Developing helical peptide antagonists of the growth hormone receptor

项目摘要 生长激素（GH）是一种单链多肽，其作为细胞生长、增殖和增殖的关键刺激物。 哺乳动物的新陈代谢。生长激素的作用，以促进纵向生长和适当的器官发育，在儿童- Hood和刺激胰岛素样生长因子1（IGF-1）在肝脏和其他组织中的生产 生活许多研究表明，体内GH活性降低会导致健康衰老， 事实上，最长寿的实验室小鼠是由于GH受体（GHR）基因的全面破坏。 值得注意的是，这样的GHR-/-小鼠已经表现出对GH介导的疾病的增强的抵抗力，所述疾病有助于 不健康的衰老，包括糖尿病终末器官损伤和某些类型的癌症。之间的这种连接 GH作用的降低和寿命的延长导致了抑制GH作用可能延迟发病的假设 与年龄有关的疾病肽代表了一类有吸引力的分子作为治疗的领导 因为它们可以被设计成模拟蛋白质相互作用结构域的可变结构和序列。 此外，肽是序列特异性的并且是合成上易处理的，允许它们避开许多限制性酶。 与蛋白质药物相关的生产问题。我们的团队最近发现了一种新的基于肽的 GHR拮抗剂（称为SH 1），可减轻培养细胞系中GH介导的信号传导。S1 H设计为 与GHR相互作用的GH小螺旋区（残基36-51）的直接序列模拟物。结构 S1 H的活性关系显示肽螺旋度和GHR拮抗作用之间存在强相关性， 我们假设螺旋倾向是生物活性所必需的。我们现在试图检验这一假设 通过开发折叠成稳定螺旋结构的S1 H衍生物，并使用它们来抑制GH介导的 体外和体内信号传导。在本提案的目标1中，我们将合成两类不同的结构化S1 H 衍生物第一类将通过将烯属侧链安装到野生型S1 H序列中来产生， 所述肽可以被“钉合”成α-螺旋结构。第二类将通过转置S1 H来开发 在一个实施方案中，将一个或多个残基连接到蟹毒素的a-螺旋上，蟹毒素是一种折叠成稳定的a/B基序的小蛋白质。所得肽 然后将其用于一系列针对重组GHR的直接结合和电泳迁移率变动测定。 在目标2中，我们将研究我们的结构化S1 H衍生物抑制细胞中GH信号传导的能力， 过表达GHR和体内小鼠衰老模型。首先，我们将采用基于细胞的替代测定， 确定结构化的S1 H衍生物是否可以抑制GH介导的下游 转录因子，如STAT 5。接下来，我们将评估结构化S1 H衍生物如何影响血清水平 IGF-1和IGF-BP 3的水平以及C57 BL 6小鼠的身体组成。本提案生成的数据将作为 为未来探索S1 H（及其结构）的分子机制的研究奠定了基础。 衍生物）影响GH介导的信号传导，并将确定潜在的先导化合物， 旨在减轻GH介导的导致不健康衰老的疾病的治疗剂。

PROJECT SUMMARY Growth hormone (GH) is a single-chain polypeptide that acts as a key stimulator of cell growth, proliferation and metabolism in mammals. GH acts to promote longitudinal growth and proper organ development during child- hood and stimulates production of insulin-like growth factor 1 (IGF-1) in the liver and other tissues throughout life. Numerous studies have indicated that reduced GH activity in vivo results in healthy aging and increased lifespan; in fact, the longest-lived laboratory mouse results from global disruption of the GH receptor (GHR) gene. Notably, such GHR-/- mice have demonstrated enhanced resistance to GH-mediated disorders that contribute to unhealthy aging, including diabetic end organ damage and certain types of cancer. This connection between reduced GH action and extended lifespan has led to the hypothesis that inhibiting GH action may delay the onset of age-related morbidities. Peptides represent an attractive class of molecule to serve as therapeutic leads because they can be designed to mimic the variable structures and sequences of protein interaction domains. Moreover, peptides are sequence specific and synthetically tractable, allowing them to circumvent many of the production problems associated with protein-based drugs. Our group recently identified a novel peptide-based GHR antagonist (termed SH1) that mitigates GH-mediated signaling in cultured cell lines. S1H was designed as a direct sequence mimic of a small helical region (residues 36-51) of GH that interacts with the GHR. Structure activity relationships of S1H showed a strong correlation between peptide helicity and GHR antagonism, leading us to hypothesize that helical propensity is required for biological activity. We now seek to test this hypothesis by developing S1H derivatives that fold into stable helical structures and use them to inhibit GH-mediated signaling in vitro and in vivo. In aim 1 of this proposal, we will synthesize two separate classes of structured S1H derivative. The first class will be generated by installing olefinic side chains into the wild-type S1H sequence so the peptides can be ‘stapled’ into a-helical structures. The second class will be developed by transposing S1H residues onto the a-helix of scyllatoxin, a small protein that folds into a stable a/b motif. The resulting peptides will then be used in a series of direct binding and electrophoretic mobility shift assays against recombinant GHR. In aim 2, we will investigate the ability of our structured S1H derivatives to inhibit GH signaling in cells that overexpress the GHR and in vivo mouse models of aging. First, we will employ a cell-based surrogate assay to determine whether structured S1H derivatives can inhibit GH-mediated phosphorylation of downstream transcription factors, such as STAT5. Next, we will assess how structured S1H derivatives affect the serum levels of IGF-1 and IGF-BP3, and body composition of C57BL6 mice. Data generated from this proposal will serve as a foundation for future studies that explore the molecular mechanisms through which S1H (and its structured derivatives) affects GH-mediated signaling and will identify potential lead compounds to be used as emerging therapeutics designed to mitigate GH-mediated disorders that contribute to unhealthy aging.