Periodic Reporting for period 1 - ToBeLi-for-GPR3-6-12 (Towards high-affinity ligands for orphan receptors GPR3, GPR6 & GPR12)
Reporting period: 2022-06-01 to 2024-07-31
Summary of the context and overall objectives of the project
Members of the protein superfamily of G protein-coupled receptors (GPCRs) are targeted by more than 30% of FDA-approved drugs. However, more than two-thirds of all non-olfactory GPCRs remain untapped for disease therapy, including many orphan GPCRs – receptors with yet unknown endogenous ligands. One of the class A orphan GPCRs, GPR3, has only recently been deorphanized but lacks drug-like ligands. GPR3, together with GPR6 and GPR12, is part of a cluster of orphan GPCRs that is phylogenetically related to receptors that bind sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), cannabinoids and proopiomelanocortin-derived peptides. GPR3 activity is involved in both, benign and malig-nant physiological processes. In the central nervous system (CNS), GPR3 mediates neurite outgrowth and neuronal cell survival but has also been implicated in Alzheimer’s disease. In the periphery, GPR3 regulates oocyte maturation and drives thermogenic programs in adipocytes. These examples demonstrate that both, agonists and inverse agonists of GPR3 may be of therapeutic value for various pathologies. However, although researchers have been trying to discover molecules that target GPR3 for more than two decades, only a limited set of ligands, that were validated in independent laboratories, is currently available.
The overall goal of ToBeLi-for-GPR3-6-12 was to identify new molecules targeting these orphan class A GPCRs and we used GPR3 is the primary target in our study.
The limited number of currently availabe ligands illustrates that today’s GPR3-targeted drug discovery is still hampered by a poor understanding of the role of this receptor in cellular signaling and by a limited panel of assays that reveal GPR3 activity in living cells. Hence, we reasoned that an innovative sensing approach is needed to facilitate tailored GPR3 ligand screening with higher success rates and developed a conformational biosensor that detects compound-induced GPR3 dynamics in a pathway-independent manner and in a medium- to high-throughput screening (HTS) assay format. This biosensor presents the first conformational biosensor for an orphan GPCR with HTS-compatible sensitivity and robustness and we successfully combined this optical tool computer-aided virtual compound screening to identify new GPR3 ligands. A subsequent classic medicinal chemistry approach revealed our most potent GPR3 ligand, which is a receptor inverse agonist inducing conformational changes in GPR3 with low micromolar potency and reducing basal Gs activity downstream of GPR3 with nanomolar potency.
The scientific impact of our new GPR3 ligand needs to be tested in further preclinical studies, including toxicology studies and animal models, before its suitability for potential Phase I clinical trials can be assessed. Regardless of their direct use in humans, the ligands discovered through our work are likely to be promising lead structures for further chemical and computational studies aimed at developing even better GPR3 ligands. These future molecules can have a significant economic and societal impact, as GPR3/6/12-targeting therapeutics would represent very promising agents for the treatment of major diseases such as metabolic syndrome and Alzheimer's disease.
Our discoveries were made possible by our unprecedented research approach, which combines computational virtual screening with a pathway-independent readout of receptor activity. Our study thus provides a blueprint for other researchers attempting to identify ligands for challenging GPCRs.
The overall goal of ToBeLi-for-GPR3-6-12 was to identify new molecules targeting these orphan class A GPCRs and we used GPR3 is the primary target in our study.
The limited number of currently availabe ligands illustrates that today’s GPR3-targeted drug discovery is still hampered by a poor understanding of the role of this receptor in cellular signaling and by a limited panel of assays that reveal GPR3 activity in living cells. Hence, we reasoned that an innovative sensing approach is needed to facilitate tailored GPR3 ligand screening with higher success rates and developed a conformational biosensor that detects compound-induced GPR3 dynamics in a pathway-independent manner and in a medium- to high-throughput screening (HTS) assay format. This biosensor presents the first conformational biosensor for an orphan GPCR with HTS-compatible sensitivity and robustness and we successfully combined this optical tool computer-aided virtual compound screening to identify new GPR3 ligands. A subsequent classic medicinal chemistry approach revealed our most potent GPR3 ligand, which is a receptor inverse agonist inducing conformational changes in GPR3 with low micromolar potency and reducing basal Gs activity downstream of GPR3 with nanomolar potency.
The scientific impact of our new GPR3 ligand needs to be tested in further preclinical studies, including toxicology studies and animal models, before its suitability for potential Phase I clinical trials can be assessed. Regardless of their direct use in humans, the ligands discovered through our work are likely to be promising lead structures for further chemical and computational studies aimed at developing even better GPR3 ligands. These future molecules can have a significant economic and societal impact, as GPR3/6/12-targeting therapeutics would represent very promising agents for the treatment of major diseases such as metabolic syndrome and Alzheimer's disease.
