Targeting receptor tyrosine kinase MET in cancer: small molecule inhibitors and clinical progress.

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Targeting Receptor Tyrosine Kinase MET in Cancer: Small Molecule Inhibitors and Clinical Progress J. Jean Cui* TP Therapeutics, Inc., 6150 Lusk Boulevard, Suite B100, San Diego, California 92121, United States ABSTRACT: The HGF/MET signaling pathway is critical in mediating a wide range of normal physiological functions including embryological development, wound healing, and tissue regeneration. Aberrant activation of the pathway has frequently been found in human cancers via protein overexpression, mutation, gene amplification, and also paracrine or autocrine up-regulation. In addition, the activation of HGF/ MET signaling confers resistance to the effects of cancer treatments. Therefore, inhibition of the HGF/MET signaling pathway has great potential for therapeutic intervention in cancer. Currently, there are three approaches toward modulating HGF/MET signaling in human clinical studies of cancer: anti-HGF monoclonal antibodies, MET monoclonal antibodies, and small molecule MET inhibitors. Preliminary clinical benefit from inhibition of HGF or MET has been reported. This Perspective will provide an overview of the HGF/MET signaling pathway in cancer and then will review the development of small molecule MET inhibitors and their progress in clinical applications. and progression of many types of cancer.3 A number of RTK inhibitors have been successfully discovered and developed to inhibit aberrant RTK signaling in various cancers, including sunitinib and axitinib (Chart 1) which target the VHL-

1. INTRODUCTION Cancer is the uncontrolled growth of abnormal cells in the body. Protein kinases are key regulators for cell growth, proliferation, and survival. The activity, localization, and function of many proteins are tightly controlled by protein kinases via the modulation of protein phosphorylation. Genetic and epigenetic alterations accumulate in cancer cells leading to abnormal activation of signal transduction pathways that drive malignant processes. Pharmacological inhibition of these signaling pathways presents promising intervention opportunities for targeted cancer therapies. There are more than 518 protein kinases encoded by the human genome, and many of them are associated with human cancers.1 The successes of the HER2/neu antibody trastuzumab for the treatment of patients with HER2-positive metastatic breast cancer and of the ABL inhibitor imatinib for the treatment of chronic myelogenous leukemia patients bearing BCR-ABL translocation validated the critical roles of protein kinases in cancers and the “druggability” of the protein kinase family. Identification of abnormal protein kinases that are key drivers of cancer progression and development of pharmacological inhibitors of those kinases are the forefront of research for molecularly targeted therapy. Receptor tyrosine kinases (RTKs) play fundamental roles in cellular processes, including cell proliferation, migration, metabolism, differentiation, and survival. Humans have 58 known RTKs that can be further classified into 20 subfamilies.2 All RTKs share a similar molecular architecture, including a ligand binding extracellular region, a single transmembrane helix, an intracellular regulatory domain (juxtamembrane), and a cytoplasmic tyrosine kinase domain. Enhanced RTK activities caused by point mutation, amplification or rearrangement of the corresponding genes, and autocrine or paracrine liganddependent activation have been implicated in the development © XXXX American Chemical Society

Chart 1. Examples of Receptor Tyrosine Kinase Drugs

dependent VEGF pathway in renal cell carcinoma, erlotinib and gefitinib (Chart 1) which target mutant EGFR in nonsmall-cell lung cancer (NSCLC), and crizotinib (Chart 2) which targets MET, ALK, and ROS1 in NSCLC. Receptor tyrosine kinase MET, also called hepatocyte growth factor receptor (HGFR), belongs to a unique subfamily of receptor tyrosine kinases that also contains the protein kinase RON. Hepatocyte growth factor (HGF), also known as scatter Received: September 16, 2013

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dx.doi.org/10.1021/jm401427c | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Chart 2. Class Ia of Selective MET Inhibitors

Figure 1. Structures of MET and HGF/SF.4b

resistance to the effects of anticancer kinase inhibitors, highlighting another important role for HGF/MET inhibitors in cancer therapy.7 Activation of HGF/MET signaling confers resistance to EGFR and BRAF kinase inhibitors.8−10 Because of the role of aberrant HGF/MET signaling in human oncogenesis, invasion/metastasis, and resistance to cancer therapies, the inhibition of HGF/MET signaling presents great potential in cancer treatment. There are three approaches toward modulating HGF/MET signaling currently in human clinical studies of cancer: the anti-HGF monoclonal antibodies, MET monoclonal antibodies, and small molecule MET inhibitors.4 This Perspective will focus on the development of MET small molecule inhibitors and their clinical applications.

factor (SF), is the high-affinity natural ligand of MET. The HGF/MET signaling pathway is critical for invasive growth during embryo development and postnatal organ regeneration. In adults, it is normally only fully active during wound healing and tissue regeneration processes. However, the HGF/MET axis is frequently reactivated by cancer cells for tumorigenesis, invasive growth, and metastasis.4 HGF and/or MET are expressed at abnormally high levels in a wide variety of solid tumors such as liver, breast, pancreas, lung, kidney, bladder, ovary, brain, prostate, and many other cancers.5 Overexpression of MET and/or HGF is often associated with a metastatic phenotype and poor prognosis.6 In addition, the activation of HGF/MET signaling confers resistance to cancer therapies. HGF up-regulation in the tumor microenvironment may cause B



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Figure 2. HGF/MET signaling.22b

of MET.17 HGF/SF belongs to the plasminogen-related growth factor family (PRGF-1), and is synthesized in cells of mesenchymal origin as a single-chain, inactive precursor (proHGF), which is subsequently converted proteolytically into a disulfide linked active α−β heterodimer HGF. Three serine proteases contribute to the activation of pro-HGF: the soluble HGF activator and the membrane-bound matriptase and hepsin found on MET-expressing cells. Activation of HGF is further balanced by the expression of HGF activator inhibitor 1 (HAI1)18 and HGF activator inhibitor 2 (HAI2).19 The structure of HGF consists of six domains (Figure 1b): the Nterminal domain (N), the four kringle domains (K1−K4) in the α-chain, and the serine protease homology domain (SPH) in the β-chain. The N and K1 domains in the α-chain recognize the Ig3 and Ig4 domains of MET with high affinity that is independent of HGF processing and maturation. Following HGF activation and maturation, the SPH domain in the β-chain interacts with the sema domain of MET with low affinity.20,21 2.3. HGF/MET Signaling. HGF/MET signaling is important in mediating a wide range of normal physiological activities, e.g., embryological development, wound healing, and tissue regeneration. HGF is secreted from cells of mesenchymal origin, is fully activated on the surface of epithelial cells expressing MET, and acts in a paracrine manner with MET kinase. Upon HGF binding, MET is dimerized and transphosphorylated at two catalytic tyrosine residues (Tyr-1234 and Tyr-1235) in the activation loop (A-loop). This leads to autophosphorylation of Tyr-1349 and Tyr-1356 in the multifunctional docking site at the C-terminus and recruitment of multiple cytoplasmic effectors such as GAB1, GRB2, PLC, and SRC.22 GAB1 is a universal docking protein of MET. The phosphorylated GAB1 bound to MET at the plasma membrane further attracts docking molecules such as SHP2, PI3K, CRKL, and others.23 The resulting multiprotein signaling complex activates a series of intracellular downstream signal transduction

2. MET SIGNALING 2.1. MET Structure. MET was first discovered in 1984 as a transforming fusion protein, TPR-MET, in a human osteogenic sarcoma (HOS) cell line after prolonged exposure to the chemical carcinogen N-methyl-N′-nitro-N-nitrosoguanidine.11 The new transforming gene was named as MET after the chemical carcinogen’s name. In TPR-MET, the 3′ region of MET is rearranged with the 5′ region of TPR (translocation promoter region) to produce a hybrid transcript.11 Cloning of the full-length MET cDNA revealed that the encoded protein is a receptor tyrosine kinase.12,13 MET is produced mainly in cells of epithelial origin, first as a single chain precursor of 170 kDa, which undergoes co-translational glycosylation and is then cleaved to yield a mature 190 kDa transmembrane receptor, MET. The mature MET exists as an α−β heterodimer with an extracellular α-chain (50 kDa) disulfide linked to the membrane-spanning β-chain (145 kDa) containing the intracellular tyrosine kinase domain.14 The extracellular region of MET has three functional domains: the N-terminal sema domain (residues 25−514) that covers the whole α-subunit and part of the β-subunit; the cystine-rich (CR) domain (residues 515−561) with four conserved disulfide bonds followed by four immunoglobin-like (Ig) domains (residues 562−922) that connect with transmembrane helix (Figure 1a).15 After the transmembrane helix (residues 923−956), the intracellular segment (residues 957−1390) is composed of three regulation sites: a juxtamembrane domain with a phosphorylation site Tyr1003 that down-regulates kinase activity via internalization; a catalytic region that positively modulates kinase activity via transphosphorylation of Tyr-1234 and Tyr-1235; and a carboxy-terminal multifunctional docking site with two docking tyrosines (Tyr-1349 and Tyr-1356) for the recruitment of transducers and adaptors to relay MET signaling.16 2.2. HGF Structure. Hepatocyte growth factor, also known as scatter factor, is the only known high-affinity natural ligand C



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growth, and survival of sensory neurons.34 The signal transduction adaptor GAB1 plays an essential role in METbased signal transduction pathway. The GAB1-null mice have similar phenotypes to HGF and MET knockout embryos, and the mechanism of recruitment of GAB1 to MET is tissuespecific for different biological outcomes in vivo.35 HGF/MET signaling plays an important role in branching morphogenesis, also known as invasive growth, of epithelial cells. The complex morphogenetic program includes cell spreading, cell−cell dissociation, acquisition of a motile phenotype, migration, settling in a new environment, proliferation, and generation of a new pseudopodium structure.36 The role of HGF/MET signaling in organ regeneration has been studied both in vitro with morphogenetic assays under three-dimensional culture conditions and in vivo with conditional deletion of MET in selected tissues. Although hepatocyte-specific loss of MET appeared not to be detrimental to hepatocyte function under physiological conditions, the adaptive responses of the liver to injury were dramatically affected.37 HGF/MET is activated in hepatocytes for DNA synthesis and cytokine production shortly after partial hepatectomy, indicating the important role of HGF/MET in liver regeneration.38 HGF/MET displays a similar protective activity in the kidney by inducing intense proliferative and antiapoptotic responses in renal epithelial cells to prevent acute renal failure because of tubular necrosis and accelerating kidney regeneration.39 In skin, it is shown both in vitro and in vivo that MET is essential for wound repair.40 Overall, HGF/MET signaling mediates invasive growth and plays important roles in embryonic development and organ formation. In adults, METmediated invasive growth becomes quiescent; however, it can be reactivated during wound healing and organ damage to expand and migrate residual cells to the injured tissues.

