ABSTRACT Research Component (RC) 4 includes the development of pharmacodynamic (PD) markers that indicate the molecular target engagement of the pain assets, typically representing engagement of a molecular component in the pathway mediating the biological effects of therapeutic target modulation, such as receptor occupancy or gene transcription. Our major goals here in RC4 are to develop pre-clinical PD markers to test target engagement by the novel NTSR1 asset generated in RC3 and tested for in vivo efficacy in RC5, and to develop a PD marker that will also be suitable to measure NTSR1 occupancy in the human brain. GPCRs, including our target NTSR1, typically undergo internalization following activation and signaling. In Aim 1, we will take advantage of a novel Ntsr1Venus knockin mouse published very recently to develop an NTSR1 internalization PD assay for profiling, in NTSR1-expressing amygdalar neurons, the activation of NTSR1 by the new asset developed in RC3. Noxious stimuli induce activation of neurons throughout CNS pain circuits to generate pain perception, including its affective-motivational dimension. In Aim 2, we therefore develop a transformative unbiased PD assay based on the inhibition of c-fos transcription that combines mouse genetics, whole-brain tissue clearing, and light-sheet microscopy to mark, in thousands to millions of individual neurons throughout the brain, the antinociceptive activity of new pain assets, such as the NTSR1 asset generated in RC3. A third goal of RC4 is to develop a PD marker to track binding of our novel asset to NTSR1 in the CNS in real time through positron emission tomography (PET). Although other PET imaging assets have been reported for NTSR1, these agents are antagonists and/or do not cross the blood-brain barrier (BBB). Instead, a brain-penetrant radioactive close analog of the NTSR1 allosteric modulator SBI-553 is desired for the present project to reflect the pharmacodynamics and pharmacokinetics of the agent that we use in other RCs. Our team has extensive experience with developing NTSR1-targeted PET agents. In Aim 3, we synthetize and validate a radiolabeled SBI-553 analog to evaluate CNS NTSR1 occupancy in rodents and humans. For Aim 1, we will use a novel Ntsr1Venus knockin mouse that expresses an NTSR1 receptor with the Venus fluorescent protein attached to its C-terminus. We will develop a PD marker to quantify target engagement based on NTSR1 internalization in amygdalar neurons following systemic administration of NTSR1 assets. For Aim 2, will develop a PD marker that quantifies the inhibition of noxious stimulus–induced c-fos transcription throughout the brain following systemically administered NTSR1 assets, using mouse genetics, tissue-clearing, and light-sheet microscopy techniques. For Aim 3, two medicinal chemistry approaches, one using 11C isotopic labeling and the other focused on 18F, will be pursued to generate radiolabeled SBI-553 analogs. Small-animal PET imaging of the n...