Identifying CXCR4 Receptor Agonists to Improve Diabetic Healing

摘要：糖尿病患者受伤后创面愈合受损是一个主要的临床问题，导致 延长住院时间和高昂的医疗费用。三分之二的非创伤性截肢手术是 之前有一处糖尿病伤口。糖尿病周围神经病变的发展增加了易感性 糖尿病皮肤的损伤。我们已经证明，小鼠和人类的糖尿病皮肤也会损害皮肤完整性。 在基线，这进一步使糖尿病皮肤容易受到伤害。糖尿病创面缺乏基质来源 因子-1(SDF-1)，一种强大的趋化因子，参与祖细胞募集、血管生成和肉芽形成 通过与CXCR4受体结合和建立趋化作用而介导的组织形成 渐变。我们已经证明，过表达sdf-1可以纠正糖尿病创面愈合障碍，并且在 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， 本建议的目的是开发一种靶向SDF-1受体CXCR4的小分子疗法，以 改善糖尿病皮肤的完整性，防止受伤，并在受伤时改善伤口愈合。我们有 开发和优化了一种创新的筛选方法来识别新的选择性CXCR4类 受体激动剂，可局部应用并渗透皮肤，改善皮肤完整性和伤口 治愈。我们建议筛选NIH SMR文库并进行Hit-to-Lead研究以确定新的CXCR4 可以针对这些糖尿病并发症开发的受体激动剂。 目标1.我们将筛选NIH化合物集合，以确定能使cAMP痛苦的分子 通过CXCR4受体的信号通路。我们已经开发和优化了初步的检测方法来筛选 一流的CXCR4小分子激动剂，并扩大了我们的中试筛选，纳入了6400种化合物 从我们的内部收集，以证明使用我们强大的测试漏斗筛选整个NIH SMR文库。 目标2.我们将实施剂量反应研究，使用计数器筛查验证所有“命中”，并 二次趋化/迁移分析，确认直接受体结合，并优化我们的最大 通过目录和药物化学方法使用合成孔径雷达的前景分子。来自Aim 1的点击数将是 对相关的CXCR5、CCR6、CXCR6受体和无关APJ的选择性进行反筛选 感受器。使用人体细胞的功能分析将评估趋化和迁移。基于单元的绑定 研究将确定与受体的直接结合、结合参数，并通过SAR优化有希望的候选分子。 目标3.我们将检查来自目标2的有效靶分子纠正异常的能力 调控炎症、血管生成和胶原合成的microRNAs在人体内的表达 糖尿病成纤维细胞，其表达被sdf-1纠正。来自Aim 2的目标分子将是 筛选它们纠正microRNA-146a、15b和29a表达的能力，这些RNA-146a、15b和29a在 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.