A Novel Ogremorphin Group of First-in-Class GPR68 Inhibitors Upregulate Endothelial Barrier Function and Rescue Bacterial Pathogens or Acidosis-Induced Inflammation in Lung Endothelium

ABSTRACT

Rationale An increase in endothelial permeability resulting from the disruption of endothelial barrier and aggravated inflammatory responses are two major pathological hallmarks of various lung disorders including the current global pandemic COVID-19. Drugs that enable the preservation and restoration of endothelial function represent attractive therapeutic targets to treat endothelial dysfunction-derived cardiopulmonary diseases. A role of G protein-coupled receptors (GPCRs), especially a sub-family of proton-sensing GPCRs including GPR4 and GPR68, has been suggested in modulation of endothelial function. In this study, we analyzed the barrier protective and anti-inflammatory effects of two recently developed novel class of GPR68 inhibitors ogremorphins OGM8345 and OGM-1.Methods: Transendothelial electrical resistance (TER) was monitored in human pulmonary arterial endothelial cells (HPAECs) to evaluate endothelial barrier function. Quantitative real time PCR and western blot analyses were performed to determine mRNA and protein expression of endothelial inflammation markers, respectively. Acidic pH (6.5) medium was used to induce acidosis, and luciferase-based Tango assay was employed to evaluate GPR68 activation. C57BL/6 mice were exposed to lipopolysaccharide (LPS from Escherichia coli) or heatkilled Staphylococcus aureus (HKSA), and vascular leak/inflammation was assessed by determining the extravasation of intravenously injected Evans blue tracer into lungs and total cells/protein count in bronchoalveolar lavage samples. Results: A robust dose-dependent increase in basal EC barrier function was observed with OGM8345 (1-5 μM) and OGM-1 (0.3-1.5 μM) evident by an 150-200% increase in TER values. Both inhibitors also effectively rescued LPS- and HKSA-induced EC hyperpermeability. RT-PCR analysis demonstrated that LPS or HKSA-induced upregulation of inflammatory cytokines/chemokines genes TNF-α, ICAM-1, VCAM-1, IL-6, IL-8, IL- 1β, and CXCL5 was significantly attenuated by OGMs. Consistently, both OGMs suppressed LPSand HKSA-induced protein expression of VCAM-1 and ICAM-1. In contrast, pharmacologic inhibition of GPR4 by NE 52-QQ57 failed to alleviate LPS or HKSA-induced EC barrier dysfunction and inflammation. Importantly, LPS, HKSA or acidosis stimulation resulted in increased GPR68 mRNA expression and GPR68 activity that was inhibited by OGMs. Intratracheal injection of LPS or HKSA in C57BL/6 mice caused vascular leak and lung inflammation that was attenuated by both OGMs as illustrated by reduced Evans blue accumulation in the lungs and significant inhibition of accumulation of inflammatory cells and protein content in bronchoalveolar lavage samples. Conclusion: These results establish a critical role of GPR68 in endothelial dysfunction and strongly suggest a therapeutic potential of GPR68-selective inhibitors in improving endothelial dysfunction caused by bacterial infections and acidosis associated with acute and chronic lung injury.