Targeting accessory proteins of alpha2C adrenergic receptor in Raynaud Phenomenon

描述（由申请人提供）：雷诺现象（RP）的特征在于对寒冷、情绪压力和振动的反应过度的血管收缩，并且当与另一种结缔组织疾病如红斑狼疮或硬皮病相关时，包括雷诺病（原发性或特发性）和雷诺综合征。虽然经常被低估和诊断不足，RP是一种常见的临床遭遇，影响3%至12%的男性和6%至20%的女性。不幸的是，一个特定的治疗RP仍然是失踪的今天，治疗方法仅包括在管理一般平滑肌松弛剂。此外，尽管该疾病在150多年前就被描述，但RP的发病机制仍然不清楚，并且没有生物标志物或临床旁研究被验证用于早期检测和治疗。临床证据表明，育亨宾，α 2-肾上腺素能拮抗剂（A2）是有效的预防RP血管痉挛发作。基于解剖分布，A2 C是最有可能引起这些效应的肾上腺素能受体亚型，实验证据支持这一观点，表明暴露于低温后功能部位的受体定位增强。然而，目前没有特异性A2 C阻断剂可用，并且这些拮抗剂也可能具有许多副作用，从而妨碍了它们在RP治疗中的使用。另一种方法是靶向控制A2 C合成、成熟和细胞内运输的辅助蛋白。在过去的几年中，我们确定了与A2 C相互作用并以温度敏感方式调节其功能的特定蛋白质。首先，我们已经表明，HSP 90抑制剂选择性地减少A2 C靶向功能位点。此外，我们比较了细胞内运输和功能 人和大鼠A2 C的反应，我们发现，即使它们超过90%同源，人A2 C显示出更大的温度依赖性，表明在RP中观察到的过度血管收缩可以仅使用人受体来研究。我们进一步确定了负责这种特殊模式的氨基酸残基，由于差异， 与RuVBL 1蛋白的相互作用。因此，RuVBL 1细胞水平的变化诱导温度依赖性A2 C功能反应的改变。类似地，另一种名为nucleophosmin的辅助蛋白的细胞水平的增加，大大增强了温度依赖性A2 C激活。因此，基于这些原始的发现，本研究的主要目标是确定使用正常和RP供体的人血管平滑肌细胞，如果HSP 90，RuVBL 1和nucleophosmin可用作RP的早期诊断和治疗的生物标志物。由于HSP 90抑制剂目前正处于治疗无关疾病的二期临床试验中，预期结果可能具有直接的临床意义。此外，我们的目标是建立一个新的模型，以测试目前和未来的治疗方法在RP的有效性。

DESCRIPTION (provided by applicant): Raynaud Phenomenon (RP) is characterized by exaggerated vasoconstriction in response to cold, emotional stress and vibrations and comprises from Raynaud's Disease (primary or idiopathic) and Raynaud's Syndrome when is associated with another connective tissue disorder like Lupus erythematosus or scleroderma. Although often underestimated and under diagnosed, RP is a common clinical encounter affecting between 3 to 12% of men and 6 to 20 % of women. Unfortunately, a specific treatment of RP is still missing today, the therapeutic approaches consisting only in administration of general smooth muscle relaxants. Moreover, although the disease was described more than 150 years ago, the RP pathogenesis is still not understood and no biological markers or paraclinical investigations were validated for early detection and treatment. Clinical evidence indicate that yohimbine, an alpha2-adrenergic antagonist (A2) is effective in preventing the vasospastic attacks in RP. Based on the anatomical distribution, A2C is the most probable adrenergic receptor subtype responsible for these effects and this view is supported by experimental evidence indicating that the receptor localization at the functional site is enhanced after exposure to low-temperature. However, currently no specific A2C blockers are available and also these antagonists may have numerous side effects precluding their use in the RP's treatment. An alternative approach is to target the accessory proteins controlling the synthesis, maturation and intracellular trafficking of A2C. Pursuing this idea, during the last few years we identified specific proteins interacting with A2C and regulating its function in temperature-sensitive manner. First, we have shown that HSP90 inhibitors are selectively reducing A2C targeting to the functional site. Further, we compared the intracellular trafficking and functional responses of human and rat A2C and we found that even if they are over 90 % homologous, human A2C displays a much greater temperature-dependence, indicating that the exaggerated vasoconstriction observed in RP can be studied using only the human receptor. We further identified the amino acid residues responsible for this particular pattern, due to the differential interactions with RuVBL1 protein. Accordingly, changes in the RuVBL1 cellular levels induced alterations in the temperature- dependent A2C functional responses. Similarly, increases in the cellular levels of another accessory protein named nucleophosmin, greatly enhanced the temperature-dependent A2C activation. Therefore, based on these original findings, the main goal of the present investigation is to determine using human vascular smooth muscle cells from normal and RP donors if HSP90, RuVBL1 and nucleophosmin may be used as biomarkers for early diagnostic and therapy of RP. As HSP90 inhibitors are currently in second phase clinical trial for the treatment of unrelated diseases, the expected results may have an immediate clinical relevance. Also, we aim to establish a new model to test the effectiveness of current and future therapeutic approaches in RP.