Validation of Therapeutic Target and Underlying Biology

ABSTRACT Research Component (RC) 1 includes the validation of novel therapeutic targets for pain and the study of their biology. In our previous work, we discovered an ensemble of neurons in the amygdala that encodes pain unpleasantness across pain modalities (heat, cold, mechanical) and pain types (acute, chronic neuropathic).

Furthermore, altering the activity of these amygdalar neurons using artificially expressed GPCRs (DREADDs, Roth lab) significantly diminished pain affective-motivational behaviors in mice, in both acute and chronic neuropathic pain models, and without altering withdrawal reflexes, anxiety or reward. Consequently, engaging a

target in the nociceptive amygdalar neurons that produce pain unpleasantness may be an effective approach to manage many, if not all, pain types, including those with different peripheral localizations and mechanisms, those of CNS origin, and even those for which we lack sufficient mechanistic understanding to treat the pain at its

source. Building on these exciting findings, we launched an analgesic target discovery project in which we combined mouse genetic tools, single-cell RNA sequencing, and bioinformatics methods to label, sequence, and catalog GPCRs present in nociceptive amygdalar neurons. After generating this catalog, we validated expression

of a dozen of GPCRs in the mouse amygdala using spatial transcriptomics. Next, we conducted a behavioral screen in mice to test the ability of known ligands for these GPCRs, either purchased or synthesized by our team, to reduce pain affective-motivational behaviors. This screen identified five amygdalar GPCRs with

antinociceptive properties. Among these, we focused our attention on neurotensin receptor 1 (NTSR1) because previous studies have established that NTSR1 agonists are not rewarding and that engaging NTSR1 can even reduce addictive behaviors in rodents. Both PD149163, a balanced/unbiased, brain-penetrant neurotensin (NTS)

peptide fragment 8-13 analog, and SBI-553, an NTSR1 negative allosteric modulator (NAM) at Gq and weaker positive allosteric modulator with intrinsic agonist activity at beta-arrestin (PAM-agonist), reduced pain affective- motivational behaviors in male mice. However, balanced/unbiased activation of NTSR1 with NTS or PD149163

produces dose-limiting side effects that could both confound antinociceptive readouts and hinder translation. In contrast, SBI-553 lacks these side effects. We thus continued our evaluation of NTSR1 as an antinociceptive target with SBI-553, and found that SBI-553 antinociceptive properties were lost in NTSR1 knockout mice,

validating on-target antinociceptive activity. In RC1, we expand validation studies to include other pain models and further elucidate the biology of our target, NTSR1, focusing on signaling and expression in the human brain, to inform optimized novel small molecule development and testing in RCs 3–5 and evaluate translation potential.

In Aim 1, we validate NTSR1 antinociceptive properties in mouse models of clinically relevant pain. In Aim 2, we establish which NTSR1 signaling pathways produce antinociception. In Aim 3, we resolve NTSR1 comparative expression in mouse and human tissues.