Project Summary We hypothesize that high affinity synthetic agonists of TLX may enhance adult hippocampal neurogenesis (AHN) and improve cognitive function in patients with dementia including Alzheimer's disease (AD). Here, we focus on developing, refining, and validation of TLX assays for the purposes of performing a screen for novel, synthetic TLX ligands that can be used as points to initiate drug discovery optimization efforts to develop TLX agonists towards potential clinical evaluation for treatment of dementia including AD. This is an application for a R61/R33 grant which focuses on the development and validation of assays that can be used to “identify and characterize potential therapeutic agents for neurological or neuromuscular disorders”. We are focused on identification of ligands targeting the nuclear receptor TLX that may hold utility to treat neurological disorders associated with cognitive dysfunction. The R61 phase is focused on development and validation of the key in vitro assays to identify putative TLX ligands. This is in alignment with the NIH's description of R61 activities which include (as specifically indicated in PAR-21-124): (1) Development and validation of assay(s) (including phenotypic assays) to support a succinct testing funnel…, (2) Development of in vitro or ex vivo potency/efficacy assays designed to indicate the specific ability of an agent to achieve a desired biological effect, and (3) Assay development and/or optimization for High-Throughput Screening (HTS). The R33 phase is initiated upon successful development of the assays and testing funnel and specifically is focused on (as specifically indicated in PAR-21-124): (1) HTS, comprising screening of large libraries for activity against biological targets via the use of automation, miniaturized assays and large-scale data analysis, (2) Preparation and screening of select series of therapeutic agents using…medicinal chemistry…, (3) Assessment of therapeutic agent's properties using computational analysis and early physicochemical measurements, polar surface area, solubility, cell permeability and efflux, (4) Assessment of initial in vitro pharmacokinetic parameters such as absorption, distribution, metabolism, and excretion (ADME), (5) Assessment of potential off target activities, and (5) Optimization of therapeutic agent(s) for FUTURE in vivo testing. To address these goals, we propose the following Specific Aims: R61 phase: AIM 1– Develop, refine, and validate a biochemical assay to detect TLX ligands in an HTS format and AIM 2 – Develop and validate secondary/tertiary assays for the purpose of characterizing “hits” identified in a TLX screen. R33 phase: AIM 1 – Screen both chemoinformatic driven nuclear receptor-biased chemical libraries and chemical diversity libraries using the validated assays from the R61 Phase to identify putative TLX agonists. AIM 2 – Evaluate the hits from the screens for their potential for further optimization as TLX agonists and...