pathways including the RAS-MAPK cascade, PI3K-AKT axis, and signal transducer and activator of transcription proteins (STATs) (Figure 2). HGF/MET signaling is regulated tightly under normal physiological conditions. MET expression at the cell surface is finely controlled by extracellular shedding, intracellular cleavage, and ubiquitin-mediated degradation. Similar to other RTKs, HGF-activated MET is internalized through endocytosis.24 The internalized MET, still capable of signaling, is delivered to endosomal and lysosomal compartments and then either degraded or recycled to the plasma membrane.25 The phosphorylated Tyr-1003 residue within the juxtamembrane domain of MET is recognized by the ubiquitin E3 ligase CBL, causing monoubiquitylation at multiple sites of MET. This leads to the delivery of activated MET to the endosomal network and to accumulation on the limiting and internal membranes of multivesicular bodies. The fusion of multivesicular bodies with the lysosomes causes the activated MET to undergo proteolytic degradation.26 Another mechanism of down-regulation of MET expression is ligand- and ubiquitinindependent proteolytic cleavages. The shedding of the MET extracellular domain via ADAM metalloproteases releases a soluble decoy MET fragment, leaving behind a membraneanchored cytoplasmic tail that then undergoes intracellular proteolysis by γ-secretase to release a labile intracellular fragment, followed by further rapid proteasome-mediated degradation.27 The decoy MET fragment interacts with both HGF and full-length MET, leading to further inhibition of MET signaling.28 2.4. Biology and Physiology of MET. High levels of MET have been found in human liver, gastrointestinal tract, and kidney.29 Although a high level of MET mRNA has been detected in the thyroid, the protein was barely detectable. MET protein was also detected in the brain.29 The tissue distribution of MET in adult rats was studied using 125I-labeled rat hepatocyte growth factor as a ligand. High affinity binding (Kd of 20−30 pM) of the labeled HGF was found in the plasma membranes of the liver, spleen, kidney, lung, adrenal gland, pituitary glands, and the thyroid.30 The maximum number of binding sites was 400−3000 sites per nanogram of plasma membrane protein of rat, and the liver had the highest number.30 The broad distribution of MET in several rat organs, and not only in the liver, implies an important role for HGF/ MET signaling in growth control of epithelial cells in tissues other than hepatocytes. The physiological functions of HGF/MET signaling have been elucidated with genetic studies in mice. HGF/MET signaling is essential for the growth and survival of hepatocytes and placental trophoblast cells during development. In HGFand MET-null mutant embryos, the liver size is reduced significantly,31 and the labyrinthine trophoblast cells are also markedly reduced, leading to severely impaired placentas and prenatal death.32 Ablation of the HGF or MET gene results in a complete absence of muscle groups in the mouse embryo, indicating the decisive role of HGF/MET signaling in the control of migration and growth of muscle precursor cells and the generation of skeletal muscle.33 In the embryo, myogenic progenitor cells are released from the epithelial dermomyotome in a MET- and GAB1-dependent manner. HGF/MET signaling controls the epithelial-to-mesenchymal transition (EMT) of these epithelial dermomyotome cells and drives their migration over long distances in embryo.33 In addition, HGF/MET signaling is required for sensory nerve development, axonal

3. MET AND CANCER Sustaining proliferative signaling, evading growth suppression, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis are the well-established hallmarks of cancer.41 Reprogramming of energy metabolism, evading immune destruction, and recruitment of the tumor microenvironment are emerging as additional hallmarks of cancer.41 Receptor tyrosine kinases are the main mediators of the signaling network that transmit extracellular signals into the cell and control many fundamental cell functions. Dysregulation of RTK activities is pivotal to tumorigenesis and cancer progression, which is underscored by the success of anti-RTK drugs, such as trastuzumab, gefitinib, and crizotinib.42 3.1. Up-Regulation of HGF/MET Signaling in Cancers. Cancer cells often hijack the signaling pathways and the genetic programs that orchestrate embryonic development and tissue morphogenesis for the neoplastic dissemination of tumors.43 The HGF/MET signaling pathway is important in mediating a wide range of biological activities via a complex biological program called “invasive growth”.44 Under normal physiological conditions, HGF/MET signaling is tightly controlled by ligand activation at the cell surface, ligand-activated receptor internalization/degradation, and paracrine ligand delivery. Aberrant MET activation is reported in almost all of human malignancies and is often caused by receptor overexpression, gene amplification, gene translocation, gain-of-function mutations, and autocrine or paracrine activation.45 MET activation can cause tumor cells to grow, dissociate from their neighbors, leave D



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MET-activating point mutation Y1253D in patients with advanced squamous cell carcinoma of the head and neck (HNSCC) was associated with distant metastasis-free survival.65 These studies illustrate the importance of activating MET mutations in tumorigenesis, and the mutations within the kinase domain distinctly affect downstream signaling. Some cell lines with point mutations resulting in MET kinase activation are also sensitive and dependent on the presence of HGF for ligand-induced receptor dimerization.66 It remains challenging to distinguish “addictive” oncogenic mutations from “passenger” mutations. In lung cancer, the mutations of MET were predominantly present in the extracellular sema domain and intracellular juxtamembrane domain. It is MET and/or HGF overexpression, but not MET mutations, which correlate with progression of non-small-cell lung cancer.67 The JM domain regulates ligand-dependent MET internalization via phosphorylation of Y1003 to recruit c-CBL, leading to MET ubiquitination and degradation. Mutations of the MET JM domain, e.g., deletion of exon 14, may prevent MET degradation and result in persistent HGF-stimulated signaling that leads to increased transforming activity and tumorigenic potential.68 Overall, MET mutations occur at a lower frequency than other mechanisms of pathway activation, like overexpression, and the mutation’s oncogenic potential for proliferation, survival, invasive growth, and metastasis requires further investigation to guide clinical interventions. Autocrine or paracrine stimulation is one of mechanisms for aberrant MET activation. MET autocrine activation has been shown to play a causal role in the development of malignant melanoma and acquisition of the metastatic phenotype.69 For glioblastoma (GBM), HGF autocrine expression correlated with MET phosphorylation levels in HGF autocrine cell lines and showed high sensitivity to MET inhibition in vivo, while an HGF paracrine environment could enhance glioblastoma growth in vivo but did not demonstrate sensitivity to MET inhibition.70 HGF autocrine GBM tumors with constitutively activated MET could serve as a biomarker for GBM patients who would benefit from MET inhibitor therapy.70 The aberrant expression of HGF is a crucial element in acute myeloid leukemia (AML) pathogenesis that leads to autocrine activation of MET in nearly half of the AML cell lines and clinical samples.71 Although MET is normally expressed on cells of epithelial origin, the MET protein was immunohistochemically detected in myofibroblasts in the invasive area for 53% of the lung adenocarcinomas investigated.72 A significant relationship between myofibroblast MET expression and shortened patient survival was observed, suggesting that the autocrine activation loop of HGF/MET in cancer-stromal myofibroblasts may play a role in invasion and metastasis of lung adenocarcinoma.72 3.2. MET and Anticancer Therapy Resistance. Emerging evidence indicates the important roles of HGF/MET signaling in mediating primary and secondary resistance to cancer therapies. Cancer therapies that deprive tumors of oxygen (e.g., antiangiogenic therapies) can be very effective at reducing tumor burden initially, but this can also cause further tumor hypoxia, leading to diminished therapeutic response and malignant progression, such as regional spread and distant metastases that escape the oxygen-deficient environment and establish new (and more aggressive) clones.73 As described previously, HGF/MET signaling plays important roles in embryonic development. During early pregnancy, the invasion of trophoblast cells in a relatively low-oxygen environment into the deciduas of the uterus is stimulated by the up-regulation of

their original environment by degrading the basement membrane, reach the circulation, survive in the bloodstream, extravasate, and eventually colonize new sites.46 HGF and/or MET are expressed at abnormally high level in neoplastic tissue compared with normal surrounding tissue, especially in the invasive front.47,48 Overexpression of MET frequently results from transcriptional up-regulation in the absence of gene aberrations. Hypoxia is a characteristic of cancer, especially late stage cancer. It has been demonstrated that hypoxia promotes tumor invasion by sensitizing cells to HGF stimulation and activating transcription of the MET protooncogene to produce higher levels of MET.49 Inhibition of MET expression prevents hypoxia-induced invasive growth. Receptor tyrosine kinases have a crucial role in establishing highly choreographed patterns of cellular organization, proliferation, and movement during development and tissue homeostasis.50 Spatial dysregulation of RTKs is likely to contribute broadly to cancer development and may affect the sensitivity and resistance of cancer to pharmacological RTK inhibitors.50 Therefore, MET overexpression can be oncogenic and essential for the maintenance of the cancer phenotype. Overexpression of MET can convert primary human osteoblasts into osteosarcoma cells with the transformed phenotype in vitro and with the distinguishing features of human osteosarcomas in vivo.51 MET amplification has been reported in different human cancers including gastresophageal carcinomas,52 colorectal cancers,53 NSCLC,54 medulloblastomas,55 and glioblastomas.56 An increased MET gene copy number is an independent negative prognostic factor and is associated with aggressiveness and metastasis of cancers.57,58 Although MET activation has primarily been linked with tumor cell migration and invasiveness, MET gene amplification causes protein overexpression and constitutive activation of the kinase domain that is required for cell survival.59 A diverse set of both germline and somatic MET mutations in the tyrosine kinase domain (21 mutations), juxtamembrane domain (5 mutations), and extracellular domain (16 mutations) have been described in many solid tumors, including hereditary and sporadic human papillary renal carcinomas, lung cancer, ovarian cancer, childhood hepatocellular carcinomas, squamous cell carcinoma of the head and neck, and gastric cancer.60,61 Even though next generation sequencing technology is accelerating the discovery of sequence variants, understanding the impact of specific MET mutations on tumorigenesis and tumor development at the molecular, cellular, and organismal levels remains a major challenge. Activating mutations of MET in the tyrosine kinase domain have been identified in both hereditary and sporadic forms of papillary renal carcinoma (PRC)62 and exhibit distinct biological effects. For example, D1228H/N and M1250T mutants showed enhanced kinase activity followed by RAS pathway activation and focus formation, while L1195V and Y1230C mutants activated PI3K more effectively to promote cell survival, soft agar colony formation, and matrix invasion.63 Mutations of RTKs often result in increased kinase activity and transforming potential. However, the requirement of the mutated RTKs for the transformation process may vary. The transforming ability of MET mutations in papillary renal carcinoma has been investigated in genetically engineered mice.64 Mice harboring D1226N, Y1228C, and M1248T/L1193V mutations developed sarcomas with high frequency and some lymphomas, whereas the M1248T mice developed carcinomas and lymphomas. The E



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acquired anoikis resistance and restored the chemosensitivity.82 MET is also characterized as a marker of pancreatic cancer stem cells (CSC), playing a key role in metastasis and chemoresistance in the most lethal pancreatic ductal adenocarcinoma (PDAC).83 The MET inhibitor crizotinib synergistically enhanced the antiproliferative and proapoptotic activity of gemcitabine against PDAC cells, including gemcitabineresistant capan-1 cells.83 Molecularly targeted therapies with genomically defined targets in human tumors have produced remarkable clinical responses in genomically stratified patient populations, even in those with advanced diseases. However, acquired resistance to such treatments in virtually all cases overshadows the success of targeted therapy. The clinical implementation of cancer genome sequencing is leading to a deeper understanding of the genetic basis of acquired drug resistance. The mechanism of drug resistance can be either intrinsic (altering the original target) or extrinsic (compensatory signaling through other pathways).84 Common intrinsic resistance mechanisms to abrogate the effectiveness of kinase inhibitor drugs include target gene amplification and the development of secondary mutations.85 In extrinsic resistance, cancer cells respond to chronic drug treatment by adapting their signaling circuitry, taking advantage of pathway redundancy and routes of feedback and cross-talk to maintain their function and eventually develop drug resistance.86 Up-regulation of HGF/MET signaling has been frequently reported as a compensatory signaling pathway that confers resistance to kinase targeted therapies. MET amplification has been detected in 4−20% of NSCLC patients with the EGFR mutations who acquired resistance to gefitinib or erlotinib treatment.8,87 The resistance mechanism is associated with the activation of ERBB3/PI3K/AKT signaling. Analysis of lung cancer patients and cell lines using the highthroughput FISH method indicated that before exposure to EGFR inhibitor drugs, the subpopulation of cells with MET gene amplification was less than 1% of the entire population and that clonal selection of cells with pre-existing MET gene amplification occurs during treatment with the drugs, conferring resistance.88 Up-regulation of ligand HGF represents another mechanism of EGFR-TKI resistance. High HGF expression was discovered among clinical specimens with acquired resistance that did not have a T790M mutation or MET amplification, as well as among cases that exhibited primary resistance despite having EGFR-TKI sensitive activating EGFR gene mutations.89 HGF mediates EGFR TKI resistance in a distinct mechanism by rescuing both PI3K/ AKT and ERK signaling through the signaling adaptor GAB1.88,89 In addition, HGF accelerates MET amplification in HCC827 cells both in vitro and in vivo in the presence of EGFR inhibition.88 EGFR-targeted monoclonal antibodies cetuximab and panitumumab are an effective treatment for KRAS wild type colon cancer, which develop drug resistance through emergence of KRAS mutations in approximately 50% of the cases.90 Amplification of MET is associated with acquired resistance to cetuximab or panitumumab in metastatic colorectal cancer patients who do not develop KRAS mutations during anti-EGFR therapy.90 In addition, patient-derived xenografts demonstrated that MET amplification plays an important role in mediating primary and secondary resistance to anti-EGFR therapies in colorectal cancer. Growth-factordriven resistance from the tumor microenvironment represents a potential common mechanism for reducing the efficacy of anticancer kinase inhibitors.7 HGF is present in stromal cells of

