Pharmacological chaperones for the oxytocin receptor increase oxytocin responsiveness in myometrial cells

Manasi Malik,Yingye Fang,Monali Wakle-Prabagaran,Michelle Roh,Kevin Prifti,Antonina I Frolova,Princess I Imoukhuede,Sarah K England

doi:10.1016/j.jbc.2022.101646

Manasi Malik, Yingye Fang + Show 6 more

Open Access

https://doi.org/10.1016/j.jbc.2022.101646

Copy DOI

Abstract

Oxytocin is a potent uterotonic agent administered to nearly all patients during childbirth in the United States. Inadequate oxytocin response can necessitate Cesarean delivery or lead to uterine atony and postpartum hemorrhage. Thus, it may be clinically useful to identify patients at risk for poor oxytocin response and develop strategies to sensitize the uterus to oxytocin. Previously, we showed that the V281M variant in the oxytocin receptor (OXTR) gene impairs OXTR trafficking to the cell surface, leading to a decreased oxytocin response in cells. Here, we sought to identify pharmacological chaperones that increased oxytocin response in cells expressing WT or V281M OXTR. We screened nine small-molecule agonists and antagonists of the oxytocin/vasopressin receptor family and identified two, SR49059 and L371,257, that restored both OXTR trafficking and oxytocin response in HEK293T cells transfected with V281M OXTR. In hTERT-immortalized human myometrial cells, which endogenously express WT OXTR, treatment with SR49059 and L371,257 increased the amount of OXTR on the cell surface by two- to fourfold. Furthermore, SR49059 and L371,257 increased the endogenous oxytocin response in hTERT-immortalized human myometrial cells by 35% and induced robust oxytocin responses in primary myometrial cells obtained from patients at the time of Cesarean section. If future studies demonstrate that these pharmacological chaperones or related compounds function similarly in vivo, we propose that they could potentially be used to enhance clinical response to oxytocin.

Highlights

The nonapeptide hormone oxytocin modulates social behavior, mediates the lactation reflex, and induces and strengthens uterine contractions
We transiently transfected HEK293T cells with V281M oxytocin receptor (OXTR) tagged with hemagglutinin and green fluorescent protein (HA-OXTR-GFP), treated them with 10 μM of each candidate drug for 16 hours, performed quantitative flow cytometry to measure cell surface OXTR [16,19]
Our results indicate that two oxytocin/vasopressin antagonists, SR49059 and L371,257, act as pharmacoperones for both variant and wild type (WT) OXTR

Summary

Introduction

The nonapeptide hormone oxytocin modulates social behavior, mediates the lactation reflex, and induces and strengthens uterine contractions. One strategy could be to identify pharmacological chaperones (pharmacoperones) that increase cell surface localization and function of the oxytocin receptor (OXTR), which is a G protein coupled receptor (GPCR). Studies on other GPCRs, show that antagonists can increase cell surface levels of wild type (WT) or variant receptors. Pharmacoperones have been investigated for therapeutic use in patients with genetic variants in the arginine vasopressin receptor 2 (AVPR2), a GPCR with high similarity to OXTR [10,11]. Previous investigators have shown that antagonists, agonists, and allosteric ligands are effective in rescuing AVPR2 trafficking and function: one such antagonist, SR49059, showed promise in a small clinical trial of patients with NDI [13,14]. Similar to pathogenic variants in AVPR2, V281M impairs OXTR trafficking to the cell surface and significantly decreases cellular response to oxytocin [16]. We evaluated the ability of pharmacoperones to augment oxytocin response in primary human myometrial cells from pregnant patients

Results

Discussion

Experimental Procedures