Developing Antagonists for the Met-HGF/SF Protein Protein ... · Small Molecule Therapeutics Developing Antagonists for the Met-HGF/SF Protein–Protein Interaction Using a Fragment-Based

Small Molecule Therapeutics

Developing Antagonists for the Met-HGF/SFProtein–Protein Interaction Using a Fragment-Based ApproachAnja Winter1, Anna G. Sigurdardottir1, Danielle DiCara2,3, Giovanni Valenti4,Tom L. Blundell1, and Ermanno Gherardi2

Abstract

In many cancers, aberrant activation of the Met receptortyrosine kinase leads to dissociation of cells from the primarytumor, causing metastasis. Accordingly, Met is a high-profiletarget for the development of cancer therapies, and progress hasbeen made through development of small molecule kinaseinhibitors and antibodies. However, both approaches posesignificant challenges with respect to either target specificity(kinase inhibitors) or the cost involved in treating large patientcohorts (antibodies). Here, we use a fragment-based approachin order to target the protein–protein interaction (PPI) betweenthe a-chain of hepatocyte growth factor/scatter factor (HGF/SF;the NK1 fragment) and its high-affinity binding site located onthe Met Sema domain. Surface plasmon resonance was used forinitial fragment library screening and hits were developed into

larger compounds using substructure (similarity) searches. Weidentified compounds able to interfere with NK1 binding toMet, disrupt Met signaling, and inhibit tumorsphere genera-tion and cell migration. Using molecular docking, we con-cluded that some of these compounds inhibit the PPI direct-ly, whereas others act indirectly. Our results indicate thatchemical fragments can efficiently target the HGF/SF-Metinterface and may be used as building blocks for generatingbiologically active lead compounds. This strategy may havebroad application for the development of a new class of Metinhibitors, namely receptor antagonists, and in general for thedevelopment of small molecule PPI inhibitors of key thera-peutic targets when structural information is not available.Mol Cancer Ther; 15(1); 1–12. �2015 AACR.

IntroductionReceptor tyrosine kinases (RTK) mediate intercellular signals

that are essential for the development and maintenance ofthe cells of multicellular organisms. Met, the RTK encoded bythe c-Met proto-oncogene (1, 2), is the receptor for hepatocytegrowth factor/scatter factor (HGF/SF; ref. 3), a large polypeptidegrowth factor discovered as a liver mitogen (HGF; refs. 4, 5), and

as a protein causing dispersion of epithelial colonies and cellmigration (SF; 6, 7). HGF/SF and Met are essential for thedevelopment of several tissues and organs, including the placenta(8, 9), liver (8), and skeletal muscle (10). HGF/SF and Met alsoplay a major role in the abnormal migration of cancer cells as aresult of Met overexpression or Met mutations (11). Moreover,these are strongly associated with poor prognosis, for instance inurothelial carcinoma of the bladder (12), prostate cancer (13),non–small cell lung cancer (14), and ovarian cancer (15). It istherefore not surprising thatHGF/SF andMet have emerged as keytherapeutic targets for the treatment of metastatic cancer.

Although Met kinase inhibitors such as cabozantinib,SAR125844, and tivantinib have been shown to be effective,resistance to Met kinase inhibitors develops rapidly (16–18).Therefore, interfering with the assembly of the HGF/SF-Metcomplex could constitute a new and promising route in thesearch for novel, cost-effective, and selective inhibitors of HGF/SF-Met signaling, but it has its own challenges. Protein–proteininteractions (PPI) are generally large and lack the large cavitiesthat characterize many enzyme active sites, such as proteinkinases and proteases, and receptors such as G-protein-coupledreceptors. However, the observation that protein surfaces oftendisplay small deep pockets (19, 20), sometimes closely clus-tered, offers a basis for alternative design approaches (21).Examples of such inhibitors can be found in the literature,although the approaches by which they were discovered arediverse (21) ranging from virtual screening (22) to fragment-screening campaigns (23, 24) and mimicking the interactionpartner by rational design (25). A further and major advantage

1Department of Molecular Cell Biology, The University of Cambridge,Cambridge, United Kingdom. 2MRC Centre, Cambridge, United King-dom. 3Department of Oncology, The University of Cambridge, Cam-bridge Biomedical Campus, Cambridge, United Kingdom. 4Depart-ment of Cancer Research, Max Delbrueck Center for Molecular Med-icine (MDC), Berlin, Germany.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Current address for A. Winter: University of Leicester, Department of MolecularCell Biology, Leicester, UK; current address for A.G. Sigurdardottir: MRC Lab-oratory of Molecular Biology, Department of Neurobiology, Cambridge, UK;current address for E. Gherardi: Universit�a di Pavia, Department of MolecularMedicine, Pavia, Italy; and current address for D. DiCara: Genentech Inc., SouthSan Francisco, California.

Corresponding Authors: Anja Winter, Department of Molecular Cell Biology,University of Leicester, Lancaster Road, Leicester LE1 9HN, UK. Phone: 44-116-299-7074; Fax: 44-116-229-7123; E-mail: [email protected]; and Tom Blundell,Department of Biochemistry, Tennis Court Road, The University of Cambridge,CB2 1GA Cambridge, UK. E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0446

�2015 American Association for Cancer Research.

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of targeting the extracellular domain of Met is that the com-pounds would not have to cross the cell membrane, thereforeallowing for more flexibility in drug design.

The interactions between the extracellular portion of Metand HGF/SF are intricate due to the complex, multidomainstructure of these proteins (4, 26). However, biochemical,biophysical, and structural analysis of individual HGF/SFdomains (27) and mutagenesis studies (28) have revealed thatHGF/SF binds Met through a high-affinity binding site locatedin the N-terminal N and K1 (NK1) domains and a low-affinitybinding site located in the C-terminal serine-proteinase homol-ogy (SPH) domain. Solution and low-resolution crystal struc-tures have subsequently defined the high-affinity binding siteof the NK1 fragment of HGF/SF on the Sema domain of the Metreceptor (29). The low-affinity binding site between the twoproteins has been defined in the crystal structure of the Semaand cysteine-rich domains of Met in complex with the SPHdomain of HGF/SF (30).

Here, we use fragment-based methods to find chemicalbuilding blocks for developing small-molecule Met receptorantagonists. We show that compound optimization in con-junction with biologic activity assays and in silico methods canlead to the discovery of compounds that are able to disrupt Metsignaling, interfere with NK1 binding to Met, and inhibittumorsphere generation as well as cell migration. These com-pounds constitute the starting points for the development ofselective, non-kinase inhibitors of the HGF/SF-Met signalingpathway.

