European Journal of Pharmacology 599 (2008) 44–53

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European Journal of Pharmacology

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Molecular and Cellular Pharmacology

Inhibition of collagen-induced discoidin domain receptor 1 and 2 activation byimatinib, nilotinib and dasatinib

Elizabeth Day a,1, Beatrice Waters a,1, Katrin Spiegel b, Tanja Alnadaf a, Paul W. Manley c,Elisabeth Buchdunger c, Christoph Walker a, Gabor Jarai a,⁎a Novartis Institutes of Biomedical Research, Respiratory Disease Area, Wimblehurst Road, Horsham, RH12 5AB, UKb Novartis Institutes of Biomedical Research, Global Discovery Chemistry, Wimblehurst Road, Horsham, RH12 5AB, UKc Novartis Institutes of Biomedical Research, Oncology Disease Area, Novartis Pharma AG, Klybeckstrasse 141, CH-4002 Basel, Switzerland

⁎ Corresponding author.E-mail address: gabor.jarai@novartis.com (G. Jarai).

1 Equal contribution.

0014-2999/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ejphar.2008.10.014

a b s t r a c t

a r t i c l e i n f o

Article history:

Imatinib, nilotinib and dasa Received 24 June 2008Received in revised form 15 September 2008Accepted 5 October 2008Available online 11 October 2008

Keywords:Chronic myelogenous leukemiaDiscoidin domainFibrosisGleevecReceptor tyrosine kinaseTasigna

tinib are protein kinase inhibitors which target the tyrosine kinase activity of theBreakpoint Cluster Region-Abelson kinase (BCR-ABL) and are used to treat chronic myelogenous leukemia.Recently, using a chemical proteomics approach another tyrosine kinase, the collagen receptor DiscoidinDomain Receptor1 (DDR1) has also been identified as a potential target of these compounds. To furtherinvestigate the interaction of imatinib, nilotinib and dasatinib with DDR1 kinase we cloned and expressedhuman DDR1 and developed biochemical and cellular functional assays to assess their activity against DDR1and the related receptor tyrosine kinase Discoidin Domain Receptor2 (DDR2). Our studies demonstrate thatall 3 compounds are potent inhibitors of the kinase activity of both DDR1 and DDR2. In order to investigatethe question of selectivity among DDR1, DDR2 and other tyrosine kinases we have aligned DDR1 and DDR2protein sequences to other closely related members of the receptor tyrosine kinase family such as MuscleSpecific Kinase (MUSK), insulin receptor (INSR), Abelson kinase (c-ABL), and the stem cell factor receptor (c-KIT) and have built homology models for the DDR1 and DDR2 kinase domains. In spite of high similarityamong these kinases we show that there are differences within the ATP–phosphate binding loop (P-loop),which could be exploited to obtain kinase selective compounds. Furthermore, the potent DDR1 and DDR2inhibitory activity of imatinib, nilotinib and dasatinib may have therapeutic implications in a number ofinflammatory, fibrotic and neoplastic diseases.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Imatinib (Gleevec®, STI571), is an inhibitor of the tyrosine kinaseactivity of BCR-ABL and is the first-line therapy for chronicmyelogenous leukemia. Although most patients respond very wellto imatinib therapy, resistance can develop in a subpopulation ofadvanced stage chronic myelogenous leukemia patients. Resistance isfrequently due to the emergence of clones expressingmutant forms ofBCR-ABL which are not sensitive to imatinib. Two second-generationagents, nilotinib (Tasigna®, AMN107) and dasatinib (Sprycell®, BMS-354825), which maintain activity against many imatinib-resistant,mutant forms of BCR-ABL, have been introduced to treat imatinib-intolerant and -resistant chronic myelogenous leukemia (Weisberget al., 2005). Several other compounds are also being developed forimatinib-resistant chronic myelogenous leukemia (Weisberg et al.,2007). Whereas imatinib and nilotinib are relatively selective tyrosine

l rights reserved.

kinase inhibitors, dasatinib is a multi-targeted kinase inhibitor, whichin addition to inhibiting BCR-ABL, potently inhibits many additionalkinases, including those of the SRC kinase family (Shah et al., 2004;Weisberg et al., 2007).

A recent chemical proteomics study of Abelson kinase inhibitorshas identified Discoidin Domain Receptor1 as an additional target ofimatinib in K562 leukemia cells (Bantscheff et al., 2007). Morerecently, by generating drug–protein interaction profiles it has alsobeen demonstrated that DDR1 can also bind nilotinib and dasatinib,which, in turn, can inhibit DDR1 activity (Rix et al., 2007).

DDR1 is one of two, non-integrin tyrosine kinase receptorsactivated by collagen. Although DDR1 has five isoforms (1a, 1b, 1c,1d, 1e) generated by alternative splicing, only DDR1a and 1b haveactive kinase domains, whereas DDR2, encoded by a distinct gene, hasone isoform (Vogel et al., 2006). Collagen binding requires dimeriza-tion of the extra-cellular domains and results in receptor auto-phosphorylation.

DDR1 is widely expressed during embryonic development and inadult tissues, with expression in the epithelium of a variety of tissues,particularly in skin, kidney, lung, gut, and brain (Vogel et al., 2006).

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DDR2 is expressed primarily in mesenchymal cells including fibro-blasts, myofibroblasts, smooth muscle, and skeletal muscle in severaltissues including skin, kidney, lung, heart and connective tissues.

