c-Met Inhibitors (c-Met 抑制剂)

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c-Met Inhibitors

c-Met is a proto-oncogene that encodes a receptor tyrosine kinase known as hepatocyte growth factor receptor (HGFR). It is a trans-membrane receptor and is essential for the development of embryo and the healing of wound. The activation of abnormal MET induces the production of the tumor cells, and triggers anigogenesis in around cancers. Thus, cancers are metastasized to other organs from originally produced sites. As far as we know, the only ligand of the tyrosine kinase receptor is hepatocyte growth factor (HGF). The HGF/c-Met signaling transduction pathway was found to be active in patients with different types of tumors. It is hypothesized that c-Met is a potential molecular target for treating multiple cancers. Several high selective small c-met tyrosine kinase inhibitors were used to block the activity of c-Met to treat various cancers.

c-Met signaling pathway has been validated to be a potential target for the treatment of multiple cancers. A plentiful of c-Met inhibitors were synthesized and profoundly studied, such as AMG-208, Axitinib, Foretinib (GSK1363089, XL880),JNJ-38877605 and MGCD-265. Here, I will introduce their biological and chemical characteristics.

AMG-208(AMG 208, AMG208) is a potent small molecular c-Met inhibitor with an IC50 of 9.3 nM. AMG-208 is a potent small molecular c-Met inhibitor with an IC50 of 9.3 nM. C-Met is a well-characterized receptor tyrosine kinase expressed on the surface of epithelial cells. C-Met receptor signaling has been shown to play a key role in the survival of cancer cells. AMG-208 inhibits both ligand-dependent and ligand-independent c‑Met cellular growth regulation. Inhibition of c-Met signaling with AMG-208 provides a potential mechanism for blocking tumor growth and survival. AMG 208 revealed a tendency to inhibit cytochrome P450 3A4 (CYP3A4) in a time-dependent manner. Pre-incubation of AMG 208 with human liver microsomes for 30 min resulted in an eightfold decrease in IC50 for CYP3A4 metabolic activity relative to the IC50 (32 μM) without preincubation. Such a decrease in IC50 (4.1 μM) indicated the presence of potent time-dependent inhibition. [1][2]

References on AMG-208
[1] Albrecht BK et al. J Med Chem. 2008 May 22;51(10):2879-82.
[2] Boezio AA et al. Bioorg Med Chem Lett. 2009 Nov 15;19(22):6307-12.

Axitinib (AG-013736) blocked phosphorylation of VEGFR-2 and VEGFR-3 with average IC50s of 0.2 and in a range of 0.1 to 0.3 nM. Axitinib is a receptor kinase inhibitor with IC50 of 0.1 nM (VEGFR-1), 0.2 nM (VEGFR-2), 0.1–0.3 nM (VEGFR-3), 1.6 nM (PDGFR-β) and 1.7 nM (c-KIT). [1] Axitinib is a substituted indazole derivative and now developed by Pfizer. [2] Axitinib could block the cellular autophosphorylation of VEGFR and VEGF-mediated endothelial cell viability, tube formation, and downstream signaling. Axitinib inhibits the proliferation of variable cell lines with IC50 of >10,000 (IGR-N91), 849 (IGR-NB8), 274 (SH-SY5Y) and 573 (non-VEGF stimulated HUVEC) nmol. [3] In vivo study also shows that Axitinib delays the tumor growth with 11.4 days compared to the controls (p.o. 30mg/kg) and decreases the Mean Vessels Density (MVD) to 21, compared to 49 in controls, in IGR-N91 ﬂank xenografts. [3] Hu-Lowe DD et al. also indicates that Axitinib exhibits primary inhibition to orthotopically transplanted models such as M24met (melanoma), HCT-116 (colorectal cancer), and SN12C (renal cell carcinoma).[1] Axitinib has shown single-agent activity in variable tumors, including renal cell carcinoma, thyroid cancer, non-small cell lung cancer, and melanoma. Axitinib is currently in phase III clinical studies, which combined with other chemotherapeutics.

