新型螺环取代的氨基吡啶类化合物SMU-B抗肿瘤作用及其作用的分子机理研究
|
摘要
肝细胞生长因子受体(hepatocyte growth factor receptor, HGFR),又称c-Met (mesenchymal-epithelial transition, MET),是在80年代发现的原癌基因产物,现已确定为受体酪氨酸激酶家族中的成员。HGF最初是作为大鼠肝细胞丝裂原(mitogen)而被发现,其作用与扩散因子(scatter factor)相似。现已证明,HGF主要由基质细胞产生,通过其受体c-Met,能刺激上皮细胞等细胞的增殖,运动,形态发生(morphogenesis)和血管生成。
在正常生理情况下,HGF与受体c-Met结合导致受体激酶区的酪氨酸发生磷酸化,进而导致受体及其下游信号通路的激活,在维持机体器官的发育和组织内环境稳定中发挥重要的作用。然而,HGF异常分泌,c-Met突变,扩增(amplification)以及高表达导致的c-Met通路的异常激活,可引起c-Met受体的持续激活与磷酸化,从而持续不断地激活下游信号通路,导致细胞的异常增殖,诱导正常细胞向癌细胞转化。
有许多方法用来抑制异常的HGF/c-Met信号通路,包括：HGF环状异构体拮抗剂(HGF variant antagonist),HGF抗体拮抗剂(HGF antibody antagonist), c-Met抗体拮抗剂(c-Met antibody antagonist)以及小分子c-Met抑制剂。目前发现的小分子c-Met抑制剂绝大多数都是作用在受体酪氨酸激酶的抑制剂。绝大多数化合物都通过与c-Met受体激酶区ATP口袋(ATP binding site)结合,抑制酪氨酸残基的磷酸化,抑制c-Met通路持续异常激活导致的肿瘤细胞异常增殖和生长。
间变性淋巴瘤激酶(anaplastic lymphoma kinase, ALK)是胰岛素受体酪氨酸激酶家族成员之一。ALK在肿瘤中的异常改变主要是基因移位,异常的移位导致许多ALK融合蛋白(ALK fusion protein)产生,并持续激活ALK,导致肿瘤细胞的异常增殖和生长。同时抑制c-Met和ALK已经证明在胶质母细胞瘤等肿瘤细胞中有协同效应。因此,研究同时作用于c-Met和ALK的双重激酶抑制剂(c-Met/ALK dual inhibitor)是非常有前景的抗肿瘤药物。美国FDA于2011年批准了c-Met/ALK双重抑制剂克唑替尼(Crizotinib)用于治疗有EML4-ALK融合蛋白形成的非小细胞肺癌患者。
虽然c-Met和ALK的双重激酶抑制剂是非常有前景的抗肿瘤策略,但在文献中报道的此类抑制剂很少,除Crizotinib外,还有一个是Xcovery公司研发的X-396,目前在进行一期临床试验。我们实验室多年来一直开展c-Met抑制剂的研究,本论文是在实验室前期的工作基础上,综合文献报道的结果以及临床上应用c-Met/ALK双重抑制剂克唑替尼(Crizotinib)成功的策略,设计和合成新的作用于c-Met和ALK的化合物,旨在发现新的高活性和高选择性的c-Met/ALK双重抑制剂。
主要的实验方法和实验结果
一.SMU系列螺环化合物的设计与合成以及分子对接模拟
在我们实验室之前有关c-Met抑制剂的研发中,发现了一类螺环化合物是有效的高选择性c-Met抑制剂,这些化合物分子中的螺环片段能很好地适应c-Met的ATP结合口袋,而c-Met/ALK双重抑制剂克唑替尼分子中含有典型的激酶铰链区结合基团,即2-氨基吡啶。据此,我们的设计思路是：将螺环片断与激酶铰链区结合基团2-氨基吡啶片断结合成一个新的化合物,可能同时具有c-Met/ALK的双重阻断作用?因此,我们设计了3-位由带手性取代基的苄氧基取代的2-氨基吡啶化合物SMU-A及SMU-B;考虑到2-氨基吡嗪基与2-氨基吡啶基的相似性,又设计了毗嗪化合物SMU-C及SMU-D。上述四个化合物分子中都具有一个手性基团,化学合成难度高。为了减小化学合成的难度,我们除去3-位取代苄氧基中的手性甲基再设计了非手性化合物SMU-E及SMU-F。
然后,我们对设计的化合物与c-Met激酶晶体结构进行了分子对接模拟,发现这些化合物都能与激酶晶体结构进行对接。6个化合物根据Surflex-Dock程序的打分值依次如下：8.68,8.67,9.11,8.95,6.70和6.91,克唑替尼为8.67。以化合物SMU-B为例,c-Met激酶的ATP口袋能够很好地接纳SMU-B,并与其形成多个包括氢键、π-π堆积作用、范德华力等相互作用。在c-Met激酶晶体结构中,SMU-B能与克唑替尼很好地叠合,除了靠近溶剂区的部分,SMU-B与克唑替尼几乎达到完美的重叠一致。此外,SMU-B也能与ALK激酶晶体结构的ATP口袋很好地对接。由此预测,SMU-B应该具有对c-Met和ALK激酶的双重抑制作用。于是,我们合成了六个SMU系列的螺环化合物拟进行下一步的研究。
二.SMU系列螺环化合物对c-Met和ALK激酶高选择性活性的确认
为了验证上述计算机分子模拟的结果,我们首先检测了化合物对细胞中c-Met激酶活性的影响。结果发现,六个化合物对MKN45胃癌细胞中c-Met激酶有显著抑制活性,IC50值依次为0.020,0.019,0.023,0.012,>10和9.0μM,克唑替尼为0.020gM,与计算机分子对接模拟的预测一致。进一步对上述化合物的生物利用度研究结果表明,SMU-B的口服生物度达到48%,因此确定该化合物进入下一步研究。
首先采用美国KINOMEscanTM公司的激酶高通量筛选技术,评价了SMU-B对人96种蛋白激酶生化活性的影响,发现SMU-B在100nM浓度下仅对c-Met,ALK和AXL三种受体酪氨酸激酶活性有显著的抑制活性,抑制率分别为94.4%,91.2%和95.4%,表明SMU-B对受体酪氨酸激酶家族的c-Met、ALK和AXL三种蛋白激酶表现出高选择性。然后,采用美国Reaction BiologyCorporation的放射蛋白激酶活性检测方法进一步测定了SMU-B对三种激酶生化活性抑制的IC50值。结果表明,SMU-B对c-Met, ALK和AXL三种激酶生化活性都有显著的抑制作用。在激酶底物ATP的存在下,c-Met酶生化活性抑制的IC50值为1.87nM,ALK的IC50值<0.5nM, AXL的IC50值为28.9nM。SMU-B对三种激酶抑制活性的强度以ALK最强,其次为c-Met, AXL最弱。最后,我们选择c-Met扩增的人胃癌MKN45细胞,ALK激活的人T淋巴瘤Karpas299细胞和AXL高表达的鼠胚胎成纤维MEF细胞来分别评价SMU-B对细胞中c-Met, ALK和AXL三种激酶的抑制活性,结果表明化合物对MKN45细胞c-Met磷酸化抑制的IC50为22nnM,对Karpas299淋巴瘤细胞ALK磷酸化抑制的IC50为39nM,对MEF鼠胚胎成纤维细胞AXL磷酸化抑制的IC50为300nM；而阳性化合物克唑替尼对MKN45细胞c-Met磷酸化抑制的IC50为20nM,对Karpas299细胞ALK磷酸化抑制的IC50为110nM,高于SMU-B的39nM。SMU-B对上述三个激酶细胞活性的作用与生化活性结果一致。而SMU-B对AXL细胞磷酸化抑制的ICso为300nM,远高于生化活性的28.9nM,进一步证明了该化合物对AXL激酶活性弱,为高选择性c-Met/ALK双重抑制剂。
三.SMU-B抑制体外培养的c-Met异常的人肿瘤细胞的增殖和生长我们采用MTT法研究了SMU-B在体外对四种c-Met异常的人肿瘤细胞增殖的影响,发现化合物在体外能显著抑制c-Met扩增的人胃癌GTL-16细胞,c-Met扩增的人肺癌H1993细胞,c-Met高表达人肺癌H441细胞以及有HGF自分泌功能的人脑胶质瘤U87MG细胞的增殖和生长,对GTL-16细胞生长抑制的IC50值为0.020gM,对H1993的IC50值为1.58gM,对H441的IC50值为2.02-tM,但对脑胶质瘤U87MG的生长抑制作用较弱,抑制的IC50>10μM。上述SMU-B在体外抗c-Met异常细胞增殖的筛选结果为下一步体内抗肿瘤实验提供了初步的依据。
四.SMU-B抑制c-Met异常的人GTL-16胃癌,U87MG脑胶质瘤和H441肺癌裸鼠异体移植瘤的生长
根据体外抗增殖作用的结果,我们采用肿瘤异体移植裸鼠模型进一步评价了SMU-B对c-Met扩增的人胃癌GTL-16细胞,c-Met高表达的人肺癌H441细胞以及有HGF自分泌功能的人脑胶质瘤U87MG细胞的生长抑制作用。结果发现,SUM-B虽然对上述三种c-Met异常的人肿瘤裸鼠异体移植瘤都有显著的抗肿瘤作用,但在裸鼠体内的作用与体外抗增殖结果不完全一致。SMU-B以20mg/kg口服灌胃给药一日一次连续14天,GTL-16胃癌的抑瘤率为56.6%,当剂量增加至40mg/kg时,抑瘤率增加至84.8%；在相同剂量下,对H441的抑瘤率分别为58.9%和78.7%,对U87MG的抑瘤率分别为60.7%和92.3。SMU-B在体外抑制U87MG细胞增殖的IC50值>101.tM,高于GTL-16和H441细胞,但在裸鼠体内的抑瘤作用却比二者好。
五.SMU-B抑制HGF诱导的人肺癌A549细胞的迁移和侵袭作用
由于HGF/c-Met具有增加肿瘤细胞移动和侵袭的能力,我们以外源性HGF诱导c-Met激活的人A549肺癌细胞为模型,研究SMU-B对HGF诱导的A549细胞的迁移和侵袭的影响。A549细胞在正常条件下表达低水平的c-Met,仅在外源性HGF的刺激下c-Met才能被激活发生磷酸化,诱导细胞的移动和迁移。我们的细胞迁移实验结果表明,在无HGF刺激时,A549细胞基本上不发生迁移,但在培养细胞中加入25ng/mL HGF作用48h后,能诱导肿瘤细胞发生移动；在此模型基础上同时加入不同浓度的SMU-B后,能明显抑制肿瘤细胞的移动。此外,细胞侵袭实验结果也表明,在无HGF刺激下,大量A549肿瘤细胞穿过滤膜,表现出明显的侵袭特性,SMU-B能显著抑制HGF诱导的肿瘤细胞的穿膜运动,显示化合物具有抗肿瘤细胞侵袭和迁移的作用。
六.SMU-B在体外能显著抑制外源性HGF诱导的A549肺癌细胞c-Met.AKT及ERK1/2蛋白的磷酸化；也能在体外抑制GTL-16胃癌细胞和在体内抑制GTL-16胃癌裸鼠异体移植瘤的c-Met、AKT和ERK1/2蛋白的磷酸化,通过抑制c-Met信号通路发挥抗肿瘤作用
为了探讨SMU-B对c-Met异常肿瘤产生抗肿瘤作用时,是否同时能抑制c-Met和下游信号分子的激活,我们以外源性HGF激活的A549肺癌和c-Met扩增的GTL-16胃癌细胞模型,研究了化合物对c-Met及其下游介导细胞增殖的信号分子AKT(蛋白激酶B)和介导细胞存活的信号分子ERK1/2(extracellular signal-regulated kinase)的磷酸化的影响。我们发现,A549细胞在HGF的作用下,c-Met, AKT和ERK1/2的磷酸化明显增加；SMU-B能显著抑制HGF诱导的c-Met、AKT及ERKl/2蛋白的磷酸化。同样,SMU-B也能在体外抑制GTL-16胃癌细胞c-Met的磷酸化以及抑制GTL-16裸鼠异体移植瘤c-Met、AKT以及ERK1/2蛋白的磷酸化。上述结果表明,SMU-B通过抑制裸鼠移植肿瘤的c-Met信号通路发挥抗肿瘤作用。
结论
1.本文在设计和合成的一系列3-位苄氧基取代的2-氨基吡啶化合物中,首次发现SMU-B对c-Met生化和细胞激酶活性抑制的IC50值分别为1.87nM和22nM,对ALK激酶生化和细胞活性抑制的ICso值分别为<0.5和39nM,表明该化合物一种高选择性c-Met/ALK双重抑制剂。
