# Nucleophile-Fragment Screening for Site-Specific Covalent Ligand Discovery in Opioid Receptor Signaling

> **NIH NIH R21** · UNIVERSITY OF FLORIDA · 2024 · $28,798

## Abstract

PROJECT SUMMARY
Substance abuse disorders including opioid dependence are characterized by brain oxidative stress and neu-
roinflammation. In addition to dopamine autooxidation, chronic drug consumption increases the expression of
pro-oxidant enzymes such as NADPH-oxidase (NOX), leading to excessive production of reactive oxygen spe-
cies (ROS) primarily in the form hydrogen peroxide (H2O2). Endogenous H2O2 can transiently and/or irreversibly
oxidize cysteinyl residues depending upon its concentration and duration of exposure. In proteins, H2O2 reacts
with redox-sensitive cysteine thiols to form sulfenic acid (Cys-SOH). Cysteine S-sulfenation has emerged as a
major post-translational modification that exerts significant effects on protein function, analogous to phosphory-
lation. A small but compelling literature suggests that changes in cysteine redox state affect mu opioid receptor
(MOR) function, as thiol alkylation, site-directed mutagenesis, and redox-modulating agents can alter ligand
binding and downstream signaling events. In addition to structural and regulatory roles, oxidation of the cysteine
thiol blocks the reaction of this residue with a,b-unsaturated carbonyls and alkyl halides, significantly limiting the
utility of conventional electrophile-fragment screening as a tool for covalent ligand discovery. To address this
issue, we have recently developed a strategy that employs nucleophilic covalent fragments to target S-sulfenated
(oxidized) cysteines. These sulfenic acid-reactive activity-based protein profiling (ABPP) probes have been cou-
pled with state-of-the-art quantitative proteomics to identify S-sulfenated cysteines in human cells, which presage
the development of covalent fragments therapeutically targeting redox-active cysteines. Here, the following Spe-
cific Aims are proposed: 1) Increase the size and structural diversity of our nucleophile-fragment libraries; 2) Map
cysteine redox reactivity changes and S-sulfenated (oxidized) cysteine ligandability in differentiated mature SH-
SY5Y neurons that are unstimulated (control) or treated with morphine agonist (stimulated). These studies are
rationalized based on proof-of-concept experiments which demonstrate that unique ligandable sites are identified
when fragments are functionalized with nucleophilic reactive groups that react with S-sulfenated (oxidized) cys-
teine residues. Deliverables from these studies are a novel chemoproteomic method, chemical matter that can
be mined as a source of small-molecule probes and as starting points for drug discovery.

## Key facts

- **NIH application ID:** 10770127
- **Project number:** 1R21DA059363-01
- **Recipient organization:** UNIVERSITY OF FLORIDA
- **Principal Investigator:** Kate Suzanne Carroll
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $28,798
- **Award type:** 1
- **Project period:** 2024-04-01 → 2024-08-02

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10770127

## Citation

> US National Institutes of Health, RePORTER application 10770127, Nucleophile-Fragment Screening for Site-Specific Covalent Ligand Discovery in Opioid Receptor Signaling (1R21DA059363-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10770127. Licensed CC0.

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