Kinase Assay:
PAE cells, which overexpress full-length VEGFR-2, PDGFR-β, KIT, and NIH-3T3, which overexpress murine VEGFR-2 (Flk-1) or PDGFR-α, are generated. The 96-well plates are coated with 100 µL/well of 2.5 µg/mL anti-VEGFR-2 antibody, 0.75 µg/mL anti-PDGFR-β antibody, 0.25 µg/mL anti-PDGFR-α antibody, 0.5 µg/mL anti-KIT antibody, or 1.20 µg/mL anti-Flk-1 antibody to prepare ELISA capture plates. Then phosphorylation of RTK by ELISA is measured. [1]

Cell Assay:
HUVEC, SH-SY5Y, IGR-N91 and IGR-NB8 cell lines are seeded in a 96-well plate at a density of 5x104 and cultured for 24 hours. Axitinib is added to the cells at concentrations ranging from 1 nmol to 10 µmol. Cell viability is measured after 72 hours by MTS tetrazolium substrate and IC50 values are calculated. [3]

Animal Studies:
Immune-deficient female mice (Nu/nu; age 8–12 weeks) are implanted subcutaneously with 17β estradiol pellets (0.1 mg/pellet) 24–48 h prior to tumor cell injection. Human BT474 breast cancer cells are implanted subcutaneously into the axillary flank at a density of 2xl07. Tumor volumes are measured by caliper measurements. The tumor volumes are calculated by this: V(mm3) = width2(mm2) x length(mm)/2. Then tumor-bearing mice are randomized into groups (10–12 mice/group), when tumors approximate 250 mm3. Animals receive daily oral (p.o.) at a dosage of 10, 30 or 100 mg/kg in 0.5% carboxymethylcellulose (CMC) or an equivalent volume of the vehicle. [4]

References on Axitinib:
[1] Hu-Lowe DD, et al., Clin Cancer Res, 2008, 14(22), 7272-7283.
[2] www.pfizer.com/files/news/asco/axitinib_fact_sheet.pdf
[3] Rössler, J. et al., Int J Cancer, 2011, 128(11), 2748-2758.
[4] Wilmes, L.J. Magn Reson Imaging 2007, 25 (3), 319-327

Foretinib (GSK1363089, XL880) is a novel MET and VEGFR2/KDR kinases inhibitor with an IC50 of 0.4 and 0.8 nM for MET and KDR, respectively. It inhibits HGF receptor family tyrosine kinases with an IC50 of 3 nM for Ron. Foretinib (GSK1363089, XL880) also inhibits KDR, Flt-1, and Flt-4 with IC50 of 0.9, 6.8 and 2.8 nM, respectively. In addition, EXEL-2880 inhibits members of the platelet-derived growth factor receptor family and the angiopoietin-1 receptor Tie-2. Foretinib(GSK1363089, XL880) exhibits modest activity against fibroblast growth factor receptor 1 and epidermal growth factor receptor and is inactive against 50 serine/threonine kinases, including cyclin-dependent kinases and protein kinase C isoforms. To delineate the cellular effect of foretinib (GSK1363089, XL880), VEGF- induced extracellular signal-regulated kinase phosphorylation was used to assess the effect of the compound on phosphorylation of KDR in human umbilical vein endothelial cells that resulted in an IC50 of 16 nM. Foretinib(GSK1363089, XL880) is the 1st orally available small molecule inhibitor of Met to enter the clinic and appears to be generally well tolerated. Anti-tumor activity has been observed and Foretinib may represent an active treatment option for patients with papillary renal-cell carcinoma. [1][2]

References on Foretinib (GSK1363089, XL880):
[1] Qian F et al. Cancer Res. 2009 Oct 15;69(20):8009-16.
[2]http://www.asco.org/ascov2/Meetings/Abstracts?&vmview=abst_detail_view&confID=55&abstractID=36164