DDRs have been proposed to play important roles in a number ofdiseases. High levels of DDR1 and 2 expression have been observed inseveral tumours of breast, ovarian, lung and brain origin (Barker et al.,1995; Nemoto et al., 1997; Weiner et al., 2000). DDR1 receptor over-expression has also been implicated in cell survival and invasiveness inhepatocellular carcinoma, pituitary adenoma and prostate cancer(Park et al., 2007; Yoshida and Teramoto, 2007; Shimada et al., 2008).DDR1, particularly the 1b isoform has also been implicated inidiopathic pulmonary fibrosis. Selective induction of DDR1b in CD14+

cells from idiopathic pulmonary fibrosis patients has been shown tolead to production of several inflammatory chemokines upon collageninduction (Matsuyama et al., 2005a). Suppression of DDR1 in thebleomycin model in vivo using siRNA or in DDR1-deficient mice haslead to the attenuation of fibrosis and inflammation (Avivi-Green et al.,2006; Matsuyama et al., 2006). In sarcoidosis, DDR1 expression levelsin CD14+ bronchoalveolar lavage cells appear to correlate withdeterioration of the disease (Matsuyama et al., 2005b). Both DDR1and 2 are expressed in atherosclerotic and lymphangioleiomyomatoticlesions and appear to participate in the regulation of collagen turnoverby smooth muscle cells (Ferri et al., 2004). DDR1 null mice areprotected in a mouse model of kidney fibrosis and implicate thereceptor in both inflammatory and fibrotic components of the disease(Flamant et al., 2006). The over-expression of DDR2 has been observedin the cartilage in patients with osteoarthritis (Xu et al., 2007) and inthe context of liver fibrosis it has been shown that the induction offibrosis in an animal model resulted in up-regulation of DDR2 instellate cells (Olaso et al., 2001).

In order to gain deeper insight into the inhibitory activity ofimatinib, nilotinib and dasatinib against DDR1 kinase we cloned andexpressed DDR1 and developed biochemical and cellular functionalassays to test their activity. Furthermore, we also evaluated the effectsof imatinib, nilotinib and dasatinib on the related receptor tyrosinekinase DDR2. Our studies demonstrate that all 3 compounds arepotent inhibitors of both DDR1 and DDR2whichmay have therapeuticimplications in a number of inflammatory, fibrotic and neoplasticdiseases. Furthermore, in order to identify possible strategies to obtaindiscoidin domain receptor specific compounds we have aligned DDR1and DDR2 protein sequences to other members of the receptortyrosine kinase family and we have built homology models for theDDR1 and DDR2 kinase domains. Most misalignments are foundwithin the ATP–phosphate binding loop (P-loop), which could befurther exploited to obtain kinase selective compounds.

2. Materials and methods

2.1. Cell lines, tissue culture and antibodies

HEK293 cells were grown in MEM (minimal essential medium)medium supplemented with 20% foetal bovine serum (FBS) and 2 mML-glutamine. For transfection, cells were seeded in poly-D-lysinecoated flasks. HEK293-DDR cells were grown in the above mediumsupplemented with 250 µg/ml geneticin. THP1 cells were grown inRPMI-1640 medium, 10% FBS and 2 mM L-glutamine. THP1-DDR1bcells were grown in the above media supplemented with 6 µg/mlblasticidin. The anti-His monoclonal antibody used in Western blotexperiments were obtained from GE Biosciences. For the FACS(Fluorescence-activated Cell Sorting) analysis of DDR1 expressingcells monoclonal antibodies from R&D Systems were used. Neutralis-ing anti-β-integrin antibodies were obtained from Millipore. For theELISA assays, DDR1 and DDR2 antibodies were obtained from R&DSystems. Anti-phosphotyrosine antibody clone 4G10 was obtainedfrom Millipore and an HRP conjugated anti-mouse secondary anti-body from Sigma was used.

2.2. Cloning of full length and intracellular domains of DDR1 and DDR2

cDNA clones for DDR1 isoforms were amplified by the polymerasechain reaction (PCR) using the following primers: forward-CACCATGG-GACCAGAGGCCCTGTCATCTTTACTGCTGCT, including CACC at the 5′ endto allow directional cloning into pENTR/D-TOPO; and reverse-TCA-CACCGTGTTGAGTGCATCCTCTGCCAGGAACCGA. DDR1a cDNA (DDR1splice variant 2, accession number NM_001954) was amplified fromhuman colon cDNA using Pfu Turbo polymerase (Stratagene). Touch-downPCRreactionswereperformedusingdenaturing conditionsof 95 °Cfor 30 s and extension conditions of 72 °C for 3 min. Annealing wasperformed for 8 touch-down cycles from 72 °C to 65 °C followed by 30cycles at 65 °C, each cycle for 1min. The amplified band of approximately2.6 kb was isolated and cloned into pENTR/D-TOPO (Invitrogen). Cloneswere isolated and purified using theQiaprep SpinMiniprep kit (Quiagen)and analyzed by restriction digestion and DNA sequencing using anABI3100 sequencer. The transcript encoding DDR1b (DDR1 variant 1,accessionnumberNM_013993) contains an additional 111basepairs (bp)long insert encoding37 additional aminoacids. To cloneDDR1b, the sameprimers and PCR conditions as above were used to amplify primaryhumanbronchial epithelial cell cDNA.Cloneswere screenedwith internalprimers and those shown toharbor the additional 111bp region andwerethen confirmed by DNA sequencing. DDR2 cDNA (accession numberNM_006182) was PCR amplified from human brain cDNA using thefollowing primers: forward-CACCATGATCCTGATTCCCAGAATGCTCTTGGT,including CACC at the 5′ end to allow directional cloning into pENTR/D-TOPO; reverse-TCACTCGTCGCCTTGTTGAAGGAG and the same PCR con-ditions as used for DDR1. The obtained 2.6 kb fragment was cloned intopENTR/D-TOPO (Invitrogen) and fully sequenced using an ABI3100sequencer. To express the kinase domains the full length cDNA cloneswere used as templates and the following PCR primers to amplify theintracellular domains of DDR1 and DDR2. The forward primers weredesigned to include anN-terminal 6xHis-tag for proteinpurification anda CACC at the 5′ end to allow directional cloning into pENTR/D-TOPO.DDR1b, forward-CACCATGCATCATCATCATCATCATCAGGAGCCC-CGGCCTCGTGGGAAT; DDR1b-reverse-TCACACCGTGTTGAGTGCATCCTCT;DDR2, forward-CACCATGCATCATCATCATCATCATTCCAACTCGAC-TTACGATCGCATCTT; and DDR2, reverse-TCACTCGTCGCCTTGTTGAA-GGAGCA. The amplified fragments encode amino acids 485–913 forDDR1band486–855 forDDR2. The amplifiedDDR sequenceswere clonedinto pENTR/D-TOPO and then transferred into the baculovirus expressionvector pDEST8 via an LR recombination reaction following the manufac-turer's recommendations (Invitrogen).

