Merestinib (LY2801653) is an orally available, small molecule inhibitor of the proto-oncogene c-Met (Ki: 2 nM) with potential antineoplastic activity. Merestinib electively binds to c-Met, thereby inhibiting c-Met phosphorylation and disrupting c-Met signal transduction pathways. This may induce cell death in tumor cells overexpressing c-Met protein or expressing the constitutively activated c-Met protein.

MET:2 nM(Ki)

Merestinib在体外展示对MET途径依赖的细胞散布和细胞增殖的影响。在拥有MET基因扩增的细胞系(MKN45, Hs746T 和 H1993)中，Merestinib展现出更强的抗增殖活性，相较于没有MET基因扩增的细胞系(U-87 mg, KATO-III)。Merestinib还针对13种每种都带有单点突变的MET变体，保持了效力。此外，它对包括MST1R, FLT3, AXL, MERTK, TEK, ROS1, DDR1/2以及丝氨酸/苏氨酸激酶MKNK1/2在内的几种其他受体酪氨酸激酶表现出强效活性。Merestinib抑制HGF激活的H460细胞中MET自磷酸化的平均IC50值为35.2±6.9 nM，而在S114细胞中MET自磷酸化的IC50为59.2 nM[1]。

Merestinib在多个异种移植模型中显示出体内抗肿瘤效果，包括MET扩增(MKN45)、MET自分泌(U-87 mg、KP4)以及MET过表达(H441)；同时也表现出体内血管正常化效应。该化合物能够在异种移植肿瘤中诱导血管正常化。在所研究的物种中，Merestinib在小鼠体内的消除半衰期最短，为2.9小时，与非人灵长类动物的14.3小时相比。目前，Merestinib正处于针对晚期癌症患者的第一阶段临床试验中[1]。

The Ki value and mode of inhibition of LY2801653 for the MET kinase activity are determined using a radiometric filter-binding assay. Reactions are carried out in 96-well plates in Enzyme dilution buffer (EDB) compose of 50 mM Tris HCl pH 7.5, 2 mM DTT, 0.005% Triton X-100, 10 mM MgCl2, and 250 ?M EDTA. Serially diluted LY2801653 (final concentration 250 to 0 nM) are followed by the addition of a series of 8 concentrations of 33P-γ-ATP (final concentration 400 to 10 μM ATP), and 5 nM enzyme (final concentration). After a 2-hour incubation, PolyGluTyr synthetic protein substrate (final 150 μg/mL) is added to initiate the 30-minute kinase reaction. Reactions are quenched with 10% H3PO4, transfer to a pre-wetted Multiscreen anionic phosphocellulose 96-well filter plate, and washed; radioactivity is measured with a scintillation counter. The experimental data are fit to a global mix model inhibition equation using GraphPad Prism softwar to generate an alpha value to determine the modality of inhibition and to calculate the Ki value for LY2801653[1].

2×103 DU145 cells/well on poly-D-lysine 96-well black/clear plates are treated with LY2801653 (in 0.4 % DMSO), immediately followed by the addition of human HGF (20 ng/ml), and incubated for 48 h at 37 °C. 2 % formaldehyde fixed cells are stained with AlexaFluor 488 Phalloidin and counterstained with Propidium Iodide. Colony counts are quantified on Acumen Explorer? laser-scanning fluorescence microplate cytometer. A colony is defined as≥4 cells.(Only for Reference)