Materials and MethodsExpression and purification of Met proteins

Two silent mutations were introduced in codons coding foramino acids Q559 and I560 of a full-length humanMet cDNA toremove a BglII site. Met deletions lacking the endogenous leader(amino acids 1–24) were generated by PCR as MluI–BglII insertsand cloned in-frame between a 21-aa Ig leader and a hexa- or octa-histidine sequence. For expression, Met constructs Met567 (aminoacids 26–567) and Met928 (amino acids 26–928) in plasmidpA71d were transfected in mouse Lec 3.2.8.1 cells (31). Stabletransfectants were selected in 0.75 mg/mL hygromycin, screenedfor expression, and positive cultures were cloned and expandedfor protein production. Monomeric Met proteins were producedin 10% a-MEM/DMEMmedia containing 2.5% FCS and purifiedon a Ni-NTA agarose column (catalog no. MG3398; Qiagen)equilibrated in PBS. The protein was eluted with 0.4 mol/Limidazole and was further purified on a Mono S column (Amer-sham Biosciences).

Expression and purification of NK1 proteincDNA encoding the NK1 fragment was obtained from full-

length humanHGF/SF and then cloned into the vector pPIC9K asdescribed before (32). This vector was transformed into Pichiapastoris strainGS115 andNK1 produced in BMMYmedia contain-ing 100 mg/mL ampicillin in aliquots of 500 mL in 2 L conicalflasks. Methanol (2.5 mL) was added to each flask to induceexpression and every 24 hours afterward. The purification of NK1was carried out in two steps following Chirgadze and colleagues(1999), except that the MES buffer was removed from the puri-fication process and 50 mmol/L sodium phosphate, pH7.4 wasused instead. Expression yields were 5 mg/L media.

Expression and purification of SPH proteinThe SPH fragment (Val495-Ser728) was cloned using cDNA of

full-length human HGF/SF and cloned into the baculovirussecretion vector pAcGP67A (BD Biosciences) with the additionof a C-terminal hexahistidine tag and aCys604Sermutation. Viralstocks were amplified using Sf9 insect cells (BD Biosciences), andprotein was produced usingHigh Five (BTI-TN-5B1-4) insect cells(Invitrogen) and ESF 921 media (þ2.5% FCS). SPH domain waspurified using a 5 mL HP HisTrap (GE Healthcare) column and agradient of imidazole from 0 to 500 mmol/L in 50 mmol/L Tris,pH7.7. Protein-containing fractions were pooled, concentrated,and injected onto a 16/60 Superdex 200 prep grade column(Amersham Pharmacia Biotech) equilibrated with 10 mmol/LHEPES, pH7.2, 150mmol/L NaCl. Expression yields were 4mg/Lmedia.

Surface plasmon resonance experimentsSurface plasmon resonance (SPR) measurements were per-

formed on a Biacore T100 instrument using research-grade CM5sensor chips. The reagents 1-ethyl-3-(3-diaminopropyl) carbodii-mide hydrochloride (EDC), N-hydroxysuccinimide (NHS), andethanolamine (pH 8.5) were purchased from Biacore and usedaccording to recommended protocols. Met, NK1, and SPHdomain were immobilized on a CM5 sensor chip using theamine-coupling method. Immobilization was done selecting atarget immobilization level of up to 10,000 RU in the immobi-lization wizard of the Biacore control software in order to saturatethe chip surface with protein. Met was diluted with 10 mmol/Lacetate buffer (pH5.5) to a final concentration of between 1 and2 mmol/L, and response levels of around 6,000 RU were immo-bilized on the activated surface. NK1 was diluted into 10mmol/Lphosphate buffer, pH7.0 for immobilization, and the SPHdomain was immobilized using 10 mmol/L sodium acetatebuffer, pH5.5. NK1 and the SPH domain were immobilized ona separate CM5 chip in flow channels 2 and 3, respectively, andresponse levels of about 2,500 RU for NK1 and 5,700 RU for SPHdomain were achieved.

Fragment library and compoundsFragments were taken from a 1,338-membered library consist-

ing of fragments taken fromMaybridge fragment libraries and in-house compounds. Fragments were dissolved in DMSO to a finalconcentration of 100 mmol/L. All compounds were purchasedfrom Enamine (www.enamine.net) and dissolved in DMSO tofinal concentrations of either 100 mmol/L or 50 mmol/L.

Fragment screening and data analysisSPR binding experiments were performed in PBS buffer

(pH7.4) as the running buffer supplemented with 0.05% surfac-tant P20 or Tween 20 and 1% DMSO to aid fragment solubility.Experiments were performed at 25�C and at a flow rate of30 mL/minute. Samples were injected for 30 seconds followedby 60-second dissociation. The sensor surface was regeneratedbetween experiments by injecting 1 MNaCl for 30 seconds. If thedifference of response before and after analyte injection wasgreater than 10 RU, 50% ethylene glycol was injected for 30seconds to remove residual sample. Fragments from our in-housefragment library were diluted 1:100 (for Met) or 1:200 (for NK1and SPH) from a 100 mmol/L stock (dissolved in 100% DMSO)into running buffer to give final concentrations of 1 mmol/L or0.5 mmol/L, respectively. All samples were measured at one

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concentration, sensorgrams were recorded at 1 Hz and screenswere done in 96-well plate format. To monitor the bindingcapacity of the protein surfaces during the screening, injectionsof control samples were carried out every 40 samples. Met567(0.25 mmol/L) and HEPES (500 mmol/L) were used as controlsamples for NK1 and the SPH domain. 200 nmol/L NK1 and 1%DMSO were used as control samples for Met. Solvent correctioncycleswere included every 40 samples to account for differences inDMSO content of the samples.

After solvent correction, binding levels of each fragment andcontrol samples were determined near the end of the injection.Instrument variations between runs were normalized usingresponse levels of the respective positive controls. A maximumof 400 samples were run consecutively followed by storing thechip in running buffer for a few days. Responses of controlsamples recovered remarkably, and one chip could be used toscreen approximately 1,400 samples.

Raw data were normalized against ligand-coupling density,ligand molecular weight, and molecular weight of each injectedfragment by calculating RUmax, which represents the theoreticalmaximum response for each fragment. RUrel is the normalizedrelative SPR response of a fragment that was calculated using themeasured binding level of the fragment and the theoreticalmaximum response RUmax.