2.3. Expression and purification of DDR1b and DDR2 kinase domains

DDR constructs were transposed into bacmid DNA to generaterecombinant bacmids by transforming pDEST8 vectors into DH10BACcompetent cells. After 48 h recombinant bacmid DNA was isolatedfrom 3 ml cultures by resuspending pelleted cells in a buffercontaining 15 mM Tris–HCl pH 8, 10 mM EDTA and 100 µg/ml RNaseA. An equal volume of 0.2 M NaOH, 1% SDS was added followed by K-acetate (pH 5.5) at a final concentration of 1 M. After 10 min on ice thetubes were centrifuged and the bacmid DNA in the supernatantprecipitated with 0.8 ml isopropanol at −20 °C overnight. Sampleswere centrifuged, washed using 70% ethanol, air-dried and dissolvedin 40 µL TE, pH 8. Sf21 insect cells were grown in Sf900 II serum-freemedium containing 5 µg/ml Gentamycin and 0.5 µg/ml Fungizone. Forthe transfection of SF21 cells 0.8×106 cells/well were seeded in 6-wellplates. 2 µg bacmid DNA was used for transfection using Cellfectin(Invitrogen) as recommended by the manufacturer. After transfectionthe plates were incubated at 27 °C for 72 h and the virus containingsupernatant collected (passage 1). After 2–3 rounds of virusamplification DDR expression cultures were set up using 300 mlSf21 cells at a cell density of 0.5×106 cells and 2.5 ml passage 3 virusstock. Cells were grown in a shaking incubator at 27.5 °C, 90 rpm for

Fig. 1. Activity testing of the purified DDR1 and DDR2 recombinant enzymes. Theactivity of DDR1 (panel A) and DDR2 (panel B) kinases was tested in the LanthaScreenusing 1:2 serial dilutions of the enzymes. The EC50 value for DDR1 was found to be1.1 μg/ml and for DDR2 1.9 μg/ml. Panel C: the effect of the nonspecific kinase inhibitorstaurosporine on DDR1 and DDR2 activity was tested using a 1:2 serial dilution ofstaurosporine in the range of 50 μM to 0.05 μM. Staurosporinewas shown to inhibit bothDDR1 and DDR2 activity with IC50 values of 2 μM for DDR1 and 2.4 μM for DDR2.Experiments were performed using at 100 μM ATP.

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3 days before being harvested by centrifugation and resuspended innative lysis buffer supplemented with 0.2% NP-40 and proteaseinhibitors. Cells were lysed on ice for 30 min and, centrifuged at20,000 ×g for 40 min. His-tagged recombinant proteins were purifiedfrom the supernatant using the QIAexpress® Ni-NTA Fast Start Kit(Qiagen) as recommended by the manufacturer. Eluted proteins wererun through a desalting column (Pierce) in order to exchange theelution buffer with a buffer containing 100 mM Tris pH 7.5, 300 mMNaCl, 0.04% triton X-100, 4 mM dithiothreitrol (DTT) and proteaseinhibitors. The protein containing fractions were pooled and glycerolwas added to a final concentration of 50% and stored at −80 °C.

2.4. Western blotting

HEK293 wild type and HEK293-DDR cells were lysed in buffercomprising 1% NP-40, 150 mM NaCl, 50 mM Tris pH 8.0, 1 mM sodiumorthovanadate, 5mMNaF and protease inhibitor cocktail. Lysateswerecentrifuged to remove debris and supernatants denatured at 70 °C in4x NuPAGE sample buffer (Invitrogen) containing 50 mM DTT prior toseparation on a 4–10% Bis–Tris (MOPS) NuPAGE gel. Staining ofacrylamide gels was performed using the Gelcode Blue Stain Reagentfrom Pierce according to the manufacturer's recommendations. Thegels were fixed in 50% methanol, 7% acetic acid and proteinstransferred onto nitrocellulose membranes. Membranes were blockedfor 1 h in TBS, 0.1% (v/v) Tween-20, 5% (w/v) non-fat dried milk.Immuno-detection was carried out by incubation with primaryantibody for 1 h at room temperature followed by washing of themembranes and incubation with a horse radish peroxidase-conju-gated anti-mouse secondary antibody for 1 h at room temperature.Membranes were developed using the Supersignal West PicoChemiluminescent substrate (Pierce) according to the manufacturer'srecommendations.

2.5. Time-resolved fluorescence resonance energy transfer (TR-FRET)kinase assay

The LanthaScreen (Invitrogen) assay format was used. DDR kinaseswere incubated with 400 nM fluorescein labeled poly-GAT substrate(poly glu:ala:tyr, 6:3:1), and 100 µM ATP in a total volume of 10 µl in384-well plates in kinase dilution buffer (50mMHEPES pH 7.5, 10 mMMgCl2, 1 mM EGTA, 0.01% Brij-35). After 60 min incubation at roomtemperature, 10 µl of TR-FRET dilution buffer containing EDTA and Tb-labeled phosphopeptide-specific antibody Tb-PY20 was added andmixed to give a final volume of 20 µl/well. After 60 min incubation atroom temperature the plate was read on LJL Analyst HT (MolecularDevices) using a 330/80 excitation filter, a 490/10 emission filter 1(donor) and a 520/10 emission filter 2 (acceptor). Compound dilutionswere added to the substrate-ATP mix prior to the addition of DDRenzymes. After the addition of DDR enzyme the reactions wereincubated at room temperature for 60 min as described above.

