Developing Antagonists for the Met-HGF/SF Protein–Protein Interaction Using a Fragment- Based Approach Anja Winter1, Anna G

Published OnlineFirst December 28, 2015; DOI: 10.1158/1535-7163.MCT-15-0446

Small Molecule Therapeutics Molecular Cancer Therapeutics Developing Antagonists for the Met-HGF/SF Protein–Protein Interaction Using a Fragment- Based Approach Anja Winter1, Anna G. Sigurdardottir1, Danielle DiCara2,3, Giovanni Valenti4, Tom L. Blundell1, and Ermanno Gherardi2

Abstract

In many cancers, aberrant activation of the Met receptor larger compounds using substructure (similarity) searches. We tyrosine kinase leads to dissociation of cells from the primary identified compounds able to interfere with NK1 binding to tumor, causing metastasis. Accordingly, Met is a high-profile Met, disrupt Met signaling, and inhibit tumorsphere genera- target for the development of cancer therapies, and progress has tion and cell migration. Using molecular docking, we con- been made through development of small molecule kinase cluded that some of these compounds inhibit the PPI direct- inhibitors and antibodies. However, both approaches pose ly, whereas others act indirectly. Our results indicate that significant challenges with respect to either target specificity chemical fragments can efficiently target the HGF/SF-Met (kinase inhibitors) or the cost involved in treating large patient interface and may be used as building blocks for generating cohorts (antibodies). Here, we use a fragment-based approach biologically active lead compounds. This strategy may have in order to target the protein–protein interaction (PPI) between broad application for the development of a new class of Met the a-chain of hepatocyte growth factor/scatter factor (HGF/SF; inhibitors, namely receptor antagonists, and in general for the the NK1 fragment) and its high-affinity binding site located on development of small molecule PPI inhibitors of key thera- the Met Sema domain. Surface plasmon resonance was used for peutic targets when structural information is not available. initial fragment library screening and hits were developed into Mol Cancer Ther; 15(1); 1–12. 2015 AACR.

Introduction as a protein causing dispersion of epithelial colonies and cell migration (SF; 6, 7). HGF/SF and Met are essential for the Receptor tyrosine kinases (RTK) mediate intercellular signals development of several tissues and organs, including the placenta that are essential for the development and maintenance of (8, 9), liver (8), and skeletal muscle (10). HGF/SF and Met also the cells of multicellular organisms. Met, the RTK encoded by play a major role in the abnormal migration of cancer cells as a the c-Met proto-oncogene (1, 2), is the receptor for hepatocyte result of Met overexpression or Met mutations (11). Moreover, growth factor/scatter factor (HGF/SF; ref. 3), a large polypeptide these are strongly associated with poor prognosis, for instance in growth factor discovered as a liver mitogen (HGF; refs. 4, 5), and urothelial carcinoma of the bladder (12), prostate cancer (13), non–small cell lung cancer (14), and ovarian cancer (15). It is 1Department of Molecular Cell Biology, The University of Cambridge, therefore not surprising that HGF/SF and Met have emerged as key Cambridge, United Kingdom. 2MRC Centre, Cambridge, United King- therapeutic targets for the treatment of metastatic cancer. dom. 3Department of Oncology, The University of Cambridge, Cam- Although Met kinase inhibitors such as cabozantinib, 4 bridge Biomedical Campus, Cambridge, United Kingdom. Depart- SAR125844, and tivantinib have been shown to be effective, ment of Cancer Research, Max Delbrueck Center for Molecular Med- icine (MDC), Berlin, Germany. resistance to Met kinase inhibitors develops rapidly (16–18). Therefore, interfering with the assembly of the HGF/SF-Met Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). complex could constitute a new and promising route in the search for novel, cost-effective, and selective inhibitors of HGF/ Current address for A. Winter: University of Leicester, Department of Molecular – Cell Biology, Leicester, UK; current address for A.G. Sigurdardottir: MRC Lab- SF-Met signaling, but it has its own challenges. Protein protein oratory of Molecular Biology, Department of Neurobiology, Cambridge, UK; interactions (PPI) are generally large and lack the large cavities current address for E. Gherardi: Universita di Pavia, Department of Molecular that characterize many enzyme active sites, such as protein Medicine, Pavia, Italy; and current address for D. DiCara: Genentech Inc., South kinases and proteases, and receptors such as G-protein-coupled San Francisco, California. receptors. However, the observation that protein surfaces often Corresponding Authors: Anja Winter, Department of Molecular Cell Biology, display small deep pockets (19, 20), sometimes closely clus- University of Leicester, Lancaster Road, Leicester LE1 9HN, UK. Phone: 44-116- tered, offers a basis for alternative design approaches (21). 299-7074; Fax: 44-116-229-7123; E-mail: [email protected]; and Tom Blundell, Examples of such inhibitors can be found in the literature, Department of Biochemistry, Tennis Court Road, The University of Cambridge, although the approaches by which they were discovered are CB2 1GA Cambridge, UK. E-mail: [email protected] diverse (21) ranging from virtual screening (22) to fragment- doi: 10.1158/1535-7163.MCT-15-0446 screening campaigns (23, 24) and mimicking the interaction 2015 American Association for Cancer Research. partner by rational design (25). A further and major advantage

