Bradykinin (BK) B1 receptor (BKB1R) activity is involved in a wide range of pathological conditions including edema, inflammation, asthma, chronic pain, vasculopathy, neuropathy, obesity, diabetic retinopathy and cancer. Antagonists of these receptors would be expected to be of great therapeutic utility, however, although several BKB1R antagonists have entered clinical trials, none have emerged successfully from Phase II. BKB1Rs are generally not expressed in most tissues unless exposed to inflammatory mediators and when expressed, have a high level of constitutive (agonist-independent) receptor activity. Although the reasons for the failure of BKB1R antagonists in clinical trials have not been reported, it is likely that the high level of constitutive activity of the BKB1R, which is not reduced by standard antagonists, is responsible for efficacy failures. Reduction of constitutive receptor activity requires an orthosteric ligand with inverse agonist activity or a negative allosteric regulator. Unfortunately, the only known orthosteric inverse agonists for the BKB1R are very weak and there are no allosteric regulators that reduce constitutive activity. The premise underlying this project is that reduction of constitutive BKB1R activity is an important component of a therapeutically effective antagonist. The goal of this project is to identify novel compounds that reduce BKB1R function by both antagonizing the endogenous agonist (des-Arg10-kallidin) and reducing constitutive BKB1R activity. This effect can be obtained with an orthosteric inverse agonist or a negative allosteric modulator (NAM). In Aim 1a, we propose to screen a library of ~170,000 small molecules to identify compounds that reduce agonist-activated and constitutive activity of the human BKB1Rs expressed in HEK cells. In Aim 1b, we will screen hits from Aim 1a to eliminate compounds that have non-specific effects or that alter BKB2R activity. In Aim 1c, compounds from Aim 1b will be rigorously tested pharmacologically (e.g. Schild analysis experiments) to identify allosteric versus orthosteric mechanisms of action. As NAMs have several advantages over orthosteric ligands, including specificity, safety and the ability to fine-tune receptor function along with reducing constitutive receptor activity, we will prioritize NAMs over orthosteric inverse agonists. Finally, in Aim 1d we will confirm that the BKB1 NAMs and inverse agonists function as expected when BKB1Rs are naturally expressed using human lung microvascular endothelial cells. This work will provide the preliminary data and candidate molecules needed to move forward to IND-enabling studies. Although this project is high risk, the identification of first-in-class negative allosteric regulators that reduce constitutive activity of human BKB1Rs is highly desirable.