NVP-AEW541 (AEW541), a potent inhibitor of IGF-1R(IC50=150 nM) and InsR(IC50=140 nM), exhibits excellent efficiency and specificity for IGF-1R in a cell-based assay.

Insulin receptor:0.14 μM, IGF-1R:0.15 μM

NVP-AEW541可抑制InsR、Tek、Flt1和Flt3的活性，其IC50分别为140 nM、530 nM、600 nM和420 nM，表现在纯化激酶/重组激酶域测定中。其对细胞水平上InsR的选择性更高，效力提高了27倍。NVP-AEW541能够抑制IGF-I介导的MCF-7细胞存活、软琼脂生长及增殖，相应IC50为0.162 μM、0.105 μM和1.64 μM。同时，NVP-AEW541还能降低NWT-21细胞中的磷酸化IGF-1R和磷酸化PKB水平。[1] NVP-AEW541显示出在低血清培养基及含10% FBS培养基中对TC-71肌肉骨骼肉瘤细胞的生长抑制效果，并在肉瘤细胞系（TC-71, SK-N-MC, SaoS-2, RD/18和RH4）中抑制细胞周期进程，诱导特异性G1阶段阻滞。[2] NVP-AEW541能抑制人类神经母细胞瘤细胞的生长，IC50范围为0.4-6.8 μM。这些细胞系可检测到次二倍体分数增加以及S和G2-M部分的耗尽。NVP-AEW541驱动的IGF-1R抑制导致Akt磷酸化减少，但不影响Erk1和Erk2。[3] NVP-AEW541抑制胶质瘤细胞生长并破坏由HIF1α稳定化引起的自分泌回路。[4] 最近的研究显示，NVP-AEW541抑制PC3、DU145和22Rv1前列腺癌细胞的增殖和活力，而不必然导致相关细胞死亡。NVP-AEW541在22Rv1和DU415细胞中降低磷酸化Akt水平，但PC3细胞除外，不影响总Akt水平，表明PTEN状态可能决定了含必需Akt的NVP-AEW541的有效性。NVP-AEW541诱导的增敏作用依赖于Akt激活状态。NVP-AEW541能增加PC3、DU145和22Rv1细胞中H2AX磷酸化（DSB的一个衡量指标）。[5]

NVP-AEW541（50 mg/kg，口服）能够抑制基础及IGF-I诱导的受体以及PKB和MAPK的磷酸化，NWT-21肿瘤移植物中的T/C值为14%。[1] NVP-AEW541（50 mg/kg）在HTLA-230和SK-N-BE2c肿瘤移植物中均能引起肿瘤缩小，且未见全身毒性迹象。NVP-AEW541能够在Matrigel-coated室和HTLA-230肿瘤移植物中抑制肿瘤侵袭。[3]

In vitro kinase assays: NVP-AEW541 is dissolved in DMSO (10 mM) and stored at -20 °C. Dilutions are freshly made in DMSO/water 1:1. The final concentration of DMSO in the enzyme assays is <0.5 %. The protein kinase assays are carried out in 96-well plates at RT and terminated by the addition of 20 μL of 125 mM EDTA. Subsequently, 30 μL (c-Abl, c-Src, IGF-1R) of the reaction mixture are transferred onto Immobilon-PVDF presoaked for 5 min with methanol, rinsed with water, then soaked for 5 min with 0.5 % H3PO4 and mounted on vacuum manifold. After spotting all samples, vacuum is connected and each well rinsed with 200 μL 0.5 % H3PO4. Membranes are removed and washed 4× on a shaker with 1.0 % H3PO4, once with ethanol. After drying, mounting in Packard TopCount 96-well frame, and adding of 10 μL/well of Microscint, membranes are counted. IC50 values are calculated by linear regression analysis of the percentage inhibition of NVP-AEW541 in duplicate, at four concentrations (usually 0.01, 0.1, 1, and 10 μM). One unit of protein kinase activity is defined as 1 nmol of 33P transferred from [γ33P]ATP to the substrate protein per minute per mg of protein at 37 °C.

Between 3 × 103 and 6 × 103 cells/well are seeded in 96-well plates with a total media volume of 100 μL/well. Increasing concentrations of NVP-AEW541 is added 24 hours thereafter in quadruplicate. 72 hours later, cells are fixed by addition of 25 μL/well Glutaraldehyde (20%) and incubation for 10 min at RT. Cells are then washed 2× with 200 μL/well Water and 100 μL Methylene Blue (0.05%) is added. After incubation for 10 min at RT, cells are washed 3× with 200 μL/well Water. 200 μL/well HCl (3%) is added, and following incubation for 30 min at RT on a plate shaker, absorbance is measured at 650 nm.(Only for Reference)