Aggregation Properties of Hemopressin

说明书(申请人提供)：内源性大麻素系统由两个大麻素受体CB1和CB2组成，这两个受体是G蛋白偶联受体，主要由脂类化合物激活。CB1是一个理想的治疗靶点，因为它参与了与成瘾障碍和肥胖相关的通路。血加压素(Hp)是一种来源于血红蛋白a链的九个氨基酸的多肽，已被证明对CB1受体具有选择性反向激动剂活性。尽管血压素作为一种安全有效的治疗方法具有巨大的潜力，但由于合成血压素在药理检测中的多样性，其进一步发展受到了阻碍。我们假设，血加压素在生物测定中作用的可变性是由于这种多肽在生理相关的条件下在溶液中形成自组装纳米结构的趋势。因此，我们提出了两个具体的目标：1)设计和合成血加压素的人工偶联物和类似物，并进行详细的核磁共振波谱和透射电子显微镜实验，以评估它们的聚集和自组装特性。2)用四种不同的检测系统来评估新设计的hp结合物和类似物对cb1的亲和力和有效性：i)cb1受体结合实验，ii)[35S]GTPgS结合实验监测G蛋白激活，iii)受体激活的细胞cAMP抑制，以及iv) B-arrestin招聘。这项应用的中心目标是评估血加压素形成纳米纤维的能力对其药理学特性的影响。自组装的生物肽，如b-淀粉样蛋白，已经深刻地提高了我们对神经生物学许多方面的理解。同样，如果我们能够证明幽门螺杆菌的自组装和聚集调节其药理活性，这一发现将具有良好的治疗应用前景 在药物滥用研究中。 与公共健康相关：内源性大麻素系统是指一组神经调节脂质及其受体，参与多种生物学过程，包括成瘾、食欲、记忆、痛感、认知和癌细胞增殖。内源性大麻素系统由两个大麻素受体CB1和CB2组成。血加压素是一种血红蛋白衍生的多肽，已被证明是CB1受体的选择性反向激动剂，CB1受体主要在大脑的动力回路中表达。CB1是一个理想的治疗靶点，因为它参与了与成瘾障碍和肥胖相关的通路。血加压素是一种潜在的候选药物，但其药理活性一直不一致和可重复性，可能是因为它在生理条件下容易形成聚集体。这项研究的目的是研究血加压素的聚集特性，并开发这种多肽的类似物和/或结合物作为治疗线索，显示出可重复的药理学特征。

DESCRIPTION (provided by applicant): The endocannabinoid system consists of two cannabinoid receptors CB1 and CB2, which are G-protein coupled receptors and activated by mainly lipidic compounds. CB1 is a desirable therapeutic target due to its involvement in pathways related to addictive disorders and obesity. Hemopressin (HP), a nine-amino acid- peptide derived from the a chain of hemoglobin, has been shown to have selective inverse agonist activity against the CB1 receptor. In spite of hemopressin's tremendous potential as a safe and effective therapeutic, its further development has been hampered due to the variability of synthetic hemopressin in pharmacological assays. We hypothesize that the variability of hemopressin's effects in biological assays is due to the tendency of this peptide to form self-assembled nanostructures in solution under physiologically relevant conditions. Accordingly, we propose two specific aims: 1) To design and synthesize synthetic conjugates and analogs of hemopressin and conduct detailed nuclear magnetic resonance spectroscopy and transmission electron microscopy experiments to assess their aggregation and self-assembly properties. 2) To assess the affinity and efficacy of the newly designed conjugates and analogs of HP towards CB1 using four different assay systems: i) CB1 receptor binding assay, ii) [35S]GTPgS binding assay to monitor G-protein activation, iii) receptor-activated inhibition of cellular cAMP, and iv) b-arrestin recruitment. The central objective of this application is to evaluate the impact of hemopressin's ability to form nanofibrils on its pharmacological properties. Self-assembling biological peptides such as b-amyloid have profoundly improved our understanding of many aspects of neurobiology. Similarly, if we are able to show that self-assembly and aggregation of HP modulate its pharmacological activity, the finding will have promising therapeutic applicability in drug abuse research. PUBLIC HEALTH RELEVANCE: The endocannabinoid system refers to a group of neuromodulatory lipids and their receptors that are involved in a variety of biological processes, including addiction, appetite, memory, pain sensation, cognition and cancer cell proliferation. The endocannabinoid system consists of two cannabinoid receptors CB1 and CB2. Hemopressin is a hemoglobin-derived peptide that has been shown to act as a selective inverse agonist for the CB1 receptor, which is predominantly expressed in the motivational circuitry of the brain. CB1 is a desirable therapeutic target due to its involvement in pathways related to addictive disorders and obesity. Hemopressin is a potential drug candidate but its pharmacological activity has not been consistent and reproducible, possibly due to its propensity to form aggregates under physiological conditions. The goal of this study is to investigate the aggregation properties of hemopressin and develop analogs and/or conjugates of this peptide as therapeutic leads that show reproducible pharmacological profiles.