2. SMU-B在体外对四种c-Met异常的人肿瘤细胞生长有显著的抑制作用。对c-Met扩增的人胃癌GTL-16细胞生长抑制的IC50值为0.020gM,对c-Met扩增的人肺癌H1993细胞为1.58μM,对c-Met高表达人肺癌H441细胞为2.02μM,但对人HGF自分泌的脑胶质瘤U87MG细胞生长抑制的IC50>10μM抑制作用较弱。
3.SMU-B对c-Met扩增的人胃癌GTL-16,c-Met高表达人肺癌H441以及HGF自分泌的脑胶质瘤U87MG细胞三种裸鼠异体移植肿瘤有显著的抗肿瘤生长作用。化合物在20mg/kg和40mg/kg给药时,对U87MG的抑瘤率分别为60.7%和92.3%,对GTL-16为56.6%和84.8%,而对H441的抑瘤率为58.8%和78.7%,具有比较强的抗肿瘤作用。
4. SMU-B在体外能抑制外源性HGF诱导的人A549肺癌细胞的迁移和跨膜移动,具有抗肿瘤细胞的迁移和侵袭作用。
5. SMU-B在体外能显著抑制外源性HGF诱导的A549肺癌细胞c-Met、AKT及ERKl/2蛋白的磷酸化；也能在体外抑制GTL-16胃癌细胞和在体内抑制GTL-16胃癌裸鼠异体移植瘤的c-Met、AKT和ERK1/2蛋白的磷酸化,通过抑制c-Met信号通路发挥抗肿瘤作用。
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 Results
1. Design and synthesis of the SMU series spiro compounds and molecular docking
In 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 compounds
In 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 cells
We 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 models
Since 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 carcinomas
The 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 carcinomas
In 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.
Conclusion
1. 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.
在正常生理情况下,HGF与受体c-Met结合导致受体激酶区的酪氨酸发生磷酸化,进而导致受体及其下游信号通路的激活,在维持机体器官的发育和组织内环境稳定中发挥重要的作用。然而,HGF异常分泌,c-Met突变,扩增(amplification)以及高表达导致的c-Met通路的异常激活,可引起c-Met受体的持续激活与磷酸化,从而持续不断地激活下游信号通路,导致细胞的异常增殖,诱导正常细胞向癌细胞转化。
有许多方法用来抑制异常的HGF/c-Met信号通路,包括：HGF环状异构体拮抗剂(HGF variant antagonist),HGF抗体拮抗剂(HGF antibody antagonist), c-Met抗体拮抗剂(c-Met antibody antagonist)以及小分子c-Met抑制剂。目前发现的小分子c-Met抑制剂绝大多数都是作用在受体酪氨酸激酶的抑制剂。绝大多数化合物都通过与c-Met受体激酶区ATP口袋(ATP binding site)结合,抑制酪氨酸残基的磷酸化,抑制c-Met通路持续异常激活导致的肿瘤细胞异常增殖和生长。
间变性淋巴瘤激酶(anaplastic lymphoma kinase, ALK)是胰岛素受体酪氨酸激酶家族成员之一。ALK在肿瘤中的异常改变主要是基因移位,异常的移位导致许多ALK融合蛋白(ALK fusion protein)产生,并持续激活ALK,导致肿瘤细胞的异常增殖和生长。同时抑制c-Met和ALK已经证明在胶质母细胞瘤等肿瘤细胞中有协同效应。因此,研究同时作用于c-Met和ALK的双重激酶抑制剂(c-Met/ALK dual inhibitor)是非常有前景的抗肿瘤药物。美国FDA于2011年批准了c-Met/ALK双重抑制剂克唑替尼(Crizotinib)用于治疗有EML4-ALK融合蛋白形成的非小细胞肺癌患者。
虽然c-Met和ALK的双重激酶抑制剂是非常有前景的抗肿瘤策略,但在文献中报道的此类抑制剂很少,除Crizotinib外,还有一个是Xcovery公司研发的X-396,目前在进行一期临床试验。我们实验室多年来一直开展c-Met抑制剂的研究,本论文是在实验室前期的工作基础上,综合文献报道的结果以及临床上应用c-Met/ALK双重抑制剂克唑替尼(Crizotinib)成功的策略,设计和合成新的作用于c-Met和ALK的化合物,旨在发现新的高活性和高选择性的c-Met/ALK双重抑制剂。
主要的实验方法和实验结果
一.SMU系列螺环化合物的设计与合成以及分子对接模拟
在我们实验室之前有关c-Met抑制剂的研发中,发现了一类螺环化合物是有效的高选择性c-Met抑制剂,这些化合物分子中的螺环片段能很好地适应c-Met的ATP结合口袋,而c-Met/ALK双重抑制剂克唑替尼分子中含有典型的激酶铰链区结合基团,即2-氨基吡啶。据此,我们的设计思路是：将螺环片断与激酶铰链区结合基团2-氨基吡啶片断结合成一个新的化合物,可能同时具有c-Met/ALK的双重阻断作用?因此,我们设计了3-位由带手性取代基的苄氧基取代的2-氨基吡啶化合物SMU-A及SMU-B;考虑到2-氨基吡嗪基与2-氨基吡啶基的相似性,又设计了毗嗪化合物SMU-C及SMU-D。上述四个化合物分子中都具有一个手性基团,化学合成难度高。为了减小化学合成的难度,我们除去3-位取代苄氧基中的手性甲基再设计了非手性化合物SMU-E及SMU-F。
然后,我们对设计的化合物与c-Met激酶晶体结构进行了分子对接模拟,发现这些化合物都能与激酶晶体结构进行对接。6个化合物根据Surflex-Dock程序的打分值依次如下：8.68,8.67,9.11,8.95,6.70和6.91,克唑替尼为8.67。以化合物SMU-B为例,c-Met激酶的ATP口袋能够很好地接纳SMU-B,并与其形成多个包括氢键、π-π堆积作用、范德华力等相互作用。在c-Met激酶晶体结构中,SMU-B能与克唑替尼很好地叠合,除了靠近溶剂区的部分,SMU-B与克唑替尼几乎达到完美的重叠一致。此外,SMU-B也能与ALK激酶晶体结构的ATP口袋很好地对接。由此预测,SMU-B应该具有对c-Met和ALK激酶的双重抑制作用。于是,我们合成了六个SMU系列的螺环化合物拟进行下一步的研究。
二.SMU系列螺环化合物对c-Met和ALK激酶高选择性活性的确认
为了验证上述计算机分子模拟的结果,我们首先检测了化合物对细胞中c-Met激酶活性的影响。结果发现,六个化合物对MKN45胃癌细胞中c-Met激酶有显著抑制活性,IC50值依次为0.020,0.019,0.023,0.012,>10和9.0μM,克唑替尼为0.020gM,与计算机分子对接模拟的预测一致。进一步对上述化合物的生物利用度研究结果表明,SMU-B的口服生物度达到48%,因此确定该化合物进入下一步研究。
首先采用美国KINOMEscanTM公司的激酶高通量筛选技术,评价了SMU-B对人96种蛋白激酶生化活性的影响,发现SMU-B在100nM浓度下仅对c-Met,ALK和AXL三种受体酪氨酸激酶活性有显著的抑制活性,抑制率分别为94.4%,91.2%和95.4%,表明SMU-B对受体酪氨酸激酶家族的c-Met、ALK和AXL三种蛋白激酶表现出高选择性。然后,采用美国Reaction BiologyCorporation的放射蛋白激酶活性检测方法进一步测定了SMU-B对三种激酶生化活性抑制的IC50值。结果表明,SMU-B对c-Met, ALK和AXL三种激酶生化活性都有显著的抑制作用。在激酶底物ATP的存在下,c-Met酶生化活性抑制的IC50值为1.87nM,ALK的IC50值<0.5nM, AXL的IC50值为28.9nM。SMU-B对三种激酶抑制活性的强度以ALK最强,其次为c-Met, AXL最弱。最后,我们选择c-Met扩增的人胃癌MKN45细胞,ALK激活的人T淋巴瘤Karpas299细胞和AXL高表达的鼠胚胎成纤维MEF细胞来分别评价SMU-B对细胞中c-Met, ALK和AXL三种激酶的抑制活性,结果表明化合物对MKN45细胞c-Met磷酸化抑制的IC50为22nnM,对Karpas299淋巴瘤细胞ALK磷酸化抑制的IC50为39nM,对MEF鼠胚胎成纤维细胞AXL磷酸化抑制的IC50为300nM；而阳性化合物克唑替尼对MKN45细胞c-Met磷酸化抑制的IC50为20nM,对Karpas299细胞ALK磷酸化抑制的IC50为110nM,高于SMU-B的39nM。SMU-B对上述三个激酶细胞活性的作用与生化活性结果一致。而SMU-B对AXL细胞磷酸化抑制的ICso为300nM,远高于生化活性的28.9nM,进一步证明了该化合物对AXL激酶活性弱,为高选择性c-Met/ALK双重抑制剂。
三.SMU-B抑制体外培养的c-Met异常的人肿瘤细胞的增殖和生长我们采用MTT法研究了SMU-B在体外对四种c-Met异常的人肿瘤细胞增殖的影响,发现化合物在体外能显著抑制c-Met扩增的人胃癌GTL-16细胞,c-Met扩增的人肺癌H1993细胞,c-Met高表达人肺癌H441细胞以及有HGF自分泌功能的人脑胶质瘤U87MG细胞的增殖和生长,对GTL-16细胞生长抑制的IC50值为0.020gM,对H1993的IC50值为1.58gM,对H441的IC50值为2.02-tM,但对脑胶质瘤U87MG的生长抑制作用较弱,抑制的IC50>10μM。上述SMU-B在体外抗c-Met异常细胞增殖的筛选结果为下一步体内抗肿瘤实验提供了初步的依据。
四.SMU-B抑制c-Met异常的人GTL-16胃癌,U87MG脑胶质瘤和H441肺癌裸鼠异体移植瘤的生长
根据体外抗增殖作用的结果,我们采用肿瘤异体移植裸鼠模型进一步评价了SMU-B对c-Met扩增的人胃癌GTL-16细胞,c-Met高表达的人肺癌H441细胞以及有HGF自分泌功能的人脑胶质瘤U87MG细胞的生长抑制作用。结果发现,SUM-B虽然对上述三种c-Met异常的人肿瘤裸鼠异体移植瘤都有显著的抗肿瘤作用,但在裸鼠体内的作用与体外抗增殖结果不完全一致。SMU-B以20mg/kg口服灌胃给药一日一次连续14天,GTL-16胃癌的抑瘤率为56.6%,当剂量增加至40mg/kg时,抑瘤率增加至84.8%；在相同剂量下,对H441的抑瘤率分别为58.9%和78.7%,对U87MG的抑瘤率分别为60.7%和92.3。SMU-B在体外抑制U87MG细胞增殖的IC50值>101.tM,高于GTL-16和H441细胞,但在裸鼠体内的抑瘤作用却比二者好。
五.SMU-B抑制HGF诱导的人肺癌A549细胞的迁移和侵袭作用
