# Assay Development, Screening and Early Optimization

> **NIH NIH U19** · UNIV OF NORTH CAROLINA CHAPEL HILL · 2024 · $1,576,534

## Abstract

ABSTRACT
Research Component (RC) 3 includes the 1) development of new in vitro and/or ex vivo assays, and 2) screening
and/or rational design efforts to identify and characterize novel assets for pain conditions/disorders. Our team
has already established binding as well as G protein and arrestin signaling in vitro assays to comprehensively
profile NTSR1 ligands, including ligands with complex allosteric modulator activities such as SBI-553. Here we
propose to 1) develop a novel ex vivo assay enabling electrophysiological recording of NTSR1 ligand modulation
of NTSR1-expressing amygdalar neuron excitability in brain slices (Aim 1), and 2) perform an ultra-large-scale
computational screen and structure-guided early optimization of NTSR1 assets (Aim 2). For Aim 1, we will cross
mice in which expression of the DNA recombinase Cre is driven by the prompter of the Ntsr1 gene (Ntsr1Cre
mice) with Ai14 reporter mice to generate Ntsr1Cre::Ai14 mice, in which NTSR1-expressing neurons are labeled
with the red fluorescent protein tdTomato. We will then slice the brains and perform whole-cell patch-clamp
recordings, in both voltage- and current-clamp modes, on visually identified fluorescent neurons, to record
standard parameters of neuronal excitability (e.g., membrane potential, rheobase, action potential firing
frequency), as in our previous studies, in this case focusing on amygdalar neurons. For Aim 2, we develop an
ultra-large-scale computational screen and structure-guided early optimization of NTSR1 assets. We will dock a
library of 5 billion make-on-demand molecules against the NTSR1 allosteric site, seeking novel chemotypes with
the best possible physical properties (e.g., cLogP <3.5, molecular weight <350). High-ranking molecules will be
synthesized and tested in vitro. In addition, we will perform directed medicinal chemistry at the allosteric site to
optimize signaling selectivity, potency, and pharmacokinetic properties, both for SBI-553 itself, and new
chemotypes emerging from the large-library docking studies. SBI-553 analogs will be profiled in vitro using our
(Roth) TRUPATH bioluminescence resonance energy transfer (BRET) platform, which enables an unbiased
interrogation of individual G protein subunit combinations and arrestins at individual GPCRs like NTSR1, as we
have shown recently (Krumm et al., Biochemistry, 2023). Active molecules are optimized for affinity, signaling
selectivity, and physical properties in a structure-based approach. Multiple previous studies by our team
(Shoichet, Roth, Scherrer) have employed this approach to optimize GPCR ligands (e.g., Manglik et al., Nature,
2016; Alon et al., Nature, 2021; Fink et al., Science, 2022; Kaplan et al., Nature, 2022). This ample experience,
combined with the characteristics of the NTSR1 site, make us optimistic about prospects for success.

## Key facts

- **NIH application ID:** 10974397
- **Project number:** 1U19NS138975-01
- **Recipient organization:** UNIV OF NORTH CAROLINA CHAPEL HILL
- **Principal Investigator:** Gregory Scherrer
- **Activity code:** U19 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $1,576,534
- **Award type:** 1
- **Project period:** 2024-09-19 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10974397

## Citation

> US National Institutes of Health, RePORTER application 10974397, Assay Development, Screening and Early Optimization (1U19NS138975-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10974397. Licensed CC0.

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