Project Summary. The V1A-specific arginine vasopressin receptor has recently become the focus of CNS research when an association between the receptor gene and autism spectrum disorder (ASD) is identified. Dysregulation of V1A activity has been suggested as a fundamental mechanism underlying ASD pathophysiology. Recent studies revealed that V1A antagonists can regulate the effect of the AVP hormone, thereby tackling a potential root cause of ASD development. PET is capable of quantifying biochemical processes in vivo, and a suitable V1A PET ligand would substantially improve our understanding of V1A-mediated AVP signaling under different pathophysiological conditions, otherwise inaccessible by ex vivo (destructive) analysis, particularly in higher species. Quantification of V1A in living brain by PET would provide the assessment of distribution and expression and target engagement of new V1A- targeted neurotherapeutics. To date, no successful examples have been demonstrated to image V1A-specific AVP for human use, representing a significant deficiency of our ability to study this target in vivo for V1A. Therefore, we propose to develop a novel PET ligand that can fill this void, as the first translational imaging tool. The PI has recently developed a novel V1A-specific AVP ligand [11C]PF-184563 at MGH in 2021. While this compound showed high potency and excellent selectivity, it is not likely pursued due to low brain penetration. In our 2nd generation, we identified a lead molecule, V1A-214, which showed substantially-improved binding affinity and excellent target selectivity among all other major CNS targets. An 11C-isotopologue of V1A-214 was synthesized and preliminary PET imaging studies confirmed that we have overcome two major obstacles for V1A-specific PET ligand development by achieving: 1) high brain uptake (>1 SUV) and 2) high target specificity validated by human V1A cell lines and blocking studies in vivo, and heterogenous uptake consistent with V1A regions in the CNS. Though V1A- 214 is a promising lead molecule for the development of new V1A-targeted PET ligands, due to species difference, further optimization for translational cross-species (naïve/humanized V1A mice and nonhuman primates) imaging studies are sought to achieve optimal AVP (V1A subunit) quantification in the living brain for drug discovery and clinical biomarker for ASD patients. On the basis that V1A-214 serves a validated lead for medicinal chemistry optimization, as specific objectives, we will design and prepare a focused library of V1A-specific antagonists amenable for labeling with 11C or 18F, and evaluate their ability to quantify V1A-specific AVP activity and changes in cellular experiments, naïve and humanized V1A mice and nonhuman primates, as well as autoradiography and biological validation in postmortem NHP/human brain tissues. The impact of this work is not only to develop the first successful high-affinity and selective V1A-specific PET ligand for th...