Abstract The AKT family of serine-threonine kinases is a critical signal transduction node serving a variety of cellular functions including survival, proliferation, protein synthesis, and glucose metabolism. Small molecule inhibitors of AKT are emerging as potential targeted agents to treat cancers that exhibit aberrant AKT pathway signaling. A screening strategy was employed to identify inhibitors which utilize the intrinsic negative regulatory function of hydrophobic clusters in the ATP-binding cleft to inhibit the kinase activity of AKT1. ARQ 092 was identified through this screening paradigm following in vitro and in vivo optimization and was selected for further development. Crystallographic studies provided evidence that this mechanism of inhibition of AKT had been achieved. ARQ 092 binds to inactive, unphosphorylated AKT1 with sub-nanomolar affinity (Kd = 0.28 nM measured by surface plasma resonance) and biochemically inhibits all three isoforms (IC50 values of 3 nM, 4.5 nM, and 5.5 nM respectively for AKT1, AKT2, and AKT3). ARQ 092 displayed inhibition kinetics against AKT1 which were not affected by ATP concentrations up to 1000 M. When screened against a panel of over 300 kinases, ARQ 092 inhibited only three kinases within 100-fold of its IC50 against AKT1. ARQ 092 potently inhibited AKT phosphorylation (Ser473 & Thr308) in AN3CA human endometrial carcinoma cells (EC50 values of 39 nM and 61 nM, respectively for p-Ser473 & p-Thr308) and demonstrated concentration-dependent inhibition of phosphorylation of the downstream AKT substrate PRAS40. ARQ 092 inhibited growth of AN3CA cells (GI50 = 555 nM), A2780 cells (GI50 = 400 nM), and IGROV-1 cells (GI50 = 66 nM), in addition to LNCaP, ZR-75–1, and BT-474 human cancer cell lines (GI50 values ranging from 1 to 4 μM). Following a single oral dose of ARQ 092, inhibition of AKT and PRAS40 phosphorylation was documented in vivo in both AN3CA human endometrial tumor and BT474 human breast tumor xenograft models. The growth of AN3CA tumor xenografts was markedly suppressed after daily oral administration of ARQ 092 for 10 days. Finally, ARQ 092 was shown to have good pharmaceutical properties and was advanced into preclinical development. Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A230.