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，导致长期住院和巨额医疗费用。在所有非创伤性截肢手术中，有三分之二的截肢手术是在糖尿病伤口之前进行的。糖尿病周围神经病变的发展和感觉减退增加了对损伤的易感性。我们已经证明，小鼠和人类的糖尿病皮肤在基线水平上损害了皮肤的完整性，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衍生因子-1(SDF-1)，这是一种参与祖细胞的有效趋化因子 通过与CXCR4受体的结合和趋化梯度的建立而介导的募集、血管生成和肉芽组织的形成。我们已经证明，慢病毒过表达sdf-1可以纠正糖尿病伤口愈合的损害，并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，本建议的目的是开发一种以SDF-1受体CXCR4为靶点的小分子疗法，以改善糖尿病皮肤的完整性，防止损伤，并在发生损伤时促进伤口愈合。我们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CXCR4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文库，并进行Hit-to-Lead研究，以确定可以为这些糖尿病并发症开发的新型CXCR4受体激动剂。目的1.我们将进行MLSMR文库筛选，以确定可通过CXCR4受体激活cAMP信号通路的分子。利用表达CXCR4受体的CHO-K1细胞和一种简单的tr-fret方法检测cAMP反应，我们建立和优化了筛选新型CXCR4小分子激动剂MLPCN文库的初步方法。目的2.我们将实施剂量反应研究，使用反筛选验证所有的“命中”，使用基于二次细胞的趋化/迁移分析，确认直接受体结合，并通过目录和药物化学方法使用SAR优化我们最有希望的分子。来自Aim 1的HITS将对相关CXCR5受体的选择性进行反筛选，并通过趋化/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优化有希望的候选分子。目的3.我们将检测Aim 2中已证实的靶分子对调节炎症、血管生成、胶原合成和皮肤完整性的microRNA的表达的能力，这些microRNA在糖尿病皮肤、伤口和成纤维细胞中调节失调，其表达被sdf-1纠正。来自Aim 2的靶分子将被筛选，以确定它们是否有能力纠正糖尿病中调节失调的microRNA-146a、15b和29a的表达，并用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.