominic Helm1, Huichao Qiao1, Giovanna Bergamini6, Hiroshi Handa7, Mikhail M Savitski6, Marcus Bantscheff6, Guillaume Médard1, Robert Preissner3,4,5, Bernhard Kuster*1,2,3,8,9 1

Chair of Proteomics and Bioanalytics, Technical University of Munich, Freising, Germany

2

German Cancer Consortium (DKTK), Munich, Germany

3

German Cancer Research Center (DKFZ), Heidelberg, Germany

4

Structural Bioinformatics Group, Charité-Universitätsmedizin Berlin, Germany

5

German Cancer Consortium (DKTK), Berlin, Germany

6

Cellzome GmbH, a GSK company, Heidelberg, Germany

7

Department of Nanoparticle Translational Research, Tokyo Medical University, Tokyo, Japan

8

Bavarian Biomolecular Mass Spectrometry Center, Technical University of Munich, Freising,

Germany 9

Center for Integrated Protein Science Munich (CIPSM), Freising, Germany

* corresponding author: Bernhard Kuster, [email protected]

Keywords: chemical proteomics, Kinobeads, Ferrochelatase, FECH, kinase inhibitor, photosensitivity, Vemurafenib

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Abstract Many protein kinases are valid drug targets in oncology because they are key components of signal transduction pathways. The number of clinical kinase inhibitors is on the rise but these molecules often exhibit polypharmacology, potentially eliciting desired and toxic effects. Therefore, a comprehensive assessment of a compounds’ target space is desirable for a better understanding of its biological effects. The enzyme Ferrochelatase (FECH) catalyzes the conversion of protoporphyrin IX into heme and was recently found to be an off-target of the BRAF inhibitor Vemurafenib, likely explaining the phototoxicity associated with this drug in melanoma patients. This raises the question if FECH binding is a more general feature of kinase inhibitors. To address this, we applied a chemical proteomics approach using kinobeads to evaluate 226 clinical kinase inhibitors for their ability to bind FECH. Surprisingly, low or submicromolar FECH binding was detected for 29 of all compounds tested and isothermal dose response measurements confirmed target engagement in cells. We also show that Vemurafenib, Linsitinib, Neratinib and MK-2461 reduce heme levels in K562 cells, verifying that drug binding leads to loss of FECH activity. Further biochemical and docking experiments identified the protoporphyrin pocket in FECH as one major drug binding site. Since genetic loss of FECH activity leads to photosensitivity in humans, our data strongly suggests that FECH inhibition by kinase inhibitors is the molecular mechanism triggering photosensitivity in patients. We therefore suggest that a FECH assay should generally be part of the pre-clinical molecular toxicology package for the development of kinase inhibitors.


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Introduction Protein kinases have emerged as a major class of drug targets in oncology as they play central roles in signal transduction cascades.1, 2 Today, more than 200 small molecule kinase inhibitors are in clinical trials, and about 30 such drugs have been approved for use in humans.3, 4 Typically, these molecules target the ATP-binding site within the kinase domain. As this domain is quite conserved across the 518 human protein kinases, it is challenging to discover highly selective drugs against one specific protein. Thus, any given kinase inhibitor may not only act on the intended target, but may have off-targets within the kinase space and also beyond. This so-called polypharmacology may increase the therapeutic spectrum of a drug but can also lead to undesirable consequences. Kinase inhibitor selectivity is traditionally assessed using in vitro assay panels containing large numbers of recombinant kinases.5-7 Albeit powerful, the recombinant enzymes do not possess all the molecular features of the endogenous target proteins such as activity regulating posttranslational modifications, cofactors or interaction partners present in the cell. With chemical proteomics, the selectivity of kinase inhibitors can be evaluated in cell and tissue lysates or intact cells8 and, therefore, lead to a better understanding of a drugs’ target spectrum or mode of action in its biological context. In a typical chemical proteomic experiment, immobilized small molecule inhibitors are used as affinity probes and, when configured into a competition binding assay using the free inhibitor and combined with a quantitative mass spectrometry readout, lead to quantitative binding information for a drug and the full spectrum of proteins captured by the immobilized inhibitor.9-11 Using either of such assays, many novel and sometimes quite surprising protein-drug interactions have been identified in recent years. To name a few, FLT3 or MAP4K4 appear to be frequently hit by kinase inhibitors,7 DDR1 was discovered as a new target for Imatinib and other BCR-ABL inhibitors10, 12 and Pazopanib as well as Ponatinib were identified as inhibitors of cellular necroptosis.13 Interestingly, enantiomers of kinase inhibitors can also have different targets.



