Targeting the longevity regulator PAPP-A with small molecule inhibitors

摘要： 哺乳动物的衰老可以通过饮食、基因突变和微小的 分子。小鼠中最有效的长寿突变调节生长激素和/或其受体的水平或作用。 下游介质，IGF1。PAPP-A蛋白酶在特定组织中通过切割 IGF结合蛋白（IGFBPs），主要是IGFBP4和IGFBP5。Pappa KO（PKO）小鼠寿命长， 保护免受多种衰老病理学，例如癌症、肾病和动脉粥样硬化。PAPP-A缺乏症 在成年小鼠中诱导的PAPP-A也能延长寿命，这意味着PAPP-A的药理学抑制可能代表了 一种有效且耐受性良好的抗衰老策略。尽管针对PAPP-A的抑制性抗体已经被发现， 在动脉粥样硬化和肾脏疾病的临床前模型中， 已经描述了针对PAPP-A的小分子抑制剂。这代表了一个关键的知识差距， 这一领域，因为对于衰老研究，使用小分子PAPP-A抑制剂而不是 抗体试剂本申请的目的是设计、合成和开发有效的、选择性的、 生物可利用的PAPP-A抑制剂，以阐明其作用机制，并在体内测试它们。我们的初步 数据显示，我们的主要PAPP-A抑制剂P100有效地抑制IGFBP-4的蛋白水解裂解， IGF信号传导，后者通过测量IGF受体磷酸化来评估。拟议的工作是基于 大量的基础数据，并将在两个具体目标的背景下进行。在目标1中，我们 基于我们的PAPP-A抑制剂设计和合成新的类似物，以提高其效力，选择性， 和药物样性质，最终目标是开发具有体内活性的化合物。一系列测定 将用于在生物化学、生物物理、结构和功能水平上表征新类似物， 从而建立这类新的PAPP-A抑制剂的结构-活性关系。在目标2中，优化的PAPP- 将使用一系列新型细胞和小鼠PAPP-A功能测定评价A抑制剂。活动 将测试PAPP-A抑制剂在抑制细胞IGF1信号传导中的作用。抑制剂诱导一种 核仁面积的减少，我们已经发现的PKO细胞的新表型，将被测试。这些测定 将全部在野生型和PKO细胞中进行，以测试观察到的表型的PAPP-A依赖性。在体内， 将评估PAPP-A抑制剂对PAPP-A依赖性基因表达变化的影响， 通过qRT-PCR和RNA-seq在脂肪、骨骼肌和胸腺中未发现。最有前途的小 将评价分子诱导Pappa KO小鼠的新表型的能力：白色的"beiging" 脂肪组织（WAT），改变WAT中M1/M2巨噬细胞的比例，增加FNDC5的表达， 具有有益生理作用激素。这些研究将首次产生有效的和有选择性的 小分子PAPP-A抑制剂。这些分子将作为探索IGF 1生物学的化学工具 信号传导和PAPP-A功能，并作为未来候选抗衰老化合物的基础。

Abstract: Aging can be slowed, lifespan extended, and diseases postponed in mammals by diet, mutations, and small molecules. The most potent longevity mutations in mice modulate levels or actions of growth hormone and/or its downstream mediator, IGF1. The PAPP-A protease activates IGF1 signaling in specific tissues by cleavage of IGF binding proteins (IGFBPs), principally IGFBP4 and IGFBP5. Pappa KO (PKO) mice are long-lived, and show protection from diverse aging pathologies, e.g. cancer, kidney disease, and atherosclerosis. PAPP-A deficiency induced in adult mice also provokes longevity, implying that pharmacologic inhibition of PAPP-A might represent an effective and well-tolerated anti-aging strategy. Although inhibitory antibodies against PAPP-A have been developed, and shown to be effective treatments in preclinical models of atherosclerosis and kidney disease, no small molecule inhibitors against PAPP-A have been described. This represents a critical knowledge gap in this field, since, for aging studies, it would be highly desirable to use small molecule PAPP-A inhibitors, rather than antibody reagents. The objective of this application is to design, synthesize and develop potent, selective, and bioavailable PAPP-A inhibitors, to elucidate their mechanism of action, and test them in vivo. Our preliminary data have shown that our lead PAPP-A inhibitor, P100, effectively inhibits proteolytic cleavage of IGFBP-4 and IGF signaling, the latter assessed by measuring IGF receptor phosphorylation. The proposed work is based on a large body of foundational data, and will be performed in the context of two Specific Aims. In Aim 1, we will design and synthesize new analogs based on our lead PAPP-A inhibitor, to improve their potency, selectivity, and drug-like properties, with the ultimate goal of developing compounds with in vivo activity. A series of assays will be used to characterize the new analogs at the biochemical, biophysical, structural and functionals level, and thus to establish structure-activity relationships of this new class of PAPP-A inhibitors. In Aim 2, optimized PAPP- A inhibitors will be evaluated using a battery of novel cellular and mouse assays of PAPP-A function. The activity of PAPP-A inhibitors in suppressing cellular IGF1 signaling will be tested. The ability of inhibitors to induce a reduction in nucleolar area, a novel phenotype of PKO cells we have uncovered, will be tested. These assays will all be performed in wild-type and PKO cells, to test for PAPP-A dependency of phenotypes observed. In vivo, the impact of PAPP-A inhibitors will be evaluated on PAPP-A-dependent gene expression changes we have uncovered in adipose, skeletal muscle, and thymus, by qRT-PCR and RNA-seq. The most promising small molecules will be evaluated for their ability to induce novel phenotypes of Pappa KO mice: “beiging” of white adipose tissue (WAT), altered ratio of M1/M2 macrophages in WAT, and increased expression of FNDC5, a pro- hormone with beneficial physiological effects. These studies will generate, for the first time, potent and selective small molecule PAPP-A inhibitors. Such molecules will serve as chemical tools to explore the biology of IGF1 signaling and PAPP-A function, and as the basis for future candidate anti-aging compounds.