2.6. Generation of stable DDR1 and 2 transfectant clones in HEK293 cellsand DDR auto-phosphorylation assay

Full length cDNAs encoding DDR1b and DDR2 in pENTR/D-TOPOvectors were transferred into pcDNA-DEST40 using LR recombinationreactions according to the manufacturer's recommendation (Invitro-gen) and resulting recombinant constructs used for the transfection ofHEK293 cells. 48 h after transfection geneticin was added to theculture medium at a concentration of 250 µg/ml. Single cell cloneswere isolated by cell sorting into 96-well plates using a MoFlo cellsorter and characterized for DDR expression using quantitative PCR,Western blotting and FACS analysis. To detect collagen-dependentphosphorylation HEK293-DDR cells were plated in poly-D-lysinecoated 96-well plates, grown overnight then serum-starved for 24 h.Cells were incubated with compounds for 30 min and then stimulated

with 10 µg/ml collagen for 16 h before being lysed with 15 µl ice-coldlysis buffer as described forWestern Blotting. ELISA plateswere coatedovernight at 4 °C using 50 µl/well of 1 µg/ml DDR1 antibody or 8 µg/mlDDR2 antibody in carbonate coating buffer. Wells were washed inELISA wash buffer (PBS/0.05% Tween (v/v)) and blocked using 200 µlblocking buffer (PBS with Ca2+ and Mg2+/0.1% (w/v) BSA/5% (w/v)sucrose) at 4 °C overnight. 50 µl/well of 1:20 diluted DDR1 cell lysate(PBS/0.1% BSA) or 1:10 diluted DDR2 cell lysatewas added to the ELISAplates and incubated overnight at 4 °C. Plates were washed and 50 µlof 0.25 µg/ml horse radish peroxidase-labelled anti-phosphotyrosine4G10 added. After incubation for 1 h in the dark at room temperatureplates were developed using the Supersignal ELISA Femto MaximumSensitivity substrate (Pierce).

Fig. 2. Inhibition of DDR1 and DDR2 kinases by imatinib, nilotinib and dasatinib. Panel A: the molecular structures of imatinib, the structurally related nilotinib and of dasatinib areshown. All three compounds were used in the TR-FRET assay and shown to inhibit both DDR1 (panel B) and DDR2 (panel C) activity (n=3). The IC50 values of imatinib were found tobe 337±56 nM for DDR1 and 675±127 nM for DDR2 and nilotinib and dasatinib appeared to bemore potent with IC50 values of 43±3 nM and 0.5±0.2 nM for DDR1 and 55±9 nM and1.4±0.3 nM for DDR2, respectively. Experiments were performed using at 100 μM ATP and representative inhibition curves are shown.

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2.7. Generation of stable DDR transfectant clones in THP1 cells andmacrophage chemoattractant protein-1 (MCP-1) release assay

The full length cDNA encoding DDR1b in the pENTR/D-TOPO vectorwas transferred into pLenti6/V5-DEST using LR recombinationaccording to the manufacturer's recommendations (Invitrogen).Lentivirus was generated using the ViraPower™ Lentiviral ExpressionSystem (Invitrogen) and viral titer determined by blasticidin selectionof transduced A549 cells. Stable DDR1b clones were generated bytransducing 5×104 THP1 cells at a multiplicity of infection of 1 in thepresence of 2 µg/ml polybrene. Culture medium was supplementedwith 6 µg/ml blasticidin at 48 h post-transduction and clones isolatedby single cell sorting of THP1 cells stained with DDR1 antibodies usinga MoFlo cell sorter. FACS analysis was used to determine DDR1expression levels in resultant clones. In order to detect collagen-inducedMCP-1 secretion, stable THP1-DDR1b cells were seeded in 96-well plates in culture medium supplemented with 10 nM phorbol 12-

myristate 13-acetate (PMA) and 5 µg/ml anti-β-integrin antibody andincubated for 1 h at 37 °C. Compound dilutions were prepared inmedia supplemented with 0.5% (v/v) di-methyl sulfoxide (DMSO) and10 nM PMA and added to cells 60 min prior to the addition of calf skincollagen I (Sigma-Aldrich) at a final concentration of 50 µg/ml. Controlwells were supplemented with media alone or media plus collagen.Cells were incubated at 37 °C for 48 h and the MCP-1 content in cellculture supernates determined by sandwich ELISA according to themanufacturer's recommendations (R&D Systems).

2.8. Homology models of DDR kinases

The amino acid sequences of human DDR1 and DDR2 wereretrieved from the SWISSPROT database (Swissprot ID: Q08345 andQ16832, respectively) and aligned to sequences of other tyrosinekinases using ClustalW. In the initial alignment, we used the followingphylogenetically related kinases: Muscle Specific Kinase (MUSK),

Fig. 3. Collagen-induced DDR auto-phosphorylation. Collagen-induced dose-dependentreceptor auto-phosphorylation was analyzed in HEK-DDR1 (panel A), HEK-DDR2(panel B) and HEK293 control cells (panel C) usingWestern blot analysis. Lane 1: no-cellcontrol; lane 2: no collagen control; lanes 3–12: 10-point collagen dose responsebetween 0.1 and 50.0 µg/ml (1:2 dilution); lane 13: molecular weight marker. Eachcell lysate was then re-run on an identical gel and probed with an anti-glyceraldehyde3-phosphate dehydrogenase (GAPDH) antibody to control for loading as shown belowfor each experiment.

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Nerve Growth Factor Receptor Kinase, Insulin Receptor Kinase (INSreceptor), Insulin-like Growth Factor Receptor (IGF-1 receptor),Abelson Kinase (c-ABL), and the stem cell factor receptor tyrosinekinase (c-KIT). The alignment was manually adjusted using the actualand predicted secondary structure (using SSPro, Cheng et al., 2005).The overall sequence similarity of these kinases to DDR1 and DDR2 isapproximately 37%, whereas sequence identity in the binding pocketrises up to 61% for c-ABL and 53% for insulin receptor and IGF-1receptor.