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Huber and co-workers have found that the (S)-enantiomer of the approved MET/ALK inhibitor Crizotinib selectively inhibits the 7,8-dihydro-8-oxoguanine triphosphatase MTH1 while the actual (R)-enantiomer drug does not.14 Surprisingly, even proteins without obvious nucleotide binding sites have been identified as off-targets of kinase inhibitors. One such example is the oxidoreductase NQO2, which is very potently inhibited by the BCR-ABL inhibitors Imatinib and Nilotinib.10, 12 A more recent such case is Ferrochelatase (FECH, EC: 4.99.1.1) which has been identified as an off-target of the approved BRAF inhibitor Vemurafenib15 that is used for the treatment of unresectable or metastatic melanoma carrying a BRAFV600E mutation.16 This interaction is noteworthy as it may explain one of the clinically observed side effects of the drug. Indeed, despite being generally well tolerated, patients receiving Vemurafenib may experience a number of side effects, including photosensitivity.17, 18 It has been noted, that some patients suffer from severe sunburn reactions even after short exposure to sunlight. Further examination of these patients revealed that UVA-radiation is responsible for this effect and that these patients show elevated porphyrin levels in erythrocytes.19, 20, 21 FECH is the last enzyme in the heme biosynthesis pathway and converts protoporphyrin IX (PPIX) to functional heme. Interestingly, genetic loss of function of FECH leads to protoporphyrin accumulation in a number of cell types and affected patients also suffer from photosensitivity.22 It is therefore not farfetched to speculate that FECH inhibition by Vemurafenib is the molecular mechanism that explains the photosensitive phenotype in patients treated with this drug. In this study, we asked if the interaction of FECH with kinase inhibitors is a more general phenomenon. To do so, we employed a chemical proteomics approach using the kinobead technology10, 11 and measured FECH binding for 226 kinase inhibitors that are currently evaluated in clinical trials or approved drugs. The data show that about 13% of all kinase inhibitors are indeed also reasonably potent FECH binders. Furthermore, we demonstrate that inhibitor binding leads to loss of FECH activity in cells and we provide structural data which 4 ACS Paragon Plus Environment

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reveal the protoporphyrin pocket as a clear, and the dimerization domain as a potential second binding site for kinase inhibitors. All experimental evidence points towards FECH as a bona fide target of some kinase inhibitors with potential clinical significance.



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Results and Discussion Kinobead profiling identifies FECH as an off-target binder of many kinase inhibitors In order to systematically profile clinical kinase inhibitors for FECH binding, we used a chemical proteomics approach consisting of affinity capture of proteins on kinobeads and protein identification and quantification by quantitative mass spectrometry (Figure 1). Briefly, kinobeads (version γ, see methods for details) consist of five immobilized broad spectrum kinase inhibitors and allow for the enrichment of more than 300 human protein kinases and about 1,000 other proteins including FECH (captured by compound 7 of KBγ) from lysates of cells or tissues.11 Inlysate competition using the unmodified inhibitor in increasing concentrations, leads to a dose dependent loss of specific protein binding to the beads. For each dose of competitor (or vehicle) proteins including FECH bound to kinobeads can be identified and quantified by label free mass spectrometry (Figure 1a). Label free quantification is based on the fact that the intensity of peptides scales linearly with the quantity of the peptide present in the analysis. The more protein is bound by an inhibitor in solution, the less can bind to kinobeads and the lower the intensity of the respective peptides is detected by the mass spectrometer. The relative quantity of each protein per dose of competitor or vehicle determined in this way can then be used to derive EC50 values from a dose response plot using nonlinear regression analysis (Figure 1b) and the determined EC50 values for each protein can be converted to a binding constant Kd by applying a correction factor that accounts for the depletion of a protein from the lysate in the affinity pulldown.11, 23 The kinobead profile of Vemurafenib using a mixed cell lysate treated with the drug showed a Kd value for its cognate target BRAF of about 1 µM (Figure 1b) confirming earlier cellular thermal shift assay data reporting the interaction of Vemurafenib and FECH.15 To rule out indirect FECH binding as part of a protein complex with BRAF, we analyzed Dabrafenib in the same assay and found a potent interaction of Dabrafenib and BRAF (60 nM; Figure 1c)


