HTS Assay for Inhibitors of Akt Phophorylation: Primary Screen
The serine/threonine protein kinase Akt (protein kinase B) regulates various cellular processes, including cell survival, growth, proliferation, migration and differentiation. It has been well established that Akt is activated through two essential steps involving membrane translocation and phosphorylation. Cytosolic Akt is recruited to the plasma membrane via the interaction between the PH more ..
BioActive Compounds: 1139
The serine/threonine protein kinase Akt (protein kinase B) regulates various cellular processes, including cell survival, growth, proliferation, migration and differentiation. It has been well established that Akt is activated through two essential steps involving membrane translocation and phosphorylation. Cytosolic Akt is recruited to the plasma membrane via the interaction between the PH domain and membrane PIP3 produced by phosphoinositide-3 kinase (PI3K) upon growth factor receptor stimulation. The membrane-Akt interaction results in conformational changes of Akt, enabling its activation through phosphorylation at T308 in the kinase domain and at S473 in the C-terminal hydrophobic motif by PDK1 and mTOR-rictor complex (mTORC2), respectively. Although T308 phosphorylation by PDK1 is indispensible for Akt activation, T308 phosphorylation is significantly boosted by prior phosphorylation of S473. Consistently, S473 phosphorylation has been shown to enhance Akt kinase activity by 4-5 fold. Conversely, knock-down of mTORC2 significantly reduces T308 phosphorylation by PDK1. It has been suggested that the non-phosphorylated C-terminal hydrophobic motif is involved in sustaining the inactive conformer of Akt. These findings indicate that S473 phosphorylation, as an important modulator of Akt activation, may be a valuable drug target for cell growth, survival, proliferation and differentiation. These inhibitors also may have significant therapeutic potential with fewer side effects, especially for the conditions involving hyperactive Akt signaling, such as cancer and Alzheimer's disease.
To this end, a miniaturized assay was developed to be screened against the Molecular Libraries Small Molecule Repository (MLSMR).
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: MH093214
Assay Submitter (PI): Hee-Yong Kim, National Institute on Alcohol Abuse and Alcoholism
3 uL of Neuro2A cell suspension in DMEM containing 0.5% FBS and Penicillin-Streptavidin antibiotics mix was plated in a white solid bottom tissue culture-treated 1536-well plate (Greiner Bio-One, Monroe, NC) at 4,500 cells per well and incubated overnight at 37 deg C. 23 nL/well of compound solutions in DMSO was added with Pintool transfer (Kalypsis, San Diego, CA) at single 50M final concentration. Then, the cells were stimulated with 1.5 uL of IGF (6 nM final concentration) and incubated at 37 deg C for 20 min. The reaction was terminated by the addition of 1.5 uL blocking reagent mixed with lysis buffer (1:25 correspondingly) provided with HTRF(R) Phospho Akt (Ser473) kit (Cisbio US, Bedford, MA), with following incubation for 50 min at ambient temperature. The detection HTRF conjugates from the kit were prepared according to the protocol, pre-mixed 1:1 prior to dispensing, and 2 uL/well was added with BioRAPTR FRD dispenser (Beckman Coulter). The assay plates were incubated for 24 hours at ambient temperature, and the HTRF fluorescence was measured using Envision plate reader (PerkinElmer) with excitation at 330nm, emission at 615nm (Eu(3)-Cryptate donor signal) and 665nm (d2 acceptor signal). The ratio of 665/615*10;4 was calculated for each well.
Compounds are considered "active" if percent of activity <= -80% and are assigned a score of 90; compounds are considered "inconclusive" if percent of activity > -80% and <= -50% and are assigned a score of 50; compounds with percent activity > -50% are considered "inactive" and are assigned a score of 10.
Data Table (Concise)