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of targeting the extracellular domain of Met is that the com- Expression and purification of SPH protein pounds would not have to cross the cell membrane, therefore The SPH fragment (Val495-Ser728) was cloned using cDNA of allowing for more flexibility in drug design. full-length human HGF/SF and cloned into the baculovirus The interactions between the extracellular portion of Met secretion vector pAcGP67A (BD Biosciences) with the addition and HGF/SF are intricate due to the complex, multidomain of a C-terminal hexahistidine tag and a Cys604Ser mutation. Viral structure of these proteins (4, 26). However, biochemical, stocks were amplified using Sf9 insect cells (BD Biosciences), and biophysical, and structural analysis of individual HGF/SF protein was produced using High Five (BTI-TN-5B1-4) insect cells domains (27) and mutagenesis studies (28) have revealed that (Invitrogen) and ESF 921 media (þ2.5% FCS). SPH domain was HGF/SF binds Met through a high-affinity binding site located purified using a 5 mL HP HisTrap (GE Healthcare) column and a in the N-terminal N and K1 (NK1) domains and a low-affinity gradient of imidazole from 0 to 500 mmol/L in 50 mmol/L Tris, binding site located in the C-terminal serine-proteinase homol- pH7.7. Protein-containing fractions were pooled, concentrated, ogy (SPH) domain. Solution and low-resolution crystal struc- and injected onto a 16/60 Superdex 200 prep grade column tures have subsequently defined the high-affinity binding site (Amersham Pharmacia Biotech) equilibrated with 10 mmol/L of the NK1 fragment of HGF/SF on the Sema domain of the Met HEPES, pH7.2, 150 mmol/L NaCl. Expression yields were 4 mg/L receptor (29). The low-affinity binding site between the two media. proteins has been defined in the crystal structure of the Sema and cysteine-rich domains of Met in complex with the SPH Surface plasmon resonance experiments domain of HGF/SF (30). Surface plasmon resonance (SPR) measurements were per- Here, we use fragment-based methods to find chemical formed on a Biacore T100 instrument using research-grade CM5 building blocks for developing small-molecule Met receptor sensor chips. The reagents 1-ethyl-3-(3-diaminopropyl) carbodii- antagonists. We show that compound optimization in con- mide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and junction with biologic activity assays and in silico methods can ethanolamine (pH 8.5) were purchased from Biacore and used lead to the discovery of compounds that are able to disrupt Met according to recommended protocols. Met, NK1, and SPH signaling, interfere with NK1 binding to Met, and inhibit domain were immobilized on a CM5 sensor chip using the tumorsphere generation as well as cell migration. These com- amine-coupling method. Immobilization was done selecting a pounds constitute the starting points for the development of target immobilization level of up to 10,000 RU in the immobi- selective, non-kinase inhibitors of the HGF/SF-Met signaling lization wizard of the Biacore control software in order to saturate pathway. the chip surface with protein. Met was diluted with 10 mmol/L acetate buffer (pH5.5) to a final concentration of between 1 and 2 mmol/L, and response levels of around 6,000 RU were immo- Materials and Methods bilized on the activated surface. NK1 was diluted into 10 mmol/L Expression and purification of Met proteins phosphate buffer, pH7.0 for immobilization, and the SPH Two silent mutations were introduced in codons coding for domain was immobilized using 10 mmol/L sodium acetate amino acids Q559 and I560 of a full-length human Met cDNA to buffer, pH5.5. NK1 and the SPH domain were immobilized on remove a BglII site. Met deletions lacking the endogenous leader a separate CM5 chip in flow channels 2 and 3, respectively, and (amino acids 1–24) were generated by PCR as MluI–BglII inserts response levels of about 2,500 RU for NK1 and 5,700 RU for SPH and cloned in-frame between a 21-aa Ig leader and a hexa- or octa- domain were achieved. histidine sequence. For expression, Met constructs Met567 (amino acids 26–567) and Met928 (amino acids 26–928) in plasmid Fragment library and compounds pA71d were transfected in mouse Lec 3.2.8.1 cells (31). Stable Fragments were taken from a 1,338-membered library consist- transfectants were selected in 0.75 mg/mL hygromycin, screened ing of fragments taken from Maybridge fragment libraries and infor expression, and positive cultures were cloned and expanded house compounds. Fragments were dissolved in DMSO to a final for protein production. Monomeric Met proteins were produced concentration of 100 mmol/L. All compounds were purchased in 10% a-MEM/DMEM media containing 2.5% FCS and purified from Enamine (www.enamine.net) and dissolved in DMSO to on a Ni-NTA agarose column (catalog no. MG3398; Qiagen) final concentrations of either 100 mmol/L or 50 mmol/L. equilibrated in PBS. The protein was eluted with 0.4 mol/L imidazole and was further purified on a Mono S column (Amer- Fragment screening and data analysis sham Biosciences). SPR binding experiments were performed in PBS buffer (pH7.4) as the running buffer supplemented with 0.05% surfac- Expression and purification of NK1 protein tant P20 or Tween 20 and 1% DMSO to aid fragment solubility. cDNA encoding the NK1 fragment was obtained from full- Experiments were performed at 25C and at a flow rate of length human HGF/SF and then cloned into the vector pPIC9K as 30 mL/minute. Samples were injected for 30 seconds followed described before (32). This vector was transformed into Pichia by 60-second dissociation. The sensor surface was regenerated pastoris strain GS115 and NK1 produced in BMMY media contain- between experiments by injecting 1 M NaCl for 30 seconds. If the ing 100 mg/mL ampicillin in aliquots of 500 mL in 2 L conical difference of response before and after analyte injection was flasks. Methanol (2.5 mL) was added to each flask to induce greater than 10 RU, 50% ethylene glycol was injected for 30 expression and every 24 hours afterward. The purification of NK1 seconds to remove residual sample. Fragments from our in-house was carried out in two steps following Chirgadze and colleagues fragment library were diluted 1:100 (for Met) or 1:200 (for NK1 (1999), except that the MES buffer was removed from the puri- and SPH) from a 100 mmol/L stock (dissolved in 100% DMSO) fication process and 50 mmol/L sodium phosphate, pH7.4 was into running buffer to give final concentrations of 1 mmol/L or used instead. Expression yields were 5 mg/L media. 0.5 mmol/L, respectively. All samples were measured at one

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concentration, sensorgrams were recorded at 1 Hz and screens value of <3 for Log P was chosen, which is thought to reduce were done in 96-well plate format. To monitor the binding toxicity and increase ease of formulation and bioavailability for capacity of the protein surfaces during the screening, injections optimal oral absorption (35). Twenty compounds derived from of control samples were carried out every 40 samples. Met567 MB621 were mostly substituted at the nitrogen atom of 1,2,3,4 (0.25 mmol/L) and HEPES (500 mmol/L) were used as control tetrahydroquinoline and lacked the methyl group at R2. Com- samples for NK1 and the SPH domain. 200 nmol/L NK1 and 1% pound 24 possesses an additional methyl group for R3. Fragment DMSO were used as control samples for Met. Solvent correction MB1271 is a tri-substituted benzene ring. All compounds pur- cycles were included every 40 samples to account for differences in chased as derivatives of this fragment retain the fluoroform group. DMSO content of the samples. In addition, all compounds harbor a morpholine group in para- After solvent correction, binding levels of each fragment and position relative to the fluoroform group. Residue R1 is variable in control samples were determined near the end of the injection. all compounds and replaces the NH2 group of the fragment. Instrument variations between runs were normalized using MB1297 is also a tri-substituted benzene ring. Apart from a cyano response levels of the respective positive controls. A maximum group it also harbors a methyl group in para-position to the cyano of 400 samples were run consecutively followed by storing the group and a NH2 group in ortho-position. The cyano group is chip in running buffer for a few days. Responses of control retained in all derived compounds, and a hydrogen atom replaces samples recovered remarkably, and one chip could be used to the methyl group in all compounds except compound 8, where a screen approximately 1,400 samples. chlorine replaces it. The NH2 group is substituted in all com- Raw data were normalized against ligand-coupling density, pounds with diverse chemical groups and features. ligand molecular weight, and molecular weight of each injected fragment by calculating RUmax, which represents the theoretical Competition assays maximum response for each fragment. RUrel is the normalized SPR competition experiments were performed as essentially relative SPR response of a fragment that was calculated using the described previously (36). Met928 was immobilized to a CM5 chip measured binding level of the fragment and the theoretical using the amine coupling method. Compounds were diluted maximum response RUmax. from their 100 mmol/L stock solution in DMSO into running buffer (PBS buffer with 0.05% Tween 20) supplemented with Kinetic analysis of fragment binding 250 nmol/L NK1 keeping the DMSO content constant at 1%. This Experiments for binding kinetics were performed by SPR using mix was passed over the Met928 sensor surface for 2 minutes at PBS buffer (pH7.4) as the running buffer supplemented with 30 mL/minute and allowed to dissociate for 3 minutes. The sensor 0.05% surfactant P20. Experiments were performed at 17 C and at surface was regenerated between experiments using 20 mmol/L a flow rate of 30 mL/minute. Samples were injected for 30 seconds Sodium acetate buffer pH 5.0 with 4 mol/L NaCl for 30 seconds. followed by 60-second dissociation. The sensor surface was Binding levels for each sample were determined 4 seconds before regenerated between experiments by injecting 1 mol/L NaCl for injection stop and plotted against the respective compound 25 seconds. A concentration series of the fragments ranging from concentration. The responses for competition assays were calcu- 7 mmol/L to 500 mmol/L in 1:2 dilutions into running buffer was lated by subtracting the response incurred from compound bind- typically run in these experiments. Sensorgrams were recorded at ing from the decreasing response levels in NK1-supplemented 10 Hz. Solvent correction cycles were included every 40 samples to samples. The resulting binding curves were fitted using a 1:1 account for DMSO content in the samples. Equilibrium affinity- Langmuir binding model or heterogeneous analyte supplied with binding data were obtained by fitting blank-subtracted and sol- the Biacore T100 evaluation software where the on- and off rate fi vent-corrected binding levels using a global t provided within the constants for NK1 binding to Met928 were predetermined and Biacore T100 instrument software. fixed for the fitting process.