Kinetic analysis of fragment bindingExperiments for binding kinetics were performed by SPR using

PBS buffer (pH7.4) as the running buffer supplemented with0.05%surfactant P20. Experimentswereperformed at 17�Candata flow rate of 30 mL/minute. Samples were injected for 30 secondsfollowed by 60-second dissociation. The sensor surface wasregenerated between experiments by injecting 1 mol/L NaCl for25 seconds. A concentration series of the fragments ranging from7 mmol/L to 500 mmol/L in 1:2 dilutions into running buffer wastypically run in these experiments. Sensorgrams were recorded at10Hz. Solvent correction cycleswere included every 40 samples toaccount for DMSO content in the samples. Equilibrium affinity-binding data were obtained by fitting blank-subtracted and sol-vent-correctedbinding levels using a globalfit providedwithin theBiacore T100 instrument software.

Differential scanning fluorimetryThermal shift (differential scanning fluorimetry) assays were

carried out bymixing buffer (50mmol/L Tris, pH7.5, 150mmol/LNaCl) and protein to a final concentration of 5 mmol/L. Fragment(5 mL; 100 mmol/L in DMSO) and 12.5 mL Sypro Orange of a1:2,000 dilution in water were added to give a final volume of50 mL. Fluorescence values were recorded at temperature intervalsof 0.5 K from25�C to 80�Cusing an iQ5Multicolor real-time PCRdetection system from BioRad. Data were analyzed, fitted, andmelting temperatures determined using Excel.

Substructure searchesSubstructure searches were carried out using ZINC, a free

database of commercially available compounds for virtual screen-ing (33). Themolecular weightwas chosen to be between 250 and350 Da. Values such as xLogP, number of hydrogen bond accep-tors, and donors were set in accordance with Lipinski's rules (34).Thirty-five compounds were purchased from Enamine with 264Da <molecular weight > 404Da, 1.2 <�LogP > 3.1, 0 <H-donors>3, 3<H-acceptors>8generously following the "rule-of-three." A

value of <3 for Log P was chosen, which is thought to reducetoxicity and increase ease of formulation and bioavailability foroptimal oral absorption (35). Twenty compounds derived fromMB621 were mostly substituted at the nitrogen atom of 1,2,3,4tetrahydroquinoline and lacked the methyl group at R2. Com-pound 24 possesses an additional methyl group for R3. FragmentMB1271 is a tri-substituted benzene ring. All compounds pur-chased as derivatives of this fragment retain the fluoroform group.In addition, all compounds harbor a morpholine group in para-position relative to the fluoroform group. Residue R1 is variable inall compounds and replaces the NH2 group of the fragment.MB1297 is also a tri-substituted benzene ring. Apart from a cyanogroup it also harbors amethyl group in para-position to the cyanogroup and a NH2 group in ortho-position. The cyano group isretained in all derived compounds, and a hydrogen atom replacesthe methyl group in all compounds except compound 8, where achlorine replaces it. The NH2 group is substituted in all com-pounds with diverse chemical groups and features.

Competition assaysSPR competition experiments were performed as essentially

described previously (36).Met928was immobilized to aCM5chipusing the amine coupling method. Compounds were dilutedfrom their 100 mmol/L stock solution in DMSO into runningbuffer (PBS buffer with 0.05% Tween 20) supplemented with250 nmol/L NK1 keeping the DMSO content constant at 1%. Thismix was passed over the Met928 sensor surface for 2 minutes at30 mL/minute and allowed to dissociate for 3minutes. The sensorsurface was regenerated between experiments using 20 mmol/LSodium acetate buffer pH 5.0 with 4 mol/L NaCl for 30 seconds.Binding levels for each sample were determined 4 seconds beforeinjection stop and plotted against the respective compoundconcentration. The responses for competition assays were calcu-lated by subtracting the response incurred from compound bind-ing from the decreasing response levels in NK1-supplementedsamples. The resulting binding curves were fitted using a 1:1Langmuir bindingmodel or heterogeneous analyte supplied withthe Biacore T100 evaluation software where the on- and off rateconstants for NK1 binding to Met928 were predetermined andfixed for the fitting process.

Phosphorylation assaysPhosphorylation assayswere carriedout as essentially described

by Ferraris and colleagues (37). Vero cells were grown to con-fluency and starved for 42 to 52 hours before treatment. Cellswere treated by incubating with either 1 mmol/L, 0.5 mmol/L,0.25 mmol/L, or 0.125 mmol/L of compound in 1% DMSO for5 minutes in the presence of 0.1 nmol/L HGF at 37�C. HGF(0.1 nmol/L) in 1% DMSO only was used as positive control and1% DMSO only was used as negative control. After a brief washwith ice-cold PBS, lysis buffer (50 mmol/L Hepes, pH7.5, 150mmol/L NaCl, 1.5 mmol/L MgCl2, 1 g/L Triton X-100, 10 g/Lglycerol, 1mmol/L EGTA, 50mmol/Lorthovanadate, 100mmol/LNaF, Roche cocktail of protease inhibitors (cat. 11 873 580 001,2 tablets in 100 mL of buffer) was added to each well. Cells werethen incubated on ice for 30minutes, scraped from the bottom ofthe well, and the lysate was immediately frozen. Lysates wereanalyzed by SDS-PAGE, blotted, and probed for levels of phos-phorylated and total proteins Met, Akt and Erk using anti-phos-phoMet (Tyr1234/1235) XP antibody (D26; Cell Signaling Tech-nology; #3077) or anti-Met antibody (L41G3; Cell Signaling

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Technology; #3148), anti-phospho Akt (Ser473) antibody (CellSignaling Technology; #9271) or anti-Akt (Cell Signaling Tech-nology; #9272) and anti-phospho Erk antibody (Sigma #M8159)or anti-Erk1/2 (Promega #9PIV114). For quantitative analysisof phosphorylation levels, fluorescent anti-mouse (a-mouseIRDye800CW, Rockland, #800-656-7625) and anti-rabbit anti-bodies (a-rabbit IRDye 680LT, LI-COR, #926-68023) were used.Band intensities were determined using the Odyssey InfraredImaging system (LI-COR). After background subtraction, phos-phorylation levels of Akt and Erk were normalized against totalamount of protein (HGFonly ¼ 100% phosphorylation, positivecontrol) and 1% DMSO (0% phosphorylation, negative control)as solvent control.