We used Modeller (Sali and Blundell, 1993) to build a multipletemplatemodel of DDR1 and DDR2 in the inactive conformation basedon structural information of MUSK (pdb-entry 1LUF), Insulin receptor(in-house structure), IGF-1 receptor (pdb-code 1 M7 N), c-ABL boundto imatinib (pdb-code 1IEP), and c-KIT tyrosine kinase bound toimatinib (pdb-code 1T46). No crystal structure of Nerve Growth FactorReceptor Kinase 1 and 2 was available. The four templates showconsiderable structural differences, especially in the glycine rich loop(P-loop), the activation loop, and to a lesser extent in the C-helix.Sequence identity in the P-loop is relatively poor for all templates.Furthermore, c-ABL shows a particular fold in this domainwith part ofthe loop bent into the ATP-binding site, forming a cage-like pocket.Disruption of this fold due to point mutation has been implied as acause for development of imatinib resistance (Cowan-Jacob et al.,2004). The P-loop of c-KIT, adopts the more common hairpin fold. Thecrystal structure of c-KIT in complex with imatinib shows thatimatinib also binds to this P-loop conformation, thus showing that thec-ABL P-loop conformation is not crucial for imatinib binding. Wetherefore built two different models for this region, one with the c-ABL conformation and one with the more common fold observed inMUSK, IGF-1 receptor, INS receptor and c-KIT.

TheDFG segment (aspartate, phenylalanine, glycin), activation loopand C-helix have been modeled using c-ABL as a template, since thesequence identity is slightly higher in this region. For the other regions,where structural differences are less pronounced, all four templateshave been considered. No particular effort was made to optimize theinserts in DDR1 and DDR2 occurring between β2 and β3, and α2 andα3, even though we are aware that especially the first insert mighthave a significant effect on the conformation of the P-loop.

The initial model was further refined in the presence of nilotinib byperforming 2000 steps of minimization and 100 ps of restrainedmolecular dynamics simulation at 300 K, using the parm99 force fieldand the generalized Born model to treat solvent effects. During thisshort molecular dynamics simulation, the backbone was constrainedusing harmonic restraints of 5 kcal/mol, and restraints were put on thehydrogen bonds between the ligand and the hinge-binder, gatekeeper,and DFG-loop.

The final model was analyzed in terms of steric clashes, bond,angle, and torsion violations, as well as a phi-psi-chart, using theprotein geometry analysis tool of moe. No steric clashes or bond andangle violations were observed. There are a few outliers in theRamachandran plot, which either lie in the loop regions or were alsopresent in the templates.

3. Results

3.1. Inhibition of human DDR1b and DDR2 kinases in a cell-free TR-FRETkinase assay by imatinib, nilotinib and dasatinib

The intracellular domains comprising the kinase domains ofDDR1b and DDR2 were expressed and purified as described inmaterials and methods. The enzymatic activity of purified batches ofDDR1 and DDR2 kinases was tested using 1:2 serial dilutions ofenzyme in a TR-FRET biochemical assay. Both purified DDR1b andDDR2 enzyme preparations were found to contain constitutivelyactive enzymes with an EC50=1.1 μg/ml for DDR1b and with anEC50=1.9 μg/ml for DDR2 (Fig. 1A and B). The assay windowwas found

to be N4-fold and the assay reproducible and robust betweenexperiments (n=3). We have also determined the Km of therecombinant enzymes for ATP, which was found to be 17 μM forDDR1b and 14 μM for DDR2. Both DDR1b and DDR2 enzymes werethen used at EC80 in subsequent experiments. To further validate theassay the broad specificity kinase inhibitor staurosporine was used ina 1:2 serial dilution in the range of 50 μM to 0.05 μM. Staurosporineinhibited both DDR1b and DDR2 activity in a dose-dependent mannerwith IC50 values of 2 μM for DDR1b and 2.4 μM for DDR2 (Fig. 1C). Inthe validated assay we evaluated the inhibitory activity of imatinib,nilotinib and dasatinib upon both DDR1b and DDR2 kinases. As shownin Fig. 2 all three compounds inhibited the activity of both kinaseswith no significant selectivity between DDR1 and DDR2, withdasatinib and nilotinib being more potent than imatinib. In threeindependent experiments the IC50 of imatinib was determined to be337±56 nM and 675±127 nM for DDR1b and DDR2 respectively,whereas nilotinib inhibited DDR1b and DDR2 activity with an IC50values of 43±3 nM and 55±9 nM, respectively, and dasatinibwith IC50values of 0.5±0.2 nM and 1.4±0.3 nM, respectively when using 100 μMATP as described in materials and methods. The activity of imatiniband dasatinib against DDR1 and DDR2 kinases was also determined ina recent study using commercial screening services (Bantscheff et al.,2007) and similar to our results both compoundswere found to inhibitboth DDRs although with somewhat different IC50 values. We thendetermined the IC50 shift when using 1 mM ATP for all 3 compoundsfor both DDR1 and DDR2. Increasing the ATP concentration 10-foldresulted in an increase in IC50 values in each case as follows: 3- and 4-fold for imatinib, 5- and 2-fold for nilotinib and 3- and 3-fold fordasatinib for DDR1 and DDR2, respectively.