Differential scanning fluorimetry Phosphorylation assays Thermal shift (differential scanning fluorimetry) assays were Phosphorylation assays were carried out as essentially described carried out by mixing buffer (50 mmol/L Tris, pH7.5, 150 mmol/L by Ferraris and colleagues (37). Vero cells were grown to con- NaCl) and protein to a final concentration of 5 mmol/L. Fragment fluency and starved for 42 to 52 hours before treatment. Cells (5 mL; 100 mmol/L in DMSO) and 12.5 mL Sypro Orange of a were treated by incubating with either 1 mmol/L, 0.5 mmol/L, 1:2,000 dilution in water were added to give a final volume of 0.25 mmol/L, or 0.125 mmol/L of compound in 1% DMSO for 50 mL. Fluorescence values were recorded at temperature intervals 5 minutes in the presence of 0.1 nmol/L HGF at 37C. HGF of 0.5 K from 25Cto80C using an iQ5 Multicolor real-time PCR (0.1 nmol/L) in 1% DMSO only was used as positive control and detection system from BioRad. Data were analyzed, fitted, and 1% DMSO only was used as negative control. After a brief wash melting temperatures determined using Excel. with ice-cold PBS, lysis buffer (50 mmol/L Hepes, pH7.5, 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 g/L Triton X-100, 10 g/L Substructure searches glycerol, 1 mmol/L EGTA, 50 mmol/L orthovanadate, 100 mmol/L Substructure searches were carried out using ZINC, a free NaF, Roche cocktail of protease inhibitors (cat. 11 873 580 001, database of commercially available compounds for virtual screen- 2 tablets in 100 mL of buffer) was added to each well. Cells were ing (33). The molecular weight was chosen to be between 250 and then incubated on ice for 30 minutes, scraped from the bottom of 350 Da. Values such as xLogP, number of hydrogen bond accep- the well, and the lysate was immediately frozen. Lysates were tors, and donors were set in accordance with Lipinski's rules (34). analyzed by SDS-PAGE, blotted, and probed for levels of phos- Thirty-five compounds were purchased from Enamine with 264 phorylated and total proteins Met, Akt and Erk using anti-phos- Da < molecular weight > 404 Da, 1.2 < LogP > 3.1, 0 < H-donors pho Met (Tyr1234/1235) XP antibody (D26; Cell Signaling Tech- > 3, 3 < H-acceptors > 8 generously following the "rule-of-three." A nology; #3077) or anti-Met antibody (L41G3; Cell Signaling

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Technology; #3148), anti-phospho Akt (Ser473) antibody (Cell Boyden chamber (AC96 Migration Chamber; Neuroprobe). Signaling Technology; #9271) or anti-Akt (Cell Signaling Tech- Lower chambers containing 30 pmol/L HGF/SF and nology; #9272) and anti-phospho Erk antibody (Sigma #M8159) 1 mmol/L compound in assay media (RPMI, 0.25% BSA or anti-Erk1/2 (Promega #9PIV114). For quantitative analysis diluted 1:1 with PBS) were separated from upper chambers by of phosphorylation levels, fluorescent anti-mouse (a-mouse aporousmembrane(8mm, PVP-free) that had been coated IRDye800CW, Rockland, #800-656-7625) and anti-rabbit anti- with 100 mg/mL collagen (Purecol, Nutacon) for 2 to 3 hours at bodies (a-rabbit IRDye 680LT, LI-COR, #926-68023) were used. room temperature. SK-OV-3 cells were labeled with the fluo- Band intensities were determined using the Odyssey Infrared rescent dye Calcein AM (Life Technologies) at a concentration Imaging system (LI-COR). After background subtraction, phos- of 5 mmol/L for 30 minutes at 37Cpriortowashingand phorylation levels of Akt and Erk were normalized against total resuspension in assay media; 25,000 cells were then added to amount of protein (HGFonly ¼ 100% phosphorylation, positive each upper well and the chamber was incubated at 37 Cfor4 control) and 1% DMSO (0% phosphorylation, negative control) hours to allow cell migration to occur. After removal of non- as solvent control. migrated cells from the membrane, the degree of cell migration was assessed by quantification of the residual fluorescence, Cytotoxicity assay indicative of migrated cells, on a Typhoon instrument (GE Life The relative cytotoxicity of the compounds was determined Sciences), using excitation/emission settings of 488 nm/526 using the alamarBlue Assay. Cells were seeded at 1,000 cells/mL in nm, respectively. Data were analyzed with ImageQuant soft- 96-well plates. The assay was conducted following the manufac- ware and background fluorescence subtracted. Statistical anal- turer's instructions. ysis was performed with GraphPad Prism 6.0 (GraphPad Soft- ware, Inc.). Tumorsphere (mammosphere) assay Female mice of strain FVB/N coexpressing activating muta- Docking using GOLD tionsinboththeWnt/b-catenin and HGF/Met signaling path- The cocrystal structure of Met567 with HGF/SF was used as ways under the control of the pregnancy-induced whey acidic protein template for the docking runs. The 3D coordinates of the protein (WAP) promoter were used at 2 weeks postpartum. enzymes were obtained from the PDB (PDB code 1SHY) and were Single-cell suspensions derived from mouse mammary gland processed by removing HGF/SF, water molecules, and other tumors were plated at a density of 80,000 cells/mL in 24-well ligands and adding hydrogen atoms to the remaining Met protein. plates coated with 1.2% Poly 2-hydroxyethyl methacrylate All 35 compounds were docked into all three pockets using GOLD (polyHEMA) and grown as nonadherent tumorspheres, as 5.2.2 (www.ccdc.cam.ac.uk). The docking centers were set as previously described (38). To test the activity, small inhibitor follows: for pocket 1 CD of Glu267, for pocket 2 CZ of Arg426, compounds were included in the growth medium at the indi- and for pocket 3 NH1 of Arg469. All docking spheres were set to cated concentrations and supplemented every 3 days. Tumor- 14 Å and protein side chains in the docking spheres were kept spheres were counted and imaged after 7 days of culture with a flexible during docking. GOLD was run using the default settings Leica microscope. The number and size of spheres was deter- for protein and ligand bonds and functional groups. ChemScore mined as assay outcomes. Controls for 100 mmol/L and was used to generate 10 docking poses for each ligand, and the 250 mmol/L were measured in four repeats, whereas the control solutions were re-scored using ChemPLP. for 1000 mmol/L compound was measured in two repeats. PHA665752 and crizonitib were used as positive controls in this assay and showed a clear reduction in mammosphere Results numbers at 0.5 mmol/L and 1 mmol/L (Supplementary Fig. Identification of Met-binding hits from a library of chemical S1). Experiments with 1% DMSO as solvent (negative) control fragments using SPR screening showed no major effect on mammosphere numbers with A fragment library of 1,338 compounds was screened by SPR average numbers ranging from 11 mammospheres (control for against amine-coupled Met567 [a soluble Met construct contain- compounds at 250 mmol/L) to 13.5 mammospheres (control ing the N-terminal b-propeller (Sema) domain and the small for compounds at 1,000 mmol/L and 100 mmol/L). Initial cysteine-rich domain] and against two fragments of HGF/SF, NK1, experiments were conducted at 100 mmol/L, 250 mmol/L, and and SPH domains. In the absence of a validated inhibitor that we 1 mmol/L compound concentration in 1% DMSO. However, could use as positive control, we defined relative response levels of poor solubility of some compounds in media resulted in each fragment by comparing the actual response with the expected precipitation as observed by eye and under the microscope; response in a 1:1 binding scenario. Expected responses were this would reduce the effective concentration of the compound calculated by taking into account the protein's immobilization in the medium. Compounds 10, 25, 33,and34 precipitated at level, molecular weight, and the molecular weight of the respec- 100 mmol/L, 250 mmol/L, and 1 mmol/L and were excluded tive fragment (Supplementary Table S1). From this initial screen, a from analysis. Compound 27 showed precipitation at the two total of 698 fragments were deemed to be Met binders with highest concentrations, 250 mmol/L and 1 mmol/L. For com- relative response levels between 25% and 300% and desired pounds 3, 6, 10, 15, 18, 26, 27,and31, mammosphere assays square sensorgrams (Fig. 1A). This generous cutoff filter allowed were extended to include concentrations of 25 mmol/L and inclusion of fragments that bind in more than one place on the 10 mmol/L. protein and accounted for variations in concentration due to solubility problems. In a second data reduction step, we identified Boyden chamber assays (migration assay) promiscuous binders by comparing relative binding levels for Met HGF/SF-induced cell migration on SKOV-3 cells (a kind gift with levels for SPH and NK1. Unique fragments, defined as those from David Allard, CRUK) was assayed using a modified that bind to one protein with at least a three times greater binding