Cytotoxicity assayThe relative cytotoxicity of the compounds was determined

using the alamarBlue Assay. Cells were seeded at 1,000 cells/mL in96-well plates. The assay was conducted following the manufac-turer's instructions.

Tumorsphere (mammosphere) assayFemale mice of strain FVB/N coexpressing activating muta-

tions in both the Wnt/b-catenin and HGF/Met signaling path-ways under the control of the pregnancy-induced whey acidicprotein (WAP) promoter were used at 2 weeks postpartum.Single-cell suspensions derived from mouse mammary glandtumors were plated at a density of 80,000 cells/mL in 24-wellplates coated with 1.2% Poly 2-hydroxyethyl methacrylate(polyHEMA) and grown as nonadherent tumorspheres, aspreviously described (38). To test the activity, small inhibitorcompounds were included in the growth medium at the indi-cated concentrations and supplemented every 3 days. Tumor-spheres were counted and imaged after 7 days of culture with aLeica microscope. The number and size of spheres was deter-mined as assay outcomes. Controls for 100 mmol/L and250 mmol/L were measured in four repeats, whereas the controlfor 1000 mmol/L compound was measured in two repeats.PHA665752 and crizonitib were used as positive controls inthis assay and showed a clear reduction in mammospherenumbers at 0.5 mmol/L and 1 mmol/L (Supplementary Fig.S1). Experiments with 1% DMSO as solvent (negative) controlshowed no major effect on mammosphere numbers withaverage numbers ranging from 11 mammospheres (control forcompounds at 250 mmol/L) to 13.5 mammospheres (controlfor compounds at 1,000 mmol/L and 100 mmol/L). Initialexperiments were conducted at 100 mmol/L, 250 mmol/L, and1 mmol/L compound concentration in 1% DMSO. However,poor solubility of some compounds in media resulted inprecipitation as observed by eye and under the microscope;this would reduce the effective concentration of the compoundin the medium. Compounds 10, 25, 33, and 34 precipitated at100 mmol/L, 250 mmol/L, and 1 mmol/L and were excludedfrom analysis. Compound 27 showed precipitation at the twohighest concentrations, 250 mmol/L and 1 mmol/L. For com-pounds 3, 6, 10, 15, 18, 26, 27, and 31, mammosphere assayswere extended to include concentrations of 25 mmol/L and10 mmol/L.

Boyden chamber assays (migration assay)HGF/SF-induced cell migration on SKOV-3 cells (a kind gift

from David Allard, CRUK) was assayed using a modified

Boyden chamber (AC96 Migration Chamber; Neuroprobe).Lower chambers containing 30 pmol/L HGF/SF and1 mmol/L compound in assay media (RPMI, 0.25% BSAdiluted 1:1 with PBS) were separated from upper chambers bya porous membrane (8 mm, PVP-free) that had been coatedwith 100 mg/mL collagen (Purecol, Nutacon) for 2 to 3 hours atroom temperature. SK-OV-3 cells were labeled with the fluo-rescent dye Calcein AM (Life Technologies) at a concentrationof 5 mmol/L for 30 minutes at 37�C prior to washing andresuspension in assay media; 25,000 cells were then added toeach upper well and the chamber was incubated at 37�C for 4hours to allow cell migration to occur. After removal of non-migrated cells from the membrane, the degree of cell migrationwas assessed by quantification of the residual fluorescence,indicative of migrated cells, on a Typhoon instrument (GE LifeSciences), using excitation/emission settings of 488 nm/526nm, respectively. Data were analyzed with ImageQuant soft-ware and background fluorescence subtracted. Statistical anal-ysis was performed with GraphPad Prism 6.0 (GraphPad Soft-ware, Inc.).

Docking using GOLDThe cocrystal structure of Met567 with HGF/SF was used as

protein template for the docking runs. The 3D coordinates of theenzymeswere obtained from the PDB (PDB code 1SHY) andwereprocessed by removing HGF/SF, water molecules, and otherligands and adding hydrogen atoms to the remainingMet protein.All 35 compoundswere docked into all three pockets usingGOLD5.2.2 (www.ccdc.cam.ac.uk). The docking centers were set asfollows: for pocket 1 CD of Glu267, for pocket 2 CZ of Arg426,and for pocket 3 NH1 of Arg469. All docking spheres were set to14 Å and protein side chains in the docking spheres were keptflexible during docking. GOLD was run using the default settingsfor protein and ligand bonds and functional groups. ChemScorewas used to generate 10 docking poses for each ligand, and thesolutions were re-scored using ChemPLP.

ResultsIdentification of Met-binding hits from a library of chemicalfragments using SPR screening

A fragment library of 1,338 compounds was screened by SPRagainst amine-coupled Met567 [a soluble Met construct contain-ing the N-terminal b-propeller (Sema) domain and the smallcysteine-rich domain] and against two fragments ofHGF/SF,NK1,and SPH domains. In the absence of a validated inhibitor that wecould use as positive control, wedefined relative response levels ofeach fragment by comparing the actual responsewith the expectedresponse in a 1:1 binding scenario. Expected responses werecalculated by taking into account the protein's immobilizationlevel, molecular weight, and the molecular weight of the respec-tive fragment (Supplementary Table S1). From this initial screen, atotal of 698 fragments were deemed to be Met binders withrelative response levels between 25% and 300% and desiredsquare sensorgrams (Fig. 1A). This generous cutoff filter allowedinclusion of fragments that bind in more than one place on theprotein and accounted for variations in concentration due tosolubility problems. In a second data reduction step, we identifiedpromiscuous binders by comparing relative binding levels forMetwith levels for SPH and NK1. Unique fragments, defined as thosethat bind to one protein with at least a three times greater binding

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level than to the other two proteins, numbered 121 (Met; Fig. 1A),71 (NK1), and 2 (SPH). Further analysis of binders to NK1 ispublished elsewhere (39).

False positives andnon-stoichiometric binderswere eliminatedfrom the hit list by determining binding levels as a function offragment concentration (Fig. 1B). Equilibrium affinity constantswere determined for each fragment, and 16 showed KD of betterthan 100 mmol/L, whereas 26 compounds exhibited KD between100 mmol/L and 500 mmol/L. Fragments that did not display abinding curve but a linear concentration dependency wererejected from further analysis as these fragments are likely tostack at the surface of the chip or protein.