JNJ-38877605 is a inhibitor of c-MET and Phospho-MET with an IC50 of 4 nM and 50 nM, respectively. JNJ-38877605 is an orally bioavailable, highly specific MET inhibitor (selective over other 229 kinases tested). In addition, JNJ-38877605 induces regression of U87-MG xenografts in vivo. [1]

References on JNJ-38877605:
[1] Paolo M. Comoglio et al. Nature Reviews Drug Discovery.2008 June;7:504-516

MGCD-265 is a multi-targeted tyrosine kinase inhibitor that targets the MET, VEGFR-1, VEGFR-2, VEGFR-3, RON and TIE2 receptor tyrosine kinases, which appear to play key roles in tumour development and blood vessel formation (angiogenesis) and tumour survival. MGCD265 is currently in phase I single-agent clinical trials for solid tumour cancers and in phase II trials for solid tumours and NSCLC . [1]

References on MGCD-265:
[1] Cañadas I et al. Clin Transl Oncol. 2010 Apr;12(4):253-60

PF-04217903is c-met inhibitor with an IC50 ranging from 3.1 to142 nM. Its formula is CH16N8O. Its molecular weight is 372.38. PF-04217903 is an orally bioavailabe, small-molecule MET tyrosine kinase inhibitor with potential antineoplastic activity. [1] PF-04217903 demonstrates exquisite kinase selectivity. Inhibition studies of the c-Met mutants show that PF-04217903 is more susceptible to oncogenic mutations that attenuate potency than PF-02341066. [1] PF-04217903 was evaluated against multiple kinase selectivity screening panels: Invitrogen Inc. (125 kinases), Millipore/Upstate Ltd. (105 kinases), University ofDundee (51 kinases), and Pfizer in-house (48 kinases) (Supporting Information). On the basis of the percent inhibition or IC50 values generated from each of these screens, PF-04217903 was estimated to be>1000-fold selective for c-Met compared with each of the other kinases included in these collective screening assays. [1]

References on PF-04217903:
[1] Sergei L. et al. Biochemistry. 2009, 48 (23):5339–5349

PF-2341066 (Crizotinib) is a inhibitor of c-Met kinase and the NPM-ALK (nucleophosmin-anaplastic lymphoma kinase) fusion protein.[1] Its IC50 is 30 nM for the inhibition of ALK-positive ALCL cell proliferation. Its formula is C21H22Cl2FN5O and molecular weight is 450.34. Inhibitor of the catalytic activity of c-Met kinase and the NPM-ALK A t(2;5) chromosomal translocation resulting in expression of an oncogenic kinase fusion protein known as NPM-ALK has been implicated in the pathogenesis of anaplastic large-cell lymphoma (ALCL). [2] After incubation with PF-2341066, cells were washed once with HBSS supplemented with 1 mmol/L Na3VO4 and protein lysates were generated. Subsequently, phosphorylation of ALK was assessed by a sandwich ELISA method using an immobilized anti–total-ALK antibody and an anti–phospho-ALK antibody (pY1604) as a detec ion antibody. PF-2341066 inhibited NPM-ALKphosphorylation in Karpas299 or SU-DHL-1 ALCL cells (mean IC50 value=24 nM). PF-2341066 potently inhibited cell proliferation, which was associated with G1-S–phase cell cycle arrest and induction of apoptosis in ALK-positive ALCL cells (IC50 values=30 nM) but not ALK-negative lymphoma cells. The induction of apoptosis was confirmed using terminal deoxyribonucleotide transferase–mediated nick-end labeling and Annexin V staining (IC50 values=25–50 nM). [3]

References on PF-2341066:
[1] Deininger M et al.Blood 2005,105:2640–53
[2] Duyster J et al.Oncogene 2001,20:5623–37
[3] James G. Christensen1 et al. Mol Cancer Ther December. 2007,6:3314

