Project Summary/Abstract: Neuropathic pain is estimated in the general population to range between 3% and 17%, and lacks an effective treatment, as most of the available treatments have only moderate efficacy and present significant side effects including addiction that limit their use. Therefore, other therapeutic approaches are needed to replace the addictive opioids that are the mainstay of current treatment. 5-HT7R agonists including AS-19, E-55888, LP-12, LP-44, LP-211, AGH-192 and compound 1g have been reported in the literature and have been used to characterize the involvement of the 5-HT7R in CNS conditions including pain, memory and cognition. More recently, 5-HT7R agonists have been implicated in the treatment of neuropathic pain, sleep disorders, and alcohol and drug abuse. However, there are divergent views reported in the literature regarding the role of the 5-HT7R ligands in their pharmacological actions. While these controversial roles of 5-HT7R may depend on non-selectivity at other CNS receptors, and/or on different brain regions and the neurochemical environment in which the receptors are located, or even receptor-receptor interactions, some of the inconsistent pharmacological effects may be explained by the different signaling pathways associated with the 5-HT7R, including G protein biased, β-arrestin biased or balanced signaling at both pathways. Current evidence indicates that 5-HT7R ligands activate not only G protein but also β-arrestin signaling pathways and that no β-arrestin-biased ligands that selectively interact with 5-HT7R have been disclosed other than compound 1g reported in a JMC paper in 2018. In addition, a recent PNAS paper reported serodolin behaves as an antagonist/inverse agonist on Gs signaling while inducing ERK activation through a β-arrestin–dependent signaling mechanism that requires activation of the non-receptor tyrosine kinase, c-Src. Serodolin was also shown to decrease hyperalgesia and pain sensation in response to inflammatory, thermal, and mechanical stimulation. Thus, in this proposal, we hypothesize that 5-HT7R ligands that recruit β-arrestin to the receptor would produce antinociceptive effects and may serve as alternatives to the use of opioids. To begin addressing several of these issues, we have proposed three specific aims as follows: Specific Aim 1 will optimize the very high affinity (5-HT7R, Ki = 0.16 nM) lead compound (55933) by designing, synthesizing, and evaluating the binding affinities of new agents that are more drug-like and capable of improving 55933’s metabolic profile. Specific aim 2, will investigate the drug-like characteristics of optimized lead compounds identified and/or proposed in specific aim 1 including their brain permeability. And in specific aim 3, the in vivo efficacy evaluation of test compounds for antinociception and neuropathic pain will be carried out.