Summary. The tens of thousands of microbial metabolites produced by the trillions of bacterial cells that colonize our intestines (our microbiota) are poorly understood, and current estimates suggest that we only know the biological functions of 0.1% of all small molecules derived from the microbiota. Some of the metabolites in this so called “dark matter” of the metabolome will undoubtedly have profound impacts on human physiology. Here, we will establish how the human microbiome “communicates” with the host via dark-matter-derived molecular modulation of host G protein-coupled receptors (GPCRs), including GPCR signaling effects mediated by novel small molecules encoded by the microbiota (Aim 1a), GPCR signaling effects derived from endogenous host chemicals (Aim 1b), and gut bacterial transformations of GPCR-targeted medical drugs that rewire chemical signaling programs at the host-microbiota interface (Aim 2). A major focus will be the structural, functional, and biosynthetic characterization of novel metabolites associated with “orphan” GPCR signaling where only limited chemical and biological information is currently available. Our proposal takes advantage of the results from two high-throughput screens and one targeted screen, which each focused on different aspects of how microbial or host metabolism impacts host GPCR signaling at a detailed molecular level. In preliminary high-throughput studies: 1) We have analyzed ~150 diverse human gut bacteria for secretion of metabolites that activate conventional GPCRs (314 GPCRs) using a high-throughput GPCR screening system (Aim 1a). We have characterized bacterial metabolites identified by this screen that activate three orphan GPCRs to date and have thus established a pipeline for “GPCR deorphanization.” 2) We have screened human tissues for small molecules that activate “orphan” GPCRs with no reported ligand information available (Aim 1b), providing a basis to elucidate the structure and distribution of endogenous orphan GPCR signals. 3) We have evaluated the processing of 271 FDA-approved drugs (including 62 GPCR-targeted drugs) by a panel of dozens of gut microbiota-derived bacteria. This preliminary data includes 585,000 drug-by-microbiota measurements. Many drug metabolism products and bacterial responses to drugs can be observed in the metabolomics data, guiding the discovery of novel GPCR drug transformations and “specialized” metabolites from the gut microbiota whose production is induced in response to GPCR drugs (Aim 2). The proposed studies will represent a heretofore unprecedented structure-function-based exploration of the “dark matter” of the microbiota metabolome and how it is “sensed” by the host. Such studies may illuminate novel GPCR- and microbiota-targeted therapeutic strategies for a diversity of human diseases.