MET that is directly induced by hypoxia-inducible factor 1 (HIF-1) α.74 The transcription factor HIF-1α is a major regulator of tumor cell adaptation to hypoxic stress. MET is upregulated by HIF-1α in a hypoxic environment and is associated with advanced cancer and metastasis. MET up-regulation has been associated with resistance to various cancer treatments including radiation, chemotherapies, and kinase targeted therapies. Because its location can be carefully controlled, radiotherapy is one of the most effective and least toxic treatments for many solid tumors. However, some of tumor cells become resistant to radiation and cause relapse and systemic dissemination of disease through induction of the epithelial−mesenchymal transition.75 Elevation of MET expression after radiotherapy has been noted in various tumors and is known to contribute to treatment resistance.75 MET expression has been shown to increase up to 5-fold after radiation treatment, and tumor cells that survived irradiation subsequently became more invasive. MET inhibition counteracted this increased invasiveness and promoted apoptotic death of tumor cells.76 MET expression was reported to be correlated to the resistance of oropharyngeal cancer to ionizing radiation and inversely correlated with failure-free survival.77 As a prognostic marker, high levels of MET expression predicted poor 5-year overall survival rates for patients with nasopharyngeal carcinoma treated with radiotherapy.78 Cytotoxic chemotherapy was the dominant cancer treatment paradigm in the 1970s and 1980s and is used as first-line treatment against many different forms of cancer even today. The relative sensitivity of neoplastic cells to DNA damaging agents varies and changes over the course of cancer therapy. Several distinct molecular mechanisms have been reported to be involved with chemoresistance, including drug efflux by ATP binding cassette (ABC) transporters, increased DNA repair, dysregulation of the apoptotic machinery, enhanced cytoplasmic inactivation of drug metabolites, and overexpression of oncogenes or down-regulation of tumor suppressor genes. A growing body of evidence has linked HGF/MET signaling with resistance to DNA-damaging agents. For example, HGF protects Burkitt’s lymphoma cell lines which express MET from death induced by DNA damaging agents commonly used in tumor therapy.79 The mechanism is associated with the inhibition of doxorubicin- and etoposide-induced suppression of the antiapoptotic proteins Bcl-X(L). Pretreating U373 human glioblastoma cells with recombinant HGF partially abrogated their cytotoxic responses to γ irradiation, cisplatin, camptothecin, adriamycin, and paclitaxel in vitro because MET receptor activation led to activation of the PI3K- and AKTdependent antiapoptotic pathways.80 Ovarian cancer is one of the most aggressive female reproductive tract tumors, and paclitaxel is widely used for its treatment. It was found that lower levels of miR31 and higher expression of MET in human ovarian cancer specimens were significantly correlated with paclitaxel chemoresistance and poor prognosis.81 MiR31 was found to bind to the 3′-UTR of mRNA of MET to regulate MET expression. This miR31-dependent regulation of MET for chemoresistance of ovarian cancer raises the possibility that combination therapy of paclitaxel with a MET inhibitor will increase paclitaxel efficacy.81 Ovarian cancer cells were shown to acquire a remarkable resistance to anoikis and apoptosis after exposure to clinically relevant doses of two front-line chemotherapeutic drugs, cisplatin and paclitaxel, when grown in three-dimensional cultures.82 Inhibition of MET blocked the F



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Figure 3. apo-MET crystal structures: (A) PDB code 1R1W and (B) PDB code 2G15.

strategies for ERBB2+ breast cancer patients with high MET expression. Harbinski et al. reported a high-throughput platform for systematically screening a cDNA library encoding 3432 secreted proteins in cellular assays for the potential RTKs to compensate in driving cancer growth.93 Ligand-mediated activation of alternative receptor tyrosine kinases was observed in cancer cells originally dependent on MET, FGFR2, or FGFR3. The studies indicated a broad and versatile potential for RTKs from the HER and FGFR families as well as MET to compensate for loss of each other and indicate that combined inhibition of simultaneously active RTKs can lead to an added anticancer effect.93

melanoma patients and correlates with a poor response to treatment with the BRAF inhibitor vemurafenib. The upregulation of stromal HGF also confers resistance to the BRAF inhibitor ramurafenib in BRAF mutant melanoma cells.10 Coactivation of HGF and/or MET with other protein kinases has been reported in many cancers and may represent innate or intrinsic resistance to cancer therapies. Kentsis et al. used a loss-of-function RNA interference genomic screen on AML cell lines and on clinical samples to identify aberrant expression of HGF as a crucial element in AML pathogenesis, and the autocrine activation of MET occurred in nearly half of the studies.71 Treatment of the leukemic cells having aberrant HGF expression and MET activation with crizotinib led to acquired resistance in a short period of time because compensatory up-regulation of HGF expression allowed restoration of MET signaling. It was discovered that FGFR1 activity was required for compensatory up-regulation of HGF in response to MET inhibition in KG-1 cells bearing FOP2FGFR1 chromosomal translocation. The combination of crizotinib with the FGFR1 inhibitor N-[2-[[4-(diethylamino)butyl]amino-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin7-yl]-N′-(1,1-dimethylethyl)urea (PD-173074)91 led to sustained inhibition of MET phosphorylation in KG-1 cells and to near-complete regression of AML in vivo, whereas crizotinib or the FGFR1 inhibitor as single agents only produced very moderate inhibitory effects. The simultaneous blockade of MET activation with crizotinib and of compensatory HGF upregulation with the FGFR1 inhibitor generates highly synergistic efficacy both in vitro and in xenograft models in vivo.71 Clearly, blocking adaptive cellular responses that drive compensatory ligand expression is necessary to achieve optimal and sustained antitumor effects. Trastuzumab is an effective treatment for ERBB2 positive breast cancer patients. However, near half of the breast cancer patients that overexpress ERBB2 are either nonresponsive to trastuzumab or develop resistance. Paulson et al. reported that MET and ERBB2 are coexpressed in ERBB2+ breast cancers and contribute to innate resistance to trastuzumab treatment.92 A significant percentage (45%) of tumors coexpress MET and ERBB2 in ERBB2+ breast cancers. MET depletion resulted in increased ERBB2 activation, and conversely ERBB2 depletion resulted in increased MET activation. Activation of EGFR and ERBB3 was not observed after MET or ERBB2 depletion.91 These studies have significant clinical implications on potential combination

4. PROTEIN STRUCTURE OF THE UNPHOSPHORYLATED MET KINASE DOMAIN MET is a receptor tyrosine kinase with activity that is highly dependent on the phosphorylation state of its catalytic domain. Autophosphorylation of the catalytic domain proceeds through sequential phosphorylation events on the activation loop at Tyr-1235 and then Tyr-1234, leading to a large enhancement in catalytic power (kcat/Km = 425 000 s−1 M−1).94 The Km of ATP for the activated MET was 36 μM for the immunoprecipitated MET from cells and 70.4 μM for the recombinantly expressed cytoplasmic domain of MET.94a MET variants with activating mutations have been shown to autophosphorylate faster than the wild type and to achieve the same maximum activity because the second autophosphorylation at Tyr-1234 is no longer required for the full activation of mutant MET, suggesting a lower threshold for kinase activation than wildtype MET.94 The increased autophosphorylation rate is based on the enhancement in kcat, but not Km for any substrate, which indicates that the effects are not substrate-specific.94a Therefore, the mutant MET provides an oncogenic predisposition, and sustained ligand stimulation or receptor overexpression is required to achieve a fully transformed phenotype.94b The crystal structure of the unphosphoryalted MET kinase domain was solved by two groups using different protein constructs (Figure 3).95,96 In general, the MET kinase domain follows the canonical bilobal protein kinase architecture with an N-terminal, mainly β-sheet-containing domain linked through a hinge segment to the mainly α-helical C lobe. Schiering et al. reported a unique autoinhibitory conformation of the MET kinase domain using the unphosphorylated but enzymatically G



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type I kinase inhibitors with a U-shaped binding mode at the ATP binding site. In general, class Ia MET inhibitors exhibit relatively good cellular selectivity profiles with a limited number of off-target kinase hits. Crizotinib (3), a prototype compound of class Ia, is a multitargeted kinase inhibitor of MET, ALK, ROS1, and RON.99 The U.S. Food and Drug Administration granted fast-track approval of crizotinib in August 26, 2011, and full approval in November 20, 2013, based on the marked efficacy by patients with ALK-positive advanced NSCLC and its good safety profile. 3-Substituted indolin-2-ones were discovered as a class of potent kinase inhibitors with a representative drug sunitinib approved for GIST and RCC treatment.100 The selectivity of indolin-2-ones against particular kinases is mediated by substituents on the indolin-2-one core. 4-Substituted indolin2-ones101 and 5-substituted indolin-2-ones102 were explored for MET inhibition leading to the discovery of 1 (SU11274) with an enzymatic IC50 of 10 nM against MET and a complete inhibition of HGF-induced MET autophosphorylation at 1 μM.103 Compound 1 was an important tool in late 1990s to validate MET biology and establish assays to measure inhibition of a variety of HGF/MET-dependent cellular processes, including cell proliferation, motility, invasion, and morphogenesis.103 Structural modifications of 1 led to the discovery of 2 (PHA-665752), a selective MET inhibitor with significantly improved cellular potency (IC50 = 9 nM in GTL16 cell line) and selectivity (>50-fold for MET compared to a panel of diverse tyrosine and serine/threonine kinases).104 Studies of 2 in vitro showed it potently inhibited MET phosphorylation, in either HGF-stimulated or constitutively activated MET cell lines. It also inhibited downstream signal transduction of MET and development of HGF/MET driven phenotypes (e.g., cell growth, cell motility, invasion, and morphology) in a variety of tumor cells.104 The MET kinase domain has unique structural features that can be used for the design of selective MET kinase inhibitors. The cocrystal structure of 2 bound to the unphosphorylated MET KD revealed unique binding interactions (Figure 4, PDB code 2WKM).99 In the complex with 2, the MET KD adopts the same autoinhibitory conformation observed previously in the crystal structures of the apo-enzyme and in a complex with the staurosporine analogue K252a.95 In these MET crystal