In a secondary screen, binding of fragment hits to Met wasanalyzed using differential scanning fluorimetry (thermal shiftassay), which determines the shift of the melting temperature(DTm) of a protein upon fragment binding. Out of 134 frag-ments screened in this assay, 63 fragments showed a thermalshift of >0.5 K, 40 fragments showed no significant shift(between 0.5 K and �0.5 K thermal shift), 31 showed a shifttoward lower temperatures. Shifts toward lower temperaturesor uncharacteristically high thermal shifts might be caused byprotein aggregation in the presence of high concentrations ofcompound, low fragment solubility, or adverse interaction ofthe fragment with the fluorescent dye. Accordingly, these frag-ments were excluded from further analysis. Fragments thatshowed a DTm of 1.5 K or 2 K and a low KD in SPR are likelyto be good binders, and five fragments were taken forward.

Elaboration of fragment hits resulted in enhanced bindingaffinity

Five structurally different fragments were selected for substruc-ture searches to find similar but larger compounds: MB1271(KD ¼ 49 mmol/L, DTm ¼ 2 K), MB783 (KD n.d., DTm ¼ 2 K),MB1297 (KD ¼ 137 mmol/L, DTm ¼ 1.5 K), MB621 (KD ¼72 mmol/L, DTm ¼ 1.5 K), and MB690 (KD ¼ 172 mmol/L, DTm¼ 1.5 K; Table 1). Thirty-five compounds derived from MB621,MB1271, or MB1297 were purchased based on their structuraldiversity and availability (Supplementary Table S2), and bindingaffinities of these compoundswere determined using SPR (Fig. 2Aand Supplementary Table S2). None of the compounds related toMB1297 showed a higher affinity to the Met Sema domain thantheir parental compound, indicating that MB1297 may bind in a

small pocket that does not allow the binding of larger mole-cules. In the case of MB1271 and MB621, the increase in affinityof related compounds was generally moderate (SupplementaryTable S2), indicating that the selection of compounds withoutstructural knowledge of the fragments' binding modes hasresulted in limited numbers of additional interactions withthe protein. Nonetheless, appreciable increases in bindingaffinity were also observed, for example, compound 31 boundMet with an affinity 36-fold higher than its parental fragmentMB621, but compound 28 with an affinity about five timeslower (Supplementary Table S2).

However, an improved KD alone is not an indication for animproved antagonist as the compound could bind to a part ofMetthat is not involved in the interaction with NK1. Therefore, it isessential to determine whether the compound can interfere withNK1 binding and moreover to assess its biologic activity.

Figure 1.Fragment screening campaign for Met. A, 1,338 fragments were screened using SPR, and the number of fragments that were excluded at each analysisstep is circled. As many as 121 hits were taken forward to the hit validation step where 16 high-affinity binders were identified. B, fragments MB621 (closed circles),MB1271 (open circles), and MB1297 (closed triangles) show suitable binding curves in SPR and were selected for substructure searches.

Table 1. Structures, DTm and KD values of parental fragments

Fragment Structure DTm (K) KD (mmol/L)

MB1271 2 49

MB738 2 n.d.

MB621 1.5 72

MB1297 1.5 137

MB690 1.5 172

Abbreviation: n.d., not determined.






Selected small molecule Met binders interfere with HGF/SF-induced Akt and Erk1/2 phosphorylation and bindingof NK1 to Met928

Compound-induced inhibition of Akt and Erk1/2 phosphor-ylation in HGF/SF-stimulated Vero cells was determined usinginfrared dyes that could be quantitatively analyzed using anOdyssey scanner (Fig. 2B). Eight out of 35 compounds (23%)were able to inhibit Akt phosphorylation after stimulation of cellswith HGF/SF to below 10% as compared with the control (Fig.2C). These compounds were deemed "active," whereas eightcompoundswere deemed "not active" (Akt phosphorylation levelabove 80%) and 19 compounds were "weakly active" (Akt phos-phorylation levels 10%–80%). Toxicity data revealed only com-

pounds 16 and 23 to have appreciable toxicity beyond thatincurred by the DMSO control (Supplementary Fig. S2). Com-pounds that successfully inhibited Akt phosphorylation weregenerally also able to inhibit Erk phosphorylation considerably,although inhibition of Erk1/2 phosphorylation varied appreci-ably with some compounds (Supplementary Table S2). Never-theless, the results obtained clearly demonstrated that eightcompounds (6, 10, 15, 25, 26, 27, 31, and 33) were able toinhibit HGF/SF-induced, Met-dependent Akt and Erk phosphor-ylation behaving as bona fide small-molecule Met antagonists.

To investigate whether these biologically active compoundsmight compete with NK1 for its binding site on Met, we carriedout in vitro competition assays using SPR. Generally, compounds

Figure 2.Elaborated fragments show an improvement in affinity, biologic activity and compete with NK1 for Met binding. A, binding of MB621-related compounds 28(closed rectangle), 23 (open circle), 31 (closed triangle), and 25 (open triangle) to Met567 as measured in SPR experiments. Compounds that displayed aconcentration-dependent binding curvewere fitted using a global fit to determine KD values. The binding of the parental fragment MB621 (closed circle) is shown forcomparison. B, HGF/SF-stimulated Vero cells show a reduction in phosphorylation levels of Akt and Erk upon compound treatment. Western blots of celllysates were probed for total protein (left) and phosphorylated protein (right). C, classification of compounds into "active," "weakly active," and "inactive." Areduction in Akt phosphorylation levels of at least 90% can be seen for "active" compounds. "Weakly active" compounds are able to prevent Akt phosphorylation byat least 20%. Shown are average Akt phosphorylation levels and standard deviations from three independent experiments. D, competition of compoundswith NK1 for binding toMet928 in SPR. Average values and standard deviations from three experimentswere calculated for each compound concentration (1mmol/L,500 mmol/L, 250 mmol/L, and 125 mmol/L). Sigmoidal fits gave rise to Ki values for five compounds: 10 (open circles, solid line, KD ¼ 904 mmol/L), 15 (opentriangles, long dashed line, KD ¼ 679 mmol/L), 25 (closed circle, short dashed line, KD ¼ 877 mmol/L), 26 (closed triangles, dash–dotted line, KD ¼ 10,723 mmol/L),and 31 (closed square, dotted line, KD ¼ 484 mmol/L).