由于HGF/c-Met具有增加肿瘤细胞移动和侵袭的能力,我们以外源性HGF诱导c-Met激活的人A549肺癌细胞为模型,研究SMU-B对HGF诱导的A549细胞的迁移和侵袭的影响。A549细胞在正常条件下表达低水平的c-Met,仅在外源性HGF的刺激下c-Met才能被激活发生磷酸化,诱导细胞的移动和迁移。我们的细胞迁移实验结果表明,在无HGF刺激时,A549细胞基本上不发生迁移,但在培养细胞中加入25ng/mL HGF作用48h后,能诱导肿瘤细胞发生移动；在此模型基础上同时加入不同浓度的SMU-B后,能明显抑制肿瘤细胞的移动。此外,细胞侵袭实验结果也表明,在无HGF刺激下,大量A549肿瘤细胞穿过滤膜,表现出明显的侵袭特性,SMU-B能显著抑制HGF诱导的肿瘤细胞的穿膜运动,显示化合物具有抗肿瘤细胞侵袭和迁移的作用。
六.SMU-B在体外能显著抑制外源性HGF诱导的A549肺癌细胞c-Met.AKT及ERK1/2蛋白的磷酸化；也能在体外抑制GTL-16胃癌细胞和在体内抑制GTL-16胃癌裸鼠异体移植瘤的c-Met、AKT和ERK1/2蛋白的磷酸化,通过抑制c-Met信号通路发挥抗肿瘤作用
为了探讨SMU-B对c-Met异常肿瘤产生抗肿瘤作用时,是否同时能抑制c-Met和下游信号分子的激活,我们以外源性HGF激活的A549肺癌和c-Met扩增的GTL-16胃癌细胞模型,研究了化合物对c-Met及其下游介导细胞增殖的信号分子AKT(蛋白激酶B)和介导细胞存活的信号分子ERK1/2(extracellular signal-regulated kinase)的磷酸化的影响。我们发现,A549细胞在HGF的作用下,c-Met, AKT和ERK1/2的磷酸化明显增加；SMU-B能显著抑制HGF诱导的c-Met、AKT及ERKl/2蛋白的磷酸化。同样,SMU-B也能在体外抑制GTL-16胃癌细胞c-Met的磷酸化以及抑制GTL-16裸鼠异体移植瘤c-Met、AKT以及ERK1/2蛋白的磷酸化。上述结果表明,SMU-B通过抑制裸鼠移植肿瘤的c-Met信号通路发挥抗肿瘤作用。
结论
1.本文在设计和合成的一系列3-位苄氧基取代的2-氨基吡啶化合物中,首次发现SMU-B对c-Met生化和细胞激酶活性抑制的IC50值分别为1.87nM和22nM,对ALK激酶生化和细胞活性抑制的ICso值分别为<0.5和39nM,表明该化合物一种高选择性c-Met/ALK双重抑制剂。
2. SMU-B在体外对四种c-Met异常的人肿瘤细胞生长有显著的抑制作用。对c-Met扩增的人胃癌GTL-16细胞生长抑制的IC50值为0.020gM,对c-Met扩增的人肺癌H1993细胞为1.58μM,对c-Met高表达人肺癌H441细胞为2.02μM,但对人HGF自分泌的脑胶质瘤U87MG细胞生长抑制的IC50>10μM抑制作用较弱。
3.SMU-B对c-Met扩增的人胃癌GTL-16,c-Met高表达人肺癌H441以及HGF自分泌的脑胶质瘤U87MG细胞三种裸鼠异体移植肿瘤有显著的抗肿瘤生长作用。化合物在20mg/kg和40mg/kg给药时,对U87MG的抑瘤率分别为60.7%和92.3%,对GTL-16为56.6%和84.8%,而对H441的抑瘤率为58.8%和78.7%,具有比较强的抗肿瘤作用。
4. SMU-B在体外能抑制外源性HGF诱导的人A549肺癌细胞的迁移和跨膜移动,具有抗肿瘤细胞的迁移和侵袭作用。
5. SMU-B在体外能显著抑制外源性HGF诱导的A549肺癌细胞c-Met、AKT及ERKl/2蛋白的磷酸化；也能在体外抑制GTL-16胃癌细胞和在体内抑制GTL-16胃癌裸鼠异体移植瘤的c-Met、AKT和ERK1/2蛋白的磷酸化,通过抑制c-Met信号通路发挥抗肿瘤作用。
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 Results
1. Design and synthesis of the SMU series spiro compounds and molecular docking
In 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 compounds
In 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 cells
We 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 models
Since 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 carcinomas
The 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 carcinomas
In 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.
Conclusion
1. 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.
引文
[1]T. Nakamura, S. Mizuno. The discovery of hepatocyte growth factor (HGF) and its significance for cell biology, life sciences and clinical medicine. Proc Jpn Acad Ser B Phys Biol Sci 86 (2010) 588-610.
[2]D.P. Bottaro, J.S. Rubin, D.L. Faletto, et al. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 251 (1991) 802-804.
[3]J.P. Eder, G.F. Vande Woude, S.A. Boerner, et al. Novel therapeutic inhibitors of the c-Met signaling pathway in cancer. Clin Cancer Res 15 (2009) 2207-2214.
[4]T. Igawa, K. Matsumoto, S. Kanda, et al. Hepatocyte growth factor may function as a renotropic factor for regeneration in rats with acute renal injury. Am J Physiol 265 (1993) F61-69.
[5]T. Yoshida, Y. Ozawa, T. Kimura, et al. Eribulin mesilate suppresses experimental metastasis of breast cancer cells by reversing phenotype from epithelial-mesenchymal transition (EMT) to mesenchymal-epithelial transition (MET) states. Br J Cancer 110 (2014) 1497-1505.
[6]K. Polyak, R.A. Weinberg. Transitions between epithelial and mesenchymal states:acquisition of malignant and stem cell traits. Nat Rev Cancer 9 (2009) 265-273.
[7]N.P. Gunasinghe, A. Wells, E.W. Thompson, et al. Mesenchymal-epithelial transition (MET) as a mechanism for metastatic colonisation in breast cancer. Cancer Metastasis Rev 31 (2012) 469-478.
[8]J.G. Christensen, J. Burrows, R. Salgia. c-Met as a target for human cancer and characterization of inhibitors for therapeutic intervention. Cancer Lett 225 (2005) 1-26.
[9]R. Jagadeeswaran, H. Surawska, S. Krishnaswamy, et al. Paxillin is a target for somatic mutations in lung cancer:implications for cell growth and invasion. Cancer Res 68 (2008) 132-142.
[10]S. Krishnaswamy, R. Kanteti, J.S. Duke-Cohan, et al. Ethnic differences and functional analysis of MET mutations in lung cancer. Clin Cancer Res 15 (2009)5714-5723.
[11]J.H. Lee, S.U. Han, H. Cho, et al. A novel germ line juxtamembrane Met mutation in human gastric cancer. Oncogene 19 (2000) 4947-4953.
[12]D. Zaffaroni, M. Spinola, A. Galvan, et al. Met proto-oncogene juxtamembrane rare variations in mouse and humans:differential effects of Arg and Cys alleles on mouse lung tumorigenesis. Oncogene 24 (2005) 1084-1090.
[13]L. Toschi, F. Cappuzzo. Clinical implications of MET gene copy number in lung cancer. Future Oncol 6 (2010) 239-247.
[14]L. Schmidt, F.M. Duh, F. Chen, et al.Germline and somatic mutations in the tyro sine kinase domain of the MET proto-oncogene in papillary renal carcinomas. Nat Genet 16 (1997) 68-73.
[15]L. Schmidt, K. Junker, G. Weirich, et al. Two North American families with hereditary papillary renal carcinoma and identical novel mutations in the MET proto-oncogene. Cancer Res 58 (1998) 1719-1722.
[16]W.S. Park, S.M. Dong, S.Y. Kim, et al. Somatic mutations in the kinase domain of the Met/hepatocyte growth factor receptor gene in childhood hepatocellular carcinomas. Cancer Res 59 (1999) 307-310.