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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 analysis step 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.

level than to the other two proteins, numbered 121 (Met; Fig. 1A), small pocket that does not allow the binding of larger mole- 71 (NK1), and 2 (SPH). Further analysis of binders to NK1 is cules. In the case of MB1271 and MB621, the increase in affinity published elsewhere (39). of related compounds was generally moderate (Supplementary False positives and non-stoichiometric binders were eliminated Table S2), indicating that the selection of compounds without from the hit list by determining binding levels as a function of structural knowledge of the fragments' binding modes has fragment concentration (Fig. 1B). Equilibrium affinity constants resulted in limited numbers of additional interactions with were determined for each fragment, and 16 showed KD of better the protein. Nonetheless, appreciable increases in binding than 100 mmol/L, whereas 26 compounds exhibited KD between affinity were also observed, for example, compound 31 bound 100 mmol/L and 500 mmol/L. Fragments that did not display a Metwithanaffinity 36-fold higher than its parental fragment binding curve but a linear concentration dependency were MB621, but compound 28 with an affinity about five times rejected from further analysis as these fragments are likely to lower (Supplementary Table S2). stack at the surface of the chip or protein. However, an improved KD alone is not an indication for an In a secondary screen, binding of fragment hits to Met was improved antagonist as the compound could bind to a part of Met analyzed using differential scanning fluorimetry (thermal shift that is not involved in the interaction with NK1. Therefore, it is assay), which determines the shift of the melting temperature essential to determine whether the compound can interfere with (DTm) of a protein upon fragment binding. Out of 134 frag- NK1 binding and moreover to assess its biologic activity. ments screened in this assay, 63 fragments showed a thermal shift of >0.5 K, 40 fragments showed no significant shift D (between 0.5 K and 0.5 K thermal shift), 31 showed a shift Table 1. Structures, Tm and KD values of parental fragments D m toward lower temperatures. Shifts toward lower temperatures Fragment Structure Tm (K) KD ( mol/L) or uncharacteristically high thermal shifts might be caused by protein aggregation in the presence of high concentrations of MB1271 249 compound, low fragment solubility, or adverse interaction of the fragment with the fluorescent dye. Accordingly, these fragments were excluded from further analysis. Fragments that showed a DTm of 1.5 K or 2 K and a low KD in SPR are likely to be good binders, and five fragments were taken forward. MB738 2 n.d.

Elaboration of fragment hits resulted in enhanced binding affinity Five structurally different fragments were selected for substruc- MB621 1.5 72 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 ¼ 72 mmol/L, DT ¼ 1.5 K), and MB690 (K ¼ 172 mmol/L, DT m D m MB1297 1.5 137 ¼ 1.5 K; Table 1). Thirty-five compounds derived from MB621, MB1271, or MB1297 were purchased based on their structural diversity and availability (Supplementary Table S2), and binding affinities of these compounds were determined using SPR (Fig. 2A and Supplementary Table S2). None of the compounds related to MB690 1.5 172 MB1297 showed a higher affinity to the Met Sema domain than their parental compound, indicating that MB1297 may bind in a Abbreviation: n.d., not determined.

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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 a concentration-dependent binding curve were fitted using a global fit to determine KD values. The binding of the parental fragment MB621 (closed circle) is shown for comparison. B, HGF/SF-stimulated Vero cells show a reduction in phosphorylation levels of Akt and Erk upon compound treatment. Western blots of cell lysates were probed for total protein (left) and phosphorylated protein (right). C, classification of compounds into "active," "weakly active," and "inactive." A reduction in Akt phosphorylation levels of at least 90% can be seen for "active" compounds. "Weakly active" compounds are able to prevent Akt phosphorylation by at least 20%. Shown are average Akt phosphorylation levels and standard deviations from three independent experiments. D, competition of compounds with NK1 for binding to Met928 in SPR. Average values and standard deviations from three experiments were calculated for each compound concentration (1 mmol/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 (open triangles, 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).

Selected small molecule Met binders interfere with HGF/ pounds 16 and 23 to have appreciable toxicity beyond that SF-induced Akt and Erk1/2 phosphorylation and binding incurred by the DMSO control (Supplementary Fig. S2). Com- of NK1 to Met928 pounds that successfully inhibited Akt phosphorylation were Compound-induced inhibition of Akt and Erk1/2 phosphor- generally also able to inhibit Erk phosphorylation considerably, ylation in HGF/SF-stimulated Vero cells was determined using although inhibition of Erk1/2 phosphorylation varied appreci- infrared dyes that could be quantitatively analyzed using an ably with some compounds (Supplementary Table S2). Never- Odyssey scanner (Fig. 2B). Eight out of 35 compounds (23%) theless, the results obtained clearly demonstrated that eight were able to inhibit Akt phosphorylation after stimulation of cells compounds (6, 10, 15, 25, 26, 27, 31, and 33) were able to with HGF/SF to below 10% as compared with the control (Fig. inhibit HGF/SF-induced, Met-dependent Akt and Erk phosphor- 2C). These compounds were deemed "active," whereas eight ylation behaving as bona ﬁde small-molecule Met antagonists. compounds were deemed "not active" (Akt phosphorylation level To investigate whether these biologically active compounds above 80%) and 19 compounds were "weakly active" (Akt phos- might compete with NK1 for its binding site on Met, we carried phorylation levels 10%–80%). Toxicity data revealed only com- out in vitro competition assays using SPR. Generally, compounds