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3.2. Imatinib, nilotinib and dasatinib are potent inhibitors of collagen-induced DDR1b and DDR2 auto-phosphorylation

To study imatinib, nilotinib and dasatinib in a DDR dependentcellular functional assay we generated stable HEK293-DDR1b andHEK293-DDR2 cell clones as described in materials and methods. Cellclones were analyzed by quantitative PCR for their level of DDR geneexpression and clones showing a high level of expression wereselected for functional analysis (data not shown). HEK293-DDR1b celllines and wild-type HEK293 controls were analyzed for collagen-induced receptor phosphorylation using Western blotting. Dose-dependent tyrosine phosphorylation induced by collagen stimulationwas shown in DDR1b and DDR2 expressing cell lines, but not in wild-type HEK293 control cells (Fig. 3). As it had been demonstrated thatcollagen-induced DDR auto-phosphorylation was relatively slow

Fig. 4. Inhibition of collagen-induced DDR1 and DDR2 auto-phosphorylation by imatinib, niwith serial dilutions of imatinib, nilotinib and dasatinib (n=4). All three compounds potentl0.20 nM, for imatinib (panel A), nilotinib (panel B) and dasatinib (panel C), respectively. RepDDR2 cells were treated with serial dilutions of imatinib, nilotinib and dasatinib (n=3). Al33.3 nM, 5.2±3.3 nM and 5.2±2.1 nM for imatinib (panel D), nilotinib (panel E) and dasatin

compared to most tyrosine kinases (Vogel et al., 1997) we nextdetermined the optimum time required for collagen-induced DDR1auto-phosphorylation. We stimulated HEK293-DDR1b cells for 0, 4, 8and 24 h. After harvesting the cells the collagen-induced responsewasdetermined using Western blotting. Results demonstrated an increasein DDR tyrosine phosphorylation signal after 8 and 24 h compared toearlier time points (data not shown) and 16 h stimulation was thenused in further experiments. To allow the determination of accurateIC50 values the assay was then converted into a 96-well ELISA formatas described in materials and methods.

The effect of imatinib, nilotinib and dasatinib on the activity ofDDR1b and DDR2 was then investigated in the functional assay. Allthree compounds potently inhibited DDR1 and DDR2 activity asmeasured by collagen-induced receptor auto-phosphorylation. TheIC50 values for imatinib, nilotinib and dasatinib for DDR1 were found

lotinib and dasatinib. Panels A–C: collagen stimulated HEK293-DDR1 cells were treatedy inhibited the cellular response with IC50 values of 43±2.4 nM, 3.7±1.2 nM and 1.35±resentative dose response curves are shown. Panels D–F: collagen stimulated HEK293-l three compounds potently inhibited the cellular response with IC50 values of 140.7±ib (panel F), respectively. Representative dose response curves are shown.

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to be 43±2.4 nM, 3.7±1.2 nM and 1.35±0.20 nM respectively (n=4).The potency of all three compounds was 2–3 fold lower against DDR2activity with IC50 values of 140.7±33.3 nM, 5.2±3.3 nM and 5.2±2.1(n=3) for imatinib, nilotinib and dasatinib, respectively. Selectedexamples of the dose response curves for the inhibition of collagen-induced auto-phosphorylation are shown in Fig. 4.

3.3. Imatinib, nilotinib and dasatinib inhibit DDR1-dependent collagen-induced MCP-1 release in monocytic cells

In order to study the downstream effects of DDRmediated collagenstimulation, we generated stable THP1-DDR1b cells, which arereported to secrete inflammatory chemokines in response to collagenstimulation when cultured in the presence of PMA (Matsuyama et al.,2004). Cell clones were generated by lentiviral vector mediatedtransduction and analyzed for DDR1 expression by FACS (Fig. 5A) andassayed for collagen mediated MCP-1 secretion by ELISA (data notshown). Clones which demonstrated the highest integrin-indepen-dent MCP-1 secretionwere selected for use in functional assays. Dose-dependent increases in MCP-1 concentration in cell culture super-natants were seen in DDR1b expressing cells, but not in wild-typecontrols in response to collagen stimulation (Fig. 5B). DDR1b auto-phosphorylation was also shown to increase in a time and dose-dependent manner in THP1-DDR1b cells by Western blotting withsignal detected from2 h post stimulation onwards and in response to aminimum of 1 µg/ml collagen (data not shown). The effects ofimatinib, nilotinib and dasatinib on DDR1b mediated collagen-

Fig. 5. Inhibition of collagen-induced DDR1-dependentMCP-1 release by imatinib, and dasatilevels of DDR1 receptor were selected (panel A). Stimulationwith collagen lead to a dose-dep(panel B). Collagen-induced MCP-1 release was dose dependently inhibited by imatinib (panrespectively.

induced MCP-1 secretion were then tested in the functional assay(Fig. 5C and D). Imatinib and dasatinib inhibited chemokine secretionwith IC50 values of 1970±1300 nM and 300 nM±172 nM (n=3) whilstintriguingly, no inhibition was observed with nilotinib. To furtherinvestigate the lack of potency of nilotinib in this assay we performeda Western blot analysis of collagen-induced receptor auto-phosphor-ylation using the same induction time as in the HEK293 cell assay.While imatinib was shown to inhibit receptor auto-phosphorylationin THP1-DDR1 cells, nilotinib appeared to be inactive in this assay (notshown) in accordance with its lack of potency in the MCP-1 releaseassay.

3.4. Homology model of DDR1 and DDR2 kinases and binding mode ofimatinib and nilotinib

As demonstrated above, imatinib and nilotinib show a similaractivity profile against DDR1, DDR2 and c-ABL, but apart from c-KIT, donot appear to inhibit other tyrosine kinases closely related to DDR1and DDR2 (unpublished observations), such as muscle specifictyrosine kinase (MUSK), insulin-like growth factor receptor kinase(IGF-1R), and the insulin receptor kinase (INSR). A sequence alignmentof DDR1 and DDR2 to these proteins is shown in Fig. 6.

To establish the structural basis of the DDR1 and DDR2 inhibitoryactivity of imatinib and nilotinib we generated homology models forboth DDR1 and DDR2 kinases (see materials and methods). Wefocused these studies on imatinib and nilotinib which are type-IIinhibitors and which bind to the inactive kinase conformation, as

nib. THP-1 cells were transfected using lentiviral delivery and cell clones expressing highendent increase of MCP-1 release in THP1-DDR1 cells but not in untransfected THP1 cellsel C) and dasatinib (panel D) with IC50 values of 1970±1300 nM and 300±172 nM (n=3),

Fig. 6. Alignment of human DDR1 and DDR2 kinase domain sequences to their closest homologues. Alignment of human DDR1 and DDR2 to Muscle Specific Kinase (MUSK), InsulinReceptor Kinase (INS receptor), Insulin-like Growth Factor Receptor (IGF-1 receptor), and Abelson Kinase (c-ABL). Conserved residues are shown in green. The P-loop is boxed ingreen, the C-helix in blue, the DFG and activation loops in red, and the two inserts of DDR1 and DDR2 in yellow. Some of the key residues mentioned in the text are highlighted asfollows: the gatekeeper is shown in orange, the DFG-loop residues in red, and Q620 and F621 (hDDR1 numbering) which are part of the P-loop are highlighted in cyan, whereas theglutamate residues in the P-loop of DDRs are highlighted in blue. Other residues, which differ from the c-ABL sequence, such as M787 and N387 in the activation loop and M704preceding the gatekeeper residue are shown in lavender. The actual secondary structure (for MUSK) is shown at the bottom of the aligned sequences.