[17]J. Tanyi, K. Tory, J. Rigo,et al. Evaluation of the tyrosine kinase domain of the Met proto-oncogene in sporadic ovarian carcinomas*. Pathol Oncol Res 5 (1999) 187-191.
[18]H. Kuniyasu, W. Yasui, Y. Kitadai,et al. Frequent amplification of the c-met gene in scirrhous type stomach cancer. Biochem Biophys Res Commun 189 (1992) 227-232.
[19]M. Nessling, S. Solinas-Toldo, K.K. Wilgenbus, et al. Mapping of chromosomal imbalances in gastric adenocarcinoma revealed amplified protooncogenes MYCN, MET, WNT2, and ERBB2. Genes Chromosomes Cancer 23 (1998) 307-316.
[20]C. Sakakura, T. Mori, T. Sakabe, et al. Gains, losses, and amplifications of genomic materials in primary gastric cancers analyzed by comparative genomic hybridization. Genes Chromosomes Cancer 24 (1999) 299-305.
[21]C. Ponzetto, S. Giordano, F. Peverali,et al. c-met is amplified but not mutated in a cell line with an activated met tyrosine kinase. Oncogene 6 (1991) 553-559.
[22]G.A. Smolen, R. Sordella, B. Muir, et al. Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752. Proc Natl Acad Sci U S A 103 (2006) 2316-2321.
[23]F. Cappuzzo, A. Marchetti, M. Skokan, et al. Increased MET gene copy number negatively affects survival of surgically resected non-small-cell lung cancer patients. J Clin Oncol 27 (2009) 1667-1674.
[24]L. Landi, G. Minuti, A. D'Incecco,et al. Targeting c-MET in the battle against advanced nonsmall-cell lung cancer. Curr Opin Oncol 25 (2013) 130-136.
[25]J.A. Engelman, K. Zejnullahu, T. Mitsudomi, et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 316(2007)1039-1043.
[26]H.J. Chen, T.S. Mok, Z.H. Chen, et al. Clinicopathologic and molecular features of epidermal growth factor receptor T790M mutation and c-MET amplification in tyrosine kinase inhibitor-resistant Chinese non-small cell lung cancer. Pathol Oncol Res 15 (2009) 651-658.
[27]J. Bean, C. Brennan, J.Y. Shih, et al. MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci U S A104 (2007) 20932-20937.
[28]E. Ichimura, A. Maeshima, T. Nakajima, et al. Expression of c-met/HGF receptor in human non-small cell lung carcinomas in vitro and in vivo and its prognostic significance. Jpn J Cancer Res 87 (1996) 1063-1069.
[29]S. Garcia, J.P. Dales, E. Charafe-Jaufffret, et al. Poor prognosis in breast carcinomas correlates with increased expression of targetable CD 146 and c-Met and with proteomic basal-like phenotype. Hum Pathol 38 (2007) 830-841.
[30]I. Takanami, F. Tanana, T. Hashizume,et al. Hepatocyte growth factor and c-Met/hepatocyte growth factor receptor in pulmonary adenocarcinomas:an evaluation of their expression as prognostic markers. Oncology 53 (1996) 392-397.
[31]C. Birchmeier, W. Birchmeier, E. Gherardi,et al. Met, metastasis, motility and more. Nat Rev Mol Cell Biol 4 (2003) 915-925.
[32]G. Maulik, A. Shrikhande, T. Kijima, et al. Role of the hepatocyte growth factor receptor, c-Met, in oncogenesis and potential for therapeutic inhibition. Cytokine Growth Factor Rev 13 (2002) 41-59.
[33]R. Wang, L.D. Ferrell, S. Faouzi,et al. Activation of the Met receptor by cell attachment induces and sustains hepatocellular carcinomas in transgenic mice. J Cell Biol 153 (2001) 1023-1034.
[34]S. Patane, S. Avnet, N. Coltella, et al. MET overexpression turns human primary osteoblasts into osteosarcomas. Cancer Res 66 (2006) 4750-4757.
[35]G. Rossi, A. Cavazza, A. Marchioni, et al. Role of chemotherapy and the receptor tyrosine kinases KIT, PDGFRalpha, PDGFRbeta, and Met in large-cell neuroendocrine carcinoma of the lung. J Clin Oncol 23 (2005) 8774-8785.
[36]M. Olivero, M. Rizzo, R. Madeddu, et al. Overexpression and activation of hepatocyte growth factor/scatter factor in human non-small-cell lung carcinomas. Br J Cancer 74 (1996) 1862-1868.
[37]K. Date, K. Matsumoto, H. Shimura,et al. HGF/NK4 is a specific antagonist for pleiotrophic actions of hepatocyte growth factor. FEBS Lett 420 (1997) 1-6.
[38]D. Tomioka, N. Maehara, K. Kuba, et al. Inhibition of growth, invasion, and metastasis of human pancreatic carcinoma cells by NK4 in an orthotopic mouse model. Cancer Res 61 (2001) 7518-7524.
[39]T.A. Martin, C. Parr, G. Davies, et al. Growth and angiogenesis of human breast cancer in a nude mouse tumour model is reduced by NK4, a HGF/SF antagonist. Carcinogenesis 24 (2003) 1317-1323.
[40]S. Yano, T. Nakagawa. The current state of molecularly targeted drugs targeting HGF/Met. Jpn J Clin Oncol 44 (2014) 9-12.
[41]D.V. Catenacci, L. Henderson, S.Y. Xiao, et al. Durable complete response of metastatic gastric cancer with anti-Met therapy followed by resistance at recurrence. Cancer Discov 1 (2011) 573-579.
[42]T.A. Yap, D. Olmos, A.T. Brunetto, et al. Phase I trial of a selective c-MET inhibitor ARQ 197 incorporating proof of mechanism pharmacodynamic studies. J Clin Oncol 29 (2011) 1271-1279.
[43]R. Bagai, W. Fan, P.C. Ma. ARQ-197, an oral small-molecule inhibitor of c-Met for the treatment of solid tumors. IDrugs 13 (2010) 404-414.
[44]J.R. Sierra, M.S. Tsao. c-MET as a potential therapeutic target and biomarker in cancer. Ther Adv Med Oncol 3 (2011) S21-35.
[45]J.J. Cui, M. McTigue, M. Nambu, et al. Discovery of a novel class of exquisitely selective mesenchymal-epithelial transition factor (c-MET) protein kinase inhibitors and identification of the clinical candidate 2-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3] triazolo [4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl) ethanol (PF-04217903) for the treatment of cancer. J Med Chem 55 (2012) 8091-8109.
[46]S. Benvenuti, L. Lazzari, A. Arnesano, et al. Ron kinase transphosphorylation sustains MET oncogene addiction. Cancer Res 71 (2011) 1945-1955.
[47]J.R. Infante, T. Rugg, M. Gordon,et al. Unexpected renal toxicity associated with SGX523, a small molecule inhibitor of MET. Invest New Drugs 31 (2013)363-369.
[48]S. Sharma, J.Y. Zeng, C.M. Zhuang, et al. Small-molecule inhibitor BMS-777607 induces breast cancer cell polyploidy with increased resistance to cytotoxic chemotherapy agents. Mol Cancer Ther 12 (2013) 725-736.
[49]A.B. Northrup, M.H. Katcher, M.D. Altman,et al. Discovery of 1-[3-(1-methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-7-yl]-N-(pyr idin-2-ylmethyl)methanesulfonamide (MK-8033):A Specific c-Met/Ron dual kinase inhibitor with preferential affinity for the activated state of c-Met. J Med Chem 56 (2013) 2294-2310.
[50]F. Bladt, B. Faden, M. Friese-Hamim,et al. EMD 1214063 and EMD 1204831 constitute a new class of potent and highly selective c-Met inhibitors. Clin Cancer Res 19 (2013) 2941-2951.
[51]D. Viola, V. Cappagli, R. Elisei. Cabozantinib (XL 184) for the treatment of locally advanced or metastatic progressive medullary thyroid cancer. Future Oncol 9 (2013) 1083-1092.
[52]P.Y. Wen. American Society of Clinical Oncology 2010:report of selected studies from the CNS tumors section. Expert Rev Anticancer Ther 10 (2010) 1367-1369.
[53]R. Tibes, G. Fine, G. Choy, et al. A phase I, first-in-human dose-escalation study of amuvatinib, a multi-targeted tyrosine kinase inhibitor, in patients with advanced solid tumors. Cancer Chemother Pharmacol 71 (2013) 463-471.
[54]A. Belalcazar, D. Azana, C.A. Perez, et al. Targeting the Met pathway in lung cancer. Expert Rev Anticancer Ther 12 (2012) 519-528.
[55]F. Qian, S. Engst, K. Yamaguchi,et al. Inhibition of tumor cell growth, invasion, and metastasis by EXEL-2880 (XL880, GSK1363089), a novel inhibitor of HGF and VEGF receptor tyrosine kinases. Cancer Res 69 (2009) 8009-8016.
[56]W. Wang, Q. Li, S. Takeuchi, et al. Met kinase inhibitor E7050 reverses three different mechanisms of hepatocyte growth factor-induced tyrosine kinase inhibitor resistance in EGFR mutant lung cancer. Clin Cancer Res 18 (2012) 1663-1671.
[57]S.M. Malik, V.E. Maher, K.E. Bijwaard,et al. Food and Drug Administration Approval:Crizotinib for Treatment of Advanced or Metastatic Non-small Cell Lung Cancer that Is Anaplastic Lymphoma Kinase Positive. Clin Cancer Res (2014).
[58]J.Y. Zeng, S. Sharma, Y.Q. Zhou, et al. Synergistic activities of MET/RON inhibitor BMS-777607 and mTOR inhibitor AZD8055 to polyploid cells derived from pancreatic cancer and cancer stem cells. Mol Cancer Ther 13 (2014) 37-48.
[59]S.W. Morris, M.N. Kirstein, M.B. Valentine, et al. Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma. Science 263 (1994)1281-1284.
[60]T. Iwahara, J. Fujimoto, D. Wen, et al. Molecular characterization of ALK, a receptor tyrosine kinase expressed specifically in the nervous system. Oncogene 14 (1997) 439-449.
[61]E. Vernersson, N.K. Khoo, M.L. Henriksson,et al. Characterization of the expression of the ALK receptor tyrosine kinase in mice. Gene Expr Patterns 6(2006)448-461.
[62]B. Hallberg, R.H. Palmer. Mechanistic insight into ALK receptor tyrosine kinase in human cancer biology. Nat Rev Cancer 13 (2013) 685-700.
