Table 1.

IC50 of recombinant Akt activity / EC50 of endogenous Akt activation

CompoundEnzyme assay
C33a cell assay
Akti-1/2aEmbedded Image1,60012,500>50,000>50,00010,00020,000>50,000
Akti-1Embedded Image76023,340>50,000>50,0001,140>10,000>10,000
Akti-2Embedded Image19,12033021,870>50,000>10,0004,150>10,000
Akti-1/2Embedded Image602102,120>50,0003102,090>10,000
  • NOTE: Purified recombinant human Akt1, Akt2, Akt3, and ΔPH-Akt1 proteins were monitored for kinase activity in the presence or absence of the Aktis. Kinase activity was determined by homogenous time-resolved fluorescence using a europium chelate-labeled anti-phospho-(serine 21)-glycogen synthase kinase α peptide antibody and streptavidin-linked XL665 fluorophore that binds to the biotin moiety on the peptide substrate (biotin-GGRARTSSFAEPG). See ref. 22 for further details. The cervical carcinoma cell line C33a contains all three isoforms of Akt. C33a cells plated in a 96-well format were treated with Aktis for 5 hours. The media was aspirated, replaced with an equal volume of lysis buffer, and the cells lysed by freeze-thaw. A volume of lysate was identified for each Akt isoform that provided equivalent kinase activity in the absence of inhibitors. Data represent the average of at least three experiments. In all cases the SE did not exceed 22% of the reported value. Values are in nanomoles per liter.