Targeting accessory proteins of alpha2C adrenergic receptor in Raynaud Phenomenon

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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.