[63]R.H. Palmer, E. Vernersson, C. Grabbe. Anaplastic lymphoma kinase:signalling in development and disease. Biochem J 420 (2009) 345-361.
[64]A. Barreca, E. Lasorsa, L. Riera, et al. Anaplastic lymphoma kinase in human cancer. J Mol Endocrinol 47 (2011) Rl 1-23.
[65]Y.L. Choi, M. Soda, Y. Yamashita,et al. EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors. N Engl J Med 363 (2010) 1734-1739.
[66]A.K. Murugan, M. Xing. Anaplastic thyroid cancers harbor novel oncogenic mutations of the ALK gene. Cancer Res 71 (2011) 4403-4411.
[67]Y.W. Wang, P.H. Tu, K.T. Lin, et al. Identification of oncogenic point mutations and hyperphosphorylation of anaplastic lymphoma kinase in lung cancer. Neoplasia 13 (2011) 704-715.
[68]T. Sasaki, K. Okuda, W. Zheng, et al. The neuroblastoma-associated F1174L ALK mutation causes resistance to an ALK kinase inhibitor in ALK-translocated cancers. Cancer Res 70 (2010) 10038-10043.
[69]H. Caren, F. Abel, P. Kogner, et al. High incidence of DNA mutations and gene amplifications of the ALK gene in advanced sporadic neuroblastoma tumours. Biochem J 416 (2008) 153-159.
[70]Y. Chen, J. Takita, Y.L. Choi, et al. Oncogenic mutations of ALK kinase in neuroblastoma. Nature 455 (2008) 971-974.
[71]R.E. George, T. Sanda, M. Hanna, et al. Activating mutations in ALK provide a therapeutic target in neuroblastoma. Nature 455 (2008) 975-978.
[72]I. Janoueix-Lerosey, D. Lequin, L. Brugieres, et al. Somatic and germline activating mutations of the ALK kinase receptor in neuroblastoma. Nature 455 (2008) 967-970.
[73]S. De Brouwer, K. De Preter, C. Kumps, et al. Meta-analysis of neuroblastomas reveals a skewed ALK mutation spectrum in tumors with MYCN amplification. Clin Cancer Res 16 (2010) 4353-4362.
[74]A. Sahu, K. Prabhash, V. Noronha,et al. Crizotinib:A comprehensive review. South Asian J Cancer 2 (2013) 91-97.
[75]B. Solomon, M. Varella-Garcia, D.R. Camidge. ALK gene rearrangements:a new therapeutic target in a molecularly defined subset of non-small cell lung cancer. J Thorac Oncol 4 (2009) 1450-1454.
[76]E.L. Kwak, Y.J. Bang, D.R. Camidge, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 363 (2010) 1693-1703.
[77]D.R. Camidge, Y.J. Bang, E.L. Kwak, et al. Activity and safety of crizotinib in patients with ALK-positive non-small-cell lung cancer:updated results from a phase 1 study. Lancet Oncol 13 (2012) 1011-1019.
[78]Y.N. Wong, S. Litwin, D. Vaughn, et al. Phase Ⅱ trial of cetuximab with or without paclitaxel in patients with advanced urothelial tract carcinoma. J Clin Oncol 30 (2012) 3545-3551.
[79]T. Sasaki, J. Koivunen, A. Ogino, et al. A novel ALK secondary mutation and EGFR signaling cause resistance to ALK kinase inhibitors. Cancer Res 71 (2011)6051-6060.
[80]A.T. Shaw, J.A. Engelman. ALK in lung cancer:past, present, and future. J Clin Oncol 31 (2013) 1105-1111.
[81]H. Sakamoto, T. Tsukaguchi, S. Hiroshima, et al. CH5424802, a selective ALK inhibitor capable of blocking the resistant gatekeeper mutant. Cancer Cell 19 (2011)679-690.
[82]T. Seto, K. Kiura, M. Nishio, et al. CH5424802 (RO5424802) for patients with ALK-rearranged advanced non-small-cell lung cancer (AF-001JP study):a single-arm, open-label, phase 1-2 study. Lancet Oncol 14 (2013) 590-598.
[83]E. Pailler, J. Adam, A. Barthelemy, et al. Detection of circulating tumor cells harboring a unique ALK rearrangement in ALK-positive non-small-cell lung cancer. J Clin Oncol 31 (2013) 2273-2281.
[84]E. Normant, G. Paez, K.A. West, et al. The Hsp90 inhibitor IPI-504 rapidly lowers EML4-ALK levels and induces tumor regression in ALK-driven NSCLC models. Oncogene 30 (2011) 2581-2586.
[85]L.V. Sequist, S. Gettinger, N.N. Senzer, et al. Activity of IPI-504, a novel heat-shock protein 90 inhibitor, in patients with molecularly defined non-small-cell lung cancer. J Clin Oncol 28 (2010) 4953-4960.
[86]G.C.t. Wallace, Y.N. Dixon-Mah, W.A. Vandergrift, et al. Targeting oncogenic ALK and MET:a promising therapeutic strategy for glioblastoma. Metab Brain Dis 28 (2013) 355-366.
[87]Q. Xie, R. Bradley, L. Kang, et al. Hepatocyte growth factor (HGF) autocrine activation predicts sensitivity to MET inhibition in glioblastoma. Proc Natl Acad Sci U S A 109 (2012) 570-575.
[88]D.C. Stylianou, A. Auf der Maur, D.P. Kodack, et al. Effect of single-chain antibody targeting of the ligand-binding domain in the anaplastic lymphoma kinase receptor. Oncogene 28 (2009) 3296-3306.
[89]A. Zamo, R. Chiarle, R. Piva, et al. Anaplastic lymphoma kinase (ALK) activates Stat3 and protects hematopoietic cells from cell death. Oncogene 21 (2002) 1038-1047.
[90]Y.W. Zhang, L.M. Wang, R. Jove, et al. Requirement of Stat3 signaling for HGF/SF-Met mediated tumorigenesis. Oncogene 21 (2002) 217-226.
[91]M.S. Samuel, F.C. Lourenco, M.F. Olson. K-Ras mediated murine epidermal tumorigenesis is dependent upon and associated with elevated Racl activity. PLoS One6(2011)e17143.
[92]E.D. Fleuren, M.H. Roeffen, W.P. Leenders, et al. Expression and clinical relevance of MET and ALK in Ewing sarcomas. Int J Cancer 133 (2013) 427-436.
[93]C.M. Lovly, J.M. Heuckmann, E. de Stanchina, et al. Insights into ALK-driven cancers revealed through development of novel ALK tyro sine kinase inhibitors. Cancer Res 71 (2011) 4920-4931.
[94]L. Ye, Y. Tian, Z. Li, et al. Design, synthesis and molecular docking studies of some novel spiro[indoline-3,4'-piperidine]-2-ones as potential c-Met inhibitors. Eur J Med Chem 50 (2012) 370-375.
[95]L. Ye, X. Ou, Y. Tian, et al. Indazoles as potential c-Met inhibitors:design, synthesis and molecular docking studies. Eur J Med Chem 65 (2013) 112-118.
[96]A.N. Jain. Surflex:fully automatic flexible molecular docking using a molecular similarity-based search engine. J Med Chem 46 (2003) 499-511.
[97]C. Sakakura, T. Mori, T. Sakabe, et al. Gains, losses, and amplifications of genomic materials in primary gastric cancers analyzed by comparative genomic hybridization. Genes Chromosomes Cancer 24 (1999) 299-305.
[98]H. Ma, K.Y. Horiuchi, Y. Wang, et al. Diamond, Nanoliter homogenous ultra-high throughput screening microarray for lead discoveries and IC50 profiling. Assay Drug Dev Technol 3 (2005) 177-187.
[99]J. J. Cui, M. Tran-Dube, H. Shen, et al. Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK). J Med Chem 54 (2011) 6342-6363.
[100]K. Kinoshita, Y. Ono, T. Emura, et al. Discovery of novel tetracyclic compounds as anaplastic lymphoma kinase inhibitors. Bioorg Med Chem Lett 21 (2011)3788-3793.
[101]G. Rege-Carnbrin, P. Scaravaglio, F. Carozzi, et al. Karyotypic analysis of gastric carcinoma cell lines carrying an amplified c-met oncogene. Cancer Genet Cytogenet 64 (1992) 170-173.
[102]J.G. Christensen, R. Schreck, J. Burrows, et al. A selective small molecule inhibitor of c-Met kinase inhibits c-Met-dependent phenotypes in vitro and exhibits cytoreductive antitumor activity in vivo. Cancer Res 63 (2003) 7345-7355.
[103]A.B. Northrup, M.H. Katcher, M.D. Altman,et al. Discovery of 1-[3-(1-methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-7-yl]-N-(pyr idin-2-ylmethyl)methanesulfoamide (MK-8033):A Specific c-Met/Ron dual kinase inhibitor with preferential affinity for the activated state of c-Met. J Med Chem 56 (2013) 2294-2310.
[104]N.R. Michaud, J.P. Jani, S. Hillerman, et al. Biochemical and pharmacological characterization of human c-Met neutralizing monoclonal antibody CE-355621. MAbs 4 (2012) 710-723.
[106]G.M. Schroeder, Y. An, Z.W. Cai, et al. Discovery of N-(4-(2-amino-3-chloropyridin-4-yloxy)-3-fluorophenyl)-4-ethoxy-1-(4-fluor opheny1)-2-oxo-1,2-dihydropyridine-3-carboxamide (BMS-777607), a selective and orally efficacious inhibitor of the Met kinase superfamily. J Med Chem 52 (2009) 1251-1254.
[106] [107]B. Lutterbach, Q. Zeng, L.J. Davis, et al. Lung cancer cell lines harboring MET gene amplification are dependent on Met for growth and survival. Cancer Res 67 (2007) 2081-2088.
[108]L. Meng, M. Shu, Y. Chen, et al. A novel lead compound CM-118:Antitumor activity and new insight into the molecular mechanism and combination therapy strategy in c-Met-and ALK-dependent cancers. Cancer Biol Ther 15 (2014).
[109]V. Tumati, S. Kumar, L. Yu, et al. Effect of PF-02341066 and radiation on non-small cell lung cancer cells. Oncol Rep 29 (2013) 1094-1100.
[110]H. Y. Zou, Q. Li, J.H. Lee, et al. Sensitivity of selected human tumor models to PF-04217903, a novel selective c-Met kinase inhibitor. Mol Cancer Ther 11 (2012) 1036-1047.