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that showed no effect in phosphorylation assays did not reducebinding of NK1 to Met928 in this assay (Fig. 2D). This confirmsthese compounds as "not active." However, in the presence of"active" compounds 15, 10, 25, 26, and 31 binding of NK1 to

Met928 was reduced. Sigmoidal fits of competition data from five"active" compounds return Ki values between 500 mmol/L and1,000 mmol/L (Table 2).

Selected small-molecule Met binders inhibit the growth ofmouse tumorspheres and the migration of carcinoma cells

Tumorsphere assays that use single-cell suspensions derivedfrommousemammary gland tumors caused by aberrantWnt andMet signaling (38) were used to determine whether the identifiedcompounds were able to inhibit tumor growth (Fig. 3A–I).

Compound 6 showed no effect at 1,000 mmol/L and wastherefore considered "negative" in this assay. In contrast, com-pounds 18 and 31 completely abolished mammosphere forma-tion at 1,000 mmol/L. Compounds 1, 18, and 31 showed a

Table 2. Compounds active in phosphorylation assays show varyingeffectiveness in inhibiting NK1 binding to Met

CompoundAkt phosphorylationinhibition (%)

NK1 competitionKi (mmol/L)

10 �2.9 � 12.6 90415 �9.2 � 9.8 67925 �6.4 � 11.9 87726 2.2 � 12.8 10,72331 5.9 � 1.8 484

Figure 3.Compounds inhibit tumor progression and cell migration. A–I, picture of one representative mammosphere from tumorsphere assays with a 250 mmol/L compoundconcentration: 1% DMSO (A, control), compounds 3 (B), 6 (C), 31 (D), 18 (E), 27 (F), 10 (G), 26 (H), and 15 (I). Some compounds cause a dramatic decrease inmammosphere size (see E, F, and H) as compared with DMSO control (A). J, outcome of tumorsphere assay as normalized mammosphere number per testedconcentration. Lower mammosphere numbers indicate inhibition of tumor growth and progression. Averages and error bars are from three differentexperiments. IC50 values could be derived for compounds 15 (closed circle, solid line, IC50¼ 534mmol/L), 31 (closed triangle, long dashed line, IC50¼ 205mmol/L), 26(open circle, dotted line, IC50 ¼ 946 mmol/L), and 18 (open triangle, short dashed line, IC50 ¼ 116 mmol/L). K, SKOV3 cell migration was tested toward 30 pmol/LHGF/SF in the presence of 1 mmol/L compound. Residual fluorescence (mean of �4 replicates per assay) of migrating cells was quantified and normalized tomigration in the presence of diluent only. Graph shows normalized migration from three independent experiments with bars representing the mean for eachcompound. Compounds 15 and 26 resulted in statistically significant inhibition compared with diluent-only control (P < 0.05) in all three experiments (two-wayANOVA with Dunnett multiple comparisons test).






reduction in mammosphere numbers at concentrations as low as100 mmol/L. Compounds 15 and 31 showed >50% inhibition at250 mmol/L and were therefore considered as "positive." Com-pound 31was the only compound that inhibited mammospherenumbers well below 50% of control at a concentration of 100mmol/L (Fig. 3J). Compounds that reduce mammosphere forma-tion with statistical significance, as determined by a t test (P <0.05), are compounds 31 (at 250 mmol/L and 1,000 mmol/L) and3 and 18 at 1,000 mmol/L. Compound 15 has a P value of around0.1 to 0.12 at the various concentrations, which is probably due tothe differences in the replicates, which affects the standard devi-ation. In addition to an inhibition of mammosphere formationfrom single tumor cells (mammosphere number), there is also aclear effect of some compounds on mammosphere size, whichindicates an effect on cell proliferation, for instance compounds18, 27, and 26 (Fig. 3E–H, respectively). Therefore, in this assay,only compounds 15 and 31were considered to show antitumori-genic properties with preliminary IC50 values of 534 mmol/L(compound 15) and 205 mmol/L (compound 31).

In order to assess whether compounds would act on themigratory behavior of cells, migration assays were conducted at1 mmol/L and 250 mmol/L compound concentration. Com-pounds 15 and 26 inhibited cell migration to 37% and 60% at1mmol/L concentration, respectively (Fig. 3K). The ability of bothcompounds to prevent phosphorylation of Akt and Erk in a

phosphorylation assay suggests that an upstream inhibition ofMet resulted in a lack of signal transduction, which in turnprevented migration of SKOV3 cells toward HGF/SF. Compound15 was also effective in mammosphere assays, suggesting inhib-itory effects in both tumor growth and cell migration.

Docking of compounds toMet Semadomain indicates differentmodes of actions

There are three crystal structures available containing parts ofthe Met extracellular region: a Met25–567/SPH cocrystal structure(PDB code 1SHY; ref. 30) and two Met25–741/internalin B36-321cocrystal structures (PDB codes 2UZX and 2UZY; ref. 40). Cross-linking experiments withNK1 and theMet Semadomain revealedtwo cross-links involving Ser309 and Glu395 of Met (L. Kemp;unpublished results). One of the cross-links, Ser309, lies very nearto the region marked as a footprint for internalin B (as Ser309 isnot resolved in either crystal structures, Lys311 is indicatedin Fig. 4A). This region also harbors three loops that are notresolved in either crystal structure: the first loop from Ile377 toAsn382, the second from Thr301 to Lys311, and the third fromArg400 to Asp414. It is conceivable that these loops might beinvolved in NK1 binding, which is consistent with the crystalstructure of the high-affinity binding site of the NK1 fragment ofHGF/SF on the Sema domain of the Met receptor defined subse-quently (29). In separate studies it was shown that the bacterial

Figure 4.Docking of compounds into the proposed binding site for NK1 on Met Sema domain. A, the Met Sema domain from the cocrystal structure with SPH domain (1SHY) isshown as green cartoon representation. The potential binding site for NK1 lies partially within the footprint of internalin B (magenta) and two cross-linkingsites (side chains in cyan sticks). Potential pockets that have been identified by PocketFinder that could accommodate a chemical compound are highlighted in blueand red space fills. Amino acids used as centers for the docking spheres are shown as sticks. B, surface electrostatic representation of the Met surface reveal thatpocket 1 is largely electronegative, whereas pockets 2 and 3 are mostly surrounded by uncharged areas. Electronegative areas are represented in red andelectropositive areas in blue. C and D, overlay of active compounds 10 (pink) and 15 (yellow) in pocket 1 show that both compounds engage the protein in similarinteractions involving R384. In addition, compound 10 interacts with H275 at the top of the pocket, and compound 15 engages in hydrophobic interactions atthe right side of thepocket. D is a rotation ofCby90�. E andF, overlayof compounds 10 (pink) and 15 (yellow) in pocket 3 shows that both compounds formhydrogenbonds to R469 as well as hydrophobic interactions to M431 and P472. F is a rotation of E by 90� . Figures prepared with PyMol.

