Aggregation Properties of Hemopressin

内源性大麻素系统由两种大麻素受体CB1和CB2组成，这两种大麻素受体是g蛋白偶联受体，主要由脂质化合物激活。CB1是一个理想的治疗靶点，因为它参与与成瘾障碍和肥胖相关的途径。加压素（HP）是一种从血红蛋白a链中提取的9个氨基酸肽，已被证明对CB1受体具有选择性的逆激动剂活性。尽管加压素作为一种安全有效的治疗药物具有巨大的潜力，但由于合成加压素在药理学试验中的可变性，它的进一步发展受到阻碍。我们假设，在生理相关条件下，这种肽在溶液中形成自组装纳米结构的倾向导致了生物检测中加压素作用的变异性。因此，我们提出了两个具体的目标：1)设计和合成加压素的合成缀合物和类似物，并进行详细的核磁共振波谱和透射电镜实验来评估它们的聚集和自组装性质。2)使用四种不同的检测系统评估新设计的HP偶联物和类似物对CB1的亲和力和有效性：1)CB1受体结合试验；2)[35S]GTPgS结合试验以监测g蛋白的激活；3)受体激活抑制细胞cAMP；

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.