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but no FECH binding, excluding the possibility that FECH is co-purified on kinobeads as part of a BRAF-FECH complex. Having established the specificity of the chemical proteomic assay, we extended the study to 226 clinical kinase inhibitors and found FECH as an off-target of 29 kinase inhibitors with binding constants (Kd) in the range of 1 to 10 µM (Figure 2a, Table 1, Supplementary Figure S1 and Table S1). Clear interactions occur between FECH and the inhibitors AEW-541, Arry-380, Axitinib, AZD-2014, AZD-5438, AZD-8055, BGT-226, BMS-690514, Cabozantinib, CP-724714, Crenolanib, CUDC-101, Cyc-116, E-7080, Erlotinib, Gefitinib, GSK-1070916, GSK-690693, Linsitinib, MK-2461, MK-8033, Momelotinib, Neratinib, Nilotinib, OSI-027, Pelitinib, R-406, Vargatef and Vemurafenib. Figure 2 b-f show the full dose response data for five selected inhibitors and the complete set of these plots can be found in Supplementary Figure S1 and Supplementary Table S1. In our assay, Cyc-116 was found to be the most potent FECH inhibitor, followed by Gefitinib and Vemurafenib.

We confirmed the above binding data for 11 compounds using an isothermal dose response assay (ITDR; Figure 3; Supplementary Figure S2) in K562 cells. The basis of this assay is that proteins bound to a drug molecule are stabilized against heat induced unfolding and aggregation and the extent of this protection is dose dependent.15, 24 We incubated K562 cells with increasing concentrations of a particular drug and then heated the cells to 55 C followed by non-denaturing cell lysis. The actual protein stabilization was measured by quantifying the amount of soluble protein (here FECH) by western blotting. The ITDR data was mostly well aligned with the affinities determined by the kinobead assay. Strong stabilization was detected for all inhibitors except for Dabrafenib which was used as negative control. Alectinib, which was negative in the kinobead assay, scored by ITDR (Supplementary Figure S2), confirming earlier results for this drug and its interaction with FECH.15



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FECH inhibition by kinase inhibitors leads to heme reduction in K562 cells Next, we investigated, whether binding to FECH also leads to loss of enzymatic activity. The human erythroleukemic K562 cell line is capable of heme biosynthesis25. These cells contain active FECH and can therefore serve as cellular system for measuring FECH inhibition by kinase inhibitors26. Four compounds (MK-2461, Neratinib, Linsitinib and Vemurafenib) were amenable to this experiment whereas the other 8 drugs tested were too toxic to the cells over the time frame of the experiment of about one week (Figure 4a). Following exposure of differentiated K562 cells (K562*, which are slightly red owing to an elevated heme content compared to the parental K562 cells) to the respective kinase inhibitors, the heme content of the cells was measured by LC-MS.27 This allowed clear separation and independent detection of heme and PPIX in the same assay (Supplementary Figure S3a). The UV absorption trace for heme (400 nm; as well as the MS trace of m/z 616) in Figure 4b shows the analytical evidence for the assay; it is evident that K562* cells produced heme whereas HEK-293 control cells did not. Hemin alone or parental K562 cells spiked with the compound showed the same elution profile as untreated K562* cells, indicating functioning heme synthesis in K562* cells. Following treatment of cells with Vemurafenib (1 or 3 µM), the heme content of the cells decreased in a dose dependent fashion. However, PPIX accumulation could not be observed as it accumulates in the mitochondria which are not lysed under the assay conditions. We then subjected MK2461, Neratinib and Linsitinib to the same assay (1 µM for 6 days) and each drug clearly led to reduced heme levels indicating inhibition of FECH activity in K562 cells (Figure 4c and Supplementary Figure S3b) with Neratinib showing the most potent effect (60% reduction). As cellular heme changes may also be due to secondary effects, we evaluated FECH activity in a cell-free assay using the FECH R115L mutant because of its better stability and solubility in vitro. Indeed, increasing concentrations of Vemurafenib led to inhibition of FECH (Supplementary Figure 4) showing that Vemurafenib inhibits FECH activity in-vitro and in cells.