Winter et al.





protein internalin B from L. monocytogenes competes with NK1 forbinding to Met, indicating that the binding sites of internalin Band NK1 on the Met Sema domain overlap extensively (E.Gherardi, unpublished observations).

Using PocketFinder we found two potential pockets within, ornear to the proposed binding site (see blue and red space fillsin Fig. 4A). One is located just above the two helices flanked byresidues Arg469, Met431, and Pro472 (red space fill) and has apocket volume of 74 Å3. The second is larger with a volume of 125Å3 and is enclosed by the disordered region leading up to strand Eof the fourth blade, residues 382 to 400 (blue space fill). This loopalso harbors one of the cross-linking sites, Glu395, and is orderedinto a short helix in the internalin B cocrystal structure 2UZX.However, due to the anticipated disorder in this region and lack ofstructural information in parts of the proposed pocket we decidedto focus our docking efforts elsewhere. Manual inspectionrevealed two additional grooves within the proposed footprintofNK1 thatmight be able to accommodate chemical compounds.The first is a large pocket near Lys 311, surrounded by residuesArg384, Glu267, Val313, Glu297, and Pro295, and has an elon-gated shape with a slight kink (pocket 1, Fig. 4B). The second,surrounded by residues Arg469, Arg426, and Tyr369 (pocket2, Fig. 4B), is small and lies near one of the pockets found byPocketFinder. The first pocket lies within a large electronegativepatch of the protein whereas the second lies in a largely hydro-phobic region. Compounds bound to either pocket could possi-bly impair or inhibit binding of NK1 toMet and therefore preventreceptor dimerization and thus activationof the signaling cascade,including activation of Akt and Erk.

Docking of compounds into the proposed Met–NK1 interfaceWe used GOLD to dock the compounds into all three pockets.

Residues within the pockets were chosen as docking centers, forpocket 1Glu267, for pocket 2 it is Arg426, and for pocket 3Arg469(Fig. 4B). Using a docking sphere of 14 Å, 10 docking poses wereevaluated for each ligand using ChemScore and the solutions re-scored using ChemPLP. All docking results are tabulated inSupplementary Table S3, and top-scoring poses of the threehighest-ranking compounds are shown in Supplementary Fig. S3.

The first pocket is flanked by several acidic residues such asGlu267, Glu419, and Glu297. Compound 10 had the highestdocking score, followed by compounds 22 and 15. The com-pounds formed hydrogen bonds to the side chain of Arg384 andGlu297, as well as Thr276 or Thr273, and Val313, Gln425, and

Ala423 gave rise tohydrophobic interactions. Compounds10 and15 were deemed "active" in phosphorylation assays, indicatingthat this pocket might be a target for inhibitors.

The second pocket is a largely nonpolar, flat groove, presentinga single small pocket at one end. Compound 24 had the bestdocking score, followed by compounds 34 and 22. The top threescorers all show limited effectiveness in phosphorylation assays,and the highest-ranking "active" compound for this pocket iscompound 6 (sixth rank). This suggests that this pocket may notbe suitable for accommodating any of the active compounds. Thispocket also lies slightly outside the area spanned by the two cross-linking sites, Ser309 andGlu395. Therefore, this site is unlikely tobe suitable for developing an antagonist.

The third pocket (Fig. 4A, red spheres) is relatively small andshallow. It harbors an acidic aspartate residue at the bottom(Asp428) and presents a deeper, nonpolar subpocket to one side.Parts of this pocket are disordered in the crystal structures, andresidues 302 to 310 were not observed. On the other side of thispocket is a small loop containing Arg469, which was used asdocking center. Docking compounds in pocket three revealedcompound 10 as highest scoring, followed by compounds 15 and8. All three compounds are related, being filial compounds to thefragment MB1297. Moreover, compounds 10 and 15 were iden-tified as "active" in phosphorylation assays and compound 8 as"weakly active." These observations indicate that the inhibitorsinvestigated here might target this pocket.

In summary, the active compounds 10 and 15 scored high indocking to pockets 1 and 3 (Table 3). In competition assays,compounds 10 and 15 inhibited NK1 binding to 60% and17%, respectively. This indicates that pocket 1 and/or pocket3 might be situated in the NK1 binding interface and "active"compounds might bind to it to disrupt NK1 binding. Addi-tionally, compound 15 was active in a tumorsphere assay and amigration assay, indicating that this compound may act as aMet antagonist that is able to inhibit tumor formation and cellmigration. However, both compounds bind with relatively lowaffinity, namely 879 mmol/L for compound 10 and 10 mmol/Lfor compound 15. The low binding affinity measured in SPRexperiments is puzzling and might indicate very inefficientbinding and a requirement for high concentrations in orderto display its inhibitory effect. All assays used a large excess ofcompound, with some showing low solubility in assaysthat would have reduced the availability of compound mole-cules. On the other hand, it might also be possible that this

Table 3. Summary of results from different assays of active compounds

Compound KDa (mmol/L)

Level of NK1bindingb (%)

Inhibition oftumorigenesisc

Level of cellmigrationd (%)

Docking results, rankingPocket 1 Pocket 2 Pocket 3

6 63 100 No n.d. 29 6 2410 879 60 No n.d. 1 15 115 10,540 17 Yes 37 3 31 225 192 60 No No 20 10 2826 406 60 Yese 60 26 27 2927 76 100 Yese No 7 11 2631 2 48 Yes n.d. 14 18 2233 80 100 No No 23 16 9

Abbreviation: n.d., not determined.aAgainst Met567 in SPR.bTo Met928 in competition assays at 1 mmol/L compound.cActivity assessed in tumorsphere assay.dActivity assessed in migration assay.eWith additional antiproliferative properties.






compound binds Met in several different places on the protein,which would mean a reduced presence of molecules in any onepocket. Interestingly, compound 31 was not found in the top ofthe docking rankings for any pocket, although it bound Metwith low micromolar affinity, was effective in competing withNK1 for Met binding and in inhibiting tumor formation. Thismight indicate that this compound may bind to a different siteon Met and distort the NK1 binding site and may therefore beconsidered as an allosteric inhibitor. Compound 26 was alsoactive in tumorsphere assays, but its ability to compete withNK1 for Met binding was limited. This compound showed anaffinity for Met in the low- to mid-micromolar range, but itslow ranking in the docking experiments suggests that it maybind slightly outside the NK1 binding site. However, com-pound 26 is still able to inhibit Met signaling sufficiently toelicit a cellular response.

