According to the NIMH, approximately one in five Americans suffered from an anxiety disorder in the last year and nearly 1/3 will experience an anxiety disorder in their lifetime. Anxiety disorders are primarily treated with biogenic amine reuptake inhibitors which can have delayed or inadequate efficacy, so there is a great need for additional anxiolytics with different mechanisms of action. There is clear evidence that activation of CB1R cannabinoid receptors can be anxiolytic; however, direct activation of CB1Rs, with THC for example, can produce undesirable effects, such as changes in cognition and posture, and can result in dependence. On the other hand, CB1R indirect agonists, which elevate concentrations of the endogenous ligands of CB1R (endocannabinoids, eCBs), represent a rational approach with beneficial effects of CB1R enhancement and fewer off-target effects. Our team has discovered that sterol carrier protein-2 (SCP-2), which is enriched in the amygdala, binds the eCBs N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG) at nanomolar concentrations and enhances intracellular sequestration of AEA. Thus, we hypothesize that inhibition of SCP-2 will reduce eCB sequestration, enhance CB1R signaling and thereby reduce anxiety. In support of this hypothesis, SCP-2-1- mice exhibit reduced anxiety-like behaviors and enhanced extinction of conditioned fear, both reversible by CB1R blockade. SCPl-1, a previously identified small molecule inhibitor of SCP-2, has anxiolytic effects that are blocked by CB1R antagonism. However, SCPl-1 is not sufficiently potent, selective, or metabolically stable to serve as a molecular probe of SCP-2 for further mechanistic studies. The goal of the studies in this proposal is to design, synthesize and test a focused library of molecules to identify a potent inhibitor of SCP-2 binding pocket that can be used in mechanistic studies to elucidate the role of this protein in the life cycle of the eCBs, particularly in brain circuits involved in fear and anxiety. In Aim 1, we will utilize computer assisted drug design (CADD) to develop FragMaps and use this information to design and synthesize new potential SCP-2 inhibitors. CADD will also be used to predict metabolic issues and off-target effects of these compounds. In Aim 2, we will use in vitro approaches to characterize SCP-2 binding of the novel inhibitors; determine key physicochemical properties; and measure effects on cellular eCB uptake. Information from these studies will be used to refine our computational model. In Aim 3, we will use in vivo approaches to determine pharmacokinetic properties of a few novel molecules to inform us about dosing for behavioral assays, which we will complete in this aim as well. We will determine potential toxicity and metabolism of these compounds. Successful completion of these studies is expected to result in new small-molecule inhibitors of SCP-2 that will be used to further validate the role of SCP-2 within ...