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Kinase inhibitors inhibit FECH by blocking the protoporphyrin binding site FECH is not a kinase and has no obvious nucleotide binding site. Hence the question arises as to where a small molecule kinase inhibitor binds the protein. First, we examined the PPIX binding site because binding of inhibitors in this area would completely shut down the catalytic activity of the enzyme. In a published crystal structure of FECH, the PPIX site is occupied by 3 cholate molecules.28 The presence of cholate was found to be required in order to keep the purified protein soluble. A kinobead competition experiment using cholate and recombinant FECH R115L enzyme showed a decrease in binding at increasing concentrations of cholate (Figure 5a), indicating that kinobeads bind FECH via its active site. Given that kinobeads comprise immobilized kinase inhibitors, the aforementioned kinase inhibitors that bind and/or inhibit FECH may act by the same mechanism. To follow up on this hypothesis, we immobilized PPIX onto sepharose beads and performed a competition pulldown experiment using Vemurafenib as the competitor. FECH binding to the PPIX beads was monitored by Western Blotting and the results clearly show a dose dependent decrease of FECH binding to PPIX beads, demonstrating that Vemurafenib indeed binds to the PPIX site in FECH (Figure 5b). To evaluate the binding mode of further FECH inhibitors identified in this study, we performed pulldowns from K562 cell lysates using PPIX beads in competition with the respective drugs (5 µM) or PPIX (5 µM) as positive and Dabrafenib (5 µM) as negative control. In line with the Kd values obtained from kinobead experiments, Vemurafenib, CUDC 101, Cyc-116, Linsitinib, MK2461, Neratinib, GSK-690693, and Crenolanib decreased FECH binding to the PPIX beads by 50% or more compared to the DMSO control. This data clearly indicate that the PPIX binding site of FECH can be targeted by kinase inhibitors (Figure 5c and Supplementary Figure S5). However, the binding data were not entirely conclusive for the compounds Cabozantinib, MK8033, GSK-1070916, Gefitinib, Alectinib, Axitinib, Nilotinib, Vargatef and Momelotinib raising the possibility that there may be one or several other sites in the FECH structure to which these inhibitors may bind. 9 ACS Paragon Plus Environment


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Unfortunately, we failed to co-crystallize FECH with kinase inhibitors since high cholate concentrations are required for protein stabilization and cholate molecules are direct competitors of the kinase inhibitors (see above). Therefore, docking studies were performed for all FECH inhibitors identified in the study based on the published crystal structure of FECH29 (PDB 3w1w; for all docking data see Supplementary Information Table S2, Table S3 and Table S4). In this structure, three cholate molecules occupy the active site and one salicylic acid molecule is found in the dimer interface. Interestingly, for Vemurafenib, Neratinib, Linsitinib, MK2461 and CUDC-101 up to 3 drug molecules could be docked into the active site akin to the three cholate molecules in the original structure (Figure 6b and Supplementary Figure 6A). For other kinase inhibitors, GSK1070916, Gefitinib, Alectinib, Axitinib, Nilotinib, Vargatef and Momelotinib, no reasonable docking results could be obtained for the PPIX site, suggesting that these molecules may bind to an alternative site. The prime candidate for such a site is the dimerization region because active FECH is a dimer and the crystal structure of the dimer accommodates a salicylic acid molecule. Therefore, we performed additional docking studies for this dimerization site (Figure 6c, Supplementary Table S4 and Supplementary Figure 6B). Overall, FECH binders showed higher docking scores compared to those kinase inhibitors that showed no binding. Axitinib showed the highest docking score for the dimerization site and did not compete FECH binding in our PPIX pulldown assay indicating that it may indeed bind to the dimerization site. Alectinib, Gefitinib, GSK-1070916, Nilotinib and Momelotinib also scored higher at the dimerization site than kinase inhibitors that showed no FECH binding raising the possibility that some molecules actually exert their FECH inhibitory effect by disrupting the dimerization site or inducing a conformational change that renders the enzyme inactive.29 Docking data also indicate that some inhibitors, including MK-2461 or Cyc-116, appear to fit into both potential binding sites, possibly suggesting a mixed mode of FECH binding and inhibition.


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However, further biochemical and structural experimentation will be required to substantiate the significance of the presumed second binding site for kinase inhibitors on FECH.

