CHIR-99021 is a GSK-3α/β inhibitor with an IC₅₀ of 10 and 6.7 nM，showing 500-fold selectivity over its closest homologs CDC2 and ERK2, as well as other protein kinases.

CHIR 99021inhibits human GSK-3β with K_i values of 9.8 nM. CHIR 99021 is a small organic molecule that inhibits GSK3α and GSK3β by competing for their ATP-binding sites.In vitro kinase assays reveal that CHIR 99021 specifically inhibits GSK3β (IC₅₀=~5 nM) and GSK3α (IC₅₀=~10 nM), with little effect on other kinases. In the presence of CHIR-99021 the viability of the ES-D3 cells is reduced by 24.7% at 2.5 μM, 56.3% at 5 μM, 61.9% at 7.5 μM and 69.2% at 10 μM CHIR-99021 with an IC₅₀ of 4.9 μM.

In ZDF rats, a single oral dose of CHIR 99021 (16 mg/kg or 48 mg/kg) rapidly lowers plasma glucose, with a maximal reduction of nearly 150 mg/dl 3-4 h after administration. CHIR99021 (2 mg/kg) given once, 4 h before irradiation, significantly improves survival after 14.5 Gy abdominal irradiation (ABI). CHIR99021 treatment significantly blocks crypt apoptosis and accumulation of p-H2AX⁺ cells, and improves crypt regeneration and villus height. CHIR99021 treatment increases Lgr5⁺ cell survival by blocking apoptosis, and effectively prevents the reduction of Olfm4, Lgr5 and CD44 as early as 4 h.

DMSO : ≥ 5.1 mg/mL (10.96 mM)

* "≥" means soluble, but saturation unknown.