Identifying CXCR4 Receptor Agonists to Improve Diabetic Healing

总结：糖尿病患者损伤后伤口愈合受损是一个主要的临床问题， 长期住院和巨额医疗费用。三分之二的非创伤性截肢 之前是糖尿病伤口糖尿病周围神经病变的发展增加了 糖尿病皮肤损伤。我们已经表明，小鼠和人类的糖尿病皮肤也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的 SDF-1是一种参与祖细胞募集、血管生成和肉芽形成的强效趋化因子 组织形成，通过与CXCR 4受体结合和趋化作用的建立介导 梯度离心我们已经证明SDF-1的过度表达可以纠正糖尿病伤口愈合障碍， 令人兴奋的初步研究，可以恢复糖尿病皮肤的完整性。鉴于这一重要的临床问题， 该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， 改善糖尿病皮肤完整性，以防止损伤，并在发生损伤时改善伤口愈合。我们有 开发并优化了一种创新的筛选方法，以识别新的选择性CXCR 4类型 可局部施用并渗透皮肤以改善皮肤完整性和创伤的受体激动剂 治愈我们建议筛选NIH SMR文库并进行命中先导研究以鉴定新的CXCR 4 受体激动剂，可以开发这些糖尿病并发症。 目标1.我们将筛选NIH的化合物集合，以确定可以激动cAMP的分子。 通过CXCR 4受体的信号通路。我们已经开发并优化了初步试验， CXCR 4小分子激动剂，并将我们的试点筛选扩大到包括6400种化合物 从我们的内部收集，以证明筛选整个NIH SMR库使用我们强大的测试漏斗。 目标2.我们将实施剂量反应研究，使用计数器屏幕验证所有“命中”， 二级趋化性/迁移试验，确认直接受体结合，并优化我们的最 通过目录和药物化学方法使用SAR的有前途的分子。目标1的命中将是 针对相关CXCR 5、CCR 6、CXCR 6受体和不相关APJ的选择性进行反筛选 受体。使用人细胞的功能测定将评估趋化性和迁移。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。 目标3.我们将检查目标2中验证的靶分子纠正异常的能力。 调节人类炎症、血管生成和胶原合成的microRNA的表达 糖尿病成纤维细胞，并且其表达被SDF-1 β校正。目标2的目标分子将 筛选其纠正microRNA-146 a、15 b和29 a表达的能力，这些microRNA-146 a、15 b和29 a在细胞中失调。 糖尿病，并用SDF-1治疗纠正。

Summary: 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 increases the susceptibility of diabetic skin to injury. We have shown that diabetic skin in mice and humans also 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 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 therapeutic to target the SDF-1 receptor CXCR4 to improve diabetic skin integrity to prevent injury, and to 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 NIH SMR 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 screen the NIH compound collection to identify molecules that can agonize the cAMP signaling pathway via the CXCR4 receptor. We have developed and optimized the primary assay to screen for first in class CXCR4 small molecule agonists and expanded our pilot screen to include 6400 compounds from our internal collection to justify screening the entire NIH SMR library using our robust testing funnel. Aim 2. We will implement dose response studies, validate all “Hits” using a counter-screen and secondary 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, CCR6, CXCR6 receptors and unrelated APJ receptors. Functional assays using human cells will assess chemotaxis and migration. Cell-based binding studies will confirm direct binding to 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 the abnormal expression of microRNAs that regulate inflammation, angiogenesis, and collagen synthesis in human diabetic 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.