In vitro activity: CI-1040 treatment produces a reduction of pMAPK levels in multiple tumor cells including Colon 26, BX-PC3 pancreatic, A431 cervical, HT-29 colon, ZR-25-1 breast and SKOV-3 ovarian carcinomas. CI-1040 treatment doesn't inhibit the phosphorylation of Jun kinase, p38 kinase or Akt, indicating CI-1040 specifically targets MEK. Inhibition of MAPK activation by CI-1040 prevents cell cycle progression and induces a G1 block. The IC50 for inhibition of MEK1 by CI-1040 is 0.3 μM, 15-fold higher than the concentration required to inhibit the EGF-induced activation of ERK2 in Swiss 3T3 cells. These results indicate CI-1040 exerts its effects on cells by suppressing the activation of MKK1, and not by blocking its activity. 2 nM PD184352 inhibits the activation of MKK1 in Swiss 3T3 cells by 50%, while over 100-fold concentration of CI-1040 inhibits MEK1 in vitro. PD184352 also inhibits the Raf-catalysed phosphorylation of MEK1 without any effect on the Raf-catalysed phosphorylation of myelin basic protein. CI-1040 inhibits 86% of papillary thyroid carcinoma (PTC) cell growth with the RET/PTC1 rearrangement at 10μM compared with cells treated with DMSO only. CI-1040 shows potent inhibition to PTC cells (BRAF mutation) with GI50 of 52 nM, but low activity to RET/PTC1 rearrangement type with GI50 of 1.1 μM. A recent research indicates CI-1040 increases the apoptotic effect of BMS-214662 in a CML blast crisis cell line, K562, and in primary chronic phase CD34+ CML cells.

Kinase Assay: MAP kinase is activated after phosphorylation by MEK; the activated MAP kinase subsequently phosphorylates myelin basic protein (MBP).Incorporation of 32P into myelin basic protein (MBP) is assayed in the presence of glutathione S-transferase (GST) fusion proteins containing the 44-kDa MAPK (GST-MAPK) or the 45-kDa MEK (GST-MEK1). Assays are conducted in 50μL of 50 mM Tris, pH 7.4/10 mM MgCl2 /2 mM EGTA/10 μM [γ-32P]ATP containing 10 μg of GST-MEK1, 0.5 μg of GST-MAPK, and 40 μg of MBP. After incubation at 30°C for 15 minutes, reactions are stopped by addition of Laemmli SDS sample buffer. Phosphorylated MBP is resolved by SDS/10% PAGE. This screening effort leads to the discovery of several small-molecule inhibitors of MEK, i.e. CI-1040. Experiments assessing the order of addition shows that CI-1040 directly inhibits MEK1 with a 50% inhibitory concentration (IC50) of 17 nM, without affecting the activity of MAPK.

Cell Assay: Cells (Colon 26 carcinoma cells) are planted seeded in T-75 cm2 flasks and treated the next day for 24 hous with either DMSO or CI-1040. Single-cell suspensions are collected, and pellets are fixed in ice-cold ethanol (70%) for 30 minutes. After centrifugation of the samples, propidium iodide (50 μg/mL) and RNase (30 units/mL) are added to the pellets for 20 minutes at 37 °C. After filtration, samples are analyzed by flow cytometry.

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