NE 52-QQ57

The protective effects of NE 52-QQ57 against interleukin-33-induced inflammatory response in activated synovial mast cells

Cytokines-mediated immunity is essential for the pathological development of rheumatoid arthritis (RA). Inhibition of signaling has suggested a potential remedial approach to RA. G protein-coupled receptor 4 (GPR4) has been proven to possess a broad range of physiological functions, but its function in synovial mast cells and RA is less reported. In this study, the protective effects of NE 52-QQ57, a GPR4 antagonist, against interleukin (IL)-33-challenged inflammatory response in activated synovial mast cells were investigated. We report that IL-33 amplified GPR4 expression in HMC-1 mast cells. The GPR4 antagonist NE 52-QQ57 alleviated IL-33-caused secretions of IL-17, interferon-γ, and tumor necrosis factor-α in HMC-1 mast cells. Furthermore, we note that NE 52-QQ57 reduced IL-33-induced expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9. Also, NE 52-QQ57 inhibited cyclooxygenase 2 and prostaglandin E2 expression in IL-33-challenged cells. Also, NE 52-QQ57 ameliorated IL-33-induced oxidative stress by reducing mitochondrial reactive oxygen species and 4-hydroxynonenal. Mechanistically, NE 52-QQ57 mitigated IL-33-induced activation of the p38/nuclear factor-κB signaling pathway. We conclude that targeting GPR4 might be a promising strategy for RA treatment.

Antagonism of GPR4 with NE 52-QQ57 and the Suppression of AGE-Induced Degradation of Type II Collagen in Human Chondrocytes

Osteoarthritis (OA) is a common degenerative joint disease for which an effective therapeutic strategy has not yet been established. AGEs are widely recognized as a contributor to OA pathogenesis. GPR4, a recently discovered proton-sensing transmembrane receptor, has been shown to possess a wide range of physiological functions. However, the potential role of this receptor in chondrocytes and the pathogenesis of OA is unclear. In the present study, we investigated the potential of GPR4 to modulate the effects of advanced glycation end products (AGEs) in SW1353 human chondrocytes. First, we demonstrate that GPR4 is fairly expressed in SW1353 chondrocytes and that exposure to AGEs increases the expression of this transmembrane receptor. Second, we found that antagonism of GPR4 with NE 52-QQ57 significantly inhibited the AGE-induced increased expression of several key inflammatory cytokines and signaling molecules, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase 2 (COX2), and prostaglandin E₂ (PGE₂). We also found that antagonisn of GPR4 had a remarkable ability to rescue type II collagen from AGE-induced degradation by inhibiting the expression of matrix metalloproteinase (MMP)-3 and MMP-13. As a key pro-inflammatory signaling pathway, we further tested the effect of GPR4 antagonism on the activation of nuclear factor-κB (NF-κB) and found that NF-κB activation was indeed suppressed, thereby indicating that the NF-κB signaling pathway may mediate the effects of GPR4 antagonism described above. These findings provide a basis for further research into the role of GPR4 -mediated signaling in OA.

CNS distribution, signalling properties and central effects of G-protein coupled receptor 4

Information on the distribution and biology of the G-protein coupled receptor 4 (GPR4) in the brain is limited. It is currently thought that GPR4 couples to G_s proteins and may mediate central respiratory sensitivity to CO₂. Using a knock-in mouse model, abundant GPR4 expression was detected in the cerebrovascular endothelium and neurones of dorsal raphe, retro-trapezoidal nucleus locus coeruleus and lateral septum. A similar distribution was confirmed using RNAscope in situ hybridisation. In HEK293 cells, overexpressing GPR4, it was highly constitutively active at neutral pH with little further increase in cAMP towards acidic pH. The GPR4 antagonist NE 52-QQ57 effectively blocked GPR4-mediated cAMP accumulation (IC₅₀ 26.8 nM in HEK293 cells). In HUVEC which natively express GPR4, physiological acidification (pH 7.4-7.0) resulted in a cAMP increase by ?55% which was completely prevented by 1 μM NE 52-QQ57. The main extracellular organic acid, l-lactic acid (LL; 1-10 mM), suppressed pH dependent activation of GPR4 in HEK293 and HUVEC cells, suggesting allosteric negative modulation. In unanaesthetised mice and rats, NE 52-QQ57 (20 mg kg^-1) reduced ventilatory response to 5 and 10% CO₂. In anaesthetised rats, systemic administration of NE 52-QQ57 (up to 20 mg kg^-1) had no effect on hemodynamics, cerebral blood flow and blood oxygen level dependent responses. Central administration of NE 52-QQ57 (1 mM) in vagotomised anaesthetised rats did not affect CO₂-induced respiratory responses. Our results indicate that GPR4 is expressed by multiple neuronal populations and endothelium and that its pH sensitivity is affected by level of expression and LL. NE 52-QQ57 blunts hypercapnic response to CO₂ but this effect is absent under anaesthesia, possibly due to the inhibitory effect of LL on GPR4.

Pharmacological inhibition of GPR4 remediates intestinal inflammation in a mouse colitis model

Inflammatory bowel disease (IBD) is characterized by chronic, recurring inflammation of the digestive tract. Current therapeutic approaches are limited and include biologics and steroids such as anti-TNFα monoclonal antibodies and corticosteroids, respectively. Significant adverse drug effects can occur for chronic usage and include increased risk of infection in some patients. GPR4, a pH-sensing G protein-coupled receptor, has recently emerged as a potential therapeutic target for intestinal inflammation. We have assessed the effects of a GPR4 antagonist, 2-(4-((2-Ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)methyl)phenyl)-5-(piperidin-4-yl)-1,3,4-oxadiazole (GPR4 antagonist 13, also known as NE-52-QQ57) in the dextran sulfate sodium (DSS)-induced acute colitis mouse model. The GPR4 antagonist 13 inhibited intestinal inflammation. The clinical parameters such as body weight loss and fecal score were reduced in the GPR4 antagonist 13 treatment group compared to vehicle control. Macroscopic disease indicators such as colon shortening, splenic expansion, and mesenteric lymph node enlargement were all reduced in severity in the GPR4 antagonist 13 treated mice. Histopathological features of active colitis were alleviated in GPR4 antagonist 13 treatment groups compared to vehicle control. Finally, inflammatory gene expression in the colon tissues and vascular adhesion molecule expression in the intestinal endothelia were attenuated by GPR4 antagonist 13. Our results indicate that GPR4 antagonist 13 provides a protective effect in the DSS-induced acute colitis mouse model, and inhibition of GPR4 can be explored as a novel anti-inflammatory approach.