| Hepatocyte growth factor receptor (HGFR), also known as c-Met (mesenchymal-epithelial transition, MET), is an oncogene product discovered in the1980s, and has now been identified as a member of the receptor tyrosine kinase family. HGF was originally developed as a hepatocyte mitogen with similar activity to scatter factor. HGF is produced mainly by various stromal cells, and via its receptor c-Met, can stimulate cell proliferation, migration and morphogenesis.Under normal physiological conditions, the binding of HGF to c-Met leads to phosphorylation of tyrosines within the kinase domain of c-Met and activates the receptor and its downstream signaling pathways, which plays important roles in maintaining the development of organs and tissues as well as homeostasis in the body. However, the abnormal activation of the c-Met signaling pathway induced by HGF elevated secretion, c-Met mutation, c-Met amplification and c-Met overexpression can cause the sustained c-Met phosphorylation, and thus result in activation of the downstream signals, which subsequently leads to abnormal cell proliferation and the transformation of normal cells to cancerous cells.There are several methods for inhibiting abnormal HGF/c-Met signaling pathway, including the use of an HGF variant antagonist, HGF antibody antagonist, c-Met antibody antagonist, and small molecule c-Met inhibitor. Almost all small molecule inhibitors of the HGF/c-Met signaling pathway are c-Met receptor tyrosine kinase inhibitors. The majority of such inhibitors bind in the ATP binding pocket of c-Met and inhibit the phosphorylation of tyrosine residues, which leads to inhibition of c-Met activation-induced abnormal cell proliferation and growth of tumor cells.Anaplastic Iymphoma kinase (ALK) is a member of the insulin receptor tyrosine kinase family. In tumor cells, ALK rearrangement can produce ALK fusion proteins, resulting in constitutive activation of the ALK kinase and abnormal cell proliferation and tumor growth. Simultaneous inhibition of both c-Met and ALK have been shown to have synergistic effects in glioblastoma and other cancer cells. Therefore, a c-Met/ALK dual inhibitor may have therapeutic potential in cancer therapy. In2011, the U.S. FDA approved Pfizer’s Crizotinib, a c-Met/ALK dual inhibitor, for treating non-small cell lung cancer patients with the EML4-ALK fusion protein.Although the dual c-Met/ALK inhibition has been clinically proven to be a successful anti-tumor strategy, there are very few c-Met/ALK dual inhibitors known in the literature. In addition to Crizotinib, Xcovery’s X-396is another c-Met/ALK dual inhibitor currently in Phase I clinical developments. In recent years, our laboratory has been conducting research on c-Met inhibitors. On the basis of our previous experience in this area in combination of the literature reports and the successful clinical application of Pfizer’s c-Met/ALK dual inhibitor Crizotinib, the present study aimed at discovering next-generation c-Met/ALK dual inhibitors with high potencies and high selectivity.Methods and Results1. Design and synthesis of the SMU series spiro compounds and molecular dockingIn our previous research, we identified a series of spiro indolinonyl compounds that are highly selective c-Met inhibitors. The spiro fragment in those compounds fit well in the ATP binding pocket of c-Met. In addition, the2-aminopyridinyl moiety present in the c-Met/ALK dual inhibitor Crizotinib is a typical kinase hinge region binding motif. We therefore thought to combine the spiro fragment identified in this lab with the2-aminopyridinyl component to form new compounds, which might have the c-Met/ALK dual inhibitory activities. Based on this concept,2-aminopyridinyl compounds SMU-A and SMU-B bearing a chiral benzyloxy substituent at the3-position were designed. Considering the similarity between2-aminopyridinyl and2-aminopyrazinyI groups, pyrazinyl analogs SMU-C and SMU-D were also designed. All above compounds have a chiral center in the molecule, thus are relatively difficult to be synthesized. For the sake of simplicity, easily accessible achiral compounds SMU-E and SMU-F were therefore included in this study.To