A-443654, a pan-Akt inhibitor, exhibits equal potency against Akt1, Akt2, and Akt3 within cells (Ki=160 pM).

Akt1:160 pM (Ki), Src:2.6 μM (Ki), Chk1:2.3 μM (Ki), GSK-3β:41 nM (Ki), MAPK-AP2:3.3 μM (Ki), Akt3:160 pM (Ki), ERK2:340 nM (Ki), CDK2:24 nM (Ki), PKCδ:33 nM (Ki), PKA:6.3 nM (Ki), Flt1:3.6 μM (Ki), Akt2:160 pM (Ki), CK2:2.4 μM (Ki), RSK2:11 nM (Ki), c-Kit:1.2 μM (Ki), PKCγ:24 nM (Ki), KDR:3.1 μM (Ki)

A-443654 demonstrates a significant improvement in potency with a K_i of 160 pM, a 30,000-fold increase compared to the initial lead molecule. It effectively reduces P-GSK3 in a dose-dependent manner across three cell lines and exhibits 40-fold greater selectivity for Akt over PKA, equally inhibiting Akt1, Akt2, and Akt3 within cells. Morphological changes induced by A-443654 are rapid, occurring within 2 to 4 hours in 10A and 10CA1a cells, with the latter being more sensitive. The compound hinders tumor cell proliferation, with an EC50 of 0.1 μM[1], and shows marked selectivity for mutant cells, inhibiting their growth more than 3.5 times compared to wild-type (WT) cells[3]. Furthermore, A-443654, especially when combined with rapamycin, significantly decreases Bcl-2 protein levels in 10A and 10CA1a cells, exhibiting a notable reduction in 10CA1a cells by approximately 70%. At 2 μM, A-443654 alone prompts 10CA1a cells to detach from the plate after 12 hours, illustrating a differential response compared to 10A cells, which remain unaffected at the same concentration[2].

A-443654, administered subcutaneously at a dosage of 7.5 mg/kg per day, effectively inhibits tumor growth in the 3T3-Akt1 flank tumor model. At a higher concentration of 30 mg/kg, this compound increases phosphorylated Akt1 levels in MiaPaCa-2 tumors. Furthermore, a dosage of 50 mg/kg induces apoptosis in 3T3-Akt1 flank tumors [1].