The discovery that kinase inhibitors can also inhibit FECH was unexpected and the fact that more than 10% of all clinically used kinase inhibitors show this off-target effect was even more surprising. This raises the question if FECH inhibition by kinase inhibitors is also of clinical relevance. Partial FECH deficiency in humans leads to the development of a disease called erythropoietic protoporphyria (EPP)30, 31 which is characterized by cutaneous signs of acute and painful photosensitivity.32 Photosensitivity also occurs in at least 50% of all patients receiving Vemurafenib therapy,17, 18, 33 whereas photosensitivity is a much lesser issue in patients treated with Dabrafenib.34 The fact that Vemurafenib is a FECH inhibitor and Dabrafenib is not, makes FECH inhibition a strong candidate for a molecular mechanism explaining the photosensitive phenotype observed in Vemurafenib patients. It has been shown that decreased FECH activity leads to protoporphyrin accumulation in a number of tissues and cell types including erythrocytes.30, 35 Protoporphyrin IX is an endogenous photosensitizer as it absorbs light in a range from 320 to 595 nm and can induce the production of reactive oxygen species (ROS) that, in turn, may contribute to pro-inflammatory processes.36 Vemurafenib patients also show elevated PPIX levels20 further substantiating the link between FECH as an off-target and the clinical phenotype. Further evidence comes from the pharmacology of Vemurafenib. With a dose of 960 mg twice daily and a reported plasma concentration of AUC0-24 of 1741±639 µM x h, 18

the Vemurafenib concentration in the human body is certainly high enough to inhibit FECH

completely and systemically. Based on this rational and the data presented above, photosensitivity due to FECH inhibition may also be a relevant issue for Cabozantinib for which plasma concentrations in the mid micromolar range have been reported37 and similar considerations may apply to Crenolanib38, Alectinib39, Axitinib40, or Nilotinib41 for which photosensitivity has also been described as a clinical side effect. For other kinase inhibitors 11 ACS Paragon Plus Environment


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such as Neratinib or MK-2461, FECH inhibition may be irrelevant as the clinical doses of these compounds do not reach the levels required for FECH inhibition in-vivo.42 Cyc-116 is currently the most potent FECH inhibitor (Kd = 0.7 µM) but no plasma concentrations have been reported so far and there are also no reports yet on clinical observations from a completed Phase I clinical trial. We note here that the photosensitizing effect of small molecule inhibitors can also be used for photodynamic therapy of cancer. For example, the heme precursor 5-aminolevulinic acid (ALA) is a commonly used prodrug to upregulate PPIX content in cancer cells. Upon light excitation, PPIX forms reactive oxygen species that kill nearby tumor cells.43, 44 It has been shown that not only ALA treatment but also FECH inhibition leads to PPIX accumulation.45 Therefore, on a more speculative note, a kinase inhibitor that is also a FECH inhibitor may be useful for photodynamic therapy, in case the kinase inhibitor is otherwise very well tolerated.


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Conclusions The present study has shown that a substantial number of clinically used kinase inhibitors including the BRAF inhibitor Vemurafenib also target the enzyme Ferrochelatase. Biochemical and structural data revealed that several inhibitors bind to the protoporphyrin site in the enzyme whereas others potentially interact with the protein in the dimerization domain. Since the photosensitive phenotype characteristic for patients with genetic FECH deficiency resembles that of Vemurafenib treated skin cancer patients, we suggest that FECH inhibition by this drug is a likely molecular mechanism by which this toxicity occurs. Given that about 13% of all kinase inhibitors are also FECH inhibitors, it would seem prudent to consider including a FECH assay in the pre-clinical development of kinase inhibitor drugs in general.



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Methods Cell culture, affinity matrices and reagents K562, Colo205 and MV4-11 were grown in RPMI1640 medium (Biorad), SKNBE-2 were cultured in DMEM/HAM’s F-12 medium (Biorad) and HEK293 in IMDM (Biorad), all supplemented with 10% FBS and 1% antibiotic solution (Sigma). Affinity matrices were produced in house as published previously. PPIX (Sigma Aldrich) 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 acid.11 Small molecule inhibitors were purchased from Selleckchem, MedChemExpress, Active Biochem, Abmole, Merck or LC Labs. Hemin was obtained from Sigma Aldrich. The stable and soluble recombinant FECH R115L mutant was expressed and purified as described previously.29 FECH protein was detected with a mouse monoclonal antibody (sc-271434, Santa Cruz Biotechnology) in a 1:400 dilution. For secondary detection IRDye 800CW conjugated goat anti-mouse (LI-COR Biosciences) was used. Binding profiling using affinity matrices Kinobead selectivity profiling, as well as profiling with PPIX affinity matrices, was performed as described previously.11 Briefly, 5 mg of a protein mixture of the four cell lines or a single cell line were incubated with compound dilution series in DMSO (3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 µM, 3 µM, 30 µM and DMSO) or single compound dose (5 µM) for 45 min at 4°C. The preincubation step was followed by incubation with kinobeads (35 µl settled beads). Bound proteins were eluted with LDS sample buffer (NuPAGE, Invitrogen) containing 50 mM DTT. For the calculation of a correction factor, the flowthrough of the DMSO control was incubated with fresh beads for a second time (pull down of pull down). LC MS/MS analysis Reduced eluates were alkylated with chloroacetamide (55 mM) and the proteins were concentrated by a short electrophoresis on a 4-12% NuPAGE gel (Invitrogen). In-gel digestion was performed according to standard procedures. Peptides generated by in-gel trypsin digestion were analyzed via LC-MS/MS on a nanoLC-Ultra 1D+ (Eksigent) coupled to a LTQOrbitrap Elite mass spectrometer (Thermo Scientific). Peptides were delivered to a trap column


