A Novel Approach to Target Neutrophilic Airway Inflammation and Airway Hyperresponsiveness in Therapy-Resistant (Refractory) Asthma.

气道高反应性（AHR）和气道炎症是哮喘的标志。难治性哮喘 尽管使用高剂量口服皮质类固醇和长效β2-激动剂，但仍表现出持续症状 支气管扩张剂，并提出了重大的医疗挑战。了解机制和发展 克服治疗耐药性的策略提出了显著的未满足的需求。气道平滑肌（ASM） 过度收缩是AHR的关键因素，辅助性T细胞17（Th 17）促进激素不敏感的嗜酸性粒细胞 气道炎症（NAI）。我们发现，p63 RhoGEF，一个RhoA激活剂，在难治性哮喘中起着至关重要的作用。 我们还开发了CXN-8，一种p63 RhoGEF的小分子抑制剂。目的：确定重要性 以及p63 RhoGEF调节哮喘素质的机制，如果CXN-8抑制p63 RhoGEF， 改善AHR和NAI。长期目标：开发治疗难治性哮喘的新疗法。结果：1）p63- RhoGEF在哮喘患者中选择性上调，并在控制ASM的RhoA激活中起关键作用 过度收缩和Th 17细胞分化。2)CXN-8抑制ASM收缩并诱导平滑肌松弛。 气道。3)CXN-8改善小鼠哮喘模型中的AHR和NAI。4)CXN-8与p63 RhoGEF相互作用， 阻断RhoA激活。假设：靶向p63 RhoGEF刺激的ASM过度收缩和Th 17细胞 与CXN-8的分化改善了AHR和NAI，并且CXN-8治疗代表了一种新的策略， 难治性哮喘我们将在体外和体内测试这一假设。目标1.来定义潜在的机制 p63 RhoGEF和CXN-8对AHR的调控。我们将使用RNAi和抑制剂来确定关键作用， p63 RhoGEF在哮喘人ASM细胞（HASM）过度收缩中的机制。我们将分析CXN- 8 RhoA活性的调节，Ca 2+信号传导，β2-激动剂敏感/不敏感HASM的收缩/舒张 细胞我们将结合联合收割机RNAi、诱变和表面等离子体共振来阐明特异性和 CXN-8调控p63 RhoGEF的机制。p63 RhoGEF缺失是否能改善AHR并降低 将在屋尘螨（HDM）驱动的哮喘鼠模型中检测CXN-8的作用。目标二。到 阐明p63 RhoGEF和CXN-8调控NAI的机制。我们将研究 CXN-8对Th 17细胞分化的影响。我们将沉默RhoA或表达活性RhoA突变体，以建立其表达。 CXN-8抑制Th 17细胞分化的重要性。我们还将确定p63 RhoGEF的丢失是否 减弱RhoA活化和细胞中的Th 17分化以及小鼠中HDM诱导的NAI，并降低CXN-8 体外和体内的抑制作用。目标3。为了检查CXN-8在小鼠模型中的治疗潜力， 难治性哮喘。我们将确定吸入CXN-8是否是一种急性和有效的支气管扩张剂， β2-激动剂不敏感AHR模型。我们将测试肺靶向的长效CXN-8微粒，以缓解 AHR/NAI在皮质类固醇不敏感性哮喘小鼠模型中具有有限的全身副作用的影响 这些研究还将检查p63 RhoGEF丢失对AHR/NAI的影响以及CXN-8的治疗作用。

Airway hyperresponsiveness (AHR) and airway inflammation are hallmarks of asthma. Refractory asthma manifests with persistent symptoms despite use of high-dose oral corticosteroids and long-acting β2-agonist bronchodilators and poses a major healthcare challenge. Understanding the mechanisms and developing strategies to overcome therapeutic resistance poses a significant unmet need. Airway smooth muscle (ASM) hypercontraction is a key factor of AHR, and T-helper 17 (Th17) cells promote steroid-insensitive neutrophilic airway inflammation (NAI). We found p63RhoGEF, a RhoA activator, plays crucial roles in refractory asthma. We also developed CXN-8, a small molecule inhibitor of p63RhoGEF. Objective: To determine the importance and mechanisms by which p63RhoGEF modulates the asthma diathesis and if CXN-8 inhibits p63RhoGEF to ameliorate AHR and NAI. Long-term goal: To develop new therapies for refractory asthma. Findings: 1) p63- RhoGEF is selectively upregulated in asthmatics and plays a critical role in RhoA activation that controls ASM hypercontraction and Th17 cell differentiation. 2) CXN-8 inhibits ASM contraction and induces relaxation of airway. 3) CXN-8 ameliorates AHR and NAI in murine asthma models. 4) CXN-8 interacts with p63RhoGEF to block RhoA activation. Hypothesis: Targeting p63RhoGEF-stimulated ASM hypercontractility and Th17 cell differentiation with CXN-8 ameliorates AHR and NAI and that CXN-8 therapy represents a novel strategy for refractory asthma. We will test this hypothesis in vitro and in vivo. Aim 1. To define the mechanism underlying p63RhoGEF and CXN-8 regulation of AHR. We will use RNAi and inhibitors to determine the pivotal role and mechanism of p63RhoGEF in hypercontractility of asthmatic human ASM (HASM) cells. We will analyze CXN- 8 regulation of RhoA activity, Ca2+ signaling, contraction/relaxation of β2-agonist-sensitive/insensitive HASM cells. We will combine RNAi, mutagenesis, and surface plasmon resonance to elucidate the specificity and mechanism for CXN-8 regulation of p63RhoGEF. Whether p63RhoGEF loss ameliorates AHR and reduces the effects of CXN-8 will be examined in a house dust mite (HDM)-driven murine model of asthma. Aim 2. To elucidate the mechanism underlying p63RhoGEF and CXN-8 regulation of NAI. We will examine the effects of CXN-8 on Th17 cell differentiation. We will silence RhoA or express an active RhoA mutant to establish its importance in CXN-8 inhibition of Th17 cell differentiation. We will also determine if loss of p63RhoGEF attenuates RhoA activation and Th17 differentiation in cells and HDM-induced NAI in mice and reduces CXN-8 inhibitory effects in vitro and in vivo. Aim 3. To examine the therapeutic potential of CXN-8 in murine models of refractory asthma. We will determine if inhaled CXN-8 is an acute and effective bronchodilator in a murine model of β2-agonist insensitive AHR. We will test lung targeted, long-acting CXN-8 microparticles to alleviate AHR/NAI with limited systemic side-effects in a murine model of corticosteroid-insensitive asthma. The impact of p63RhoGEF loss on AHR/NAI and therapeutic effects of CXN-8 will also be examined in these studies.