active Y1194F, Y1234F, and Y1235D mutant construct (1049− 1360) (Figure 3A, PDB code 1R1W).95 The mutant construct was designed to mimic the negative charge of the Y1235 phosphotyrosine. Indeed, the specific activity of the mutated protein was found to be comparable to that of the wild-type MET using gastrin as substrate, indicating that the mutant construct is able to adopt an active A-loop conformation. However, an inactive kinase conformation was observed in the apo-MET crystal structure that shares certain similarity with the inactive conformation of unphosphorylated insulin receptor kinase (IRK).97 The salt bridge between residues Lys-1110 and Glu-1127 typically observed in the active kinase conformation is not present. Similar to IRK, an inactive A-loop conformation (magenta color in Figure 3A) is observed with the segment blocking peptide substrate binding. The segment 1223−1226 at the N terminus of the A-loop forms a unique type II′ β-turn to bulge up to the N lobe that constrains the α-C helix in an inactive orientation that does not allow proper catalytic positioning of Glu-1127, and residues 1228−1230 are in a position that would interfere with nucleotide binding. The unique bulged conformation of the A-loop of the unphosphorylated apo-MET kinase domain is consistent with the reported structure of the wild-type, unphosphorylated MET in complex with an oxindole inhibitor bound in the ATP cleft in a patent application,98 indicating that this conformation is a unique structural feature of MET and could be used for the design of selective MET inhibitors. Wang et al. reported a crystal structure of the apo-MET tyrosine kinase domain at 2.15 Å resolution using the wild-type, unphosphorylated protein produced in bacteria by PTP coexpression (Figure 3B),96 which is dramatically different from the mutant MET structure A reported by Schiering et al. The unique type II′ β-turn bulge at the N-terminus of the Aloop is consistent in both structures. However, the subsequent structures of the activation loop and the whole P-loop differ significantly. In the apo-MET structure B, the A-loop forms a series of autoinhibitory interactions with the P-loop, with the αC helix, and with itself. Met-1229, a point of divergence between the A-loops in the two crystal structures, here projects into the ATP-binding pocket and is sandwiched between the Ploop residue, Phe-1089, and the side chain of Lys-1110, which pushes away the P-loop compared to the apo-MET structure A. The P-loop conformation is further stabilized through interactions with Tyr-1230 and Lys-1232, as the activation loop turns in the direction of the α-C helix and then traverses to the lower lobe. Tyr-1234 and Tyr-1235, the two phosphorylation sites at the A-loop required for MET activation, project in opposite directions, with Tyr-1234 pointing inward and forming a hydrogen bond with catalytic residue, Glu-1127, and with Tyr-1235 pointing to solvent.96

5. MET SMALL MOLECULE INHIBITORS Pharmaceutical strategies to regulate the HGF/MET pathway include HGF antagonists (NK2 and NK4), anti-HGF antibodies, anti-MET monoclonal antibodies, and MET small molecule inhibitors. Several classes of small molecule MET inhibitors with different binding modes and selectivity profiles have been discovered and progressed to clinical development. In this section, MET small molecule inhibitors are grouped into four classes (classes Ia, Ib, II, and other) according to their binding mode with MET. 5.1. Class Ia of Selective MET Inhibitors. Small molecule MET inhibitors are categorized as class Ia (Chart 2) if they are

Figure 4. Cocrystal structure of 2 bound to the kinase domain of unphosphorylated MET. The backbone trace of the activation loop is highlighted in cyan and hydrogen bonds are indicated as dashed lines (PDB code 2WKM).99 H



Perspective

structures, the beginning of the kinase activation loop (residues 1222−1227) forms a turn that wedges between the β-sheet and the α-C-helix. Consequently, the activation loop significantly displaces the α-C-helix from a catalytically competent position and the downstream activation loop residues (1228−1245) occupy a position that interferes with ATP and substrate binding. This unusual kinase activation loop conformation creates a unique inhibitor binding pocket that presents an opportunity for the design of selective inhibitors. Furthermore, the binding conformation of 2 in the MET KD is surprising. Instead of passing through the gatekeeper to the hydrophobic back pocket, the benzylsulfonyl group folded over to interact with Tyr-1230. This rationalizes the dramatic improvement in cellular potency when the N-methylsulfonamide linker in 1 was replaced with a more flexible methylenesulfonyl linker in 2. To make space for the benzyl group, the plane of the oxindolepyrrole system in 2 is tilted away from the MET Ploop significantly (Figure 4). This lower inhibitor position in MET is permitted by the flexible (and compressed) Met-1211 and is stabilized by the critical π−π stacking interaction between the inhibitor benzyl group and the Tyr-1230 of the Aloop. A hydrogen bond between the oxygen of the sulfonyl group and the backbone N−H of Asp-1222 is also stabilizing this orientation. Although 2 demonstrated potent and selective inhibition of MET autophosphorylation and related biological functions in both in vitro and in vivo studies, its poor pharmaceutical properties (low solubility, high metabolic clearance, and poor permeability) limited its further development for human clinical studies.99 On the basis of the unique crystal structures of apo-MET and the MET complex with 2, a 2-amino-5-aryl-3-benzyloxypyridine series was designed for more efficient interaction with unphosphorylated MET KD. The monoaromatic 2-aminopyridine was designed to replace the indolin-2-one of 2 as a hinge binder and to allow the flexible 3-benzyloxy group to reach the same hydrophobic pocket occupied by the 2,6dichlorophenyl group in 2 but from a more direct angle that required less molecular weight and conformational constraint. Lead optimization of the 2-amino-5-aryl-3-benzyloxypyridine series generated clinical candidate 3 (PF-02341066), which is a selective inhibitor of MET, ALK, ROS1, and RON at the cellular level. 99 Compound 3 potently inhibited MET phosphorylation and MET-dependent proliferation, migration, and invasion of human tumor cells in vitro (IC50 values of 5−20 nM) and demonstrated dose-dependent antitumor efficacy, including cytoreductive antitumor activity at well tolerated dose levels in several tumor models that express activating MET. The cocrystal structure of the unphosphorylated MET KD with 3 demonstrated a similar binding environment as 2, and the 2,6-dichloro-3-fluorophenyl group of 3 reached the same position as the 2,6-dichlorophenyl group of 2 to form the key π−π stacking interaction with Tyr-1230 in A-loop (Figure 5).99 In addition, compound 3 inhibited tumor cell growth in cell lines harboring fusion variants or activating mutations of ALK, including Karpas 299 (NPM-ALK), SU-DHL-1 (NPM-ALK), Kelly neuroblastoma (activating mutation), and NCI-H3122 (EML4-ALK variant 1) with IC50 values ranging from 74 to 566 nM.105 Compound 3, later named crizotinib, was given fast track approval by the U.S. Food and Drug Administration on August 26, 2011, to treat certain patients with late-stage (locally advanced or metastatic), non-small-cell lung cancers who express the abnormal anaplastic lymphoma kinase gene.

Figure 5. Overlay of 2 and 3 bound to MET (PDB code 2WKM, cyan colore, and PDB code 2WGJ, gray colore).99

Several newer class Ia MET inhibitors use the critical R-(2,6dichloro-3-fluorophenyl)methyl group to capture key interactions with the MET KD (Chart 2). Compound 4, the thioether analogue of 3, showed reduced potency against MET (enzymatic IC50 of 7.7 nM and cell proliferation IC50 of 190 nM in SNU-5 cell line).106 Compound 5, which has a 1′methylspiro[indoline-3,4′-piperidine]-2-one at the 5-position of the 2-aminopyridine, is a potent, highly selective, well tolerated, and orally efficacious MET (cell IC50 of 22 nM) and ALK (cell IC50 of 39 nM) dual inhibitor. Compound 5 showed pharmacodynamic effects on inhibiting MET phosphorylation in vivo and significant tumor growth inhibition (>50%) in the GTL-16 human gastric carcinoma xenograft models.107 Compound 6 (OSI-296), which has a furo[3,2-c]pyridin-6ylamine as the kinase hinge binder, is a potent and selective inhibitor of MET (cell IC50 of 40 nM) and RON (cell IC50 of 200 nM).108 Compound 6 shows in vivo efficacy in tumor xenograft models upon oral dosing and is well tolerated.108 The cocrystal structure of MET in complex with 6 (PDB code 4KNB) revealed a similar binding interaction as 4. With a quinoline as its kinase hinge binder, 7 retained the binding characteristics of 4 with MET KD and had a MET enzymatic potency of 9.3 nM and MKN-45 cell potency of 93 nM.109 Compound 8 (X-376), having the pyridazin-3-ylamine group as a hinge binder and the 6-amide extending to the solvent area, is a more potent ALK inhibitor (cell IC50 of 77 nM in H3122 cell line) than MET inhibitor (cell IC50 of 150 nM in MKN-45 cell line).110 Compound 9, a close analogue of 8 but having a less lipophilic pyridone hinge binder, inhibited MET with an IC50 of 12 nM in enzymatic assay and 2200 nM in EBC-1 cell assay.111 The reported enzymatic and primary cell potencies along with the calculated physical properties of class Ia MET inhibitors are summarized in Table 1. The ligand efficiency (LE), lipophilic ligand efficiency (LLE), and ligand-efficiencydependent lipophilicity (LELP) based on the primary cell potency are calculated as comprehensive indices for druglike properties.112 Studies on the predictive performance of LLE and LELP with multiple data sets including fragment and HTS hits and leads, development candidates, phase II compounds, and launched drugs indicate that both LLE and LELP correlate significantly with ADME and safety profiles while LELP outperforms LLE in risk assessment.113 Wager et al. analyzed 95 CNS drugs, and the median LE, LLE, and LELP values for the drug set were 0.52, 6.3, and 5.9, respectively.114 Although it I



Perspective

Table 1. Summary of MET Inhibitors of Class Ia compd 103

1 299 399 4106 5107 6108 7109 8110 9111

MW

cLogP a

cLogD b

enzyme Ki or IC50 (nM)

cell IC50 (nM) (cell line)

cell LEc

cell LLEd

cell LELPe

status

568 642 450 466 515 465 419 519 519

4.43 5.21 4.29 4.10 5.06 3.52 4.86 5.41 4.31

0.859 2.56 1.06 0.87 1.04 4.01 3.98 3.47 3.50

44.5 (IC50) 2.2 (Ki) 2.0 (Ki) 7.7 (IC50) 1.87 (IC50) 26 (IC50) 9.2 (IC50) 0.69 (IC50) 12 (IC50)

132 (A549) 7.5 (A549) 8.0 (A549) 190 (SNU-5) 22 (MKN45) 40 (MKN45) 93 (MKN45) 150 (MKN45) 2200 (EBC-1)

0.247 0.265 0.378 0.313 0.306 0.334 0.251 0.195 0.162

6.02 5.56 7.03 5.85 6.61 3.39 3.05 3.35 2.15

17.9 19.7 11.4 13.1 16.5 10.5 19.4 27.7 26.7

discovery discovery approved NA NA NA NA discovery NA

a

cLogP is the calculated logarithm of the partition coefficient in octanol/water using the BioByte program ClogP, version 4.3. bcLogD is the calculated logarithm of the octanol/water distribution coefficient using ACD pchbat, version 12.1. cCell LE = −1.4 log IC50 [M]/number of heavy atoms. dCell LLE = −log IC50 [M] − cLogD. eCell LELP = cLogP/LE.