[111]B. Li, A. Torossian, Y. Sun, et al. Higher levels of c-Met expression and phosphorylation identify cell lines with increased sensitivity to AMG-458, a novel selective c-Met inhibitor with radiosensitizing effects. Int J Radiat Oncol Biol Phys 84 (2012) e525-531.
[112]S.B. Yan, V.L. Peek, R. Ajamie, et al. LY2801653 is an orally bioavailable multi-kinase inhibitor with potent activity against MET, MST1R, and other oncoproteins, and displays anti-tumor activities in mouse xenograft models. Invest New Drugs 31 (2013) 833-844.
[113]V. Pillay, L. Allaf, A.L. Wilding, et al. The plasticity of oncogene addiction: implications for targeted therapies directed to receptor tyro sine kinases. Neoplasia 11 (2009) 448-458,442 p following 458.
[114]H. Kawakami, I. Okamoto, T. Arao, et al. MET amplification as a potential therapeutic target in gastric cancer. Oncotarget 4 (2013) 9-17.
[115]Y. Asaoka, M. Tada, T. Ikenoue, et al. Gastric cancer cell line Hs746T harbors a splice site mutation of c-Met causing juxtamembrane domain deletion. Biochem Biophys Res Commun 394 (2010) 1042-1046.
[116]M.A. Shah, Z.A. Wainberg, D.V. Catenacci, et al. Phase Ⅱ study evaluating 2 dosing schedules of oral foretinib (GSK1363089), cMET/VEGFR2 inhibitor, in patients with metastatic gastric cancer. PLoS One 8 (2013) e54014.
[117]Y.K. Kang, K. Muro, M.H. Ryu,et al. A phase Ⅱ trial of a selective c-Met inhibitor tivantinib (ARQ 197) monotherapy as a second- or third-line therapy in the patients with metastatic gastric cancer. Invest New Drugs 32 (2014)355-361.
[118]R. Schwab, I. Petak, M. Kollar, et al. Major partial response to crizotinib, a dual MET/ALK inhibitor, in a squamous cell lung (SCC) carcinoma patient with de novo c-MET amplification in the absence of ALK rearrangement. Lung Cancer 83 (2014) 109-111.
[119]L.V. Sequist, B.A. Waltman, D. Dias-Santagata, et al. Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. Sci Transl Med 3 (2011) 75ra26.
[120]K.W. Robinson, A.B. Sandler. The role of MET receptor tyrosine kinase in non-small cell lung cancer and clinical development of targeted anti-MET agents. Oncologist 18 (2013) 115-122.
[121]J.C. Lee, S.H. Jang, K.Y. Lee, et al. Treatment of Non-small Cell Lung Carcinoma after Failure of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor. Cancer Res Treat 45 (2013) 79-85.
[122]T.A. Yap, L. Vidal, J. Adam, et al. Phase I trial of the irreversible EGFR and HER2 kinase inhibitor BIBW 2992 in patients with advanced solid tumors. J Clin Oncol 28 (2010) 3965-3972.
[123]Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 455 (2008) 1061-1068.
[124]S. Koochekpour, M. Jeffers, S. Rulong, et al. Met and hepatocyte growth factor/scatter factor expression in human gliomas. Cancer Res 57 (1997) 5391-5398.
[125]M. Milanovic, S. Radtke, N. Peel, et al. Anomalous inhibition of c-Met by the kinesin inhibitor aurintricarboxylic acid. Int J Cancer 130 (2012) 1060-1070.
[126]H.E. Crosswell, A. Dasgupta, C.S. Alvarado, et al. PHA665752, a small-molecule inhibitor of c-Met, inhibits hepatocyte growth factor-stimulated migration and proliferation of c-Met-positive neuroblastoma cells. BMC Cancer 9 (2009) 411.
[127]T.K. Choueiri, U. Vaishampayan, J.E. Rosenberg, et al. Phase II and biomarker study of the dual MET/VEGFR2 inhibitor foretinib in patients with papillary renal cell carcinoma. J Clin Oncol 31 (2013) 181-186.
[2]D.P. Bottaro, J.S. Rubin, D.L. Faletto, et al. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 251 (1991) 802-804.
[3]J.P. Eder, G.F. Vande Woude, S.A. Boerner, et al. Novel therapeutic inhibitors of the c-Met signaling pathway in cancer. Clin Cancer Res 15 (2009) 2207-2214.
[4]T. Igawa, K. Matsumoto, S. Kanda, et al. Hepatocyte growth factor may function as a renotropic factor for regeneration in rats with acute renal injury. Am J Physiol 265 (1993) F61-69.
[5]T. Yoshida, Y. Ozawa, T. Kimura, et al. Eribulin mesilate suppresses experimental metastasis of breast cancer cells by reversing phenotype from epithelial-mesenchymal transition (EMT) to mesenchymal-epithelial transition (MET) states. Br J Cancer 110 (2014) 1497-1505.
[6]K. Polyak, R.A. Weinberg. Transitions between epithelial and mesenchymal states:acquisition of malignant and stem cell traits. Nat Rev Cancer 9 (2009) 265-273.
[7]N.P. Gunasinghe, A. Wells, E.W. Thompson, et al. Mesenchymal-epithelial transition (MET) as a mechanism for metastatic colonisation in breast cancer. Cancer Metastasis Rev 31 (2012) 469-478.
[8]J.G. Christensen, J. Burrows, R. Salgia. c-Met as a target for human cancer and characterization of inhibitors for therapeutic intervention. Cancer Lett 225 (2005) 1-26.
[9]R. Jagadeeswaran, H. Surawska, S. Krishnaswamy, et al. Paxillin is a target for somatic mutations in lung cancer:implications for cell growth and invasion. Cancer Res 68 (2008) 132-142.
[10]S. Krishnaswamy, R. Kanteti, J.S. Duke-Cohan, et al. Ethnic differences and functional analysis of MET mutations in lung cancer. Clin Cancer Res 15 (2009)5714-5723.
[11]J.H. Lee, S.U. Han, H. Cho, et al. A novel germ line juxtamembrane Met mutation in human gastric cancer. Oncogene 19 (2000) 4947-4953.
[12]D. Zaffaroni, M. Spinola, A. Galvan, et al. Met proto-oncogene juxtamembrane rare variations in mouse and humans:differential effects of Arg and Cys alleles on mouse lung tumorigenesis. Oncogene 24 (2005) 1084-1090.
[13]L. Toschi, F. Cappuzzo. Clinical implications of MET gene copy number in lung cancer. Future Oncol 6 (2010) 239-247.
[14]L. Schmidt, F.M. Duh, F. Chen, et al.Germline and somatic mutations in the tyro sine kinase domain of the MET proto-oncogene in papillary renal carcinomas. Nat Genet 16 (1997) 68-73.
[15]L. Schmidt, K. Junker, G. Weirich, et al. Two North American families with hereditary papillary renal carcinoma and identical novel mutations in the MET proto-oncogene. Cancer Res 58 (1998) 1719-1722.
[16]W.S. Park, S.M. Dong, S.Y. Kim, et al. Somatic mutations in the kinase domain of the Met/hepatocyte growth factor receptor gene in childhood hepatocellular carcinomas. Cancer Res 59 (1999) 307-310.
[17]J. Tanyi, K. Tory, J. Rigo,et al. Evaluation of the tyrosine kinase domain of the Met proto-oncogene in sporadic ovarian carcinomas*. Pathol Oncol Res 5 (1999) 187-191.
[18]H. Kuniyasu, W. Yasui, Y. Kitadai,et al. Frequent amplification of the c-met gene in scirrhous type stomach cancer. Biochem Biophys Res Commun 189 (1992) 227-232.
[19]M. Nessling, S. Solinas-Toldo, K.K. Wilgenbus, et al. Mapping of chromosomal imbalances in gastric adenocarcinoma revealed amplified protooncogenes MYCN, MET, WNT2, and ERBB2. Genes Chromosomes Cancer 23 (1998) 307-316.
[20]C. Sakakura, T. Mori, T. Sakabe, et al. Gains, losses, and amplifications of genomic materials in primary gastric cancers analyzed by comparative genomic hybridization. Genes Chromosomes Cancer 24 (1999) 299-305.
[21]C. Ponzetto, S. Giordano, F. Peverali,et al. c-met is amplified but not mutated in a cell line with an activated met tyrosine kinase. Oncogene 6 (1991) 553-559.
[22]G.A. Smolen, R. Sordella, B. Muir, et al. Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752. Proc Natl Acad Sci U S A 103 (2006) 2316-2321.
[23]F. Cappuzzo, A. Marchetti, M. Skokan, et al. Increased MET gene copy number negatively affects survival of surgically resected non-small-cell lung cancer patients. J Clin Oncol 27 (2009) 1667-1674.
[24]L. Landi, G. Minuti, A. D'Incecco,et al. Targeting c-MET in the battle against advanced nonsmall-cell lung cancer. Curr Opin Oncol 25 (2013) 130-136.
[25]J.A. Engelman, K. Zejnullahu, T. Mitsudomi, et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 316(2007)1039-1043.
[26]H.J. Chen, T.S. Mok, Z.H. Chen, et al. Clinicopathologic and molecular features of epidermal growth factor receptor T790M mutation and c-MET amplification in tyrosine kinase inhibitor-resistant Chinese non-small cell lung cancer. Pathol Oncol Res 15 (2009) 651-658.
[27]J. Bean, C. Brennan, J.Y. Shih, et al. MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci U S A104 (2007) 20932-20937.
[28]E. Ichimura, A. Maeshima, T. Nakajima, et al. Expression of c-met/HGF receptor in human non-small cell lung carcinomas in vitro and in vivo and its prognostic significance. Jpn J Cancer Res 87 (1996) 1063-1069.
[29]S. Garcia, J.P. Dales, E. Charafe-Jaufffret, et al. Poor prognosis in breast carcinomas correlates with increased expression of targetable CD 146 and c-Met and with proteomic basal-like phenotype. Hum Pathol 38 (2007) 830-841.
[30]I. Takanami, F. Tanana, T. Hashizume,et al. Hepatocyte growth factor and c-Met/hepatocyte growth factor receptor in pulmonary adenocarcinomas:an evaluation of their expression as prognostic markers. Oncology 53 (1996) 392-397.
[31]C. Birchmeier, W. Birchmeier, E. Gherardi,et al. Met, metastasis, motility and more. Nat Rev Mol Cell Biol 4 (2003) 915-925.
[32]G. Maulik, A. Shrikhande, T. Kijima, et al. Role of the hepatocyte growth factor receptor, c-Met, in oncogenesis and potential for therapeutic inhibition. Cytokine Growth Factor Rev 13 (2002) 41-59.