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Table 2. Compounds active in phosphorylation assays show varying Met928 was reduced. Sigmoidal fits of competition data from five effectiveness in inhibiting NK1 binding to Met "active" compounds return Ki values between 500 mmol/L and Akt phosphorylation NK1 competition 1,000 mmol/L (Table 2). Compound inhibition (%) Ki (mmol/L) 10 2.9 12.6 904 15 9.2 9.8 679 Selected small-molecule Met binders inhibit the growth of 25 6.4 11.9 877 mouse tumorspheres and the migration of carcinoma cells 26 2.2 12.8 10,723 Tumorsphere assays that use single-cell suspensions derived 31 5.9 1.8 484 from mouse mammary gland tumors caused by aberrant Wnt and Met signaling (38) were used to determine whether the identified compounds were able to inhibit tumor growth (Fig. 3A–I). that showed no effect in phosphorylation assays did not reduce Compound 6 showed no effect at 1,000 mmol/L and was binding of NK1 to Met928 in this assay (Fig. 2D). This confirms therefore considered "negative" in this assay. In contrast, com- these compounds as "not active." However, in the presence of pounds 18 and 31 completely abolished mammosphere forma- "active" compounds 15, 10, 25, 26, and 31 binding of NK1 to tion at 1,000 mmol/L. Compounds 1, 18, and 31 showed a

Figure 3. Compounds inhibit tumor progression and cell migration. A–I, picture of one representative mammosphere from tumorsphere assays with a 250 mmol/L compound concentration: 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 in mammosphere size (see E, F, and H) as compared with DMSO control (A). J, outcome of tumorsphere assay as normalized mammosphere number per tested concentration. Lower mammosphere numbers indicate inhibition of tumor growth and progression. Averages and error bars are from three different experiments. IC50 values could be derived for compounds 15 (closed circle, solid line, IC50 ¼ 534 mmol/L), 31 (closed triangle, long dashed line, IC50 ¼ 205 mmol/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/L HGF/SF in the presence of 1 mmol/L compound. Residual fluorescence (mean of 4 replicates per assay) of migrating cells was quantified and normalized to migration in the presence of diluent only. Graph shows normalized migration from three independent experiments with bars representing the mean for each compound. Compounds 15 and 26 resulted in statistically significant inhibition compared with diluent-only control (P < 0.05) in all three experiments (two-way ANOVA with Dunnett multiple comparisons test).

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reduction in mammosphere numbers at concentrations as low as phosphorylation assay suggests that an upstream inhibition of 100 mmol/L. Compounds 15 and 31 showed >50% inhibition at Met resulted in a lack of signal transduction, which in turn 250 mmol/L and were therefore considered as "positive." Com- prevented migration of SKOV3 cells toward HGF/SF. Compound pound 31 was the only compound that inhibited mammosphere 15 was also effective in mammosphere assays, suggesting inhib- numbers well below 50% of control at a concentration of 100 itory effects in both tumor growth and cell migration. mmol/L (Fig. 3J). Compounds that reduce mammosphere formation with statistical significance, as determined by a t test (P < Docking of compounds to Met Sema domain indicates different 0.05), are compounds 31 (at 250 mmol/L and 1,000 mmol/L) and modes of actions 3 and 18 at 1,000 mmol/L. Compound 15 has a P value of around There are three crystal structures available containing parts of 0.1 to 0.12 at the various concentrations, which is probably due to the Met extracellular region: a Met25–567/SPH cocrystal structure the differences in the replicates, which affects the standard devi- (PDB code 1SHY; ref. 30) and two Met25–741/internalin B36-321 ation. In addition to an inhibition of mammosphere formation cocrystal structures (PDB codes 2UZX and 2UZY; ref. 40). Cross- from single tumor cells (mammosphere number), there is also a linking experiments with NK1 and the Met Sema domain revealed clear effect of some compounds on mammosphere size, which two cross-links involving Ser309 and Glu395 of Met (L. Kemp; indicates an effect on cell proliferation, for instance compounds unpublished results). One of the cross-links, Ser309, lies very near 18, 27, and 26 (Fig. 3E–H, respectively). Therefore, in this assay, to the region marked as a footprint for internalin B (as Ser309 is only compounds 15 and 31 were considered to show antitumori- not resolved in either crystal structures, Lys311 is indicated m genic properties with preliminary IC50 values of 534 mol/L in Fig. 4A). This region also harbors three loops that are not (compound 15) and 205 mmol/L (compound 31). resolved in either crystal structure: the first loop from Ile377 to In order to assess whether compounds would act on the Asn382, the second from Thr301 to Lys311, and the third from migratory behavior of cells, migration assays were conducted at Arg400 to Asp414. It is conceivable that these loops might be 1 mmol/L and 250 mmol/L compound concentration. Com- involved in NK1 binding, which is consistent with the crystal pounds 15 and 26 inhibited cell migration to 37% and 60% at structure of the high-affinity binding site of the NK1 fragment of 1 mmol/L concentration, respectively (Fig. 3K). The ability of both HGF/SF on the Sema domain of the Met receptor defined subse- compounds to prevent phosphorylation of Akt and Erk in a 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)is shown as green cartoon representation. The potential binding site for NK1 lies partially within the footprint of internalin B (magenta) and two cross-linking sites (side chains in cyan sticks). Potential pockets that have been identiﬁed by PocketFinder that could accommodate a chemical compound are highlighted in blue and 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 that pocket 1 is largely electronegative, whereas pockets 2 and 3 are mostly surrounded by uncharged areas. Electronegative areas are represented in red and electropositive 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 similar interactions involving R384. In addition, compound 10 interacts with H275 at the top of the pocket, and compound 15 engages in hydrophobic interactions at the right side of the pocket. D is a rotation of C by 90. E and F, overlay of compounds 10 (pink) and 15 (yellow) in pocket 3 shows that both compounds form hydrogen bonds to R469 as well as hydrophobic interactions to M431 and P472. F is a rotation of E by 90. Figures prepared with PyMol.

