IQ-1S 

Kinase profiling is performed using a panel of 91 protein kinases. In brief, kinases are produced and displayed on T7 phage or expressed in HEK-293 cells. Binding reactions are performed at room temperature for 1 h, and the fraction of kinase not bind to test compound is determined by capture with an immobilized affinity ligand and quantified by quantitative polymerase chain reaction. Primary screening at fixed concentrations of compounds is performed in duplicate. Selected hit compounds are submitted for dissociation constant (K_d) determination using the same platform. For dissociation constant K_d determination, a 12-point half-log dilution series (a maximum concentration of 100 μM) is used. Assays are performed in duplicate, and their mean value is displayed^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Human PBMCs are plated in 96-well plates at a density of 2×10⁵ cells/well in culture medium supplemented with 3% (v/v) endotoxin-free FBS. PBMCs are pretreated with 20 μM IQ-1S (The sodium salt of IQ-1) or DMSO for 30 min, followed by addition of 200 ng/ml LPS for 24 h. A human cytokine MultiAnalyte ELISArray Kit is used to evaluate various cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17A, interferon (IFN)-γ, TNF-α, and granulocyte-macrophage colony-stimulating factor in supernatants of PBMCs^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[1]
For in vivo analysis, 12.5 or 30 mg/kg i.p. doses of IQ-1S (The sodium salt of IQ-1) are administered to BALB/c mice (15-20 animals/group), and the mice are sacrificed at various time points after compound administration. For quantification, a calibration curve is established using mouse serum samples spiked with known concentrations of IQ-1S (0.1-20 μM), and a linear dependence of the peak area with IQ-1S concentration is obtained (correlation coefficient r=0.997). The area under the serum concentration-time curve (AUC_0-12h) is calculated using the linear trapezoidal method up to the last measured concentration.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Schepetkin IA, et al. Identification and characterization of a novel class of c-Jun N-terminal kinase inhibitors. Mol Pharmacol. 2012 Jun;81(6):832-45.  [Content Brief]