evaluate the compounds designed, we performed molecular docking experiments utilizing a c-Met crystal structure (PDB code:2WGJ). It was found that all six compounds fitted very well into the ATP binding pocket of c-Met. The Surflex-Dock scoring function gave the following scores for SMU-A to SMU-F:8.68,8.67,9.11,8.95,6.70, and6.91, respectively, which are in the same range with the score for Crizotinib at8.67. One of the compounds, i.e., SMU-B, not only can fit very well in the ATP pocket of c-Met, but it can also overlay almost perfectly with Crizotinib in the c-Met crystal structure except for a small portion of the spiro moiety, which is close to the solvent exposed region. SMU-B can also be well docked into the ATP-binging pocket of the ALK kinase (PDB code:2XP2). The above docking experiments suggested that SMU-B should have c-Met and ALK dual inhibitory activities. To examine the computer modeling results, SMU series spiro compounds were synthesized and biologically studied.2. c-Met and ALK biochemical and cellular activities and kinase selectivity of SMU series spiro compoundsIn order to verify the results of the computer modeling, we first examined the effect of compounds on c-Met kinase activity in c-Met-amplified MKN45gastric cancer cells. All the six compounds showed inhibitory activities on cellular c-Met with IC50values at0.020,0.019,0.023,0.012,>10.0and9.0μM, respectively for SMU-A to SMU-F. Crizotinib had an IC50of0.020μM in the same assay. While SMU-A to SMU-D had very high potencies against c-Met, SMU-E and SMU-F were much less potent. These results were consistent with and could be explained by the modeling scores. We also determined the oral bioavailability of compound SMU-B in mice, which was48%. The high potency and good oral bioavailability of SMU-B warranted for further research on this compound.By using KINOMEscanTM kinase panel screening technology, the kinase selectivity of SMU-B was determined at100nM drug concentration against96protein kinases. It was found that out of all96kinases tested, only c-Met, ALK, and AXL showed significant inhibitions at94.4%,91.2%, and95.4%, respectively. To confirm the screening results, we determined the biochemical IC50values of SMU-B against c-Met, ALK, and AXL.At the ATP concentrations equal to their Km’s, the biochemical IC50values of SMU-B for c-Met, ALK, and AXL were1.87nM,<0.5nM, and28.9nM, respectively. To examine if the biochemical results could translate into the cellular activities, the cellular IC50values of SMU-B in inhibiting c-Met, ALK, and AXL were determined in c-Met-amplified MKN45gastric cancer cells, ALK-activated human T lymphoma Karpas299cells, and AXL-overexpressed mouse embryonic fibroblast MEF cells, respectively. The results showed that SMU-B could effectively inhibit the kinase activies in cancer cells with IC50values at22nM,39nM, and300nM, respectively for c-Met, ALK, and AXL. In the same assays, the c-Met and ALK ICso values for Crizotinib were20nM and110nM, respectively. Compared with Crizotinib, SMU-B’s c-Met potency was comparable and ALK potency was3-fold better. The above results were consistent with the biochemical IC50potencies and showed that SMU-B was a highly selective c-Met/ALK dual inhibitor.3. SMU-B inhibits cell proliferation and tumor growth in vitro in c-Met-activated tumor cellsWe investigated the anti-proliferative effects of SMU-B on four c-Met-activated human tumor cell lines by using the MTT assay. The cell lines used for the MTT assays were c-Met-amplified GTL-16human gastric cancer cells, c-Met-amplified H1993human lung cancer cells, c-Met-overexpressed H441human lung cancer cells, and HGF autocrine U87MG human glioma cells. The IC50values were0.020μM,1.58μM,2.02μM, and>10μM, respectively. These results provided preliminary information for the next studies for in vivo anti-tumor effects.4. SMU-B inhibits the tumor growths in the GTL-16gastric cancer, U87MG glioma, and H441lung cancer xenograft mouse modelsSince SMU-B showed potent anti-proliferative potencies and good oral bioavailability in mice, we determined the in vivo efficacies of SMU-B in the GTL-16human gastric cancer, H441human lung cancer, and U87MG