NE52-QQ57 attenuates post-ischemia protein phosphorylation in the mouse brain
- 1Tulane University
Abstract
Introduction: Cerebral ischemia induces a reduction in brain pH. GPR4, a proton-sensing G protein-coupled receptor is highly expressed in vascular endothelial cells. Pharmacological inhibition of GPR4 with NE52-QQ57 (NEQQ) reduces ischemia-induced blood-brain barrier (BBB) disruption and brain injury. To facilitate targeting GPR4 in vivo, it is important to better define the effect of NEQQ on post-ischemia signaling and the pharmacokinetics of NEQQ in vivo. Methods: We performed biochemical analysis following transient middle cerebral artery occlusion (tMCAO). We used Liquid Chromatography-Mass spectrometry (LC-MS/MS) to quantify the concentration of NEQQ in serum, brain, and peripheral tissues. Results: NEQQ administration decreased the phosphorylation of PKA substrates, PKC substrates, ERK1/2, and CREB. LC-MS/MS analysis detected NEQQ in both ipsilateral and contralateral hemisphere of the brain at 5 h and 24 h after reperfusion, demonstrating that NEQQ crosses the BBB. In peripheral tissues, NEQQ exhibited highest level in kidney and lowest in the heart. In sham controls, average serum concentrations were ~4 and 0.9 g/g at 5 and 24 h, respectively. Following tMCAO, ipsilateral brain exhibited higher levels as compared to the contralateral side at 5 h while the difference diminished at 24 h. Conclusions: NEQQ attenuated the ischemia-induced activation of multiple signaling pathways. NEQQ exhibited broad distribution in peripheral organs, and its brain level was sufficient for GPR4 inhibition at 24 h after injection. These findings support the use of NEQQ as a pharmacological inhibitor of GPR4-mediated signaling following ischemic stroke.
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BibTeX
@misc{joshi2026ne52qq57,
title = {{NE52-QQ57 attenuates post-ischemia protein phosphorylation in the mouse brain}},
author = {S. Joshi and Y. Zhou and X.-m. Zha},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.09.754558},
url = {https://www.biorxiv.org/content/10.64898/2026.10.09.754558v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - NE52-QQ57 attenuates post-ischemia protein phosphorylation in the mouse brain
AU - S. Joshi
AU - Y. Zhou
AU - X.-m. Zha
PY - 2026
DA - 2026/10/09
PB - bioRxiv
DO - 10.64898/2026.10.09.754558
UR - https://www.biorxiv.org/content/10.64898/2026.10.09.754558v1
AB - Introduction: Cerebral ischemia induces a reduction in brain pH. GPR4, a proton-sensing G protein-coupled receptor is highly expressed in vascular endothelial cells. Pharmacological inhibition of GPR4 with NE52-QQ57 (NEQQ) reduces ischemia-induced blood-brain barrier (BBB) disruption and brain injury. To facilitate targeting GPR4 in vivo, it is important to better define the effect of NEQQ on post-ischemia signaling and the pharmacokinetics of NEQQ in vivo. Methods: We performed biochemical analysis following transient middle cerebral artery occlusion (tMCAO). We used Liquid Chromatography-Mass spectrometry (LC-MS/MS) to quantify the concentration of NEQQ in serum, brain, and peripheral tissues. Results: NEQQ administration decreased the phosphorylation of PKA substrates, PKC substrates, ERK1/2, and CREB. LC-MS/MS analysis detected NEQQ in both ipsilateral and contralateral hemisphere of the brain at 5 h and 24 h after reperfusion, demonstrating that NEQQ crosses the BBB. In peripheral tissues, NEQQ exhibited highest level in kidney and lowest in the heart. In sham controls, average serum concentrations were ~4 and 0.9 g/g at 5 and 24 h, respectively. Following tMCAO, ipsilateral brain exhibited higher levels as compared to the contralateral side at 5 h while the difference diminished at 24 h. Conclusions: NEQQ attenuated the ischemia-induced activation of multiple signaling pathways. NEQQ exhibited broad distribution in peripheral organs, and its brain level was sufficient for GPR4 inhibition at 24 h after injection. These findings support the use of NEQQ as a pharmacological inhibitor of GPR4-mediated signaling following ischemic stroke.
SN - 2692-8205
ER -