SGX-523 is an exquisitely selective, ATP-competitive MET receptor tyrosine kinase inhibitor with an IC50 of 4 nM for the inhibition of HGFR. Its formula is C18H13N7S and molecular weight is 359.41. It is >1,000-fold selective versus the >200-fold selectivity of other protein kinases tested in biochemical assays. It belongs to the class of c-Met/hepatocyte growth factor receptor (HGFR) tyrosine kinase inhibitors and stabilizes MET in a unique inactive conformation that is inaccessible to other protein kinases, suggesting an explanation for the selectivity. SGX523 inhibited MET-mediated signaling, cell proliferation and cell migration at nanomolar concentrations but had no effect on signaling dependent on other protein kinases, including the closely related RON, even at micromolar concentrations. SGX523 inhibition of MET in vivo was associated with the dose-dependent inhibition of growth of tumor xenografts derived from human glioblastoma and lung and gastric cancers, confirming the dependence of these tumors on MET catalytic activity. [1][2]

References on SGX523:
[1] Buchanan SG et al. Mol Cancer Ther. 2009 Dec; 8(12):3181-90.
[2] http://nci.nih.gov/drugdictionary/?CdrID=588972

SU11274 is a potent c-Met inhibitor with an IC50 of 12 nM. Its formula is C28H30ClN5O4S and molecular weight is 568.09. SU11274 is a highly specific inhibitor of c-Met. The inhibitor did not affect other tyrosine kinase oncoproteins, including BCR-ABL, TEL-JAK2, TEL-PDGF_R, or TEL-ABL. Inhibition of the Met kinase activity by SU11274 led to time- and dose-dependent reduced cell growth. [1]
Further study shows SU11274 belongs to a class of small molecule RTK inhibitors that exert their activity by competing for the Mg-ATP complex binding pocket, which results in inhibition of kinase activity and subsequent downstream signaling. [2][3]

References on SU11274:
[1] Koon EC et al. Int J Gynecol Cancer. 2008 Sep-Oct; 18(5):976-984
[2] Berthou S et al. Oncogene. 2004 Jul 8; 23(31):5387-93
[3] Patrick C. Ma et al. Cancer Res. 2005 Feb 15; 65(4):1479-88

XL184 (Cabozantinib) is a potent multitargeted VEGFR2, Met, FLT3, Tie2, Kit and Ret inhibitor with IC50 of 0.035, 1.8, 14.4, 14.3 and 4.6 nM for VEGFR2, Met, FLT3, Tie2 and Kit, respectively. Its formula is C28H24FN3O5 and molecular weight is 501.51. In cells, XL184 (Cabozantinib) is also very potent, with IC50 under 10 nM for VEGFR2, MET, Kit and FLT3-ITD mechanistic assays. In in vitro angiogenesis assays, XL184 (Cabozantinib) exhibits single-digit nanomolar potency. In mouse xenograft models, XL184 (Cabozantinib), dosed orally, demonstrated dose-dependent inhibition of tumor growth and tumor regression, associated with disruption of the tumor vasculature and extensive tumor cell apoptosis. [1][2][3]

References on XL184:
[1] http://www.glgroup.com/News/XL-184--an-RTK-inhibitor-with-no-apparent-incraesed-cardiac-toxicity-20616.html
[2] http://www.glgroup.com/News/XL184-(Exelixis)-may-have-an-agent-that-can-be-given-with-other-targeting-agents-(EGFR)-in-Lung-Cancer.-20560.html
[3] A Study of XL184 With or Without Erlotinib in Adults With Non-Small Cell Lung Cancer.

