Translating Novel Antitumor Targets of Vitamin E into New Chemopreventive Agents

描述（由申请人提供）：该项目基于PI的新发现，即-生育酚，在更大程度上，-生育酚通过PH结构域介导的Akt和PHLPP1 （PH结构域富亮氨酸重复磷酸酶1）的膜共定位，促进Akt在Ser-473位点的选择性去磷酸化。PHLPP1是一种Ser-473 Akt磷酸酶，通过负调控Akt发挥肿瘤抑制作用。从机制的角度来看，这些发现提供了第一个证据，证明-/ -生育酚通过抵消磷脂酰肌醇3,4,5-三磷酸对Akt激活的影响，介导氧化还原非依赖性抗肿瘤作用，至少在一定程度上是这样。这一独特的机制为通过PHLPP1的膜募集调节Akt活性提供了一个范式转变，揭示了维生素E如何介导其化学预防作用以及为什么-生育酚在抑制癌细胞增殖方面比对应物更有效的谜团。鉴于PHLPP在阻断PTEN突变前列腺癌发生中的抑癌作用，这一发现为使用-生育酚作为支架开发一类新的phlpp1靶向Akt抑制剂提供了分子基础，这些抑制剂具有与其他类型的Akt抑制剂不同的作用模式。该先导优化的概念证明是由-VE5提供的，这是一种侧链截断的-生育酚衍生物，在介导Akt去磷酸化和前列腺癌细胞生长抑制方面，其效力至少是-生育酚的20倍。同样重要的是，- ve5在体内抑制PTEN-缺陷PC-3和lncap -能力的裸鼠异种移植肿瘤的生长。因此，本研究包括三个具体目标，目的是将这一新的机制发现转化为一类新的phlpp1靶向Akt抑制剂，以阻断或延迟前列腺肿瘤发生的发生。目的1是对-VE5进行基于结构的先导优化，以开发更有效的靶向phlpp1的Akt抑制剂。基于建模和突变分析，我们假设配体与结合袋中亲水性残基的极性相互作用增加将增强对PH的结合亲和力

DESCRIPTION (provided by applicant): This project is based on the PI's novel finding that -tocopherol and, to a greater extent, -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 -/ -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 -tocopherol is more potent relative to the 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 -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 -VE5, a side chain-truncated -tocopherol derivative, which exhibited at least 20-fold higher potency relative to -tocopherol in mediating Akt dephosphorylation and growth inhibition of prostate cancer cells. Equally important, -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 -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 -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 -VE5 derivatives inhibit cell proliferation of PTEN-deficient prostate cancer cells. The top 3 optimal -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)3¿, the forkhead box transcription factor Foxo3a, NF-¿B, 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 -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 -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 -VE5-derived PHLPP1-targeted Akt inhibitors.