PS-1145 (IKK Inhibitor X) is a specific IKK inhibitor with IC50 of 88 nM.

IKKϵ:88 nM

Administering 50 mg/kg of PS-1145 intravenously in male Wistar rats with DMBA-induced skin tumors resulted in the upregulation of p53 and activation of caspases, coupled with the downregulation of NF-κB and VEGF factors, thereby enhancing apoptosis in tumor cells.

PS-1145 inhibits the proliferation of primary human CD4(+) T cells and impedes the activation of NF-κB and AP-1 transcription factors through the cooperative engagement of CD3 and CD28 co-receptors. Additionally, PS-1145 blocks TNFalpha-induced NF-kappaB activation by inhibiting the phosphorylation of IkappaBalpha and disrupting the protective effect of IL-6 on Dex-induced apoptosis.

Kinase assay: PS-1145 are dissolved in DMSO and stored at ?20?°C until use. Ki value of PS-1145 against the IKK complex is determined by measuring Km ,?ATP against varying fixed concentration of the inhibitor. Briefly, partially purified IKK complex obtained from unstimulated HeLa S3 cells are pre-activated using the catalytic domain of MEKK1 expressed in sf9. Kinase activity is assessed using a biotinylated IκBα peptide (250 μM, RHDSGLDSMKD,Km ,peptide = 30 μM,K m ,?ATP = 10 μM) and phospho-[Ser32]-phosphoantibodies in an ELISA format with appropriate standard curve for quantification. For PS-1145 Ki measurement, the activated IKK complex is first preincubated in varying fixed concentration of the inhibitor (0.1–1 μM) at 25 °C for 1 h. Then apparentK m measurement for MgATP is performed at each discrete inhibitor concentration.

The inhibitory effect of PS-1145 on MM growth is assessed by measuring MTT dye absorbance of the cells. Cells from 48-h cultures are pulsed with 10 μL of 5 mg/ml MTT to each well for the last 4 h of 48-h cultures, followed by 100 μl of isopropanol containing 0.04N HCl. Absorbance is measured at 570 nm using a spectrophotometer. (Only for Reference)