51E. Day et al. / European Journal of Pharmacology 599 (2008) 44–53

shown by several co-crystal structures of c-ABL in complex with theseinhibitors (Cowan-Jacob et al., 2007; Nagar et al., 2002), whereasdasatinib binds the active kinase.

In the inactive conformation the DFG segment and the activationloop undergo a conformation change, which allows type-II inhibitors toprotrude into a newly formed back-pocket forming additional contactswith the DFG backbone and the C-helix (Fig. 7A). They form twoimportant hydrogen bonds, namely to the threonine gatekeeper residueand to the backbone of the conserved aspartate in the DFG motif. Thegatekeeper residue is a methionine in MUSK, INSR, and IGF-1R,whichmight explain the lack of activity in these kinases. The gatekeeperin c-KIT is a threonine residue as in DDR1/2 and c-ABL, but the bindingaffinity of imatinib and nilotinib is decreased by 10–100 fold in c-KIT.Thus, besides the nature of the gatekeeper residue, other factors, such asthe shape of the back-pocket and the conformation of the ATP-bindingsite play an important role in selectivity.

The P-loop of the ATP-binding site in c-ABL adopts a particularcage-like conformation. Besides a conserved H-bond with the hinge-region, the inhibitors also interact with Y253 via edge-to-face contacts(Fig. 7B) (Cowan-Jacob et al., 2007). Several mutations in the P-loop inc-ABL confer resistance towards imatinib, namely G250E, Y253H/F,and E255V, although this resistance is overcame with nilotinib anddasatinib (Shah et al., 2002; Manley et al., 2005). It has beenhypothesized that disruption of the particular fold leads to loss ofaffinity towards imatinib. Interestingly, two of the above mutations,G250E and Y253F lead to an exact match with the DDR sequences,

suggesting that these mutations do not affect imatinib-binding toDDRs. There are two possible explanations for this observation. Either(i) the particular fold is maintained in DDRs due to other stabilizinginteractions, or (ii) imatinib binds to a different conformation. Indeed,crystal structures of c-ABL in complex with other ligands point toconsiderable conformational variation of the P-loop (Modugno et al.,2007, Zhou et al., 2007). We have therefore modeled the P-loop ofDDR1 and 2 in two different conformations: conformation A ismodeled on c-ABL, whereas conformation B uses MUSK, IGF-1R andINSR as templates.

In conformation A, the side chains of Q620 (DDR1) (Q252 in c-ABL)and F621(DDR1) (Y253 in c-ABL) are flipped into the active site to form ahydrophobic cage as in c-ABL. This conformation is stabilized by stackinginteractions between Q620 and F621 and a salt bridge between K615(DDR1) (K247 in c-ABL) and E623 (DDR1) (E255 in c-ABL), which isconserved in DDRs and c-ABL, but not in other kinases.

In conformation B, Q620 (DDR1) (Q252 in c-ABL) and F621 (DDR1)(Y253 in c-ABL) are pointing away from the ATP-binding pocket andinteract with the backbone of the activation loop and side chainslocated on the C-helix (Fig. 7C). F785 (DDR1) (F382 in c-ABL) of theDFG segment can rotate towards the inhibitor and form an edge-to-face interaction with nilotinib, similar to the one observed in c-KITbound to imatinib.

In summary, sequence alignment and homology modeling haverevealed discrepancies in the hydrophobic back-pocket, the gate-keeper residue as well as the ATP-binding site. For the latter, several

Fig. 7. Modeling of the nilotinib binding site in DDR1. Panel A: binding sites in thepresence of nilotinib for c-ABL. Panel B: DDR1 model with P-loop modeled as in c-ABL.Panel C: DDR1 model with P-loop modeled as in INS receptor and MUSK. The P-loop isshown in green, the DFG segment and activation loop in red and nilotinib is highlightedin yellow. The conserved aspartate and phenylalanine of the DFG-loop are depicted inred, whereas the phenylalanine and glutamine side chains of the P-loop are shown ingreen. See text for more details.

52 E. Day et al. / European Journal of Pharmacology 599 (2008) 44–53

possible conformations can be envisaged, which might require adifferent design to introduce selectivity.

4. Discussion

Imatinib inhibits the tyrosine kinase activities of ABL, as well as thec-KIT and PDGF (platelet derived growth factor) receptor kinases, andis an effective therapy for chronic myeloid leukemia and c-KIT andPDGF receptor-dependent gastrointestinal stromal tumours. Nilotiniband dasatinib are second generation ABL kinase inhibitors that havebeen developed to treat imatinib-resistant chronic myelogenous

leukemia. The potency of the 3 compounds against c-ABL and theirselectivity profile are quite different: dasatinib is non-selective kinaseinhibitor, which potently inhibits ABL as well as the SRC-familykinases, in addition to PDGF receptor and KIT (Weisberg et al., 2007).In contrast, nilotinib is a potent and quite selective ABL inhibitor, withless activity against the KIT and PDGF receptor tyrosine kinases and noactivity against the SRC-family tyrosine kinases. Two recent chemicalproteomic studies have addressed the kinase profiles of these 3compounds and found that the compounds also bind to the DDR1,another receptor tyrosine kinase (Bantscheff et al., 2007; Rix et al.,2007).

