Identification of therapeutic molecules to treat amphetamine use disorders using our DNA-encoded library (DEL) technology

PROJECT SUMMARY Identification of therapeutic molecules to treat amphetamine use disorders using our DNA-encoded library (DEL) technology The objective of this Phase I SBIR project is to utilize our own innovative and proprietary DEL drug discovery platform to identify drug-like compounds binding the orphan receptor GPR37, a member of the G-protein coupled receptors (GPCRs) family. The overreaching goal is to restore brain functions linked to the reward system without impacting harmful neurotransmitter systems. Amphetamine effects are primarily mediated through dopaminergic pathways. We have extensively studied these pathways, and the basal ganglia signaling in general, in the Greengard lab. Amphetamines disrupt the mesocorticolimbic dopaminergic system, which plays a key role for reward, dependence and withdrawal. Amphetamine-dependency is linked to altered cAMP levels, affecting broad cAMP signaling. PKA and MAPK/ERK pathways involved in gene expression and synaptic plasticity are also activated. GPCRs constitute a crucial therapeutic category; those lacking a known ligand are called orphan GPCRs or oGPCRs. We have identified several oGPCRs selectively expressed in brain regions highly relevant to amphetamine use disorders and regulating the pathways above mentioned. Our proposal aims to focus on GPR37, which, according to our expertise and initial findings, emerges as the strongest candidate to potentially restore equilibrium to basal ganglia signaling. High-throughput screening (HTS) drug discovery is hindered by the requirement for large quantities of protein target, often obtained from E. coli and synonymous of non-physiological conditions. These and other HTS limitations are deleterious for GPCRs. Our research strategy aims to overcome these limitations by using a DNA-encoded library (DEL) screening approach that allows small amounts of protein to be used while retaining very large screening power (125 million compounds) and an assay that is activity-independent. The hypothesis is that the extensive pool of compounds, coupled with their inherent diversity, will enable the efficient and cost-effective identification of high-affinity binders for GPR37. Quantities of receptor used for DEL screening are compatible with mammalian cells. These measures will guarantee the utilization of a physiological GPR37 receptor, enhancing the reliability. Aim 1 will focus on the production of GPR37 in mammalian cells, followed by its purification and immobilization. This material will also be used for validation steps proposed in Aim 2. Aim 1 DEL screening will involve the testing of 125 million DEL compounds, utilizing either purified GPR37 or GPR37-containing membranes as the target source. Aim 1 aims to generate a list of 100 preferred hits, which we will then prioritize for further validation. Aim 2 will involve the chemical synthesis of 100 on-DNA compounds and 18 off- DNA. Each hit will be validated for binding by 2 methods, followed by activity assessment in mammalian cells. The program aims to identify and validate compounds with a KD in the three-digit nanomolar range or lower, and demonstrating receptor activity. This achievement will serve as a robust proof of concept, fully validating Phase I and providing a solid foundation for initiating Phase II with 5 compounds.