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(100 µm x 2 cm, packed in house with Reprosil-Pur C18 AQ 5 µm resin, Dr. Maisch) at a flow rate of 5 µl/min in 100% solvent A0 (0.1% FA in HPLC grade water). Peptides were then separated on an analytical column (75 µm x 40 cm, packed in-house with Reprosil-Gold c18, 3 µm resin, Dr. Maisch) using a 100 min gradient ranging from 4-32% solvent B (0.1% FA and 5% DMSO in acetonitrile) in A1 (0.1% FA and 5% DMSO in HPLC grade water) at a flow rate of 300 nL/min. The mass spectrometer was operated in data dependent mode, automatically switching between MS and MS2 spectra. Up to 15 peptide precursors were subjected to fragmentation by higher energy collision-induced dissociation (HCD) and analyzed in the Orbitrap. An inclusion list for kinase peptides was enabled. Dynamic exclusion was set to 20 s. Peptide and protein identification and quantification Peptide and protein identification and quantification was performed using MaxQuant (version 1.4.0.5)46 by searching the tandem MS data against a human Uniprot reference database (version 22.07.13, annotated in-house with PFAM domains) using the embedded search engine Andromeda47. Carbamidomethylated cysteine was used as fixed modification, phosphorylation of serine, threonine, and tyrosine, oxidation of methionine, and N-terminal protein acetylation as 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 0.05 Da. Label free quantification (LFQ)48 and match between runs were enabled within MaxQuant. Search results were filtered for a minimal length of seven amino acids, 1% peptide and protein FDR as well as common contaminants and reverse identifications. Data analysis For competition binding assays, a ratio of the LFQ intensity of each dose point to the DMSO control was calculated for every protein. EC50 values were determined based on this ratio using an in house generated R-Script10, using nonlinear regression with variable slope. A Kd was then calculated by multiplying the EC50 with a correction factor for each protein. The correction factor (r) for a protein is defined as the ratio of the amount of protein captured from two consecutive of pull downs of the same DMSO control lysate.11,

23

Selected dose dependent inhibition curves

were analyzed using GraphPadPrism (version 5.03). Isothermal dose response assay Isothermal dose response assays were performed as described before.15 Briefly, K562 cells were incubated with increasing concentrations of drug for 1h at 37 °C. Then, cells were washed 15 ACS Paragon Plus Environment


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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. Denatured proteins were precipitated by centrifugation at 20,000xg for 30 min. Supernatants were reduced in LDS Sample buffer and proteins were separated in a 4-12% NuPAGE gel (Invitrogen). FECH protein was detected by immunoblotting following standard procedures and quantified with the Odyssey infrared imaging system (LI-COR Biosciences). 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.27 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 20,000xg 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 A (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.29 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 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 16 ACS Paragon Plus Environment

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protein. Docking studies were carried out using GOLD 5.249 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 step.



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Data availability Raw mass spectrometry files for FECH inhibitors and respective MaxQuant result files are available via PRIDE and ProteomeXchange under the accession code PXD003373.

Supporting Information Supporting Information Available: This material is available free of charge via the Internet. •

Supplementary figures and tables with docking scores (PDF).

•

Table for 226 inhibitors with EC50 and Kd values and further parameters after curve fitting (XLXS).

Acknowledgements The authors want to thank A. Hubauer, M. Krötz-Fahning and A. Klaus for their technical assistance.

Competing Financial Interests Statement Jessica Perrin, Giovanna Bergamini, Mikhail M Savitski and Marcus Bantscheff are employees of Cellzome GmbH, a GSK company. Bernhard Kuster is a founder and shareholder of OmicScouts GmbH.


