CI-1040 (PD 184352) (PD184352) is an ATP non-competitive MEK1/2 inhibitor (IC50: 17 nM).

MEK2:17 nM (cell free), MEK1:17 nM (cell free)

PD 184352（CI-1040）显著降低了在血清存在条件下生长的多种肿瘤细胞系中磷酸化MAPK（pMAPK）的稳态水平。在含1 μM PD 184352 的培养基中处理结肠26细胞1小时，可使pMAPK水平降低超过75%。用10 μM PD 184352处理结肠26细胞，不会抑制Jun激酶、p38激酶或AKT的磷酸化[1]。PD 184352抑制MKK1的IC50为0.3 μM，这比抑制Swiss 3T3细胞中EGF诱导的ERK2活化所需浓度高出15倍。在2 nM PD 184352 的作用下，MKK1在细胞中的活化被50%抑制[2]。CI-1040以剂量和时间依赖的方式诱导U-937细胞凋亡和抑制增殖。CI-1040显著增加PUMA mRNA和蛋白水平。通过PUMA siRNA转染敲低PUMA，可以抑制CI-1040诱导的U-937细胞凋亡和增殖抑制[3]。

处理PD 184352（150 mg/kg，i.p. 或 p.o.）后1小时和6小时切除了肿瘤。无论通过何种给药方式，PD 184352处理至少6小时内完全抑制了MAPK磷酸化。MAPK磷酸化在给药后12小时恢复，并在24小时达到对照水平[1]。体内，MEK抑制剂CI-1040的系统给药将腺瘤形成减少至三分之一，并显著恢复了肺结构。CL-1040处理的小鼠肺细胞的增殖率降低，对肺泡细胞的分化没有明显影响[4]。

All protein kinase activities were linear with respect to time in every incubation. Assays were performed either manually for 10 min at 30 °C in 50 μl incubations using [γ-32P]ATP, or with a Biomek 2000 Laboratory Automation Workstation in a 96-well format for 40 min at ambient temperature in 25 μl incubations using [γ-33P]ATP. The concentrations of ATP and magnesium acetate were 0.1 mM and 10 mM respectively, unless stated otherwise. This concentration of ATP is 5–10-fold higher than the Km for ATP of most of the protein kinases studied in the present paper, but lower than the normal intracellular concentration, which is in the millimolar range. All assays were initiated with MgATP. Manual assays were terminated by spotting aliquots of each incubation on to phosphocellulose paper, followed by immersion in 50 mM phosphoric acid. Robotic assays were terminated by the addition of 5 μl of 0.5 M phosphoric acid before spotting aliquots on to P30 filter mats. All papers were then washed four times in 50 mM phosphoric acid to remove ATP, once in acetone (manual incubations) or methanol (robotic incubations), and then dried and counted for radioactivity [2].

Cells were planted seeded in T-75 cm2 flasks and treated the next day for 24 h with either DMSO or PD 184352. Single-cell suspensions were collected, and pellets were fixed in ice-cold ethanol (70%) for 30 min. After centrifugation of the samples, propidium iodide (50 μg/ml) and RNase (30 units/ml) were added to the pellets for 20 min at 37 °C. After filtration, samples were analyzed by flow cytometry [1].

Tumor fragments (approximately 3 mm^3 in size) were implanted subcutaneously into the right axillae of CD2F1 male mice (colon 26 studies) or female nude mice (HT-29 studies) 4–6 weeks old. Treatment was administered by gavage or intraperitoneally and was initiated either the day after tumor implantation (colon 26) or when tumors reached approximately 200 mg in size (HT-29). PD 184352 was prepared in a vehicle of 10% Cremophore EL, 10% ethanol and 80% water. Tumor size was evaluated periodically by caliper measurements, generally three times per week. Percent tumor growth inhibition was calculated as [(T–C)/number of days of treatment] × 100, with T and C being defined as the time required for treated and control tumors, respectively, to reach 750 mg (colon 26) or to reach twofold growth (HT-29) [1].