Creighton University

A Novel Approach to Target Neutrophilic Airway Inflammation and Airway Hyperresponsiveness in Therapy-resistant (refractory) Asthma. - 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 SS2-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 SS2-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 SS2-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.