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protein internalin B from L. monocytogenes competes with NK1 for Ala423 gave rise to hydrophobic interactions. Compounds 10 and binding to Met, indicating that the binding sites of internalin B 15 were deemed "active" in phosphorylation assays, indicating and NK1 on the Met Sema domain overlap extensively (E. that this pocket might be a target for inhibitors. Gherardi, unpublished observations). The second pocket is a largely nonpolar, flat groove, presenting Using PocketFinder we found two potential pockets within, or a single small pocket at one end. Compound 24 had the best near to the proposed binding site (see blue and red space fills docking score, followed by compounds 34 and 22. The top three in Fig. 4A). One is located just above the two helices flanked by scorers all show limited effectiveness in phosphorylation assays, residues Arg469, Met431, and Pro472 (red space fill) and has a and the highest-ranking "active" compound for this pocket is pocket volume of 74 Å3. The second is larger with a volume of 125 compound 6 (sixth rank). This suggests that this pocket may not Å3 and is enclosed by the disordered region leading up to strand E be suitable for accommodating any of the active compounds. This of the fourth blade, residues 382 to 400 (blue space fill). This loop pocket also lies slightly outside the area spanned by the two cross- also harbors one of the cross-linking sites, Glu395, and is ordered linking sites, Ser309 and Glu395. Therefore, this site is unlikely to into a short helix in the internalin B cocrystal structure 2UZX. be suitable for developing an antagonist. However, due to the anticipated disorder in this region and lack of The third pocket (Fig. 4A, red spheres) is relatively small and structural information in parts of the proposed pocket we decided shallow. It harbors an acidic aspartate residue at the bottom to focus our docking efforts elsewhere. Manual inspection (Asp428) and presents a deeper, nonpolar subpocket to one side. revealed two additional grooves within the proposed footprint Parts of this pocket are disordered in the crystal structures, and of NK1 that might be able to accommodate chemical compounds. residues 302 to 310 were not observed. On the other side of this The first is a large pocket near Lys 311, surrounded by residues pocket is a small loop containing Arg469, which was used as Arg384, Glu267, Val313, Glu297, and Pro295, and has an elon- docking center. Docking compounds in pocket three revealed gated shape with a slight kink (pocket 1, Fig. 4B). The second, compound 10 as highest scoring, followed by compounds 15 and surrounded by residues Arg469, Arg426, and Tyr369 (pocket 8. All three compounds are related, being filial compounds to the 2, Fig. 4B), is small and lies near one of the pockets found by fragment MB1297. Moreover, compounds 10 and 15 were iden- PocketFinder. The first pocket lies within a large electronegative tified as "active" in phosphorylation assays and compound 8 as patch of the protein whereas the second lies in a largely hydro- "weakly active." These observations indicate that the inhibitors phobic region. Compounds bound to either pocket could possi- investigated here might target this pocket. bly impair or inhibit binding of NK1 to Met and therefore prevent In summary, the active compounds 10 and 15 scored high in receptor dimerization and thus activation of the signaling cascade, docking to pockets 1 and 3 (Table 3). In competition assays, including activation of Akt and Erk. compounds 10 and 15 inhibited NK1 binding to 60% and 17%, respectively. This indicates that pocket 1 and/or pocket Docking of compounds into the proposed Met–NK1 interface 3 might be situated in the NK1 binding interface and "active" We used GOLD to dock the compounds into all three pockets. compounds might bind to it to disrupt NK1 binding. Addi- Residues within the pockets were chosen as docking centers, for tionally, compound 15 was active in a tumorsphere assay and a pocket 1 Glu267, for pocket 2 it is Arg426, and for pocket 3 Arg469 migration assay, indicating that this compound may act as a (Fig. 4B). Using a docking sphere of 14 Å, 10 docking poses were Met antagonist that is able to inhibit tumor formation and cell evaluated for each ligand using ChemScore and the solutions re- migration. However, both compounds bind with relatively low scored using ChemPLP. All docking results are tabulated in affinity, namely 879 mmol/L for compound 10 and10mmol/L Supplementary Table S3, and top-scoring poses of the three for compound 15. The low binding affinity measured in SPR highest-ranking compounds are shown in Supplementary Fig. S3. experiments is puzzling and might indicate very inefficient The first pocket is flanked by several acidic residues such as binding and a requirement for high concentrations in order Glu267, Glu419, and Glu297. Compound 10 had the highest to display its inhibitory effect. All assays used a large excess of docking score, followed by compounds 22 and 15. The com- compound, with some showing low solubility in assays pounds formed hydrogen bonds to the side chain of Arg384 and that would have reduced the availability of compound mole- Glu297, as well as Thr276 or Thr273, and Val313, Gln425, and cules. On the other hand, it might also be possible that this

Table 3. Summary of results from different assays of active compounds Level of NK1 Inhibition of Level of cell Docking results, ranking a b c d Compound KD (mmol/L) binding (%) tumorigenesis migration (%) Pocket 1 Pocket 2 Pocket 3 6 63 100 No n.d. 29 6 24 10 879 60 No n.d. 1 15 1 15 10,540 17 Yes 37 3 31 2 25 192 60 No No 20 10 28 26 406 60 Yese 60 26 27 29 27 76 100 Yese No 7 11 26 31 2 48 Yes n.d. 14 18 22 33 80 100 No No 23 16 9 Abbreviation: n.d., not determined. a Against Met567 in SPR. b To Met928 in competition assays at 1 mmol/L compound. cActivity assessed in tumorsphere assay. dActivity assessed in migration assay. eWith additional antiproliferative properties.

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compound binds Met in several different places on the protein, NK1–Met interaction whereas others are likely to be allosteric which would mean a reduced presence of molecules in any one inhibitors. pocket. Interestingly, compound 31 wasnotfoundinthetopof Our study further uncovered strategies for compound devel- the docking rankings for any pocket, although it bound Met opment and optimization. Compounds 10 and 15 undergo with low micromolar affinity, was effective in competing with similar interactions with Met in pocket 1 (Fig. 4C and D), mainly NK1 for Met binding and in inhibiting tumor formation. This forming hydrogen bonds to Arg384 and Glu297, and engaging the might indicate that this compound may bind to a different site right-hand side and top of the pocket in hydrophobic interactions. on Met and distort the NK1 binding site and may therefore be Combining both compounds could produce a compound better considered as an allosteric inhibitor. Compound 26 was also suited to occupy all available space in pocket 1. The interaction active in tumorsphere assays, but its ability to compete with between cyano-N of compound 10 with the amino-nitrogen NK1 for Met binding was limited. This compound showed an of His275 could be preserved as well as interactions with the affinity for Met in the low- to mid-micromolar range, but its main chain amide of Thr276 and main chain carbonyl of Thr421. low ranking in the docking experiments suggests that it may In pocket 3, both compounds undergo an interaction with bind slightly outside the NK1 binding site. However, com- Arg469: compound 10 with its carbonyl group and compound pound 26 is still able to inhibit Met signaling sufficiently to 15 with its nitrogens as hydrogen-bond acceptors (Fig. 4E and F). elicit a cellular response. Compound 10 also engages the protein via hydrophobic inter- Binding modes of these compound on Met need to be evalu- actions to Pro472 and Met431. Additionally, one of its carbonyl ated further using structural studies in order to assess whether groups engages Ser470 in a hydrogen-bond interaction. compound 15 is indeed a direct inhibitor of this interaction, and In conclusion, the fragment-based approach to design inhibi- whether compound 31 is an allosteric inhibitor. tors of PPIs without tethering the fragments is a new development in the field of drug discovery that has recently led to one marketed drug, vemurafenib, for treatment of metastatic melanoma (42). Discussion Fragments are well suited to efficiently explore the small pockets Modulators of protein interfaces are therapeutically relevant characteristic of protein–protein interfaces and may lead to the and selective, which makes them attractive targets in drug discovery of new scaffolds and chemotypes, as already suggested discovery (41, 42). Many essential cellular functions are carried by Wells and McClendon (47). We have demonstrated here that out by multiprotein assemblies, and so it is not surprising that fragments can be used as good starting points for a de novo drug- many human diseases, such as neurodegenerative diseases (43) discovery campaign even when no crystal structure of the target and metabolic diseases (44) as well as cancer (45), can be interface is available. The need for expanding fragment-based caused by aberrant PPI. The development of novel chemical drug discovery to targets where no crystal structure is available has moieties targeting PPIs has recently turned previously "undrug- already been recognized (51). We also showed that the applica- gable" targets into tractable macromolecular sites (42, 46, 47), tion of similarity searches enables a progression from fragment and examples for successful campaigns can be found in the hits to possible lead compounds with a modest requirement of literature (42, 48, 49). resources. We have reasons to believe that applying this approach In this study, we aimed to find small molecules that inhibit the to so-called "undruggable" targets in future might open up the interaction between Met and NK1 using a fragment-based drug-discovery field to new classes of proteins and reach out to approach. The large number of promiscuous binders found in new disease targets. our initial fragment screen reflects the challenges faced when no known inhibitor/small molecule binder or control protein con- Disclosure of Potential Conflicts of Interest taining a disabled binding site can be utilized (50). Therefore, T.L. Blundell is a member of the commercial board and the Science Advisory screening campaigns for protein interfaces are likely to require Board of Astex Therapeutics Ltd., has provided expert testimony for MedIm- additional selection steps to ensure identification of unique mune, has a role on the Science Advisory Board for UCB, reports receiving fragment hits (21). The progression from fragment hit to lead commercial research grant from UCB Celltech, is a consultant for Pfizer, and has fl compound is challenging even in projects when structural infor- provided expert testimony for Ipsen. No potential con icts of interest were disclosed by the other authors. mation is available. In this study, however, high-resolution structural data were not available to verify fragment binding and determine their binding mode. Authors' Contributions In order to overcome this, we modified the traditional drug- Conception and design: T.L. Blundell, E. Gherardi Development of methodology: A. Winter, A.G. Sigurdardottir, T.L. Blundell discovery process by including substructure searches and cell- Acquisition of data (provided animals, acquired and managed patients, based assays early on in the pipeline. We utilized substructure provided facilities, etc.): A. Winter, A.G. Sigurdardottir, D. DiCara, G. Valenti, searches to gain first insights into which type of compound Analysis and interpretation of data (e.g., statistical analysis, biostatistics, might be a promising antagonistic lead compound. This computational analysis): A. Winter, A.G. Sigurdardottir, D. DiCara, G. Valenti, enabled us to progress speedily to test compounds in cell- T.L. Blundell, E. Gherardi based and other biophysical assays. An antitumorigenic and Writing, review, and/or revision of the manuscript: A. Winter, A.G. Sigurdar- dottir, D. DiCara, G. Valenti, T.L. Blundell, E. Gherardi antimigratory effect of several of our compounds was corre- Administrative, technical, or material support (i.e., reporting or organizing lated with their ability to disrupt the interaction between NK1 data, constructing databases): A. Winter, T.L. Blundell and Met and subsequently inhibit Met signaling. These are Study supervision: T.L. Blundell encouraging findings, especially as these compounds were generated using similarity searches instead of the traditional Acknowledgments structure-based approach. Docking studies further indicated The authors thank Professor Chris Abell (Chemistry, Cambridge) and that some compounds might act as direct inhibitors of the his colleagues for giving them access to the fragment library. They also thank