complexed to 11 revealed a unique binding mode (Figure 6). Surprisingly, the oxindole ring is not interacting with the hinge

is expected that higher molecular weight and lipophilicity of the kinase drugs on average would generate lower LE and LLE and higher LELP than the average values of CNS drugs, the rank order of LLE and LELP of the inhibitors for a specific kinase target can be used as indices for overall druglike properties. Crizotinib has the highest LLE and the second best LELP among class Ia MET inhibitors, and 6 with a LELP of 10.5 has higher potential to be successful in clinic trials. 5.2. Class Ib of Exquisitely Selective MET Inhibitors. The MET tyrosine kinase possesses a unique autoinhibitory conformation in the kinase domain with a β′-turn at the beginning of the A-loop, and the A-loop could interact with inhibitors directly. Therefore, MET has a more closed and smaller adenine binding site that can be used for the design of specific MET kinase inhibitors with desired druglike properties. Class Ib MET kinase inhibitors capture these unique binding characteristics of the unphosphorylated MET conformation and have exquisitely selective profiles even though they are type I kinase inhibitors with a U-shaped binding mode. A unique and highly specific MET inhibitor 10 (Chart 3) was identified from an HTS campaign of the MET program at

Figure 6. Cocrystal structure of unphosphorylated MET kinase domain with 11 (PDB code 3ZZE).115

Chart 3. Discovery of Highly Selective MET Inhibitors115 as originally suspected, and instead the phenolic moiety of 11 functions as the hinge binder with the phenol oxygen forming a hydrogen bond with the N−H of the hinge Met-1160.115 Also, a water network links the phenol hydroxyl and Met-1160 carbonyl group. As the molecule makes a turn at the methylene linker, the large coplanar structure of the hydrazide−oxindole allows for strong π−π interactions with Tyr-1230 at the A-loop. The protein crystal structure of the unphosphorylated MET with 11 reveals a similar conformation as the unphosphorylated apo-MET (PDB code 1R1W) and the protein complex with 2 (PDB code 2WKM). A unique β-turn at the beginning of the A-loop is formed, and MET protein adopts an autoinhibitory conformation with the A-loop occupying the binding site for the triphosphate of ATP. The exquisite selectivity profile of 11 is largely a result of the unique and extensive interactions with the A-loop and fewer interactions with the common and conserved areas in the kinase domain. On the basis of the cocrystal structure of MET in the complex with 11, Vojkovsky et al. designed the tetracyclic aromatic scaffold, exemplified by 12, which proved to be an effective mimic of original oxindole hydrazide series.117 Zhang et al. further simplified the nondruglike tetracyclic scaffold down to the bicyclic triazolotriazine scaffold, exemplified by 13, which gave better ligand efficiency against MET than the

Pharmacia.115 Optimization of 10 by Koenig et al. afforded 11 which has an enzymatic Ki of 1.3 nM and an IC50 of 7 nM for the inhibition of MET autophosphorylation in A549 cell line, as well as an exquisite kinase selectivity profile.116 The cocrystal structure of the unphosphorylated MET kinase domain J



Perspective

the A-loop Tyr-1230 is a key driver for the potency and the exquisite kinase selectivity. Albrecht et al. reported an elegant hybrid design of a novel chemical series based on an analogue of a SUGEN patent compound and an Amgen quinoline MET inhibitor, which eventually led to 22 (Figure 8).127 Although the bicyclic triazolopyridazine heteroaryl group had been preserved for its interaction with Tyr-1230, the connection with quinoline at 4-position and an additional oxygen atom in the linker distinguished 22 from most of the exquisitely selective MET inhibitors in Chart 4. Boezio et al. further modified this series by replacing the oxygen linkage with NH and replacing the quinoline with [1,5]naphthyridine, thus introducing an intramolecular hydrogen bond to lock the favored conformation, resulting in 23. The new naphthyridine series also solved the issue of time dependent inhibition of CYP3A4.128 In 24, Bode et al. used a triazolopyridinone interacting with the A-loop Tyr-1230, as opposed to the bicyclic heteroaryl group found in the rest of the class Ib MET inhibitors.129 The cocrystal structure of MET complexed to 24 revealed the conserved MET selective binding mode and demonstrated that the carbonyl group of the triazolopyridinone formed an intramolecular hydrogen bond with the hydrogen of C-5 in quinoline to lock the U-shaped conformation. Meanwhile, the (S)-methyl group extended into a small, lipophilic pocket in MET (defined by the side chains of Val1092, Leu-1157, Ala-1226, and Lys-1110), boosting potency 10-fold.129 The binding mode of 25 (EMD-1204831) has not been reported, so it was grouped with the class Ib MET inhibitors based on its reported MET-specific selectivity profile.130 Overall, the class Ib MET inhibitors demonstrated potent and exquisitely selective inhibition against MET in vitro and in vivo and exhibited excellent pharmaceutical properties associated with marked tumor growth inhibition in animal models. The physical properties and reported potencies of the class Ib MET inhibitors are summarized in Table 2 with the calculated LE, LLE, and LELP. The clinical status is based on the public Web site information.131 In summary, the exquisitely selective class Ib MET inhibitors demonstrated excellent druglike properties with the average of LLE and LELP aligned with marketed drugs, and 16 has the lowest LELP. Solubility may be a common issue for the majority of the class Ib MET inhibitors that have two bicyclic aromatic rings plus another heteroaromatic ring in the molecule. Open access to the C-2 position of quinoline may be a risk factor for aldehyde oxidase (AO) catalyzed oxidation of the quinoline ring which can lead to poor human PK and PK variations among patients because of high and variable AO activity in humans.132 Compound 19 was withdrawn from phase I studies because of acute renal failure secondary to crystal nephropathy from insoluble metabolites at levels not seen in preclinical rat or dog toxicology studies.133 The formation of crystals both within the renal tubules and within giant cell macrophages was later observed in primate toxicology evaluation. Diamond et al. identified a 2-quinolinone metabolite of 19, which was formed by AO oxidation of the quinoline core at C-2 position in human and monkey liver S-9 and to a lesser extent in rat S-9 incubation but absent in the dog.134 This metabolite was found at ∼70-fold greater concentration than the parent 19 in urine and was markedly less soluble. Although the AO oxidation profiles of the other quinoline MET inhibitors in Chart 4 have not been reported, 18 was terminated early in phase I studies because of increase in serum creatinine levels and minimal PD activity.131 Additional

tetracyclic scaffold despite the loss of the hydrogen bond with the carbonyl group of Arg-1208.118 The electron deficiency of the bicyclic aromatic ring governs the strength of the interaction with Tyr-1230 at the A-loop and is a determining factor for the potency and selectivity. Therefore, the triazolopyrazine scaffold, exemplified by 14,119 and the triazolopyridazine scaffold, exemplified by 15, are less potent in general. Discovery of unique and exquisitely selective MET inhibitors at SUGEN enabled identification of a class of highly selective and potent MET inhibitors that have been developed into clinical candidates for cancer therapy. The original phenol hinge binder was successfully replaced with conventional kinase hinge binders, e.g., quinoline. Although it provided the best potency, the triazolotriazine scaffold of 13 was proved to be metabolically unstable in human hepatocyte assay.115 The less potent but metabolically stable triazolopyrazine and triazolopyridazine scaffolds were optimized to produce 16 (PF04217903)115 and 17 (PF-04254644).120 Both 16 and 17 demonstrated potent inhibition of MET in cell assays in vitro and target modulation in vivo, marked tumor growth inhibition in MET-driven tumor models, and good oral pharmacokinetic (PK) properties.115,120 When screened against a panel of 208 different protein kinases, both 16 and 17 exhibited exquisitely selective profiles.115,120 However, compound 17 was also found to be a potent phosphodiesterase family inhibitor, which led to a sustained increase in heart rate, increased cardiac output, and decreased contractility indices, as well as myocardial degeneration in rats.121,122 Thus, compound 17 was terminated as a preclinical candidate because of its cardiac safety issues and 16 was forwarded into clinical studies.120 The crystal structure of the unphosphorylated MET kinase domain complexed to 16 revealed a binding environment very similar to that seen with 11 (Figure 7).115

Figure 7. Cocrystal structure of unphosphorylated MET kinase domain with 16 (PDB code 3ZXZ).115

Several highly potent and selective MET inhibitors have been reported (Chart 4), including 18 (JNJ-38877605),123 19 (SGX523),124 20 (NVP-BUV972),125 21 (INCB-28060 or INC280),126 22 (AMG-208),127 23,128 and 24.129 The published cocrystal structures of MET in the complexes with 19 (PDB code 3DKF) and 20 (PDB code 3QTI) revealed the same binding mode as 11, where the π−π stacking interaction with K



Perspective

Chart 4. Class Ib of the Exquisitely Selective MET Inhibitors

Figure 8. Hybrid design leading to the discovery of 22.127

Table 2. Summary of the Exquisitely Selective MET Inhibitorsf compd 115

16 17120 18123 19124 20125 21126 22127 23128 24129 25130

MW

cLogP a

cLogD b


cell IC50 (nM) (cell line)

cell LEc

cell LLEd

cell LELPe

status131

372 355 377 359 340 411 387 404 465 493

0.525 1.99 1.28 2.14 2.42 2.96 1.98 2.64 1.92 3.40

1.67 2.71 1.96 3.45 2.74 2.74 3.03 2.94 3.37 1.40

5 (Ki) 10.3 (Ki) 4.0 (IC50) 2.7 (Ki) 14 (IC50) 0.13 (IC50) 9 (IC50) 5 (IC50) 1 (IC50) 4 (IC50)

4.0 (A549) 6.0 (A549) NA 12 (A549) 22 (A549) 0.7 (A549) 46 (PC3) 3 (PC3) 2 (PC3) 6 (A549)

0.420 0.426 NA 0.427 0.412 0.413 0.354 0.411 0.369 0.311

6.73 5.51 NA 4.47 4.91 6.41 4.31 5.58 5.33 6.82

1.25 4.67 NA 5.02 5.87 7.16 5.59 6.42 5.20 10.93

phase I terminated terminated phase I terminated phase I terminated NA phase I completed phase I NA NA phase I

a cLogP is the calculated logarithm of the partition coefficient in octanol/water using the BioByte program ClogP, version 4.3. bcLogD is the calculated logarithm of the octanol/water distribution coefficient using ACD pchbat, version 12.1. cCell LE = −1.4 log IC50 [M]/number of heavy atoms. dCell LLE = −log IC50 [M] − cLogD. eCell LELP = cLogP/LE. fNA, not available.

L



Perspective

Chart 5. Class II of Nonselective MET Inhibitors

the key structural motif that allows an additional phenyl group to enter the deep hydrophobic pocket for potent MET inhibition. Many teams from different companies have focused on replacing the chemically unstable acyl thiourea linkage and/ or on finding alternative heteroaryl templates to replace the quinoline/quinazoline cores that bind to the kinase hinge region. A number of compounds (Chart 5) have been discovered and taken into human clinical trials. Cabozatinib (27) was granted marketing approval by the U.S. FDA on November 29, 2012, under the name Cometriq for treatment of medullary thyroid cancer.135 The inactive form of the MET protein has a unique autoinhibitory conformation with a turn at the beginning of the A-loop which will block the inhibitor from entering into the hydrophobic back pocket. Class Ia and class Ib U-shaped MET inhibitors retain or further strengthen this autoinhibitory conformation and thus demonstrate high binding efficiency and a highly selective profile. It is expected that a high energy barrier exists to move the β′-turn at the N-terminus of the Aloop to open the “gate” to the back pocket. In general, the MET inhibitors in class II (Chart 5) have higher molecular weights than the type II VEGFR inhibitors, low ligand efficiency, and a broad spectrum of kinase acivity. 27 (XL-

human clinical observation will clarify if the renal toxicity observed with 18 and 19 is common to this scaffold. 5.3. Class II of Nonselective MET Inhibitors. The class II MET inhibitors are multitargeted MET inhibitors that pass the gatekeeper and occupy the deep hydrophobic back pocket (Chart 5). A significant movement of the A-loop is required for entering the MET back pocket which is compensated by additional interactions with the hydrophobic back pocket, often leading to compounds with high molecular weights and high lipophilicities. 4-Phenoxyquinolines with additional substituents on the 4phenyl ring are widely used as kinase inhibitor scaffolds for VEGFRs, PDGFRs, and many other kinases. The first patent application of MET inhibitors based on a 4-phenoxyquinoline/ quinazoline template was published in 2003 by Kirin.135 N-[4[(6,7-Dimethoxy-4-quinolyl)oxy]-3-fluorophenyl]-N′(phenylacetyl)thiourea (26) showed potent inhibition of MET phosphorylation (IC50 of 0.0087 μM) in epidermoid carcinoma cells (A431) stimulated by human recombinant HGF. Compound 26 inhibited tumor growth by 70% at a dose of 100 mg/kg in nude mice which had been transplanted with U87MG, a human brain tumor cell line.135 In contrast to type II PDGFR and VEGFR2 inhibitors, the acyl thiourea linkage is M



Perspective

Figure 9. Interaction surface of 28 with MET having DFG out conformation (PDB code 3LQ8).

