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.