To investigate the activity profile of imatinib, nilotinib anddasatinib against DDR1 and DDR2 kinases we developed biochemicaland functional cellular assays for these enzymes and evaluated theeffects of the drugs. Our results demonstrate that all three compoundspotently inhibit both DDR1 and DDR2 activity, which in turnmay haveimplications to the therapeutic potential of these compounds.

In the biochemical assay nilotinib was approximately 10-fold morepotent than imatinib against both kinases and dasatinib was found tobe particularly potent with IC50 values in the low nM range. None ofthe three compounds appeared to have significant selectivity betweenDDR1 and DDR2 kinases.

To study the effect of DDR inhibition by imatinib, nilotinib anddasatinib in a cellular environment we generated DDR1 and DDR2expressing HEK293 cell lines by stable transfection. Of the five DDR1splice variants we focused on the 1b variant whose over-expressionhas been associated with pulmonary disorders (Matsuyama et al.,2005a; Matsuyama et al., 2005b) although in preliminary experimentsDDR1a was also found to be active in the assay (data not shown)whereas the c, d and e variants lack kinase activity. Using receptorauto-phosphorylation as the read-out we determined the IC50 valuesfor all 3 compounds. We found the ranking order of the threecompounds to be the same as in the biochemical assay; with imatinibbeing the least and dasatinib the most potent inhibitor. Interestingly,when comparing IC50 values obtained in the two different assays, bothimatinib and nilotinib were found to be approximately 10-fold morepotent in the cellular assay than in the kinase assay. The observeddifferences can be due to the fact that in the biochemical assay theconstitutive activity of the intracellular domains was assayed for,whereas in the cellular context the fully functional, membranespanning, collagen activated receptors were tested, which were likelyto have different conformational states.

As described above DDR1 expression and activity has beenassociated with various cancers and pulmonary and fibrotic disorders.While DDR1 was primarily identified as an epithelial receptor with apotential role in various carcinomas, intriguingly a unique role forDDR1 has also been hypothesized in monocytic cells in idiopathicpulmonary fibrosis (Matsuyama et al., 2005a). It was found that CD14+

monocytes/macrophages isolated from bronchoalveolar lavage fluid ofidiopathic pulmonary fibrosis patients had elevated levels of DDR1expression, particularly of the 1b isoform. Furthermore, stimulation ofthese cells with collagen via DDR1 leads to the generation of a numberof pro-inflammatory chemokines believed to play a role in theinflammatory component of the disease (Agostini and Gurrieri,2006). In order to evaluate the inhibitory potential of imatinib,nilotinib and dasatinib in a disease relevant cellular assay wegenerated DDR1b over expressing THP-1 monocytic cell lines usinglentiviral delivery of the expression constructs. Imatinib and dasatinibboth inhibited MCP-1 release and similarly to the biochemical and thereceptor auto-phosphorylation assays, dasatinibwas found to bemorepotent although both compounds had significantly higher IC50 valuesthan in the other two assays. Intriguingly, however, nilotinib did notinhibit MCP-1 production even at the highest concentration tested. Tobetter understand this unexpected result we evaluated the effect ofnilotinib on receptor auto-phosphorylation in DDR1-THP-1 cells andfound that nilotinib appeared to be inactive in this assay, whereas

53E. Day et al. / European Journal of Pharmacology 599 (2008) 44–53

imatinib was shown to inhibit receptor auto-phosphorylation. This isin agreementwith the lack of potency of nilotinib in theMCP-1 releaseassay, however, the underlying reason is presently unclear. It isconceivable that a lack of intracellular concentrations of nilotinib inTHP-1 cells plays a role in this observation, although in systemsevaluated thus far nilotinib readily penetrates cells by passivediffusion and is not a substrate for efflux pumps and therefore furtherstudies are needed to fully understand this finding. Future experi-ments include the testing of compounds with similar chemicalstructures to imatinib and nilotinib in the MCP-1 release assay todetermine if there is a structural basis for this observation.

Although imatinib and nilotinib are relatively selective inhibitors ofABL, both compounds possess significant activity towards DDR1 andDDR2. Imatinib and nilotinib are type-II inhibitors, which bind to theinactive conformation of their target kinases. Selectivity over closelyrelated kinases, such as MUSK, INSR and IGF-1R, can be partiallyexplained by a mutation in the gatekeeper residue, which forms animportant hydrogen bond to imatinib and nilotinib. To better under-stand the binding mode of these inhibitors in DDR1 and DDR2, as wellas to elucidate the discrepancies between c-ABL and theDDRs,we builthomology models for these two kinases in an inactive conformation.Wefind thatwhile there are only fewconservedmutations in the back-pocket with respect to c-ABL, residues lining the ATP-binding pocketare less well conserved. These differences could therefore be exploitedin the design of DDR-selective compounds.

In summary, we have characterized in detail the inhibitory effect ofimatinib, nilotinib and dasatinib on the collagen receptor tyrosinekinases DDR1 and DDR2. Our findings may contribute to the betterunderstanding of the therapeutic potential of these compounds andhighlight the potential of these and related kinase inhibitors for thetreatment of diseases associated with pathological DDR1 or DDR2activity. For example, both DDR1 and DDR2 have been associated withfibrotic disorders and imatinib has been shown to be effective in animalmodels of fibrosis (Daniels et al., 2004; Aono et al., 2005). However,while imatinib is a kinase inhibitor with a relatively narrow selectivityprofile further investigations are needed to delineate whether itsobserved anti-fibrotic effect is due to its activity against a particularkinase, such as PDGF receptor or DDR1 or 2. Alternatively, the potentialof imatinib to attenuate the fibrosis in in vivo models may be due to itsactivity against more than one kinase involved in the fibrotic process.

Acknowledgements

The authors would like to thank Mohammed Akhlaq for his helpwith the Km determinations and Duncan Shaw, Peter Gedeck, LilyaSviridenko and Pascal Furet for the helpful discussions andsuggestions.

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