Figure 10. Interaction surface of an analogue of 31 with MET having DFG-in conformation (PDB code 3U6I).

184, cabozantinib), which has a novel cyclopropyl-1,1dicarboxamide linkage instead of the acyl thiourea found in 26, is a multitargeted kinase inhibitor with enzymatic activities against VEGFR2 (IC50 of 0.035 nM), MET (IC50 of 1.3 nM), RET (IC50 4 nM), KIT (IC50 of 4.6 nM), FLT1/2/3 (IC50 of 12/11.3/6 nM), TIE-2 (IC50 of 14.3 nM), and AXL (IC50 of 7 nM).136,137 Cabozantinib has relatively weak inhibition against RON (IC50 of 124 nM) and PDGFR (IC50 of 234 nM). In cellular assays, cabozantinib inhibits phosphorylation of VEGFR2 and MET, as well as KIT, FLT3, and AXL with IC50 values of 1.9, 7.8, 5.0, 7.5, and 42 nM, respectively. Cabozantinib inhibits MET and VEGFR2 phosphorylation in tumor models in vivo and demonstrates potent antimetastatic, antitumor, and antiangiogenic activity in preclinical models.137 In an experimental model of metastasis, treatment with cabozantinib did not increase lung tumor burden, which had been observed after treatment with inhibitors of VEGF signaling that do not target MET.137 Overall, cabozantinib is a promising agent for inhibiting tumor angiogenesis and metastasis in cancers with dysregulated MET and VEGFR signaling. Inhibitor 28 (XL-880, foretinib) has a very similar

structure to 27 except that it has a long morpholinylpropyl group in the solvent-exposed area. Similar to 27, 28 is also a multitargeted kinase inhibitor with enzymatic activities against MET (IC50 of 0.4 nM), VEGFR2 (IC50 of 0.86), RON (IC50 of 3 nM), FLT1/3/4 (IC50 of 6.8/3.6/2.8 nM), KIT (IC50 of 6.7 nM), PDGFRα/β (IC50 of 3.6/9.6 nM), and TIE-2 (IC50 of 1.1 nM).138 The cocrystal structure of MET with 28 shows that the inhibitor is deeply bound in the MET kinase back pocket with the 4-fluorophenylamine motif pushing Phe-1223 out of the active position (Figure 9). Compound 28 inhibits HGFinduced MET phosphorylation and VEGF-induced extracellular signal-regulated kinase phosphorylation in vitro and prevents both HGF-induced responses of tumor cells and HGF/VEGF induced responses of endothelial cells. In addition, 28 prevents anchorage-independent proliferation of tumor cells under both normoxic and hypoxic conditions. In vivo, 28 generates significant dose-dependent inhibition of tumor burden in an experimental model of lung metastasis.138 Similarly, 29 (E7050, golvatinib) with a 2-aminopyridine hinge binder also inhibits both MET (cell IC50 of 14 nM) and VEGFR2 (16 nM),139 whereas 30 (MGCD-265) has the same N



Perspective

Table 3. Summary of Class II Multitargeted MET Inhibitorsf compd 135

26 27137 28138 29139 30140 31142 32143 33145

MW

cLogP a

cLogD b


cell IC50 (nM)

cell LEc

cell LLEd

cell LELPe

status131

492 502 615 634 518 540 513 448

5.19 4.94 5.37 1.63 5.12 3.04 4.41 4.43

3.89 3.05 1.90 0.412 2.82 2.88 2.49 3.84

NA 1.3 (IC50) 0.4 (IC50) NA 1.0 (IC50) 1.2 (Ki) 3.9 (IC50) NA

8.7 (A431) 7.8 (PC3) 23 (PC3) 14 (MKN45) 20 (MKN45) 60 (PC3) 20 (GTL-16) NA

0.322 0.307 0.238 0.239 0.299 0.253 0.299 NA

4.17 5.05 5.74 7.44 6.07 4.34 5.21 NA

16.10 16.10 22.6 6.82 17.1 12.0 14.73 NA

patent approved phase I phase I phase I NA phase I phase I

a

cLogP is the calculated logarithm of the partition coefficient in octanol/water using the BioByte program ClogP, version 4.3. bcLogD is the calculated logarithm of the octanol/water distributin coefficient using ACD pchbat, version 12.1. cCell LE = −1.4 log IC50 [M]/number of heavy atoms. dCell LLE = −log IC50 [M] − cLogD. eCell LELP = cLogP/LE. fNA, not available.

acyl thiourea linker as 26 but a thienopyridine motif as a hinge binder, and it is a multitargeted kinase inhibitor with potent inhibition against MET (enzymatic IC50 of 1 nM), VEGFR1/2/ 3 (3/3/4 nM), RON (2 nM), and TIE-2 (7 nM).140 Amgen scientists successfully used a pyrazolone amide to replace the acylthiourea linker in 26 to generate a series of MET inhibitors.141,142 In contrast to typical kinase inhibitors that have a motif in the hydrophobic back pocket to make the protein adopt a DFG-out conformation (such as imatinib bound with ABL and 28 with MET in Figure 9), the fivemember pyrazolone amide delivers the phenyl group into the Ile-1145 pocket while leaving Phe-1223 of the DFG motif in its cognate hydrophobic pocket for MET (Figure 10).142 Interestingly, the VEGFR2 protein adopts a DFG-out conformation when complexed to the same molecule, showing the structural diversity in the kinase hydrophobic back pocket.142a An elegant medicinal chemistry effort aimed at achieving selectivity over the VEGFR family of kinases and removing the reactive aniline metabolite that is present in many class II MET inhibitors led to the discovery of 31 (AMG485).142 31, with a tert-butylhydroxyl motif to achieve selectivity over VEGFRs and a 2-aminopyridine group to replace the aniline, demonstrated potent inhibition against MET (Ki of 1.2 nM and cell IC50 of 60 nM), ∼350-fold selectivity over VEGFR2 in cells, and good pharmacokinetic properties.142 When dosed orally, 31 significantly inhibited tumor growth in the NIH3T3/TPR-MET and U-87MG xenograft models with no adverse effect on body weight.142 A team at Bristol-Myers Squibb used a six-member pyridonylamide to replace the acylurea linker in 26 and 3-chloro-2aminopyridine to replace the quinoline hinge binder and generated the clinical compound 32 (BMS-777607), a potent and selective kinase inhibitor with biochemical activity against MET (enzyme IC50 of 3.9 nM), RON (1.8 nM), AXL (1.1 nM), and TYRO3 (4.3 nM).143 Moderate selectivity over VEGFR2 (180 nM) is achieved with 32. Complete tumor stasis in a MET-dependent GTL-16 human gastric carcinoma xenograft model was observed following oral administration of 32. MET adopts a DFG-out conformation in the complex with 32, which is similar to the complex of 28 but distinct from the analogue of 31. Inhibitor 33 (MP470, amuvatinib) is structurally different from most of the MET inhibitors in Chart 5, as it has a piperazinyl ring to deliver the aryl group into the back pocket instead of a phenoxy group.144 Compound 33 is reported to be a multitarget tyrosine kinase inhibitor that has demonstrated in vitro and in vivo activity against multiple validated cancer targets including mutant KIT, mutant PDGFR, mutant FLT3, MET, and RET.145

Overall, class II MET inhibitors (Table 3) have the lowest LLE and the highest LELP among the three classes of MET inhibitors, which correlates with more off-target hits, at least in the protein kinase family. The more potent VEGFR activity in some analogues may lead to suboptimal dosing for MET inhibition in clinical applications because of VEGFR-related side effects. 5.4. Other MET Inhibitors. A novel orally available multitargeted kinase inhibitor, 34 (MK-2461, Chart 6), inhibits Chart 6. Other MET Inhibitors

MET (enzyme IC50 of 2 nM, GTL-16 cell IC50 of 56 nM) and several other oncology kinase targets, including RON (enzyme IC50 of 7 nM), FLT1/3/4 (enzyme IC50 of 10/22/78 nM), MER (enzyme IC50 of 24 nM), FGFR1/2/3 (enzyme IC50 of 65/39/50 nM), VEGFR2 (enzyme IC50 of 44 nM), and TRKA/B (enzyme IC50 of 46/61 nM).146 The cocrystal structure of activated MET in the complex with an analogue of 34 (which has a 4-piperidine ring on the pyrazole instead of the methyl group of 34) shows that the small molecule fully occupies the adenine pocket from the gatekeeper Leu-1157 to the solvent exposed area (Figure 11). The molecule makes a Uturn at the sulfonamide to orient the N−H for a hydrogen bond with the carbonyl of Asp-1222.146 The unique A-loop autoinhibitory conformation of the unphosphorylated MET seen in the apo-crystal structure (Figure 3) should not prefer the large, conjugated, coplanar structure of 34, so it is not surprising that 34 preferentially binds to active MET (KD of 4.4 nM) over unphosphorylated MET (KD of 27.2 nM). Compound 34 demonstrates different inhibition activities depending upon the autophosphorylation sites of MET. Compound 34 is substantially more potent against the autophosphorylation of Y1349 (IC50 of ∼100 nM) and Y1365 (IC50 of ∼26 nmol/L) in the C-terminal docking site than against the autophosphorylation of the activation loop residues (IC50 of ∼900 nmol/L and >2 μmol/L as measured by an anti-pMET [Y1234/Y1235] antibody and an anti-pMET [Y1230/1234/1235] antibody, respectively).146 Further testing showed 34 has significant in vitro and in vivo antitumor activity O



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Figure 11. Cocrystal structure of activated MET with an analogue of 34 having 4-piperidine ring on pyrazole (PDB code 3R7O).

Table 4. Summary of MET Inhibitors 34 and 35 compd

MW

cLogP a

cLogD b

enzyme IC50 (nM)

cell IC50 (nM)

cell LEc

cell LLEd

cell LELPe

status131

34146 35147

496 369

1.31 2.90

2.04 3.37

2 355

56 (GTL-16) 100−300 (HT-29)

0.290 0.35−0.326

5.21 3.63−3.15

4.52 8.28−8.89

phase I phase III

a cLogP is the calculated logarithm of the partition coefficient in octanol/water using the BioByte program ClogP, version 4.3. bcLogD is the calculated logarithm of the octanol/water distributin coefficient using ACD pchbat, version 12.1. cCell LE = −1.4 log IC50 [M]/number of heavy atoms. dCell LLE = −log IC50 [M] − cLogD. eCell LELP = cLogP/LE.

