Identifying CXCR4 Receptor Agonists to Improve Diabetic Healing

 描述（由申请人提供）：糖尿病患者受伤后伤口愈合受损是一个主要的临床问题，导致住院时间延长和医疗费用增加。三分之二的非创伤性截肢之前都有糖尿病伤口。糖尿病周围神经病变和感觉减退的发展增加了对损伤的易感性。我们已经表明，小鼠和人类的糖尿病皮肤在基线时具有受损的皮肤完整性，这进一步使糖尿病皮肤易于损伤。糖尿病伤口缺乏基质衍生因子-1（SDF-1），一种参与祖细胞分化的强效趋化因子。 募集、血管生成和肉芽组织形成，通过与CXCR 4受体结合和建立趋化梯度介导。我们已经证明，SDF-1的慢病毒过表达可以纠正糖尿病伤口愈合障碍，并且在令人兴奋的初步研究中，可以恢复糖尿病皮肤的完整性。鉴于这一重要的临床问题，本提案的目的是开发一种靶向SDF-1 β受体CXCR 4的小分子治疗剂，以改善糖尿病皮肤的完整性，防止损伤，并在发生损伤时改善伤口愈合。我们已经开发并优化了一种创新的筛选方法，以确定新类别的选择性CXCR 4受体激动剂，这些激动剂可以局部应用并渗透皮肤，以改善皮肤完整性和伤口愈合。我们建议筛选完整的MLSMR库，并进行命中铅研究，以确定新的CXCR 4受体激动剂，可以开发用于这些并发症的糖尿病。目标1.我们将进行MLSMR文库筛选，以鉴定可以通过CXCR 4受体激动cAMP信号通路的分子。使用表达CXCR 4受体的CHO-k1细胞和检测cAMP反应的简单TR-FRET方案，我们开发并优化了初步试验，以筛选MLPCN文库中的新型、首个CXCR 4小分子激动剂。目标二。我们将实施剂量反应研究，使用计数器筛选筛选所有“命中”，采用基于次级细胞的趋化性/迁移试验，确认直接受体结合，并使用SAR通过目录和药物化学方法优化我们最有前途的分子。将针对针对相关CXCR 5受体的选择性对来自Aim 1的命中进行反筛选，并使用趋化性/迁移试验评估功能活性。使用HTRF技术的基于细胞的结合研究将确认与受体的直接结合、结合参数，并通过SAR优化有希望的候选物。AIM 3.我们将检查目标2中验证的靶分子纠正调节炎症、血管生成、胶原蛋白合成和皮肤完整性的microRNA表达的能力，这些microRNA在糖尿病皮肤、伤口和成纤维细胞中失调，其表达被SDF-1 β纠正。将筛选来自Aim 2的靶分子，以确定其纠正microRNA-146 a、15 b和29 a表达的能力，这些微RNA-146 a、15 b和29 a在糖尿病中失调，并通过SDF-1治疗进行纠正。

 DESCRIPTION (provided by applicant): Impaired wound healing following injury in diabetics represents a major clinical problem, resulting in prolonged hospitalizations and significant healthcare costs. Two-thirds of all non-traumatic amputations are preceded by a diabetic wound. The development of diabetic peripheral neuropathy and decreased sensation increases the susceptibility to injury. We have shown that diabetic skin in mice and humans has impaired skin integrity at baseline which further predisposes diabetic skin to injury. Diabetic wounds are deficient in stromal derived factor-1 (SDF-1), a potent chemokine involved in progenitor cell recruitment, angiogenesis, and granulation tissue formation, mediated through binding to the CXCR4 receptor and the establishment of a chemotactic gradient. We have shown lentiviral overexpression of SDF-1 corrects the diabetic wound healing impairment, and in exciting preliminary studies, can restore the integrity of diabetic skin. Given this important clinical problem, the objective of this proposal is to develop a small molecule therapeutics to target the SDF-1 receptor CXCR4 to improve diabetic skin integrity to prevent injury, and improve wound healing should injury occur. We have developed and optimized an innovative screening approach to identify new classes of selective CXCR4 receptor agonists that can be applied topically and penetrate the skin to improve skin integrity and wound healing. We propose to screen the full MLSMR library and carry out hit-to-lead studies to identify novel CXCR4 receptor agonists that can be developed for these complications of diabetes. Aim 1. We will Perform the MLSMR Library screen to identify molecules that can agonize the cAMP signaling pathway via the CXCR4 receptor. Using CHO-k1 cells expressing the CXCR4 receptor and a simple TR-FRET protocol to detect the cAMP response, we have developed and optimized the primary assay to screen the MLPCN library for novel, first in class CXCR4 small molecule agonists. Aim 2. We will Implement dose response studies, Validate all the "Hits" using a Counter-Screen, Employ secondary cell-based chemotaxis/migration assays, Confirm direct receptor binding, and Optimize our most promising molecules using SAR by Catalog and Medicinal Chemistry approaches. Hits from Aim 1 will be counter-screened for selectivity against the related CXCR5 receptor, and functional activity assessed using chemotaxis/migration assays. Cell-based binding studies using HTRF technology will confirm direct binding to the receptor, binding parameters, and optimize promising candidates by SAR. AIM 3. We will Examine the ability of Validated target molecules from Aim 2 to correct expression of microRNAs that regulate inflammation, angiogenesis, collagen synthesis, and skin integrity, that are dysregulated in diabetic skin, wounds and fibroblasts, and whose expression is corrected by SDF-1. Target molecules from Aim 2 will be screened for their ability to correct expression of microRNA-146a, 15b, and 29a, which are dysregulated in diabetes, and corrected with SDF-1 treatment.