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Table 1 Table 1: Binding constants Kd for 29 inhibitors and FECH showing clear FECH-drug interaction.

Kinase inhibitor

FECH Kd (µM)

Kinase inhibitor

FECH Kd (µM)

AEW-541

4.4

Gefitinib

1.0

ARRY-380

6.4

GSK-1070916

1.7

Axitinib

3.1

GSK-690693

2.7

AZD-2014

3.4

Linsitinib

2.2

AZD-5438

2.6

MK-2461

2.1

AZD-8055

6.2

MK-8033

28.0

BGT-226

3.4

Momelotinib

3.4

BMS-690514

3.0

Neratinib

5.1

Cabozantinib

2.8

Nilotinib

1.7

CP-724714

23.0

OSI-027

4.5

Crenolanib

3.8

Pelitinib

10.0

CUDC-101

3.5

R-406

3.6

Cyc-116

0.7

Vargatef

3.2

E-7080

3.1

Vemurafenib

1.3

Erlotinib

9.3



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Figure legends Figure 1: (a) Kinobeads competitive binding assay workflow. The protein repertoire from lysates of four different cell lines is incubated with increasing concentrations of a drug. The affinity matrix captures kinases, nucleotide binding proteins, FECH and other proteins whose binding pocket is not occupied by the drug at a given drug concentration. LC-MS/MS analysis quantifies the amount of captured protein at each drug concentrations which allows the computation of dose dependent inhibition curves. (b)-(c) Dose response curves for Vemurafenib (b) and Dabrafenib (c) for the main drug target BRAF (black line) and the off-target FECH (red line). Figure 2: (a): Radar plot showing Kd values for FECH binding determined for 226 small molecule kinase inhibitors using the kinobead assay. Drugs that show no inhibition of FECH binding populate the inner circle. (b)-(f): Full dose response curves with EC50 values for the five selected kinase inhibitors: Cyc-116, Linsitinib, MK-2461, Neratinib and Vemurafenib. Figure 3: Validation of kinobead binding data using isothermal dose response assays in K562 cells. Axitinib, Cabozantinib, CUDC-101, Cyc-116, GSK-1070916, MK-2461, Neratinib and Vemurafenib show concentration-dependent thermal stabilization of FECH. EC50 values, if determined, are shown next to the respective graph. Dabrafenib is shown as a negative control as it does not bind to FECH. Figure 4: Assay for FECH inhibition. (a) Cell viability assay for K562 cells treated with 1 µM drug for up to 6 days. Error bars represent the standard deviation (SD) of triplicate experiments. While most drugs tested were toxic to the cells, four inhibitors could be used in the assay. (b) left panel: chromatograms for heme absorption at 400 nm for K562 lysates. Differentiated K562* cells (black line) contain high amounts of hemin, undifferentiated K562 lysates spiked with 30 µM hemin (orange line) and lysates of HEK 293 (blue line) cells show that hemin can be detected in cells with active heme biosynthesis. Right panel: increasing Vemurafenib concentrations decrease the amount of heme in K562 cells. (c) Change in K562 cells heme content after 6 days of treatment with 1 µM MK-2461, Neratinib, Linsitinib and Vemurafenib relative to vehicle treated cells. Figure 5: Binding mode of kinase inhibitors to FECH. (a) Increasing concentrations of cholate reduce binding of FECH to kinobeads. (b) Increasing Vemurafenib concentrations can compete FECH binding to PPIX immobilized on beads, demonstrating that Vemurafenib binds in the PPIX pocket of FECH. (c) Analogous binding site determination experiments for 18 kinase inhibitors in triplicates. PPIX is included as a positive control and Dabrafenib serves as a negative control. While Vemurafenib, CUDC-101, Cyc-116, Linsitinib, MK-2461, Neratinib, GSK690693, Cabozantinib, Crenolanib, MK-8033 and GSK-1070916 show decreased binding of FECH to PPIX beads, Gefitinib, Alectinib, Axitinib, Nilotinib, Vargatef and Momelotinib do not, implying the presence of an alternative binding site for these inhibitors on FECH. Error bars depict the standard error of the mean (SEM).