Table 5. Summary of Specific HGF/MET Inhibitors in Clinical Development131 target

selected clinical studies based on HGF/MET signaling

rilotumumab (AMG-102), Amgen

agent

HGF

ficlatuzumab (AV-299), Aveo TAK701, Millennium onartuzumab (MetMab), Genentech

HGF HGF MET

Phase III: +ECX first line for MET + untreated advanced gastric or GEJ adenocarcinoma. Phase II: FOLFOX alone or in combination with AMG102 or panitumumab as first-line treatment in patients with advanced gastresophageal cancer. Phase II: +avastin for recurrent malignant glioma. Phase I/II: +erlotinib for previously treated patients with advanced NSCLC. Phase Ib/II in Asian subjects with NSCLC. Phase I study in adult patients with advanced solid tumors. Phase III: +mFOLFOX6 for Her-2 negative and MET-positive stomach or gastresophageal cancer.

MET MET MET MET

Phase III: +erlotinib in patients with unresectable stage IIIb or IV, MET positive NSCLC carrying an activating EGFR mutation. Phase III: +erlotinib in patients with advanced MET positive NSCLC who have received standard chemotherapy for advanced/ metastatic disease. Phase II: +bevacizumab in comparison with bevacizumab alone or MetMab monotherapy for recurrent glioblastoma. Phase II: +paclitaxel + cisplatin or carboplatin first-line for stage IIIb or IV squamous NSCLC. Phase II: combination with either bevacizumab + platinum + paclitaxel or pemetrexed + platinum in patients with untreated stage IIIb or IV nonsquamous NSCLC Phase Ib: single or +sorafenib for advanced HCC. Phase I: prematurely discontinued due to a strategic development decision by Pfizer, not based on any safety concerns. Phase I: early termination due to increase in serum creatinine levels and minimal PD activity. Phase I: terminated due to acute renal toxicity. Phase II: advanced HCC.

MET MET

Phase Phase Phase Phase

16 (PF-04217903), Pfizer 18 (JNJ38877605), J&J 19 (SGX-523), SGX 21 (INC280) (INCB28060), Novartis/Incyte

22 (AMG-208), Amgen 25 (EMD1204831), EMD

Ib/II: +gefitinib in patients with EGFR mutated, MET-amplified NSCLC who have progressed after EGFRi treatment. I: MET dependent advanced solid tumors. I: advanced solid tumors. I: terminated for reasons other than safety

against MET and FGFR2-driven tumors and good pharmaceutical and safety profiles.146 Inhibitor 35 (ARQ-197, tivantinib, Chart 6) was discovered with a phenotype cell viability assay in metastatic cancer cell lines.147 Subsequent experiments identified MET as the molecular target of 35. Further characterization showed 35 was a highly selective and non-ATP competitive inhibitor with a Ki of 355 nM. It inhibited MET phosphorylation in HT29 and MKN-45 cells with constitutively active MET activity, and

HGF-induced MET phosphorylation in MDA-MB-231 and NCI-H441 cells with an IC50 of 100−300 nM. Exposure to 35 resulted in the inhibition of proliferation of MET-expressing cancer cell lines as well as the induction of caspase-dependent apoptosis in cell lines with constitutive MET activity. Moderate growth inhibition of human tumors following oral administration of 35 (200 mg/kg) in multiple mouse xenograft efficacy studies was observed.147 P



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Table 6. Summary of Multitargeted MET Inhibitors in Clinical Development131 agent

targeta

3 (PF-2341066) (crizotinib), Pfizer

MET/ALKROS1/RON

27 (XL184) (cabozantinib), Exelixis

VEGFR2/MET/RET/KIT/ FLTs/TIE2/AXL

28 (XL-880) (GSK-1363089) (foretinib), GSK/Exelixis

MET/VEGFR2/RON/FLTs/ KIT/PDGFRs/TIE2

29 (E7050) (golvatinib), Eisai

MET/VEGFR2

30 (MGCD-265), MethylGene 32 (BMS-777607), BMS

MET/VEGFRs/RON/TIE2 MET/AXL/RON/TYRO3

33 (MP-470) (amuvatinib), Astex

MET/KIT/PDGFRα /FLT3

34 (MK-2461), Merck 35 (ARQ-197) (tivantinib), ArQule

MET/RON/FLT1/FGFRs MET and microtubing target(s)

AMG-337 (structure undisclosed), Amgen

MET (unknown selectivity profile)

a

selected clinical studies based on MET target Phase Phase Phase Phase

I: +pazopanib or pemetrexed followed by the triplet in patients with advanced malignancies. I: +dasatinib in patients with advanced malignancies. I: +pan-Her inhibitor PF-0299804 for NSCLC. III: vs everolimus in metastatic RCC.

Phase III: vs prednisone in metastatic castration-resistant prostate cancer patients who have received prior docetaxel and prior abiraterone or MDV3100. Phase II: castration-resistant prostate cancer with visceral metastases. Phase II: castration-resistant prostate cancer with bone metastases. Phase II: HCC patients who have received prior sorafenib. Phase I/II: relapsed or refractory myeloma. Phase I: relapsed/refractory multiple myeloma with bone disease. Phase I: +gemcitabine in advanced pancreatic cancer. Multiple phase IIs (completed): patients with SCC of the head and neck; metastatic gastric cancer; papillary renal-cell carcinoma. Phase 1/2: patients with previously treated NSCLC receiving standard erlotinib. Phase 1/2: +lapatinib for HER-2 positive metastatic breast cancer. Phase 1/2: adults with HCC. Phase I/II: +sorafinib vs sorafinib alone as a first-line for HCC. Phase I/II: +cetuximab vs cetuximab alone for platinum-resistant HNSCC. Phase I/II: +E7080 for patients with recurrent glioblastoma or unresectable stage III or IV melanoma after prior systemic therapy. Phase I/II: +erlotinib or docetaxel for patients with advanced NSCLC. Phase I: multiple ascending dose study in subjects with advanced or metastatic solid tumors (ASLAN002). Phase II: +platinum-etoposide chemotherapy for small cell lung cancer who have not responded to or relapsed after standard treatment. Phase I/II (completed): advanced solid tumors. Phase II: +erlotinib vs single agent chemotherapy in previously treated KRAS mutation positive subjects with locally advanced or metastatic NSCLC. Phase II: +cetuximab in EGFRi-resistant MET high subjects with locally advanced or metastatic colorectal cancer with wild-type KRAS. Phase II: metastatic triple negative breast cancer. Phase Ib: +pazopani in patients with refractory advanced solid tumors. Phase I: advanced HCC. Phase I: advanced solid tumors.

The selectivity profile was obtained based on the literature that may not represent the complete selectivity profile.

MET small molecule inhibitors currently being studied in the clinic are summarized in Table 5 and Table 6.131 Most of the MET inhibitors in clinical development, including highly specific HGF and MET antibodies, as well as specific and multitargeted small molecule MET inhibitors, demonstrated pharmacokinetic properties suitable to cover the preclinically predicted efficacy concentrations and exhibited acceptable safety profiles in phase I studies. The relatively few serious adverse events observed with the selective HGF and MET antibodies imply noncritical roles for the HGF/MET pathway in normal tissues. Peripheral edema seems to be a common mechanism-based side effect. Although a broad spectrum of antitumor efficacies have been reported in many preclinical animal models, only moderate clinical benefits have been observed for MET inhibitors currently in clinical development as single agents for cancers associated with HGF/MET signaling abnormalities. This section will review the clinical efficacy reports pertaining to major cancers with aberrant HGF/MET signaling. 6.1. Lung Cancer. Lung cancer is the leading cause of cancer-related mortality worldwide with many patients presenting advanced disease at initial diagnosis. Overexpression of HGF and/or MET has been observed in lung cancer and associated with a poorer prognosis.60 Activated MET is

Although 35 was identified as a moderate non-ATP competitive MET inhibitor, two independent research groups reported that 35 exerted its pharmacologic action in cells not dependent on MET for growth and survival.148,149 Katayama et al. showed that 35 inhibited cell viability with similar potency in both MET-addicted and nonaddicted cells, which suggests that the antitumor activity of 35 is independent of MET status.148 Furthermore, it was shown that 35 inhibited microtubule polymerization in addition to inhibiting MET.148 Basilico et al. reported that 35 did not inhibit HGF-dependent or -independent MET tyrosine autophosphorylation in all cell models they analyzed.149 Instead, 35 perturbed microtubule dynamics, induced G2/M arrest, and promoted apoptosis. Therefore, 35 displayed cytotoxic activity that was independent of its ability to bind to MET.149 The physical properties and calculated LE, LLE, and LELP of 34 and 35 are summarized in Table 4.

6. TARGETING THE HGF/MET PATHWAY IN THE CLINIC A broad spectrum of inhibitors targeting the HGF/MET pathway have reached clinical development and have been extensively reviewed recently.150 Anti-HGF humanized antibodies, MET extracellular domain monoclonal antibodies, and Q



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detected in the invasive fronts of NSCLC tumor tissues.151 MET gene amplification (a mean increase of five or more gene copies) has been reported in approximately 4% of NSCLC and is an independent negative prognostic factor in surgically resected NSCLC.152 MET amplification is also responsible for acquired resistance to EGFR tyrosine kinase inhibitors in 5− 20% of EGFR-mutant patients.8,87 The resistance mechanism is associated with ERBB3 (HER3)-dependent activation of PI3K, a pathway thought to be specific to the EGFR/ERBB family of receptors.8 MET mutations in lung cancer occur mainly in the non-tyrosine kinase domains, namely, in the juxtamembrane and sema domains, and are found preferentially in metastatic lesions instead of primary lesions.153 Preclinical studies with the MET small molecule inhibitor crizotinib or with RNA interference-mediated MET depletion in lung cancer cells which are positive or negative for either MET amplification or MET mutation revealed that MET signaling is essential for the survival of cells with MET amplification but not for those with a MET mutation.154 Up-regulation of BIM, a proapoptotic member of the Bcl-2 family, and down-regulation of survivin, a member of the inhibitor of apoptosis protein family, contribute to the proapoptotic effect induced by crizotinib via inhibition of AKT and extracellular signal-regulated kinase phosphorylation in lung cancer cells with MET amplification but not in cells with a MET mutation or in those without amplification or mutation of MET.154 Several MET inhibitors have been studied in clinical trials for lung cancer. The first rapid and durable response from inhibition of MET signaling in lung cancer was reported in a patient treated with crizotinib as a single agent for NSCLC with de novo MET amplification but without ALK rearrangement.155 Because of the low frequency of MET amplification (10 copies per cell) was present in 3 of 23 (13%) tumor tissues.173 In addition, HGF overexpression is detected in 45% of HNSCC, suggesting a paracrine activation mechanism.173 Mutations have been detected in multiple domains, including the extracellular MET ligand-binding domain (T230M/E168D), the JM domain (R988C, T1010I), and the tyrosine kinase domain (T1275I, Y1230C, Y1235D, and V14333I) in HNSCC tumor samples.173,174 It is interesting to note that the tyrosine kinase domain mutations were often detected in metastatic sites; e.g., the activation loop Y1235D mutation is present in 50% of metastatic tumor sites versus 2− 6% in primary tumor sites, indicating the potential role of kinase domain mutations in acquiring a metastatic phenotype and having a selective advantage for growth and/or survival in metastatic sites.174 Although activating EGFR mutations are infrequent in HNSCC, EGFR gene amplification remains a strong indicator for poor patient survival, radioresistance, and locoregional failure.175 In March 2006, the U.S. Food and Drug Administration granted approval for the addition of cetuximab to radiation therapy in locally or regionally advanced HNSCC or for use as monotherapy for patients with platinum-refractory, recurrent, or metastatic HNSCC. However, the success of cetuximab has not been replicated with the small molecule EGFR inhibitors gefitinib and erlotinib. RTK crosstalk or compensatory pathway activity may contribute to resistance to the combination treatment with small molecule EGFR inhibitors.175 Preclinically, the combination of the MET inhibitor crizotinib with the EGFR inhibitor gefitinib abrogated HNSCC cell proliferation, invasion, and wound healing significantly more than inhibition of each pathway alone in