Our discoveries were made possible by our unprecedented research approach, which combines computational virtual screening with a pathway-independent readout of receptor activity. Our study thus provides a blueprint for other researchers attempting to identify ligands for challenging GPCRs.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
Training in computational techniques & virtual compound screening
At the beginning of this project, the researcher received training in computational methods, in particular in the development of receptor homology models, the preparation of compound libraries for virtual screening, and the analysis of large data sets using RStudio. Subsequently, active and inactive state homology models of GPR3 were developed and their validity was tested validity by virtually screening known GPR3 ligands (AF64394 and two structural analogs) embedded in libraries of decoy molecules. Based on receiver operating characteristic (ROC) curve analysis, one active and one inactive state model were selected. Next, a screen of about 100,000 molecules was conducted and about 500 docking poses were visually inspected and 93 molecules were purchased for in vitro testing.
Development of live cell GPR3 assays & teaching of other lab members
A conformational GPR3 biosensor based on bioluminescence resonance energy transfer (BRET) was successfully developed in work package 2. This biosensor allows for monitoring GPR3 conformational changes in a 96-well microplate format with high sensitivity and robustness, as demonstrated by a high Z-factor of approximately 0.8 (Figure 1). In addition to this conformational readout, intracellular signaling readouts using established sensors for Gs activity and cAMP accumulation were developed and verified using known GPR3 ligands, DPI and AF64394. In the course of this work package.
In-vitro testing and pharmacological profiling of virtual screening hits
First, all 93 compounds selected from the virtual screening were tested at a single concentration using the GPR3 conformational biosensor. Three molecules induced significant changes in GPR3 conformation, and 14 chemical analogs of these molecules were purchased from commercial suppliers. Of these 14 molecules, three additional compounds induced significant changes in GPR3 conformation, yielding a total of 6 active GPR3 ligands. To check for GPR3 selectivity, all six compounds were tested as a negative control on the conformational biosensor beta2-AR. From these tests, three molecules – hereafter referred to as VH1/2/3 – were selected as GPR3 hit ligands for further optimization efforts. VH1/2/3 were also tested for effects on GPR3/6/12-mediated cAMP accumulation. VH1 reduced GPR3- and GPR6-mediated cAMP signaling, while VH2 selectively blocked GPR3-mediated cAMP production. VH3 had no effect on cAMP accumulation downstream of GPR3/6/12.
Finding affinity- and efficacy-optimized ligands through a 2nd round of screening
Two independent ligand-based approaches were implemented to obtain GPR3 ligands with improved ligand potency. First, a virtual screening for shape, 3D and functional group mimetics of VH1/2/3 was performed using the ROCS software from Openeye. Approximately 500,000 molecules, pre-filtered based on physicochemical similarity to VH1/2/3, were screened for similarity to the query molecules and a total of 19 new molecules were purchased and tested with the GPR3 conformational biosensor. Unfortunately, none of the molecules tested showed GPR3-specific activity in our assay. The second approach was based on classical chemical derivatization of a hit molecule. Here, a total of 40 new molecules was synthesized and tested using the GPR3 conformational biosensor. All molecules were first tested at two different concentrations and 20 compounds were selected for full concentration-response curves. Three molecules showed enhanced GPR3-specific activity, with the analog named MR20 having the highest potency at GPR3. MR20 induced conformational changes in GPR3 with a potency of 5 µM and inhibited GPR3-mediated Gs activation with an EC50 of 300 nM (Figure 2), representing a significant improvement in ligand potency compared to the parent molecule VH1. Compared to VH1, MR20 exhibited reduced inverse agonist potency, and neither our computational models nor the structure-activity relationship data sets enabled us to understand the attenuated potency.
At the beginning of this project, the researcher received training in computational methods, in particular in the development of receptor homology models, the preparation of compound libraries for virtual screening, and the analysis of large data sets using RStudio. Subsequently, active and inactive state homology models of GPR3 were developed and their validity was tested validity by virtually screening known GPR3 ligands (AF64394 and two structural analogs) embedded in libraries of decoy molecules. Based on receiver operating characteristic (ROC) curve analysis, one active and one inactive state model were selected. Next, a screen of about 100,000 molecules was conducted and about 500 docking poses were visually inspected and 93 molecules were purchased for in vitro testing.