[33]R. Wang, L.D. Ferrell, S. Faouzi,et al. Activation of the Met receptor by cell attachment induces and sustains hepatocellular carcinomas in transgenic mice. J Cell Biol 153 (2001) 1023-1034.
[34]S. Patane, S. Avnet, N. Coltella, et al. MET overexpression turns human primary osteoblasts into osteosarcomas. Cancer Res 66 (2006) 4750-4757.
[35]G. Rossi, A. Cavazza, A. Marchioni, et al. Role of chemotherapy and the receptor tyrosine kinases KIT, PDGFRalpha, PDGFRbeta, and Met in large-cell neuroendocrine carcinoma of the lung. J Clin Oncol 23 (2005) 8774-8785.
[36]M. Olivero, M. Rizzo, R. Madeddu, et al. Overexpression and activation of hepatocyte growth factor/scatter factor in human non-small-cell lung carcinomas. Br J Cancer 74 (1996) 1862-1868.
[37]K. Date, K. Matsumoto, H. Shimura,et al. HGF/NK4 is a specific antagonist for pleiotrophic actions of hepatocyte growth factor. FEBS Lett 420 (1997) 1-6.
[38]D. Tomioka, N. Maehara, K. Kuba, et al. Inhibition of growth, invasion, and metastasis of human pancreatic carcinoma cells by NK4 in an orthotopic mouse model. Cancer Res 61 (2001) 7518-7524.
[39]T.A. Martin, C. Parr, G. Davies, et al. Growth and angiogenesis of human breast cancer in a nude mouse tumour model is reduced by NK4, a HGF/SF antagonist. Carcinogenesis 24 (2003) 1317-1323.
[40]S. Yano, T. Nakagawa. The current state of molecularly targeted drugs targeting HGF/Met. Jpn J Clin Oncol 44 (2014) 9-12.
[41]D.V. Catenacci, L. Henderson, S.Y. Xiao, et al. Durable complete response of metastatic gastric cancer with anti-Met therapy followed by resistance at recurrence. Cancer Discov 1 (2011) 573-579.
[42]T.A. Yap, D. Olmos, A.T. Brunetto, et al. Phase I trial of a selective c-MET inhibitor ARQ 197 incorporating proof of mechanism pharmacodynamic studies. J Clin Oncol 29 (2011) 1271-1279.
[43]R. Bagai, W. Fan, P.C. Ma. ARQ-197, an oral small-molecule inhibitor of c-Met for the treatment of solid tumors. IDrugs 13 (2010) 404-414.
[44]J.R. Sierra, M.S. Tsao. c-MET as a potential therapeutic target and biomarker in cancer. Ther Adv Med Oncol 3 (2011) S21-35.
[45]J.J. Cui, M. McTigue, M. Nambu, et al. Discovery of a novel class of exquisitely selective mesenchymal-epithelial transition factor (c-MET) protein kinase inhibitors and identification of the clinical candidate 2-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3] triazolo [4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl) ethanol (PF-04217903) for the treatment of cancer. J Med Chem 55 (2012) 8091-8109.
[46]S. Benvenuti, L. Lazzari, A. Arnesano, et al. Ron kinase transphosphorylation sustains MET oncogene addiction. Cancer Res 71 (2011) 1945-1955.
[47]J.R. Infante, T. Rugg, M. Gordon,et al. Unexpected renal toxicity associated with SGX523, a small molecule inhibitor of MET. Invest New Drugs 31 (2013)363-369.
[48]S. Sharma, J.Y. Zeng, C.M. Zhuang, et al. Small-molecule inhibitor BMS-777607 induces breast cancer cell polyploidy with increased resistance to cytotoxic chemotherapy agents. Mol Cancer Ther 12 (2013) 725-736.
[49]A.B. Northrup, M.H. Katcher, M.D. Altman,et al. Discovery of 1-[3-(1-methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-7-yl]-N-(pyr idin-2-ylmethyl)methanesulfonamide (MK-8033):A Specific c-Met/Ron dual kinase inhibitor with preferential affinity for the activated state of c-Met. J Med Chem 56 (2013) 2294-2310.
[50]F. Bladt, B. Faden, M. Friese-Hamim,et al. EMD 1214063 and EMD 1204831 constitute a new class of potent and highly selective c-Met inhibitors. Clin Cancer Res 19 (2013) 2941-2951.
[51]D. Viola, V. Cappagli, R. Elisei. Cabozantinib (XL 184) for the treatment of locally advanced or metastatic progressive medullary thyroid cancer. Future Oncol 9 (2013) 1083-1092.
[52]P.Y. Wen. American Society of Clinical Oncology 2010:report of selected studies from the CNS tumors section. Expert Rev Anticancer Ther 10 (2010) 1367-1369.
[53]R. Tibes, G. Fine, G. Choy, et al. A phase I, first-in-human dose-escalation study of amuvatinib, a multi-targeted tyrosine kinase inhibitor, in patients with advanced solid tumors. Cancer Chemother Pharmacol 71 (2013) 463-471.
[54]A. Belalcazar, D. Azana, C.A. Perez, et al. Targeting the Met pathway in lung cancer. Expert Rev Anticancer Ther 12 (2012) 519-528.
[55]F. Qian, S. Engst, K. Yamaguchi,et al. Inhibition of tumor cell growth, invasion, and metastasis by EXEL-2880 (XL880, GSK1363089), a novel inhibitor of HGF and VEGF receptor tyrosine kinases. Cancer Res 69 (2009) 8009-8016.
[56]W. Wang, Q. Li, S. Takeuchi, et al. Met kinase inhibitor E7050 reverses three different mechanisms of hepatocyte growth factor-induced tyrosine kinase inhibitor resistance in EGFR mutant lung cancer. Clin Cancer Res 18 (2012) 1663-1671.
[57]S.M. Malik, V.E. Maher, K.E. Bijwaard,et al. Food and Drug Administration Approval:Crizotinib for Treatment of Advanced or Metastatic Non-small Cell Lung Cancer that Is Anaplastic Lymphoma Kinase Positive. Clin Cancer Res (2014).
[58]J.Y. Zeng, S. Sharma, Y.Q. Zhou, et al. Synergistic activities of MET/RON inhibitor BMS-777607 and mTOR inhibitor AZD8055 to polyploid cells derived from pancreatic cancer and cancer stem cells. Mol Cancer Ther 13 (2014) 37-48.
[59]S.W. Morris, M.N. Kirstein, M.B. Valentine, et al. Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma. Science 263 (1994)1281-1284.
[60]T. Iwahara, J. Fujimoto, D. Wen, et al. Molecular characterization of ALK, a receptor tyrosine kinase expressed specifically in the nervous system. Oncogene 14 (1997) 439-449.
[61]E. Vernersson, N.K. Khoo, M.L. Henriksson,et al. Characterization of the expression of the ALK receptor tyrosine kinase in mice. Gene Expr Patterns 6(2006)448-461.
[62]B. Hallberg, R.H. Palmer. Mechanistic insight into ALK receptor tyrosine kinase in human cancer biology. Nat Rev Cancer 13 (2013) 685-700.
[63]R.H. Palmer, E. Vernersson, C. Grabbe. Anaplastic lymphoma kinase:signalling in development and disease. Biochem J 420 (2009) 345-361.
[64]A. Barreca, E. Lasorsa, L. Riera, et al. Anaplastic lymphoma kinase in human cancer. J Mol Endocrinol 47 (2011) Rl 1-23.
[65]Y.L. Choi, M. Soda, Y. Yamashita,et al. EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors. N Engl J Med 363 (2010) 1734-1739.
[66]A.K. Murugan, M. Xing. Anaplastic thyroid cancers harbor novel oncogenic mutations of the ALK gene. Cancer Res 71 (2011) 4403-4411.
[67]Y.W. Wang, P.H. Tu, K.T. Lin, et al. Identification of oncogenic point mutations and hyperphosphorylation of anaplastic lymphoma kinase in lung cancer. Neoplasia 13 (2011) 704-715.
[68]T. Sasaki, K. Okuda, W. Zheng, et al. The neuroblastoma-associated F1174L ALK mutation causes resistance to an ALK kinase inhibitor in ALK-translocated cancers. Cancer Res 70 (2010) 10038-10043.
[69]H. Caren, F. Abel, P. Kogner, et al. High incidence of DNA mutations and gene amplifications of the ALK gene in advanced sporadic neuroblastoma tumours. Biochem J 416 (2008) 153-159.
[70]Y. Chen, J. Takita, Y.L. Choi, et al. Oncogenic mutations of ALK kinase in neuroblastoma. Nature 455 (2008) 971-974.
[71]R.E. George, T. Sanda, M. Hanna, et al. Activating mutations in ALK provide a therapeutic target in neuroblastoma. Nature 455 (2008) 975-978.
[72]I. Janoueix-Lerosey, D. Lequin, L. Brugieres, et al. Somatic and germline activating mutations of the ALK kinase receptor in neuroblastoma. Nature 455 (2008) 967-970.
[73]S. De Brouwer, K. De Preter, C. Kumps, et al. Meta-analysis of neuroblastomas reveals a skewed ALK mutation spectrum in tumors with MYCN amplification. Clin Cancer Res 16 (2010) 4353-4362.
[74]A. Sahu, K. Prabhash, V. Noronha,et al. Crizotinib:A comprehensive review. South Asian J Cancer 2 (2013) 91-97.
[75]B. Solomon, M. Varella-Garcia, D.R. Camidge. ALK gene rearrangements:a new therapeutic target in a molecularly defined subset of non-small cell lung cancer. J Thorac Oncol 4 (2009) 1450-1454.
[76]E.L. Kwak, Y.J. Bang, D.R. Camidge, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 363 (2010) 1693-1703.
[77]D.R. Camidge, Y.J. Bang, E.L. Kwak, et al. Activity and safety of crizotinib in patients with ALK-positive non-small-cell lung cancer:updated results from a phase 1 study. Lancet Oncol 13 (2012) 1011-1019.
[78]Y.N. Wong, S. Litwin, D. Vaughn, et al. Phase Ⅱ trial of cetuximab with or without paclitaxel in patients with advanced urothelial tract carcinoma. J Clin Oncol 30 (2012) 3545-3551.
[79]T. Sasaki, J. Koivunen, A. Ogino, et al. A novel ALK secondary mutation and EGFR signaling cause resistance to ALK kinase inhibitors. Cancer Res 71 (2011)6051-6060.
[80]A.T. Shaw, J.A. Engelman. ALK in lung cancer:past, present, and future. J Clin Oncol 31 (2013) 1105-1111.
[81]H. Sakamoto, T. Tsukaguchi, S. Hiroshima, et al. CH5424802, a selective ALK inhibitor capable of blocking the resistant gatekeeper mutant. Cancer Cell 19 (2011)679-690.