human glioma cancer mouse xenograft models. In the GTL-16models, when dosed orally at20mg/kg once a day continuously for14days, the Tumor Growth Inhibition (TGI) was 56.6%. With a higher dose at40mg/kg, the TGI was increased to84.8%. In the H441lung cancer models, with the same two doses, the TGI’s were58.9%and78.7%, respectively. In the U87MG case, the TGI’s were60.7%and92.3, respectively, which were better than the GTL-16and H441tumors.5. SMU-B inhibits the migration and invasion of HGF-activated A549human lung carcinomasThe HGF/c-Met was reported to be able to increase the migration and invasion of tumor cells. We used HGF-stimulated A549lung cancer cells as a model to study the effects of SMU-B on cell migration and invasion. Under normal conditions, A549cells express low levels of c-Met, and can be activated by exogenous HGF. Our experimental results showed that in the absence of HGF stimulation, A549cells do not migrate, but after48h treatment with25ng/mL HGF, the tumor cells could be induced to migrate. SMU-B was found to be able to inhibit the migration of A549tumor cells. The cell invasion assay showed that SMU-B significantly inhibited HGF-induced transmembrane movement of tumor cells. These results indicated that SMU-B had anti-invasion and anti-migration activities.6. SUM-B inhibits the phosphorylation of c-Met and its downstream signaling molecules AKT and ERK1/2in exogenous HGF activated-A549lung cancer cells and human GTL-16gastric carcinomasIn order to know whether SMU-B could inhibit the phosphorylation of c-Met and its downstream signaling molecules, we investigated the effects of SMU-B on the phosphorylation of c-Met and its downstream signaling molecules AKT and ERK1/2in exogenous HGF activated-A549lung cancer cells and human GTL-16gastric carcinomas. Under the stimulation of HGF, the phosphorylated c-Met(p-Met), p-AKT, and p-ERKl/2were significantly increased. After the A549cells were treated with SMU-B, the levels of p-Met, p-AKT, and p-ERK1/2proteins were significantly lower. It was also found that SMU-B could lower the p-Met, p-AKT, and p-ERK1/2levels in vitro in GTL-16cells, and in vivo in GTL-16tumor xenografts in nude mice. These results indicated that SMU-B exerted its anti-tumor effects through inhibiting the c-Met signaling pathway.Conclusion1. In the present study, we for the first time designed and synthesized a series of3-benzyloxy-substituted2-aminopyridinyl spiro compounds. One of these compounds, i.e., SMU-B, was proved to be a highly selective c-Met/ALK dual inhibitor with the biochemical and cellular ICso values for c-Met at1.87nM and39nM, respectively, and ALK ICso values at<0.5nM and22nM, respectively.2. Compound SMU-B significantly inhibited the in intro proliferation of c-Met-amplified GTL-16human gastric cancer cells, c-Met-amplified H1993human lung cancer cells, c-Met-overexpressed H441human lung cancer cells with ICso values equal to0.020μM,1.58μM, and2.02μM, respectively. For the HGF autocrine U87MG human glioma cells, however, the anti-proliferative ICso was>10μM.,3. SUM-B showed good efficacies in three tumor xenograft mouse models. In the c-Met-amplified GTL-16human gastric tumor models, at20mg/kg and40mg/kg once a day oral dosing, SMU-B had Tumor Growth Inhibition (TGI) rate at56.6%and84.8%, respectively. In the c-Met-overexpressed H441human lung tumor models, at the same doses, the TGI’s were58.9%and78.7%, respectively. In the HGF autocrine U87MG human glioma models, the TGI’s were60.7%and92.3%, respectively, which were better than those for in the GTL-16and H441tumor models.4. SMU-B showed strong inhibitions for HGF-induced migration and transmembrane movement of HGF-stimulated A549lung cancer cells, indicating the compound had anti-invasion and anti-migration activities.5. SMU-B significantly inhibited HGF-induced phosphorylation of c-Met, AKT and ERKl/2proteins in A549cells, and inhibited the phosphorylation of c-Met, AKT and ERK1/2both in vitro in GTL-16cells and in vivo in GTL-16tumor xenografts in nude mice, indicating that SMU-B exerted anti-tumor effects through inhibition of the c-Met signaling pathway. |
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