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Fragment-Based Drug Design of HGF/SF-Met Antagonists

Dr. Oliver Korb (CCDC, Cambridge) and Dr. Ralf Schmid (University of The costs of publication of this article were defrayed in part by the payment of Leicester) for help with computational methods and feedback on the manuscript. page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Grant Support This work was supported by the European Union project SFMET (FP7), Gates Cambridge Scholarship (A.G. Sigurdardottir), and CRUK (C24461/A12772, Received June 2, 2015; revised October 11, 2015; accepted October 29, 2015; to A. Winter). published OnlineFirst December 28, 2015.

References 1. Cooper CS, Blair DG, Oskarsson MK, Tainsky MA, Eader LA, Vande Woude 20. Fuller JC, Burgoyne NJ, Jackson RM. Predicting druggable binding sites at GF. Characterization of human transforming genes from chemically trans- the protein–protein interface. Drug Discov Today 2009;14:155–61. formed, teratocarcinoma, and pancreatic carcinoma cell lines. Cancer Res 21. Winter A, Higueruelo AP, Marsh M, Sigurdardottir A, Pitt WR, Blundell TL. 1984;44:1–10. Biophysical and computational fragment-based approaches to targeting 2. Park M, Dean M, Kaul K, Braun MJ, Gonda MA, Vande Woude G. Sequence protein–protein interactions: applications in structure-guided drug discov- of MET protooncogene cDNA has features characteristic of the tyrosine ery. Q Rev Biophys 2012;45:383–426. kinase family of growth-factor receptors. Proc Natl Acad Sci U S A 1987;84: 22. Yang RY, Yang KS, Pike LJ, Marshall GR. Targeting the dimerization of 6379–83. epidermal growth factor receptors with small-molecule inhibitors. Chem 3. Bottaro DP, Rubin JS, Faletto DL, Chan AM, Kmiecik TE, Vande Woude GF, Biol Drug Des 2010;76:1–9. et al. Identification of the hepatocyte growth factor receptor as the c-met 23. Scott DE, Coyne AG, Venkitaraman A, Blundell TL, Abell C, Hyvonen M. proto-oncogene product. Science 1991;251:802–4 Small-molecule inhibitors that target protein–protein interactions in the 4. Nakamura T, Nishizawa T, Hagiya M, Seki T, Shimonishi M, Sugimura A, RAD51 family of recombinases. ChemMedChem 2015;10:296–303. et al. Molecular cloning and expression of human hepatocyte growth factor. 24. Muratore G, Goracci L, Mercorelli B, Foeglein A, Digard P, Cruciani G, et al. Nature 1989;342:440–3. Small molecule inhibitors of influenza A and B viruses that act by disrupt- 5. Zarnegar R, Michalopoulos G. Purification and biological characterization ing subunit interactions of the viral polymerase. Proc Natl Acad Sci U S A of human hepatopoietin A, a polypeptide growth factor for hepatocytes. 2012;109:6247–52. Cancer Res 1989;49:3314–20. 25. Christ F, Voet A, Marchand A, Nicolet S, Desimmie BA, Marchand D, et al. 6. Stoker M, Gherardi E, Perryman M, Gray J. Scatter factor is a fibroblast- Rational design of small-molecule inhibitors of the LEDGF/p75-integrase derived modulator of epithelial cell mobility. Nature 1987;327:239–42. interaction and HIV replication. Nat Chem Biol 2010;6:442–8. 7. Gherardi E, Gray J, Stoker M, Perryman M, Furlong R. Purification of scatter 26. Donate LE, Gherardi E, Srinivasan N, Sowdhamini R, Aparicio S, Blundell factor, a fibroblast-derived basic protein that modulates epithelial inter- TL. Molecular evolution and domain structure of plasminogen-related actions and movement. Proc Natl Acad Sci U S A 1989;89:5844–8. growth factors (HGF/SF and HGF1/MSP). Protein Sci 1994;3:2378–94. 8. Schmidt C, Bladt F, Goedecke S, Brinkmann V, Zschiesche W, Sharpe M, 27. Holmes O, Pillozzi S, Deakin JA, Carafoli F, Kemp L, Butler PJ, et al. Insights et al. Scatter factor/hepatocyte growth factor is essential for liver develop- into the structure/function of hepatocyte growth factor/scatter factor from ment. Nature 1995;373:699–702. studies with individual domains. J Mol Biol 2007;367:395–408. 9. Uehara Y, Minowa O, Mori C, Shiota K, Kuno J, Noda T, et al. Placental 28. Lokker NA, Mark MR, Luis EA, Bennett GL, Robbins KA, Baker JB, et al. defect and embryonic lethality in mice lacking hepatocyte growth factor/ Structure-function analysis of hepatocyte growth factor: identification of scatter factor. Nature 1995;373:702–5. variants that lack mitogenic activity yet retain high affinity receptor bind- 10. Bladt F, Riethmacher D, Isenmann S, Aguzzi A, Birchmeier C. Essential role ing. EMBO J 1992;11:2503–10. for the c-met receptor in the migration of myogenic precursor cells into the 29. Blaszczyk M, Harmer NJ, Chirgadze DY, Ascher DB, Blundell TL. Achieving limb bud. Nature 1995;376:768–71. high signal-to-noise in cell regulatory systems: Spatial organization of 11. Schmidt L, Duh FM, Chen F, Kishida T, Glenn G, Choyke P, et al. Germline multiprotein transmembrane assemblies of FGFR and MET receptors. and somatic mutations in the tyrosine kinase domain of the MET proto- Progress in Biophysics and Molecular Biology 2015;118:103–11. oncogene in papillary renal carcinomas. Nat Genet 1997;16:68–73. 30. Stamos J, Lazarus RA, Yao X, Kirchhofer D, Wiesmann C. Crystal structure 12. Lee YH, Apolo AB, Agarwal PK, Bottaro DP. Characterization of HGF/Met of the HGF beta-chain in complex with the Sema domain of the Met signaling in cell lines derived from urothelial carcinoma of the bladder. receptor. EMBO J 2004;23:2325–35. Cancers 2014;6:2313–29. 31. Oelmann S, Stanley P, Gerardy-Schahn R. Point mutations identified in 13. Humphrey PA, Zhu X, Zarnegar R, Swanson PE, Ratliff TL, Vollmer RT, et al. Lec8 Chinese hamster ovary glycosylation mutants that inactivate both the Hepatocyte growth factor and its receptor (c-MET) in prostatic carcinoma. UDP-galactose and CMP-sialic acid transporters. J Biol Chem 2001;276: Am J Pathol 1995;147:386–96. 26291–300. 14. Ichimura E, Maeshima A, Nakajima T, Nakamura T. Expression of c-met/ 32. Chirgadze DY, Hepple JP, Zhou H, Byrd RA, Blundell TL, Gherardi E. HGF receptor in human non-small cell lung carcinomas in vitro and in vivo Crystal structure of the NK1 fragment of HGF/SF suggests a novel mode for and its prognostic significance. Jpn J Cancer Res 1996;87:1063–9. growth factor dimeriztion and receptor binding. Nat Struct Biol 1999;6: 15. Sawada K, Radjabi AR, Shinomiya N, Kistner E, Kenny H, Becker AR, 72–9. et al. c-Met overexpression is a prognostic factor in ovarian cancer and 33. Irwin JJ, Shoichet BK. ZINC–a free database of commercially available an effective target for inhibition of peritoneal dissemination and compounds for virtual screening. J Chem Inf Model 2005;45:177–82. invasion. Cancer Res 2007;67:1670–9. 34. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and 16. Mahadevan D, Theiss N, Morales C, Stejskal AE, Cooke LS, Zhu M, et al. computational approaches to estimate solubility and permeability in drug Novel receptor tyrosine kinase targeted combination therapies for imati- discovery and development settings. Adv Drug Deliv Rev 2001;46:3–26. nib-resistant gastrointestinal stromal tumors (GIST). Oncotarget 2015;6: 35. Teague SJ, Davis AM, Leeson PD, Oprea T. The design of leadlike combi- 1954–66. natorial libraries. Angew Chem Int Ed Engl 1999;38:3743–8. 17. Jung KA, Choi BH, Kwak MK. The c-MET/PI3K signaling is associated with 36. Wear MA, Patterson A, Malone K, Dunsmore C, Turner NJ, Walkinshaw cancer resistance to doxorubicin and photodynamic therapy by elevating MD. A surface plasmon resonance-based assay for small molecule inhibi- BCRP/ABCG2 expression. Mol Pharmacol 2015;87:465–76. tors of human cyclophilin A. Anal Biochem 2005;345:214–26. 18. Ozasa H, Oguri T, Maeno K, Takakuwa O, Kunii E, Yagi Y, et al. Significance 37. Ferraris DM, Gherardi E, Di Y, Heinz DW, Niemann HH. Ligand-mediated of c-MET overexpression in cytotoxic anticancer drug-resistant small-cell dimerization of the Met receptor tyrosine kinase by the bacterial invasion lung cancer cells. Cancer Sci 2014;105:1032–9. protein InlB. J Mol Biol 2009;395:522–32. 19. Jubb H, Blundell TL, Ascher DB. Flexibility and small pockets at protein– 38. Holland JD, Gyorffy B, Vogel R, Eckert K, Valenti G, Fang L, et al. Combined protein interfaces: new insights into druggability. Prog Biophys Mol Biol Wnt/beta-catenin, Met, and CXCL12/CXCR4 signals characterize basal 2015;119:2–9. breast cancer and predict disease outcome. Cell Rep 2013;5:1214–27.