[82]T. Seto, K. Kiura, M. Nishio, et al. CH5424802 (RO5424802) for patients with ALK-rearranged advanced non-small-cell lung cancer (AF-001JP study):a single-arm, open-label, phase 1-2 study. Lancet Oncol 14 (2013) 590-598.
[83]E. Pailler, J. Adam, A. Barthelemy, et al. Detection of circulating tumor cells harboring a unique ALK rearrangement in ALK-positive non-small-cell lung cancer. J Clin Oncol 31 (2013) 2273-2281.
[84]E. Normant, G. Paez, K.A. West, et al. The Hsp90 inhibitor IPI-504 rapidly lowers EML4-ALK levels and induces tumor regression in ALK-driven NSCLC models. Oncogene 30 (2011) 2581-2586.
[85]L.V. Sequist, S. Gettinger, N.N. Senzer, et al. Activity of IPI-504, a novel heat-shock protein 90 inhibitor, in patients with molecularly defined non-small-cell lung cancer. J Clin Oncol 28 (2010) 4953-4960.
[86]G.C.t. Wallace, Y.N. Dixon-Mah, W.A. Vandergrift, et al. Targeting oncogenic ALK and MET:a promising therapeutic strategy for glioblastoma. Metab Brain Dis 28 (2013) 355-366.
[87]Q. Xie, R. Bradley, L. Kang, et al. Hepatocyte growth factor (HGF) autocrine activation predicts sensitivity to MET inhibition in glioblastoma. Proc Natl Acad Sci U S A 109 (2012) 570-575.
[88]D.C. Stylianou, A. Auf der Maur, D.P. Kodack, et al. Effect of single-chain antibody targeting of the ligand-binding domain in the anaplastic lymphoma kinase receptor. Oncogene 28 (2009) 3296-3306.
[89]A. Zamo, R. Chiarle, R. Piva, et al. Anaplastic lymphoma kinase (ALK) activates Stat3 and protects hematopoietic cells from cell death. Oncogene 21 (2002) 1038-1047.
[90]Y.W. Zhang, L.M. Wang, R. Jove, et al. Requirement of Stat3 signaling for HGF/SF-Met mediated tumorigenesis. Oncogene 21 (2002) 217-226.
[91]M.S. Samuel, F.C. Lourenco, M.F. Olson. K-Ras mediated murine epidermal tumorigenesis is dependent upon and associated with elevated Racl activity. PLoS One6(2011)e17143.
[92]E.D. Fleuren, M.H. Roeffen, W.P. Leenders, et al. Expression and clinical relevance of MET and ALK in Ewing sarcomas. Int J Cancer 133 (2013) 427-436.
[93]C.M. Lovly, J.M. Heuckmann, E. de Stanchina, et al. Insights into ALK-driven cancers revealed through development of novel ALK tyro sine kinase inhibitors. Cancer Res 71 (2011) 4920-4931.
[94]L. Ye, Y. Tian, Z. Li, et al. Design, synthesis and molecular docking studies of some novel spiro[indoline-3,4'-piperidine]-2-ones as potential c-Met inhibitors. Eur J Med Chem 50 (2012) 370-375.
[95]L. Ye, X. Ou, Y. Tian, et al. Indazoles as potential c-Met inhibitors:design, synthesis and molecular docking studies. Eur J Med Chem 65 (2013) 112-118.
[96]A.N. Jain. Surflex:fully automatic flexible molecular docking using a molecular similarity-based search engine. J Med Chem 46 (2003) 499-511.
[97]C. Sakakura, T. Mori, T. Sakabe, et al. Gains, losses, and amplifications of genomic materials in primary gastric cancers analyzed by comparative genomic hybridization. Genes Chromosomes Cancer 24 (1999) 299-305.
[98]H. Ma, K.Y. Horiuchi, Y. Wang, et al. Diamond, Nanoliter homogenous ultra-high throughput screening microarray for lead discoveries and IC50 profiling. Assay Drug Dev Technol 3 (2005) 177-187.
[99]J. J. Cui, M. Tran-Dube, H. Shen, et al. Structure based drug design of crizotinib (PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK). J Med Chem 54 (2011) 6342-6363.
[100]K. Kinoshita, Y. Ono, T. Emura, et al. Discovery of novel tetracyclic compounds as anaplastic lymphoma kinase inhibitors. Bioorg Med Chem Lett 21 (2011)3788-3793.
[101]G. Rege-Carnbrin, P. Scaravaglio, F. Carozzi, et al. Karyotypic analysis of gastric carcinoma cell lines carrying an amplified c-met oncogene. Cancer Genet Cytogenet 64 (1992) 170-173.
[102]J.G. Christensen, R. Schreck, J. Burrows, et al. A selective small molecule inhibitor of c-Met kinase inhibits c-Met-dependent phenotypes in vitro and exhibits cytoreductive antitumor activity in vivo. Cancer Res 63 (2003) 7345-7355.
[103]A.B. Northrup, M.H. Katcher, M.D. Altman,et al. Discovery of 1-[3-(1-methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-7-yl]-N-(pyr idin-2-ylmethyl)methanesulfoamide (MK-8033):A Specific c-Met/Ron dual kinase inhibitor with preferential affinity for the activated state of c-Met. J Med Chem 56 (2013) 2294-2310.
[104]N.R. Michaud, J.P. Jani, S. Hillerman, et al. Biochemical and pharmacological characterization of human c-Met neutralizing monoclonal antibody CE-355621. MAbs 4 (2012) 710-723.
[106]G.M. Schroeder, Y. An, Z.W. Cai, et al. Discovery of N-(4-(2-amino-3-chloropyridin-4-yloxy)-3-fluorophenyl)-4-ethoxy-1-(4-fluor opheny1)-2-oxo-1,2-dihydropyridine-3-carboxamide (BMS-777607), a selective and orally efficacious inhibitor of the Met kinase superfamily. J Med Chem 52 (2009) 1251-1254.
[106]
[108]L. Meng, M. Shu, Y. Chen, et al. A novel lead compound CM-118:Antitumor activity and new insight into the molecular mechanism and combination therapy strategy in c-Met-and ALK-dependent cancers. Cancer Biol Ther 15 (2014).
[109]V. Tumati, S. Kumar, L. Yu, et al. Effect of PF-02341066 and radiation on non-small cell lung cancer cells. Oncol Rep 29 (2013) 1094-1100.
[110]H. Y. Zou, Q. Li, J.H. Lee, et al. Sensitivity of selected human tumor models to PF-04217903, a novel selective c-Met kinase inhibitor. Mol Cancer Ther 11 (2012) 1036-1047.
[111]B. Li, A. Torossian, Y. Sun, et al. Higher levels of c-Met expression and phosphorylation identify cell lines with increased sensitivity to AMG-458, a novel selective c-Met inhibitor with radiosensitizing effects. Int J Radiat Oncol Biol Phys 84 (2012) e525-531.
[112]S.B. Yan, V.L. Peek, R. Ajamie, et al. LY2801653 is an orally bioavailable multi-kinase inhibitor with potent activity against MET, MST1R, and other oncoproteins, and displays anti-tumor activities in mouse xenograft models. Invest New Drugs 31 (2013) 833-844.
[113]V. Pillay, L. Allaf, A.L. Wilding, et al. The plasticity of oncogene addiction: implications for targeted therapies directed to receptor tyro sine kinases. Neoplasia 11 (2009) 448-458,442 p following 458.
[114]H. Kawakami, I. Okamoto, T. Arao, et al. MET amplification as a potential therapeutic target in gastric cancer. Oncotarget 4 (2013) 9-17.
[115]Y. Asaoka, M. Tada, T. Ikenoue, et al. Gastric cancer cell line Hs746T harbors a splice site mutation of c-Met causing juxtamembrane domain deletion. Biochem Biophys Res Commun 394 (2010) 1042-1046.
[116]M.A. Shah, Z.A. Wainberg, D.V. Catenacci, et al. Phase Ⅱ study evaluating 2 dosing schedules of oral foretinib (GSK1363089), cMET/VEGFR2 inhibitor, in patients with metastatic gastric cancer. PLoS One 8 (2013) e54014.
[117]Y.K. Kang, K. Muro, M.H. Ryu,et al. A phase Ⅱ trial of a selective c-Met inhibitor tivantinib (ARQ 197) monotherapy as a second- or third-line therapy in the patients with metastatic gastric cancer. Invest New Drugs 32 (2014)355-361.
[118]R. Schwab, I. Petak, M. Kollar, et al. Major partial response to crizotinib, a dual MET/ALK inhibitor, in a squamous cell lung (SCC) carcinoma patient with de novo c-MET amplification in the absence of ALK rearrangement. Lung Cancer 83 (2014) 109-111.
[119]L.V. Sequist, B.A. Waltman, D. Dias-Santagata, et al. Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. Sci Transl Med 3 (2011) 75ra26.
[120]K.W. Robinson, A.B. Sandler. The role of MET receptor tyrosine kinase in non-small cell lung cancer and clinical development of targeted anti-MET agents. Oncologist 18 (2013) 115-122.
[121]J.C. Lee, S.H. Jang, K.Y. Lee, et al. Treatment of Non-small Cell Lung Carcinoma after Failure of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor. Cancer Res Treat 45 (2013) 79-85.
[122]T.A. Yap, L. Vidal, J. Adam, et al. Phase I trial of the irreversible EGFR and HER2 kinase inhibitor BIBW 2992 in patients with advanced solid tumors. J Clin Oncol 28 (2010) 3965-3972.
[123]Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 455 (2008) 1061-1068.
[124]S. Koochekpour, M. Jeffers, S. Rulong, et al. Met and hepatocyte growth factor/scatter factor expression in human gliomas. Cancer Res 57 (1997) 5391-5398.
[125]M. Milanovic, S. Radtke, N. Peel, et al. Anomalous inhibition of c-Met by the kinesin inhibitor aurintricarboxylic acid. Int J Cancer 130 (2012) 1060-1070.
[126]H.E. Crosswell, A. Dasgupta, C.S. Alvarado, et al. PHA665752, a small-molecule inhibitor of c-Met, inhibits hepatocyte growth factor-stimulated migration and proliferation of c-Met-positive neuroblastoma cells. BMC Cancer 9 (2009) 411.
[127]T.K. Choueiri, U. Vaishampayan, J.E. Rosenberg, et al. Phase II and biomarker study of the dual MET/VEGFR2 inhibitor foretinib in patients with papillary renal cell carcinoma. J Clin Oncol 31 (2013) 181-186.