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

一种针对难治性哮喘中性粒细胞性气道炎症和气道高反应性的新方法。

• 批准号: 10659658
• 负责人: YAPING TU
• 金额: $ 41.11万
• 依托单位: CREIGHTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-20 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10659658
• 关键词: Acute; Address; Adrenal Cortex Hormones; Affinity; Agonist; Animal Model; Asthma; Attenuated; Binding; Bronchoconstrictor Agents; Bronchodilator Agents; CD4 Positive T Lymphocytes; Catalytic Domain; Cell Differentiation process; Cell model; Cells; Clinical Management; Disease susceptibility; Docking; Dose; Extrinsic asthma; Functional disorder; Goals; Health; Healthcare; Human; In Vitro; Inhalation; Libraries; Lung; Mediating; Molecular; Mus; Muscle Contraction; Mutagenesis; Oral; Pathogenesis; Pharmaceutical Preparations; Phenotype; Pirfenidone; Play; Pyroglyphidae; RNA Interference; Refractory; Regulation; Relaxation; Role; Signal Pathway; Signal Transduction; Smooth Muscle Myocytes; Specificity; Spectrum Analysis; Steroids; Surface Plasmon Resonance; System; T-Lymphocyte; Testing; Therapeutic; Therapeutic Effect; Tissue Model; airway hyperresponsiveness; airway inflammation; asthma model; asthmatic; cytokine; data integration; effectiveness testing; idiopathic pulmonary fibrosis; in vivo; inhibitor; molecular modeling; mouse model; mutant; neutrophil; novel; novel strategies; novel therapeutics; overexpression; particle; persistent symptom; pharmacologic; respiratory smooth muscle; side effect; small molecule inhibitor; structural determinants; therapy resistant

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.

气道高反应性（AHR）和气道炎症是难治性哮喘的标志。 尽管使用大剂量口服皮质类固醇和长效 β2 激动剂，但症状仍持续存在 支气管扩张剂并提出了重大的医疗保健挑战。 克服治疗抵抗的策略提出了显着的未满足的需求。 过度收缩是 AHR 的关键因素，辅助性 T 17 (Th17) 细胞可促进类固醇不敏感的中性粒细胞 我们发现 p63RhoGEF（一种 RhoA 激活剂）在难治性哮喘中发挥着至关重要的作用。 我们还开发了 CXN-8，一种 p63RhoGEF 的小分子抑制剂 目的：确定其重要性。 p63RhoGEF 调节哮喘素质的机制以及 CXN-8 是否抑制 p63RhoGEF 改善 AHR 和 NAI 长期目标：开发难治性哮喘的新疗法 研究结果：1) p63-。 RhoGEF 在哮喘患者中选择性上调，并在控制 ASM 的 RhoA 激活中发挥关键作用 过度收缩和 Th17 细胞分化 2) CXN-8 抑制 ASM 收缩并诱导松弛。 3) CXN-8 改善小鼠哮喘模型中的 AHR 和 NAI。 4) CXN-8 与 p63RhoGEF 相互作用。 阻断 RhoA 激活假设：针对 p63RhoGEF 刺激的 ASM 过度收缩和 Th17 细胞 CXN-8 的分化改善了 AHR 和 NAI，并且 CXN-8 疗法代表了一种新策略 我们将在体外和体内测试这一假设。 目的 1. 明确其潜在机制。 我们将使用 RNAi 和抑制剂来确定 p63RhoGEF 和 CXN-8 对 AHR 的关键作用和调节。 p63RhoGEF 在哮喘人 ASM (HASM) 细胞过度收缩中的机制 我们将分析 CXN-。 8 RhoA 活性、Ca2+ 信号传导、β2 激动剂敏感/不敏感 HASM 收缩/舒张的调节 我们将结合 RNAi、诱变和表面等离子共振来阐明特异性和 CXN-8 对 p63RhoGEF 的调节机制。 p63RhoGEF 缺失是否会改善 AHR 并降低 将在屋尘螨 (HDM) 驱动的小鼠哮喘模型中检查 CXN-8 的影响。 目标 2。 阐明 p63RhoGEF 和 CXN-8 对 NAI 调节的机制。 CXN-8 对 Th17 细胞分化的影响 我们将沉默 RhoA 或表达活性 RhoA 突变体以建立其活性。 我们还将确定 p63RhoGEF 是否丢失在 CXN-8 抑制 Th17 细胞分化中的重要性。 减弱细胞中的 RhoA 激活和 Th17 分化以及 HDM 诱导的小鼠 NAI 并减少 CXN-8 目的 3. 检测 CXN-8 在小鼠模型中的治疗潜力。 我们将确定吸入 CXN-8 对小鼠是否是一种急性且有效的支气管扩张剂。 我们将测试肺部靶向长效 CXN-8 微粒以缓解 β2 激动剂不敏感 AHR 模型。 AHR/NAI 在皮质类固醇不敏感哮喘小鼠模型中具有有限的全身副作用。 这些研究还将检验 p63RhoGEF 缺失对 AHR/NAI 的影响以及 CXN-8 的治疗效果。