www.aacrjournals.org Mol Cancer Ther; 15(1) January 2016 OF11

Winter et al.

39. Sigurdardottir AG, Winter A, Sobkowicz A, Fragai M, Chirgadze D, Ascher 45. Nero TL, Morton CJ, Holien JK, Wielens J, Parker MW. Oncogenic protein DB, et al. Exploring the chemical space of the lysine-binding pocket of the interfaces: small molecules, big challenges. Nat Rev Cancer 2014;14: ﬁrst kringle domain of hepatocyte growth factor/scatter factor (HGF/SF) 248–62. yields a new class of inhibitors of HGF/SF-MET binding. Chem Sci 46. Overington JP, Al-Lazikani B, Hopkins AL. How many drug targets are 2015;6:6147–57. there? Nat Rev Drug Discov 2006;5:993–6. 40. Niemann HH, Jager V, Butler PJ, van den Heuvel J, Schmidt S, Ferraris D, 47. Wells JA, McClendon CL. Reaching for high-hanging fruit in drug discovery et al. Structure of the human receptor tyrosine kinase met in complex with at protein–protein interfaces. Nature 2007;450:1001–9. the Listeria invasion protein InlB. Cell 2007;130:235–46. 48. Lund G, Dudkin S, Borkin D, Ni W, Grembecka J, Cierpicki T. Inhibition of 41. Garner AL, Janda KD. Protein–protein interactions and cancer: targeting the CDC25B phosphatase through disruption of protein–protein interaction. central dogma. Curr Top Med Chem 2011;11:258–80. ACS Chem Biol 2015;10:390–4. 42. Baker M. Fragment-based lead discovery grows up. Nat Rev Drug Discov 49. Douse CH, Vrielink N, Wenlin Z, Cota E, Tate EW. Targeting a dynamic 2013;12:5–7. protein–protein interaction: fragment screening against the malaria myo- 43. Blazer LL, Neubig RR. Small molecule protein–protein interaction inhibi- sin A motor complex. ChemMedChem 2015;10:134–43. tors as CNS therapeutic agents: current progress and future hurdles. 50. Neumann T, Junker HD, Schmidt K, Sekul R. SPR-based fragment Neuropsychopharmacology 2009;34:126–41. screening: advantages and applications. Curr Top Med Chem 2007;7: 44. Virkamaki A, Ueki K, Kahn CR. Protein–protein interaction in insulin 1630–42. signaling and the molecular mechanisms of insulin resistance. J Clin Invest 51. Hubbard RE, Murray JB. Experiences in fragment-based lead discovery. 1999;103:931–43. Methods Enzymol 2011;493:509–31.

OF12 Mol Cancer Ther; 15(1) January 2016 Molecular Cancer Therapeutics

Developing Antagonists for the Met-HGF/SF Protein−Protein Interaction Using a Fragment-Based Approach

Anja Winter, Anna G. Sigurdardottir, Danielle DiCara, et al.

Mol Cancer Ther Published OnlineFirst December 28, 2015.

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