GPR68 is an understudied G-protein coupled receptor (GPCR), responds to protons and couples to multiple signaling pathways. It is expressed in many cells and tissues and believed to play important but presently unclear roles in normal physiology and pathological conditions. While GPR68 is expressed most abundantly in the brain, its potential function in this organ are yet to be identified. Pharmacological studies with GPR68 have been difficult and controversial, mainly due to the lack of small molecule tool compounds. We took a combined approach (modeling and virtual screening, SAR, and cell-based functional assays) and successfully identified the first Gs-based positive allosteric modulator (PAM), ogerin, for GPR68. We were the first to show that ogerin suppresses learning and memory in the wild-type but not GPR68 KO mice, suggesting an important role in the brain function (Huang et al., 2015). Based on our preliminary results, we are proposing to take the same combined approach, but at much larger scale, to design and develop functionally selective (biased) allosteric modulators for individual signaling pathways mediated by protons at GPR68. Specifically, we propose to identify and optimize positive and/or negative allosteric modulators (PAMs and/or NAMs) with distinct allosteric activity (?, -values) for the following pathways (readout): Gs-cAMP (with cAMP production and CRE-Luc reporter), Gq-calcium release (Calcium release on FLIPR), Gq-PI hydrolysis (SAP PI hydrolysis), G12/13 Rho (G12/13 specific SRE-Luc reporter), hypoxia signaling pathway (HRE-Luc reporter). The proposal has three components: (i) modeling and virtual screening will be carried out in Dr. Brian Shoichet lab (UCSF); (ii) chemistry synthesis and structural modification will be carried out in Dr. Jian Jin lab (Mount Sinai School of Medicine), and (iii) cell-based functional assays and compound characterization will be carried out in Dr. Xi-Ping Huang lab and Dr. Terry Kenakin lab (UNC Chapel Hill). Dr. Xi-Ping Huang is a Research Assistant Professor in Dr. Bryan Roth lab and serves as the Assistant Scientific Director for NIMH PDSP (National Institute of Mental Health Psychoactive Drug Screening Program). The SAR studies will be guided by novel and quantitative analysis for induced functional selectivity (10?Log(?)) between two selected signaling pathways (assays) and assisted by modeling/docking studies. 1 ? 2 candidate modulators of each type (?-, -, ?-PAMs and/or -NAMs) for each individual signaling pathways will be selected for (i) selectivity assays over related proton receptors GPR4 and GPR65; (ii) binding activity at >50 common drug targets; (iii) off-target agonist activity at 318 human GPCRs (90% of human GPCRome); (iv) hERG inhibition and Caco-2 assays. At the end of the proposed project, we hopefully will identify potent, pathway specific, and functionally selective allosteric modulators as pharmacological probes for GPR68 studies.
Our ultimate goal is to design and develop pathway specific and functionally selective allosteric modulators that may be useful as pharmacological probes for investigating roles of individual signaling pathways mediated by GPR68. GPR68 is an understudied G-protein coupled receptor (GPCR), responds to protons and couples to multiple signaling pathways (e.g., Gs, Gi, Gq, G12/13). However, pharmacological studies with GPR68 have been difficult and controversial, mainly because of the lacking of small molecule tool compounds. It is not clear how individual signaling pathways of GPR68 play roles in normal physiology and pathological conditions. One unique mechanism to manipulate GPR68 activity is through allosteric modulation of proton activity. Since allosteric modulator could modify orthosteric agonist binding affinity (? activity) or efficacy ( activity) or both (? activity) to enhance agonist activity (positive allosteric modulator, PAM) or inhibit agonist activity (negative allosteric modulator, NAM) in a pathway dependent manner. In this case, since protons always exists in the receptor compartment, allosteric modulators could provide pH-range dependent allosteric modulation of proton agonism. For example, ?-PAM could potentiate proton activity at high pH (above 7.4), -PAM could potentiate proton activity at low pH (around 6.8); while ?-NAM could render GPR68 less sensitive to protons, - NAM could reduce proton efficacy. Our preliminary work identified a group of allosteric modulators for protons at GPR68. Ogerin, one lead compound, is a strong Gs-biased ?-PAM with induced functional selectivity and suppresses learning and memory at wild-type but not GPR68 KO mice, for the first time suggesting a role of GPR68 in the brain. Our initial SAR studies also indicated that small structural modifications could results in dramatic changes in allosteric activity (? and activity). These preliminary results suggest that it is possible to design and optimize pathway specific and functionally selective (aka biased) allosteric modulators as pharmacological probes that would allow us to clarify roles of individual signaling pathways mediated by GPR68. We have two specific aims to achieve the ultimate goal. Aim 1. Based on the existing modulators we have identified, we will (i) carry out extensive Structure-Activity Relationship (SAR) studies to design and develop different types of modulators (PAMs and NAMs) with distinct allosteric activity (varying ?, , or ?-values) for H+ at each individual signaling pathway (e.g. Gs, Gq, G12/13) mediated by GPR68; (ii) perform molecular modeling and docking studies to predict potential hits from ZINC library and to identify critical interactions to assist proposed SAR studies; (iii) apply novel analytic methods to quantify induced functional selectivity (bias agonism) to guide the proposed SAR studies. We will advance 1 - 2 modulators of each type (?-, -, ?-PAMs or NAMs) for every signaling pathways for further characterization. Aim 2. For selected lead modulators at each signaling pathways, we will (i) functionally characterize their selectivity against related proton receptors GPR4 and GPR65; (ii) measure binding activity against over 50 druggable targets; (iii) determine off-target agonist activity against 318 human GPCRs (>90% GPCRome); (iv) carry out early stage ADME-Tox tests (hERG binding and inhibition activity and Caco-2 permeability activity). The strength of the proposal includes: (i) GPR68 is an understudied GPCR with no small molecule ligand until we recently reported ogerin as a PAM, therefore, this is a novel molecular target; (ii) The first GPR68 modulator ogerin is a Gs-biased ?-PAM with in vivo activity to suppress learning and memory, therefore, functionally selective allosteric modulators as probes is an achievable goal; (iii) Our combined approach (virtual screening, SAR, and cell-based functional assays) successfully identified ogerin and would very likely identify more functionally selective modulators for GPR68; (iv) Targeting allosteric modulation of proton activity at GPR68 could result in a range of pH-dependent proton-agonist probes (?-PAM, -PAM, ?-PAM, ?-NAM, -NAM, or ?-NAM) for each signaling pathway, therefore this is an innovative approach for probe design and development; (v) SAR studies will be guided by novel and quantitative analysis generating system-independent parameters (induced bias agonism = 10?Log(?)) and assisted by molecular modeling and docking studies; (vi) Selected lead modulators will be profiled for selectivity among related receptors (GPR4 and GPR65), binding activity at common drug targets (>50), and off-target agonist activity (318 human GPCRs), therefore with high potential as selective probes for GPR68.