Design of Pin1 Inhibitors

项目摘要/摘要 分子伴侣和Pro异构酶Pin1是一种很有前途的肿瘤治疗靶点。PIN1基因敲除 在正常小鼠中只产生轻微的退行性表型，但在致癌的小鼠中极大地削弱了肿瘤的发生 并减少前列腺癌和乳腺癌异种移植模型中的增殖。对…的依赖 癌细胞对Pin1的活性似乎是通过其稳定多个癌基因和破坏稳定的能力而发生的 多种肿瘤抑制因子。然而，尽管平1‘S作为治疗靶点的潜力很大，但没有任何化学物质 具有特异性和生物活性的抑制剂已有报道。挑战是Pin1拥有 一种经典的“无法下药”的蛋白质的特征；它有一个浅的，极地绑定的口袋，到目前为止， 阻止了可行的抑制剂的发现。我们提出了一种以前未曾探索过的方法来传递渗透性 对现有的有效的Pin1体外抑制剂进行研究，以及使用二硫化物片段拴系来扩展这一SAR。一个 本研究的关键创新之处在于，我们设计并初步测试了第一个有潜力的细胞 使用高亲和力磷酸盐的Pin1渗透性缓蚀剂及其渗透性 通过磷酰胺笼子。这些物质渗透到脂质双层中，随后被酶作用。 被释放为一种含磷的活性代谢物。在令人兴奋的初步结果中，我们的顶级化合物(DS-2(R)) 具有个位数的纳米分子EC50对细胞周期蛋白D1在体内失稳--一个已知的Pin1抑制生物标记物。 在此，我建议(SA 1)对第一种生物活性进行严格的物理和功能表征 有效且具有细胞活性的PIN1抑制剂。由于我们还不确定这些分子的特异性，所以 作为对这一方法的补充，我们提出(SA 2)我们的特异度的表征和改进 初步先导化合物DS-2(R)。这种方法源于对Pin1能够容忍笨重的观察 取代苯并噻吩会导致脱靶亲和力降低。因此，在 与加州大学旧金山分校的先驱专家(吉姆·威尔斯、米歇尔·阿金)合作，我们将筛选出一种含有二硫化物的 用于药效团的片段文库，该片段文库与耐体积疏水货架接合以制备类似物 它将保持与Pin1的有效相互作用，同时有望减少与OFF-1的配体互补性- 目标。我们将使用在二硫化物系链筛查中获得的信息来合成和评估 定向系列DS-2(R)类似物，用于提高Pin1的特异性。总体而言，该项目的目标是 利用现代设计原理对第一种具有生物活性的Pin1化学品进行表征和改进 探测器。这些分子将促进PIN1在肿瘤学中的一致和高质量研究，并可能导致 对其作为目标的适宜性的新见解。重要的是，拟议的研究将极大地扩展我的培训，因为 化学生物学家结合了基于结构的设计，现代筛选方法，以及化合物和 目标验证方法。

Project Summary/Abstract The molecular chaperone and prolyl isomerase Pin1 is a promising target for cancer therapy. Knockout of PIN1 yields only mild degenerative phenotypes in normal mice, but greatly impairs tumorigenesis in oncogenic mouse models and decreases proliferation in prostate and breast cancer xenograft models. The reliance of cancer cells on Pin1 activity seems to occur through its ability to stabilize multiple oncogenes and destabilize multiple tumor suppressors. However, despite Pin1's great potential as a therapeutic target, no chemical inhibitor which is both specific and biologically-active has been reported. The challenge is that Pin1 has the features of a classically “undruggable” protein; it has a shallow, polar binding pocket which has, thus far, prevented discovery of viable inhibitors. We propose a previously-unexplored option for imparting permeability on existing potent in vitro inhibitors of Pin1 as well as expand this SAR using disulfide fragment tethering. A key innovation of this study is that we have designed and preliminarily tested the first potent and cell permeable inhibitors of Pin1 using high affinity phosphate-baring compounds and imparting permeability through phosphoramidate caging. These permeate the lipid bilayer and are subsequently enzymatically liberated to a phosphate-baring active metabolite. In exciting preliminary results, our top compound (DS-2(R)) has single-digit nanomolar EC50 for cyclin D1 destabilization in vivo – a known Pin1 inhibition biomarker. Herein, I propose (SA 1) rigorous physical and functional characterization of the biological activity of the first potent and cell-active Pin1 inhibitors. Since we are yet unsure of the specificity of these molecules, to supplement this approach, we propose (SA 2) the characterization and improvement of the specificity of our preliminary lead compound DS-2(R). This approach arose from observations that Pin1 is able to tolerate bulky substitutions in place of the benzothiophene which would likely lead to reduced affinity for off-targets. Thus, in collaboration with pioneering experts at UCSF (Jim Wells, Michelle Arkin), we will screen a disulfide-containing fragment library for pharmacophores which engage the bulk-tolerant hydrophobic shelf to prepare analogues which will maintain potent interaction with Pin1 while hopefully reducing ligand complementarity with off- targets. We will use the information gained in the disulfide tethering screen to synthesize and evaluate a directed series of DS-2(R) analogues for improved Pin1 specificity. Overall, the goal of this project is to leverage modern design principles to characterize and improve the first biologically active Pin1 chemical probes. These molecules will facilitate consistent and quality research of Pin1 in oncology and potentially lead to new insights into its suitability as a target. Critically, the proposed studies will greatly expand my training as a chemical biologist by combining structure-based design, modern screening methods, and compound and target validation methods.