Development of live cell GPR3 assays & teaching of other lab members
A conformational GPR3 biosensor based on bioluminescence resonance energy transfer (BRET) was successfully developed in work package 2. This biosensor allows for monitoring GPR3 conformational changes in a 96-well microplate format with high sensitivity and robustness, as demonstrated by a high Z-factor of approximately 0.8 (Figure 1). In addition to this conformational readout, intracellular signaling readouts using established sensors for Gs activity and cAMP accumulation were developed and verified using known GPR3 ligands, DPI and AF64394. In the course of this work package.
In-vitro testing and pharmacological profiling of virtual screening hits
First, all 93 compounds selected from the virtual screening were tested at a single concentration using the GPR3 conformational biosensor. Three molecules induced significant changes in GPR3 conformation, and 14 chemical analogs of these molecules were purchased from commercial suppliers. Of these 14 molecules, three additional compounds induced significant changes in GPR3 conformation, yielding a total of 6 active GPR3 ligands. To check for GPR3 selectivity, all six compounds were tested as a negative control on the conformational biosensor beta2-AR. From these tests, three molecules – hereafter referred to as VH1/2/3 – were selected as GPR3 hit ligands for further optimization efforts. VH1/2/3 were also tested for effects on GPR3/6/12-mediated cAMP accumulation. VH1 reduced GPR3- and GPR6-mediated cAMP signaling, while VH2 selectively blocked GPR3-mediated cAMP production. VH3 had no effect on cAMP accumulation downstream of GPR3/6/12.
Finding affinity- and efficacy-optimized ligands through a 2nd round of screening
Two independent ligand-based approaches were implemented to obtain GPR3 ligands with improved ligand potency. First, a virtual screening for shape, 3D and functional group mimetics of VH1/2/3 was performed using the ROCS software from Openeye. Approximately 500,000 molecules, pre-filtered based on physicochemical similarity to VH1/2/3, were screened for similarity to the query molecules and a total of 19 new molecules were purchased and tested with the GPR3 conformational biosensor. Unfortunately, none of the molecules tested showed GPR3-specific activity in our assay. The second approach was based on classical chemical derivatization of a hit molecule. Here, a total of 40 new molecules was synthesized and tested using the GPR3 conformational biosensor. All molecules were first tested at two different concentrations and 20 compounds were selected for full concentration-response curves. Three molecules showed enhanced GPR3-specific activity, with the analog named MR20 having the highest potency at GPR3. MR20 induced conformational changes in GPR3 with a potency of 5 µM and inhibited GPR3-mediated Gs activation with an EC50 of 300 nM (Figure 2), representing a significant improvement in ligand potency compared to the parent molecule VH1. Compared to VH1, MR20 exhibited reduced inverse agonist potency, and neither our computational models nor the structure-activity relationship data sets enabled us to understand the attenuated potency.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
GPR3/6/12 remain very challenging GPCRs that have not yet been exploited for drug therapy. Progress towards GPR6-targeting therapeutics has been made in the last 3 years with phase II clinical trials investigating the effect of CVN464 – an inverse agonist of GPR6 – on Parkinson's disease. However, no chemically novel ligands for GPR3 and GPR12 have been reported since 2014. To-be-Li-GPR3-6-12 revealed such chemically novel ligands for GPR3 – MR20 has no chemical similarity to any of the validated GPR3 ligands, AF64394 and DPI. Its potency in the high nanomolar to low micromolar range is similar to the potencies of AF64394 and DPI, underscoring the scientific advance that MR20 represents. However, the scientific impact of MR20 needs to be tested in further preclinical studies, including toxicology studies and animal models, before its suitability for potential Phase I clinical trials can be assessed. Regardless of its direct use in humans, MR20 is likely to be a promising lead structure for further chemical and computational studies aimed at developing even better GPR3 ligands from MR20. Certainly, potential GPR3-targeting drugs derived from MR20 can have a significant economic and societal impact, as these therapeutics would represent very promising agents for the treatment of two major diseases: metabolic syndrome and Alzheimer's disease.
The discovery of MR20 was made possible by our unprecedented research approach, which combines computational virtual screening with a pathway-independent readout of receptor activity. Our study thus provides a blueprint for other researchers attempting to identify ligands for challenging GPCRs. The design of our conformational biosensor is and will be openly communicated in our research talks and publications and can serve as a template to develop similar sensors for other poorly understood receptors. Our study will thus advance the field of GPCR-targeted drug discovery and development.
The discovery of MR20 was made possible by our unprecedented research approach, which combines computational virtual screening with a pathway-independent readout of receptor activity. Our study thus provides a blueprint for other researchers attempting to identify ligands for challenging GPCRs. The design of our conformational biosensor is and will be openly communicated in our research talks and publications and can serve as a template to develop similar sensors for other poorly understood receptors. Our study will thus advance the field of GPCR-targeted drug discovery and development.