This project is based on the PI's novel finding that α-tocopherol and, to a greater extent, γ-tocopherol facilitate the selective dephosphorylation of Akt at Ser-473 through PH domain-mediated membrane co- localization of Akt and PHLPP1 (PH domain leucine-rich repeat phosphatase 1). PHLPP1, a Ser-473 Akt phosphatase, acts as a tumor suppressor by negatively regulating Akt. From a mechanistic perspective, these findings provide the first evidence that α-/γ-tocopherol mediates redox-independent antitumor effects, at least in part, by counteracting the effect of phosphatidylinositol 3,4,5-trisphosphate on Akt activation. This unique mechanism provides a paradigm shift with respect to the regulation of Akt activity through membrane recruitment of PHLPP1, sheds light onto the enigma of how vitamin E mediates its chemopreventive effect and of why γ-tocopherol is more potent relative to the α counterpart in suppressing cancer cell proliferation. In light of the tumor suppressor role of PHLPP in blocking PTEN mutant prostate carcinogenesis, this finding provides a molecular rationale for the use of γ-tocopherol as a scaffold to develop a novel class of PHLPP1-targeted Akt inhibitors, which have a distinct mode of action from other types of Akt inhibitors. The proof-of-concept of this lead optimization is provided by γ-VE5, a side chain-truncated γ-tocopherol derivative, which exhibited at least 20-fold higher potency relative to γ-tocopherol in mediating Akt dephosphorylation and growth inhibition of prostate cancer cells. Equally important, γ-VE5 exhibited in vivo efficacy in suppressing the growth of PTEN- deficient PC-3 and LNCaP-abl xenograft tumors in nude mice. Thus, this proposal consists of three specific aims with the goal of translating this novel mechanistic finding into a novel class of PHLPP1-targeted Akt inhibitors to block or delay the onset of prostate tumorigenesis. Aim 1 is to conduct structure-based lead optimization of γ-VE5 to develop more potent PHLPP1-targeted Akt inhibitors. Based on modeling and mutational analyses, we hypothesize that increasing polar interactions of the ligand with the hydrophilic residues in the binding pocket will enhance binding affinity for the PH domain. Proof-of-concept of this premise has been established by analysis of lead γ-VE5 derivatives. Continued optimization of these leads to generate 2nd generation compounds via isosteric replacement of metabolically labile moieties is proposed. Aim 2 is to investigate the mechanisms by which optimized γ-VE5 derivatives inhibit cell proliferation of PTEN-deficient prostate cancer cells. The top 3 optimal γ-VE5 derivatives from Aim 1 will be mechanistically validated by examining their effects on the activation status of Akt and various Akt downstream targets relevant to prostate carcinogenesis and tumor progression, especially glycogen synthase kinase (GSK)3ß, the forkhead box transcription factor Foxo3a, NF-κB, and AR signaling. As PHLPP1 plays a pivotal role in mediating the effect of AR inhibition on Akt activation in PTEN- deficient prostate cancer cells, the effects of these compounds on crosstalk of AR signaling with PHLPP1- mediated regulation of Akt activation will also be explored. In vivo efficacy of three optimal γ-VE5 derivatives will be evaluated in both PTEN-deficient (LNCaP-abl and PC-3) and PTEN-functional (22RV1) xenograft tumor models, which will be correlated with changes in the aforementioned biomarkers in tumors. Aim 3 is to assess the in vivo chemopreventive efficacy of a structurally optimized γ-VE5 derivative to block prostate tumorigenesis in the PTEN-knockout and TRAMP models. In light of the role of aberrant Akt signaling in prostate carcinogenesis, these two transgenic animal models represent therapeutically relevant models to evaluate the chemopreventive activities of these γ-VE5-derived PHLPP1-targeted Akt inhibitors.