Aggregation Properties of Hemopressin

描述（由申请人提供）：内源性大麻素系统由两种大麻素受体CB 1和CB 2组成，这两种受体是G蛋白偶联受体，主要由内源性化合物激活。CB 1是一个理想的治疗靶点，因为它参与成瘾性疾病和肥胖相关的途径。血加压素（HP）是一种由血红蛋白α链衍生的9个氨基酸组成的肽，已被证明对CB 1受体具有选择性反向激动剂活性。尽管加压素作为一种安全有效的治疗剂具有巨大的潜力，但由于合成加压素在药理学测定中的可变性，其进一步发展受到阻碍。我们假设，在生物测定中的加压素的效果的变化是由于这种肽的倾向，在生理相关的条件下，在溶液中形成自组装的纳米结构。因此，我们提出了两个具体的目标：1）设计和合成合成的缀合物和类似物的hemopressin和进行详细的核磁共振光谱和透射电子显微镜实验，以评估其聚集和自组装性能。2)使用四种不同的测定系统评估新设计的HP缀合物和类似物对CB 1的亲和力和功效：i）CB 1受体结合测定，ii）监测G蛋白活化的[35 S]GTPgS结合测定，iii）细胞cAMP的受体活化抑制，和iv） b-抑制蛋白的募集。本申请的中心目的是评价加压素形成纳米原纤维的能力对其药理学性质的影响。自组装生物肽，如b-淀粉样蛋白，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HP的自组装和聚集调节其药理活性，则该发现将具有有希望的治疗应用性 在药物滥用研究中。

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.