# Deciphering biased agonistic activation of mu-opioid receptor by novel optogenetic hydrogen peroxide sensor

> **NIH NIH F31** · UNIVERSITY OF WASHINGTON · 2024 · $37,928

## Abstract

ABSTRACT: In 2017 the U.S. Department of Health and Human Services declared the ongoing opioid epidemic
a public health crisis and more than 100,000 people died due to opioid overdose in 2021. A critical part of the
solution is to understand the fundamental reaction and adaptation of brain circuits to stimulation by opioids. For
example, the desensitization of opioid receptors is a critical problem in pain management because it requires
increasing doses of analgesic compounds, which could contribute to developing a drug addiction. Recently, it
has been shown that the activation of mu and kappa opioid receptors in neurons causes the production of
reactive oxygen species (ROS) through a pathway involving NADPH oxidase and c-Jun N-terminal kinase.
Therefore, this distinct response, downstream from the receptor, could be utilized to detect specific opioid
receptor activation and modulation. However, we currently lack sensitive fluorescent sensors, which would allow
us to directly monitor pathways downstream from mu-opioid receptor (MOR) activation in real-time. Current
limitations of contemporary sensors are slow response time, low specificity, low signal output, low brightness,
and toxicity. My goal is to develop a genetically encoded sensor protein that detects ROS levels at
endogenous levels with response time and signal amplitudes that will enable monitoring of neuronal
systems upon MOR activation in brain tissue. I have developed a novel fluorescent ROS sensor with
significantly improved signaling amplitude, sensitivity, and response kinetics compared to previous sensors. I
used a newly identified insertion site on OxyR, a bacterial hydrogen peroxide binding protein, that putatively
improved allosteric coupling to the fluorescent reporter domain. We will increase the fidelity of this tool with new
green fluorescent protein (AausFP1) that exhibits exceptional quantum yield and brightness. I will optimize a
new ROS sensor at an unprecedented rate through a multifaceted approach that combines rational,
computational, and evolutionary protein engineering. My objective is to express this novel tool in MOR positive
neurons and to link ROS signals to MOR activity pharmacologically. I hypothesize that ROS signals in MOR
neurons will increase under morphine but not fentanyl through a pathway including c-Jun N-terminal Kinase,
Peroxiredoxin 6 and NADPH oxidase. Furthermore, I hypothesize that we will observe a decrease in ROS
transients under repeated drug exposure reflecting the desensitization of MORs. At the end, I will have a novel
and highly specific sensor for monitoring opioid receptor activity and adaptivity. My proposal is significant
because, for the first time, we will be able to monitor the adaptation of this clinically relevant signaling pathway
to opioid exposure. My approach is innovative because it combines novel protein engineering and monitoring
of opioid-triggered signals to dissect a difficult-to-access neuronal signaling pathway. Furt...

## Key facts

- **NIH application ID:** 10791769
- **Project number:** 5F31DA056121-02
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** Justin Lee
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $37,928
- **Award type:** 5
- **Project period:** 2023-03-01 → 2024-12-15

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10791769

## Citation

> US National Institutes of Health, RePORTER application 10791769, Deciphering biased agonistic activation of mu-opioid receptor by novel optogenetic hydrogen peroxide sensor (5F31DA056121-02). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10791769. Licensed CC0.

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