Binding modes of these compound on Met need to be evalu-ated further using structural studies in order to assess whethercompound 15 is indeed a direct inhibitor of this interaction, andwhether compound 31 is an allosteric inhibitor.

DiscussionModulators of protein interfaces are therapeutically relevant

and selective, which makes them attractive targets in drugdiscovery (41, 42). Many essential cellular functions are carriedout by multiprotein assemblies, and so it is not surprising thatmany human diseases, such as neurodegenerative diseases (43)and metabolic diseases (44) as well as cancer (45), can becaused by aberrant PPI. The development of novel chemicalmoieties targeting PPIs has recently turned previously "undrug-gable" targets into tractable macromolecular sites (42, 46, 47),and examples for successful campaigns can be found in theliterature (42, 48, 49).

In this study, we aimed to find small molecules that inhibit theinteraction between Met and NK1 using a fragment-basedapproach. The large number of promiscuous binders found inour initial fragment screen reflects the challenges faced when noknown inhibitor/small molecule binder or control protein con-taining a disabled binding site can be utilized (50). Therefore,screening campaigns for protein interfaces are likely to requireadditional selection steps to ensure identification of uniquefragment hits (21). The progression from fragment hit to leadcompound is challenging even in projects when structural infor-mation is available. In this study, however, high-resolution struc-tural data were not available to verify fragment binding anddetermine their binding mode.

In order to overcome this, we modified the traditional drug-discovery process by including substructure searches and cell-based assays early on in the pipeline. We utilized substructuresearches to gain first insights into which type of compoundmight be a promising antagonistic lead compound. Thisenabled us to progress speedily to test compounds in cell-based and other biophysical assays. An antitumorigenic andantimigratory effect of several of our compounds was corre-lated with their ability to disrupt the interaction between NK1and Met and subsequently inhibit Met signaling. These areencouraging findings, especially as these compounds weregenerated using similarity searches instead of the traditionalstructure-based approach. Docking studies further indicatedthat some compounds might act as direct inhibitors of the

NK1–Met interaction whereas others are likely to be allostericinhibitors.

Our study further uncovered strategies for compound devel-opment and optimization. Compounds 10 and 15 undergosimilar interactions with Met in pocket 1 (Fig. 4C and D), mainlyforminghydrogenbonds toArg384andGlu297, and engaging theright-hand side and topof the pocket in hydrophobic interactions.Combining both compounds could produce a compound bettersuited to occupy all available space in pocket 1. The interactionbetween cyano-N of compound 10 with the amino-nitrogenof His275 could be preserved as well as interactions with themain chain amide of Thr276 andmain chain carbonyl of Thr421.In pocket 3, both compounds undergo an interaction withArg469: compound 10 with its carbonyl group and compound15with its nitrogens as hydrogen-bond acceptors (Fig. 4E and F).Compound 10 also engages the protein via hydrophobic inter-actions to Pro472 and Met431. Additionally, one of its carbonylgroups engages Ser470 in a hydrogen-bond interaction.

In conclusion, the fragment-based approach to design inhibi-tors of PPIs without tethering the fragments is a new developmentin the field of drug discovery that has recently led to onemarketeddrug, vemurafenib, for treatment of metastatic melanoma (42).Fragments are well suited to efficiently explore the small pocketscharacteristic of protein–protein interfaces and may lead to thediscovery of new scaffolds and chemotypes, as already suggestedby Wells and McClendon (47). We have demonstrated here thatfragments can be used as good starting points for a de novo drug-discovery campaign even when no crystal structure of the targetinterface is available. The need for expanding fragment-baseddrug discovery to targets where no crystal structure is available hasalready been recognized (51). We also showed that the applica-tion of similarity searches enables a progression from fragmenthits to possible lead compounds with a modest requirement ofresources. We have reasons to believe that applying this approachto so-called "undruggable" targets in future might open up thedrug-discovery field to new classes of proteins and reach out tonew disease targets.

Disclosure of Potential Conflicts of InterestT.L. Blundell is a member of the commercial board and the Science Advisory

Board of Astex Therapeutics Ltd., has provided expert testimony for MedIm-mune, has a role on the Science Advisory Board for UCB, reports receivingcommercial research grant fromUCBCelltech, is a consultant for Pfizer, and hasprovided expert testimony for Ipsen. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: T.L. Blundell, E. GherardiDevelopment of methodology: A. Winter, A.G. Sigurdardottir, T.L. BlundellAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Winter, A.G. Sigurdardottir, D. DiCara, G. Valenti,Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Winter, A.G. Sigurdardottir, D. DiCara, G. Valenti,T.L. Blundell, E. GherardiWriting, review, and/or revision of the manuscript: A. Winter, A.G. Sigurdar-dottir, D. DiCara, G. Valenti, T.L. Blundell, E. GherardiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Winter, T.L. BlundellStudy supervision: T.L. Blundell

AcknowledgmentsThe authors thank Professor Chris Abell (Chemistry, Cambridge) and

his colleagues for giving them access to the fragment library. They also thank

Winter et al.





Dr. Oliver Korb (CCDC, Cambridge) and Dr. Ralf Schmid (University ofLeicester) forhelpwith computationalmethodsand feedbackon themanuscript.

Grant SupportThis workwas supported by the EuropeanUnion project SFMET (FP7), Gates

Cambridge Scholarship (A.G. Sigurdardottir), and CRUK (C24461/A12772,to A. Winter).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 2, 2015; revised October 11, 2015; accepted October 29, 2015;published OnlineFirst December 28, 2015.

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Winter et al.




Published OnlineFirst December 28, 2015.Mol Cancer Ther Anja Winter, Anna G. Sigurdardottir, Danielle DiCara, et al. Interaction Using a Fragment-Based Approach

Protein−Developing Antagonists for the Met-HGF/SF Protein

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