PROJECT SUMMARY Genetic variants in the TRIO gene increase risk for neurodevelopmental disorders (NDDs) including schizophrenia, autism, and related disorders. A cluster of NDD-associated variants selectively impacts TRIO guanine nucleotide exchange factor domain 1 (GEF1) activity, which activates the Rac1 and RhoG GTPases, but the downstream molecular mechanisms by which TRIO GEF1 activity regulates neuronal development and whether they can be targeted therapeutically are not known. We discovered a key autoregulatory mechanism, and showed that NDD-related genetic variants impact this mechanism to enhance or inhibit TRIO GEF1 activity. Here, we propose to employ a novel construct that includes the physiologically-relevant autoregulatory elements in a high throughput screen to discover and validate positive and negative modulators of TRIO GEF1 function. These probes will be critical to advance our understanding of TRIO GEF1 function and its regulation, to probe candidate disease mechanisms, and to potentially suggest novel therapeutic strategies to address a spectrum of NDDs. Aim 1. Identify positive and negative small molecule modulators of TRIO GEF1 activity. The GEF1 domain alone catalyzes robust GTP exchange on Rac1 in vitro, whereas the SR6-GEF1 construct which includes the adjacent spectrin repeats (SRs) 6-9 reduces GEF1 catalytic activity 7-fold. Introducing NDD-related SR variants into SR6-GEF1 increases its activity, while variants in GEF1 domain reduce its activity, indicating that small chemical changes dramatically impact GEF1 activity. We will use a HTS biochemical assay of 150,000 structurally diverse drug- and lead-like compounds to identify those that increase or decrease the SR6-GEF1 activity. Aim 2. Verify and evaluate HTS hits using secondary assays. Hit compounds will be rescreened for confirmation and run in assays lacking SR6-GEF1 to rule out false positives. We will verify hits by repurchasing selected hits as dry powders and retesting their activity against SR6-GEF1. We will measure SR6-GEF1- mediated activation of Rac1 vs. RhoG to determine the substrate specificity of the compounds and investigate isoform selectivity for TRIO GEF1 by testing confirmed hits against a panel of neuronal GEFs. We will test a select group of the most potent hits to address mechanisms of action, including KM and kcat for Rac1 and RhoG, whether compounds bind SR6-GEF1, and how they impact SR6-GEF1 binding to Rac1 and RhoG. Aim 3. Assess probe efficacy on TRIO GEF1 activity in cells and neurons. We will study a set of the most potent and selective activators and inhibitors for their ability to penetrate cells and modulate TRIO GEF1 activity using both a Rac1 biosensor and a quantitative readout of TRIO GEF1 activity on HEK293 cell morphology. We will measure whether a select group of inhibitors recapitulates defects in dendrite development and synaptic transmission resulting from reduced TRIO GEF1 activity. We have also generated CRISPR mice hete...