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Figure 6: Docking studies using the crystal structure of FECH. (a) Structure of the FECH dimer with both the PPIX and dimerization sites occupied by Vemurafenib. Three Vemurafenib molecules can be placed into the PPIX binding site. The first molecule docked into the PPIX site is shown in gold, the second molecule when the first is already in place is shown in red and orange depicts the third and final molecule of Vemurafenib in the PPIX binding pocket (b) Detail of the PPIX binding site with 3 molecules of Vemurafenib stacked inside the pocket. (c) Detail of the dimerization site with Vemurafenib docked at the dimer interface.



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Table of contents graphic 275x190mm (300 x 300 DPI)

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Figure 1: (a) Kinobeads competitive binding assay workflow. The protein repertoire from lysates of four different cell lines is incubated with increasing concentrations of a drug. The affinity matrix captures kinases, nucleotide binding proteins, FECH and other proteins whose binding pocket is not occupied by the drug at a given drug concentration. LC-MS/MS analysis quantifies the amount of captured protein at each drug concentrations which allows the computation of dose dependent inhibition curves. (b)-(c) Dose response curves for Vemurafenib (b) and Dabrafenib (c) for the main drug target BRAF (black line) and the off-target FECH (red line). 275x190mm (300 x 300 DPI)



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Figure 2: (a): Radar plot showing Kd values for FECH binding determined for 226 small molecule kinase inhibitors using the kinobead assay. Drugs that show no inhibition of FECH binding populate the inner circle. (b)-(f): Full dose response curves with EC50 values for the five selected kinase inhibitors: Cyc-116, Linsitinib, MK-2461, Neratinib and Vemurafenib. 275x190mm (300 x 300 DPI)


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Figure 3: Validation of kinobead binding data using isothermal dose response assays in K562 cells. Axitinib, Cabozantinib, CUDC-101, Cyc-116, GSK-1070916, MK-2461, Neratinib and Vemurafenib show concentration-dependent thermal stabilization of FECH. EC50 values, if determined, are shown next to the respective graph. Dabrafenib is shown as a negative control as it does not bind to FECH. 275x190mm (300 x 300 DPI)



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Figure 4: Assay for FECH inhibition. (a) Cell viability assay for K562 cells treated with 1 µM drug for up to 6 days. Error bars represent the standard deviation (SD) of triplicate experiments. While most drugs tested were toxic to the cells, four inhibitors could be used in the assay. (b) left panel: chromatograms for heme absorption at 400 nm for K562 lysates. Differentiated K562* cells (black line) contain high amounts of hemin, undifferentiated K562 lysates spiked with 30 µM hemin (orange line) and lysates of HEK 293 (blue line) cells show that hemin can be detected in cells with active heme biosynthesis. Right panel: increasing Vemurafenib concentrations decrease the amount of heme in K562 cells. (c) Change in K562 cells heme content after 6 days of treatment with 1 µM MK-2461, Neratinib, Linsitinib and Vemurafenib relative to vehicle treated cells. 275x190mm (300 x 300 DPI)


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Figure 5: Binding mode of kinase inhibitors to FECH. (a) Increasing concentrations of cholate reduce binding of FECH to kinobeads. (b) Increasing Vemurafenib concentrations can compete FECH binding to PPIX immobilized on beads, demonstrating that Vemurafenib binds in the PPIX pocket of FECH. (c) Analogous binding site determination experiments for 18 kinase inhibitors in triplicates. PPIX is included as a positive control and Dabrafenib serves as a negative control. While Vemurafenib, CUDC-101, Cyc-116, Linsitinib, MK2461, Neratinib, GSK-690693, Cabozantinib, Crenolanib, MK-8033 and GSK-1070916 show decreased binding of FECH to PPIX beads, Gefitinib, Alectinib, Axitinib, Nilotinib, Vargatef and Momelotinib do not, implying the presence of an alternative binding site for these inhibitors on FECH. Error bars depict the standard error of the mean (SEM). 275x190mm (300 x 300 DPI)



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Figure 6: Docking studies using the crystal structure of FECH. (a) Structure of the FECH dimer with both the PPIX and dimerization sites occupied by Vemurafenib. Three Vemurafenib molecules can be placed into the PPIX binding site. The first molecule docked into the PPIX site is shown in gold, the second molecule when the first is already in place is shown in red and orange depicts the third and final molecule of Vemurafenib in the PPIX binding pocket (b) Detail of the PPIX binding site with 3 molecules of Vemurafenib stacked inside the pocket. (c) Detail of the dimerization site with Vemurafenib docked at the dimer interface. 275x190mm (300 x 300 DPI)


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Chemical Proteomics Reveals Ferrochelatase as a ... - ACS Publications

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