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）和气道注射是哮喘的标志。难治性哮喘 表现出持续的症状目的地使用高剂量口服皮质类固醇和长效β2激动剂 支气管扩张剂并提出了一个重大的医疗挑战。了解机制和发展 克服热电阻的策略提出了一个很大的未满足需求。气道平滑肌（ASM） 超收集是AHR的关键因素，T馆17（Th17）细胞促进了对类固醇不敏感的中性粒细胞 气道注入（NAI）。我们发现RhoA活化剂P63Rhogef在难治性哮喘中起着至关重要的作用。 我们还开发了p63rhogef的小分子抑制剂CXN-8。目的：确定重要性 p63rhogef调节哮喘素质的机制以及CXN-8抑制p63rhogef至 改善AHR和NAI。长期目标：开发难治性哮喘的新疗法。调查结果：1）P63- Rhogef在哮喘患者中有选择地更新，并在控制ASM的RhoA激活中起着至关重要的作用 超收集和Th17细胞分化。 2）CXN-8抑制ASM收缩并影响 呼吸道。 3）CXN-8在鼠哮喘模型中改善AHR和NAI。 4）CXN-8与p63rhogef相互作用 块RhoA激活。假设：靶向p63rhogef刺激的ASM超额合同和Th17细胞 与CXN-8的分化可以改善AHR和NAI，而CXN-8治疗代表了一种新颖的策略 难治性哮喘。我们将在体外和体内检验该假设。目的1。定义基本机制。 p63rhogef和CXN-8 AHR的调节。我们将使用RNAi和抑制剂来确定关键作用和 p63rhogef的机理在哮喘的人ASM（HASM）细胞的超收缩性中。我们将分析CXN- 8调节RhoA活性，Ca2+信号传导，收缩/β-激动剂敏感/不敏感的HASM的弛豫 细胞。我们将结合RNAi，诱变和表面等离子体共振，以阐明特异性和 CXN-8调节p63rhogef的机制。 p63rhogef损失是否可以改善AHR并减少 CXN-8的效果将在由哮喘的室内尘螨（HDM）驱动的鼠模型中进行检查。目标2 阐明NAI的p63rhogef和CXN-8调节的机制。我们将研究 Th17细胞分化的CXN-8。我们将使Rhoa沉默或表达活跃的Rhoa突变体以建立其 CXN-8抑制Th17细胞分化的重要性。我们还将确定p63rhogef的损失是否 减轻细胞和HDM诱导的小鼠NAI的RHOA激活和Th17分化，并减少CXN-8 体外和体内抑制作用。目标3。检查CXN-8在鼠模型中的治疗潜力 难治性哮喘。我们将确定吸入的CXN-8是否是鼠中的急性和有效的支气管扩张剂 β2激动剂不敏感的AHR的模型。我们将测试具有长效的肺CXN-8微粒以减轻 在皮质类固醇不敏感哮喘的鼠模型中，AHR/NAI具有有限的全身副作用。影响 在这些研究中，还将检查p63rhogef对AHR/NAI的损失以及CXN-8的治疗作用。