PHA665752is another c-Met inhibitor. PHA-665752 was identified as a small molecule, ATP-competitive, active site inhibitor of the catalytic activity of c-Met kinase (Ki of 4 nM, IC50 of 9 nM). Its formula C32H34C12N4O4S and molecular weight is 641.61. In a study, after the treatment of PHA665752, the phosphoylation of c-Met was significantly blocked. Sequentially, downstream PI3K and MAPK signaling pathways were prevented. Cell proliferation was further inhibited and apoptosis was induced in c-met overpressed cells. PHA-665752 also exhibited >50-fold selectivity for c-Met compared with a panel of diverse tyrosine and serine-threonine kinases. [1] In cellular studies, PHA-665752 potently inhibited HGF-stimulated and constitutive c-Met phosphorylation, as well as HGF and c-Met-driven phenotypes such as cell growth (proliferation and survival), cell motility, invasion, and/or morphology of a variety of tumor cells. [6] In mouse tumor models, PHA-665752 inhibited Met phosphorylation and signal transduction, which correlated with tumor growth inhibition or tumor regression at well tolerated doses. [1]

In a study, PHA665752 remarkably prevented the growth of MHCC97-L and MHCC97-H tumor which up-regulatedly expressed c-Met in in vivo xenograft models. In addition, it was demonstrated that another c-met phosphorylation inhibitor SU11274 suppressed tumor proliferation and blocked the migration of the cancer cells. Conclusively, inhibition of c-met might be an efficacious therapeutic strategy for a variety of carcinomas including rhabdomyosarcoma (RMS) and hepatocellular carcinoma (HCC) et al.[1-3]

PHA-665752 was identified as a small molecule, ATP-competitive, active site inhibitor of the catalytic activity of c-Met kinase (Ki of 4 nM, IC50 of 9 nM). [1]PHA-665752 also exhibited >50-fold selectivity for c-Met compared with a panel of diverse tyrosine and serine-threonine kinases. [4]
In cellular studies, PHA-665752 potently inhibited HGF-stimulated and constitutive c-Met phosphorylation, as well as HGF and c-Met-driven phenotypes such as cell growth (proliferation and survival), cell motility, invasion, and/or morphology of a variety of tumor cells. [4]
In mouse tumor models, PHA-665752 inhibited Met phosphorylation and signal transduction, which correlated with tumor growth inhibition or tumor regression at well tolerated doses. [5]

References on PHA-665752:
[1] Christensen JG et al. Cancer Res. 2003 Nov 1;63(21):7345-55
[2] Hepatology. 2011 May 26.
[3] J Transl Med. 2011 May 16;9:64.
[4] Christensen JG et al. Cancer Res. 2003 Nov 1;63(21):7345-55
[5] Yanan Yang et al. Mol Cancer Ther. 2008 Apr; 7(4):952-60

BMS 777607 is a Small-Molecule Met Kinase Inhibitor with an IC50 of < 0.1 μM. Its treatment had little effect on tumor cell growth but inhibited cell scattering activated by exogenous HGF, with almost complete inhibition at 0.5 μmol/L in PC-3 and DU145 cells. This agent also suppressed HGF-stimulated cell migration and invasion in a dose-dependent fashion (IC50 < 0.1 μmol/L) in both cell lines. Mechanistically, nanomolar doses of BMS 777607 potently blocked HGF-stimulated c-Met autophosphorylation and downstream activation of Akt and extracellular signal-regulated kinase. In addition, both wortmannin and U0126, but not dasatinib, attenuated cell scattering and migration induced by HGF, suggesting the involvement of the phosphoinositide 3-kinase and mitogen-activated protein kinase pathways, but not of Src or focal adhesion kinase, in HGF-mediated motogenic effects. [1][2]

References on BMS 777697:
[1] Dai Y et al. Mol Cancer Ther. 2010 Jun;9(6):1554-61.
[2] Schroeder GM et al .J Med Chem. 2009 Mar 12;52(5):1251-4.

Other related References on these inhibitors:
*Future Oncol. 2011 Aug;7(8):947-53.
*J Med Chem. 2011 Aug 3.
*Drug Metab Dispos. 2011 Mar;39(3):383-93.
*J Bone Miner Res. 2011 Jun;26(6):1283-94.
*Biosci Trends. 2011 Apr;5(2):52-6.
*Biosci Trends. 2011 Apr;5(2):52-6.