Post-traumatic stress disorder (PTSD) affects 6.8% of the US population, making it the 4th most common psychiatric disorder. The following comorbidities are associated with this anxiety disorder: disturbed sleep, substance-related disorders, and increased mortality. Sertraline and paroxetine (serotonin reuptake inhibitors) are the most widely prescribed drugs; however response is limited, particularly in combat veterans, and sleep disruption is unaffected by these agents. Currently no available pharmacological agents can be administered prior to or immediately following trauma to effectively prevent the development of PTSD symptoms. As such, there remains a significant unmet need for effective treatment options for individuals with PTSD. We conducted an ultra-high throughput phenotypic screen to identify inhibitors of cortisol-mediated GR nuclear translocation due to the strong rationale for antagonism of GR signaling in the prevention of PTSD symptoms, and the need for more selective GR antagonists without intrinsic agonist activity. This approach led to the discovery of a series of highly unique compounds inhibiting (low nM EC50) agonist-induced GR nuclear translocation and stabilizing the transcriptionally inert cytosolic GR complex. These compounds are highly selective for GR and block both GR transactivation and transrepression. Unlike orthosteric GR antagonists, our compounds do not directly engage the GR ligand binding domain and display no mixed agonist activity. In addition, our compounds block several prerequisite steps in GR activation, including dissociation of heat shock factors (HSP70 and HSP90) and phosphorylation of serine 211. Compounds emerging from these efforts are starting points for lead optimization to generate in vivo tool compoundsto be tested in PTSD models. To accomplish the overall objective of this project, which is to develop novel glucocorticoid (GC) signaling antagonists for the treatment of PTSD, we will pursue three specific aims: Aim 1. Design and synthesize GR passive antagonists that are orally active in vivo. We will synthesize ~450 analogs around our lead compound, SBI-240. The objective is to maintain an IC50 of 10-50 nM with 100X selectivity against other nuclear receptors, with improved Absorption/Distribution/Metabolism/Excretion and rodent pharmacokinetics suitable for oral dosing. These efforts will yield compounds suitable for in vivo efficacy studies in models of PTSD. Aim 2. Define the molecular mechanism and target(s) of GR passive antagonists. A combination of biochemical, proteomic and tandem-affinity approaches will be employed to further characterize the mechanism of action of SBI-240 and related compounds. Aim 3. Examine the activity of GR passive antagonists in animal models of PTSD. We will employ a highly sophisticated fear conditioning and predator stress protocol that incorporates components of helplessness and social instability to faithfully model PTSD. We will determine the efficacy of GR passive antagonists in this model using several dosing paradigms.