Identification of Small Molecule RGS10 Modulators in a Microglial Cell Model of Neuroinflammation

Abstract

<b>Abstract ID 55886</b> <b>Poster Board 518</b> Regulator of G protein signaling 10 (RGS10) has recently emerged as a prominent negative regulator of neuroinflammation in microglia, resident macrophages in the central nervous system (CNS). These are highly dynamic cells that exist in various states and elicit a protective role in the CNS. Generally, microglia exist in a “resting” state where they regulate CNS homeostasis, synaptic pruning, and neurogenesis. However, upon activation by an inflammatory stimulus, microglia will polarize to a reactive state where they are highly cytotoxic, secrete cytokines, and permeabilize the blood brain barrier. Interestingly, RGS10 is highly expressed in resting microglia and, upon microglial activation, RGS10 levels are suppressed. Furthermore, reversal of this suppression returns microglia back to a resting state and protects against dopaminergic neuronal cell death. Yet, the mechanisms of this suppression and regulation remain unknown. In addition, pharmacological tools to elucidate the role of RGS10 expression in microglia are lacking. To address this gap in knowledge, we developed a high-throughput screen to identify molecular probes that will aid in investigation of RGS10 regulation. We used BV-2 cells, a widely-used murine microglial cell line, and tagged endogenously expressed RGS10 with a split luciferase using CRISPR technology. This enabled high-throughput detection of changes in relative RGS10 protein levels. To induce the reactive state of microglia, we treated cells with an endogenous cytokine, interferon-γ (IFNγ), which resulted in significant suppression of the luciferase signal. Because understanding the mechanism of RGS10 suppression is most relevant to disease pathology, we developed our high-throughput screen under conditions that identified molecular probes that reversed the IFNγ-induced RGS10 suppression. After extensive assay optimization, we obtained a Z9 of 0.51, indicating a robust screen. We screened 9,600 compounds from the ChemDiv CNS diversity library and identified 37 primary hits, that significantly reversed IFNγ-induced suppression of RGS10 protein levels. Following initial chemical analysis and clustering, 15 of these compounds were chosen for follow-up studies. These are currently being elucidated for their potency and mechanism of action. Future studies are aimed at pursuing these hits as molecular probes to elucidate regulation of RGS10 in microglia.