evaluating the efficacy and safety of the fully humanized monoclonal anti-HGF antibody rilotumumab in patients with metastatic renal cell carcinoma was reported.164 In 61 patients with metastatic renal cell carcinoma (40 at 10 mg/kg; 21 at 20 mg/kg), one patient in the 10 mg/kg group had confirmed partial response and maintained for over 2.5 years, and 43% of all patients had stable disease.164 A report of a prolonged response to the treatment of the exquisitely MET-selective small molecule inhibitor 16 in a patient with MET-mutated papillary RCC suggests an oncogenically addictive role of the MET pathway in this disease.165 A 58-year-old white man having papillary RCC with M1268T mutation had a 35% reduction of target lesions after receiving 16 for 53 weeks and continued to be treated for 26 months. The role of MET in papillary RCC is further demonstrated with foretinib, an oral multikinase inhibitor targeting MET, VEGFRs, RON, AXL, and TIE-2 receptors.166 A study of 74 patients enrolled in two dosing cohorts found an objective response rate of 13.5% and mean median PFS of 9.3 months. The presence of a germline MET mutation was highly predictive of a positive response (5 of 10 vs 5 of 57 patients with vs without germline MET mutations, respectively).166 6.4. Gastric and Esophagogastric Cancers. MET amplification has been reported in gastric and esophagogastric cancers and is associated with poor prognosis.167 A durable and complete response was reported with the MET monoclonal antibody onartuzumab in a 48 year-old female patient with chemorefractory metastatic gastric cancer.168 The primary tumor revealed high MET gene polysomy and evidence for autocrine production of HGF. A complete response following four doses from March 2008 to June 2008 was observed, which lasted 2 years even though the treatment was discontinued in September 2008. It is confounding that the patient had been treated previously with a MET small molecule inhibitor (having a broad spectrum of kinase activities including VEGFRs) for 4 months, resulting in a transient partial response after 2 months, and then disease progression occurred after the next 2 months.168 The unexpected rarity of MET amplification in GEC patients (∼2%) and the aggressive nature of GEC make it challenging to select patients who meet entry criteria for clinical trials.169 In one study, four patients with MET-amplified GEC were treated with crizotinib (250 mg b.i.d.), and two patients with stage IV junctional GEC with MET/CEP7 of greater than 5 demonstrated clinical benefit. One patient experienced a rapid symptomatic response with improvement in appetite, reduction of pain, and improvement in performance status after 1 week on crizotinib, and a partial response with a 41% reduction in tumor measurements was confirmed at 12 weeks. However, the disease progressed after approximately 112 days on crizotinib. Another patient also showed rapid clinical improvement, with decreased pain and improved performance status after 1 week on crizotinib and 16% reduction in multiple target lesions after two cycles of treatment. The time to progression on crizotinib was 105 days for this patient.169 Although a limited number of patients with clinical benefit from inhibition of the MET signaling pathway in gastric and esophagogastric cancers have been reported, the preliminary findings indicate that MET amplification has the potential to act as an oncogenic driver in gastric cancer, and a large clinical trial of patients with MET-amplified gastric cancer is necessary to understand the observed response patterns and identify the predictive biomarkers and the right combination strategies.169 S



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cancers. Increasing evidence links activation of MET to adaptive response mechanisms for resistance to certain anticancer therapies, e.g., resistance to the treatments of kinase inhibitors, chemotherapy, and radiotherapy. The combination of a MET inhibitor with kinase inhibitor or with general chemotherapy or radiotherapy in clinical trials has drawn a lot of attention with many combination clinical trials ongoing. More than three classes of small molecule MET inhibitors with distinct binding modes, selectivity, and safety profiles have been discovered and developed into clinical trials. Crizotinib, a prototype MET inhibitor of class Ia, and cabozantinib, a multitargeted MET inhibitor of class II, have been approved based on the indications of ALK and RET targets, respectively. The approved drugs provide tools to further validate the important role of HGF/MET signaling in the development of human cancer, especially in cancer metastasis. Several exquitely selective class Ib MET inhibitors are actively under development in clinical trials. The clinical results from class Ib MET inhibitors will provide unambiguous answers to MET cancer biology. The highly selective class Ib MET inhibitors provide invaluable opportunities for the combination with other therapies safely to achieve the optimal efficacy. In summary, MET remains as a promising target in cancers because of its causative roles in cell survival, growth, angiogenesis, and metastasis. There are still many challenges for the development of MET inhibitors into useful drugs. These challenges include identifying the genetically defined responsive patient subset that could benefit from MET inhibition, developing analytically accurate biomarkers for diagnostic and pharmacodynamic application, and determining the right combinations of therapies. With a rich collection of HGF/ MET inhibitors targeting HGF or MET, clinical studies with carefully characterized patients will provide the deep understanding of the roles of HGF/MET signaling in cancer needed to translate the preclinical research into effective therapeutic strategies and to accelerate the development of MET inhibitors into drugs.

HNSCC cell lines, indicating compensatory RTK signaling between MET and EGFR.176 The preclinical studies support the potential combinational strategy of dual inhibition of MET and EGFR for treating HNSCC. The clinical results of MET inhibitors for HNSCC are emerging. An open-label, single-arm, multicenter trial of foretinib, a multitargeted MET/VEGFR inhibitor, was conducted with a total of 14 patients enrolled.177 Foretinib was administered at 240 mg orally for 5 consecutive days of a 14day treatment cycle (5/9 schedule) to patients with recurrent and/or metastatic HNSCC. Under this regimen, 40% of patients (7/14) showed stable disease (SD) and 43% of patients (6/14) experienced tumor shrinkage. Among them, two patients had prolonged disease stabilization for ≥13 months. Foretinib at 240 mg on a 5-day-on and 9-day-off schedule was generally well tolerated, and the most common adverse events were fatigue, constipation, and hypertension.177

7. CONCLUSIONS Since the first discovery of the MET kinase in 1984, the number of studies and publications related to the biology of HGF/MET and the pathological relationship of the pathway to diseases has been increasing exponentially, especially in the past decade. The prevalence of HGF/MET pathway activation in human malignancies has driven a rapid growth in drug discovery programs targeting HGF/MET signaling. More than 20 different therapeutic agents, including HGF monoclonal antibodies, MET monoclonal antibodies, and small molecule MET kinase inhibitors, have entered human clinical trials which will ultimately validate the pathogenic role of HGF/MET in cancers and provide new medicines for oncology application. Dysregulation of MET and/or HGF via activating mutations, gene amplifications, or overexpression and autocrine or paracrine loop regulation evoke cell growth, proliferation, angiogenesis, invasion, survival, and metastasis and lead to tumorigenisis and tumor metastasis. Although the pathology of HGF/MET signaling together with the preclinical data suggests that the inhibition of HGF/MET signaling may have a profound effect on many cancers, the limited efficacy results from clinical trials to date highlight the clinical challenges in the right application of MET inhibitors. Marked antitumor activity of MET inhibitors as single agents has been observed in carefully characterized patients with abnormal HGF/MET signaling as in the examples of (a) the durable response of a NSCLC patient with de novo MET amplification to crizotinib, (b) the prolonged response of a papillary RCC patient with M1268T mutation to exquisitely MET-selective inhibitor 16 for up to 26 months, and (c) the patient with MET-amplified GBM who experienced rapid and durable clinical improvement when treated with MET inhibitor crizotinib. These successful individual examples highlight the importance of identifying the appropriate biomarkers to predict responsiveness to specific therapies, and the necessity of prospective molecular profiling to ensure successful clinical trials. Intratumoral heterogeneity presents major challenges to personalized medicine and biomarker development and has a substantial impact on both innate and acquired resistance to tyrosine kinase inhibitors. Overall, the frequency of MET mutations and amplification is relatively low and MET inhibitors as single agents may be important only in a subset of patients. MET and/or HGF is frequently overexpressed even without mutations or gene amplifications in a number of

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AUTHOR INFORMATION

Corresponding Author

*Phone: 858-692-6798. E-mail: [email protected]. Notes

The author declares no competing financial interest. Biography J. Jean Cui earned her Ph.D. in Organic Chemistry from The Ohio State University under the direction of Professor Derek Horton and received her B.S. and M.S. in Chemistry from University of Science and Technology of China. Jean worked at Pfizer for more than 14 years including 4 years at SUGEN/Pharmacia conducting oncology drug discovery. She recently started up TP Therapeutics focusing on turning point medicines for cancer patients. Jean is the lead inventor of crizotinib (Xalkori). Jean and the crizotinib chemistry team were selected for the 38th National Inventor of the Year Award in 2011 and for the 2013 American Chemical Society’s Heroes of Chemistry Program for the discovery of crizotinib. Jean received Pfizer Achievement Awards in 2006 and 2012 and Innovation Award in 2011.

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ACKNOWLEDGMENTS The author is grateful to Pfizer colleagues Ben Burke, Susan Kephart, Wei Tan, and Baohua Xin for their critical reading, editing, and suggestions; to Asako Nagata for help on the T



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preparation of figures; and to reviewers for their critical suggestions.

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ABBREVIATIONS USED 3′-UTR, 3′ untranslated region; ABL, Abelson murine leukemia viral oncogene homologue 1; AO, aldehyde oxidase; BCR, breakpoint cluster region protein; A-loop, activation loop; CBL, casitas B-lineage lymphoma protein; CR, cystine-rich; CRKL, crk-like protein; CSC, cancer stem cell; DCR, disease control rate; ERBB2, v-erb-b2 avian erythroblastic leukemia viral oncogene homologue 2; ECX, epirubicin, cisplatin, and capecitabine; FISH, fluorescence in situ hybridization; FGFR, fibroblast growth factor receptor; FLT3, fms-like tyrosine kinase 3; FOLFOX, folinic acid, fluorouracil, and oxaliplatin; GAB1, GRB2-associated-binding protein 1; GBM, glioblastoma; GEC, gastric and esophagogastric cancers; GEJ, gastroesophageal junction; GIST, gastrointestinal stromal tumor; GRB2, growth factor receptor-bound protein 2; HCC, hepatocellular carcinoma; HER2, human epidermal growth factor receptor 2; HGF, hepatocyte growth factor; HGFR, hepatocyte growth factor receptor; HIF-1, hypoxia-inducible factor 1; HNSCC, squamous cell carcinoma of the head and neck; HOS, human osteogenic sarcoma; HPRC, hereditary papillary renal carcinoma; Ig, immunoglobin-like; IRK, insulin receptor kinase; JM, juxtamembrane; KD, kinase domain; MAPK, mitogen-activated protein kinase; NSCLC, non-smallcell lung cancer; ORR, objective response rate; OS, overall survival; PDAC, pancreatic ductal adenocarcinoma; PDGFR, platelet-derived growth factor receptor; PFS, progression free survival; PLC, phospholipase C; PRC, papillary renal carcinoma; PRGF, plasminogen-related growth factor family; PTP, protein tyrosine phosphatase; RTK, receptor tyrosine kinase; RCC, renal cell carcinoma; ROS1, c-ros oncogene 1; SCC, squamous cell carcinoma; SF, scatter factor; SPH, serine protease homology; STAT, signal transducer and activator of transcription proteins; TKI, tyrosine kinase inhibitor; TRK, neurotrophic tyrosine kinase; TPR, translocated promotor region; VHL, von Hippel−Lindau tumor suppressor

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