PD0166285 is a potent Wee1 and Chk1 inhibitor with activity at nanomolar concentrations.PD0166285 is a novel G2 checkpoint abrogator.

Wee1:24 nM, Chk1:3.4 μM, Myt1:72 nM

PD0166285通过纳摩尔浓度抑制Wee1活性，使G2/M检查点失效，诱导提前细胞分裂。在细胞水平上，0.5 μM PD0166285显著抑制七种测试癌细胞系中的辐射诱导的Cdc2在Tyr-15和Thr-14的磷酸化。PD0166285通过废除G2检查点来杀死癌细胞。PD0166285不抑制Cdc2/cyclin B，而是以更高浓度（3433 nM）抑制Chk1激酶。用抑制剂治疗细胞与微管稳定和cyclin D转录减少有关。因此，PD0166285可能是一种有潜力的抗癌疗法[1][2]。

PD0166285在0.5 μM浓度下，能够抑制所有测试细胞系中的Cdc2Y15/T14磷酸化，这一效果与细胞的p53状态无关[1]。此外，通过药理学上靶向WEE1的PD0166285能够增强U251-FM GBM肿瘤对体内IR的敏感性[3]。

Wee1 Mass Screening: Wee1 mass screening is performed using Amersham's p34cdc2 kinase SPA kit with some modifications. Briefly, 45–60 nM full-length Wee1 kinase is incubated with 25 μM compounds, 20 μM ATP, and 122–441 nM Cdc2/cyclin B in a final volume of 50 μl of enzyme dilution buffer [50 mM Tris (pH 8.0), 10 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 0.1 mM Na3VO4]. After 30 min incubation at 30°C, 30 μl of [33P]ATP containing kinase buffer [67 mM Tris (pH 8.0), 40 mM NaCl, 13 mM MgCl2, 1 mM DTT, and 0.13 mM Na3VO4] containing 1 μM biotinylated peptide, and 0.25 μCi of [γ-33P]ATP is added to the reaction and incubated for another 30 min at 30°C. The reaction is stopped by adding 200 μl of stop buffer [50 μM ATP, 5 mM EDTA, 0.1% Triton X-100, and 1.25 mg/ml SPA beads in PBS]. After centrifugation at 2400 rpm for 15 min, the plate is counted with Wallac's Microbeta counter.

B16 cells (1 × 105 cells in 100 mm-dishes) are maintained in medium overnight. In addition, the cells are treated with (0, 0.5, 1.0, or 2.0 μM) PD0166285 (including DMSO vehicle) for indicated times. The cells are washed twice with phosphate-buffered saline (PBS). Next, the cells are trypsinized, so the cell numbers in each dish are determined by using a computed cell-counter (Sysmex CDA-500) according to manufacturer's recommendation. (Only for Reference)

E98 tumor fragments (8 mm3) were grafted subcutaneously in the flank of Balb/C nude mice (n = 10). For the experiments with the orthotopic tumors, U251 mg and E98 cells were transduced to express Fluc and mCherry, to generate U251-FM and E98-FM cells. One million cells were injected intracranially. Starting at 12 days after tumor inoculation, mice received the WEE1 inhibitor PD0166285 via intraperitoneal injections (500 μl of a 20 μM solution diluted in NaCl) or vehicle for 5 consecutive days. On days 10–15, mice were irradiated with a single dose of irradiation.