DPTIP, a newly identified potent brain penetrant neutral sphingomyelinase 2 inhibitor, regulates astrocyte-peripheral immune communication following brain inflammation

Camilo Rojas,Takashi Tsukamoto,Ying Wu,Ondrej Stepanek,Sarah C Zimmermann,Barbara S Slusher,Norman Haughey,Ajit G Thomas,Elena Barnaeva,Niyada Hin,Amrita Datta Chaudhuri,Marc Ferrer,Seung-Wan Yoo,Juan Marugan,Xin Hu,Alexandra G Gadiano,Noel Southall,Rana Rais

doi:10.1038/s41598-018-36144-2

Abstract

Brain injury and inflammation induces a local release of extracellular vesicles (EVs) from astrocytes carrying proteins, RNAs, and microRNAs into the circulation. When these vesicles reach the liver, they stimulate the secretion of cytokines that mobilize peripheral immune cell infiltration into the brain, which can cause secondary tissue damage and impair recovery. Recent studies suggest that suppression of EV biosynthesis through neutral sphingomyelinase 2 (nSMase2) inhibition may represent a new therapeutic strategy. Unfortunately, currently available nSMase2 inhibitors exhibit low potency (IC50 ≥ 1 μM), poor solubility and/or limited brain penetration. Through a high throughput screening campaign of >365,000 compounds against human nSMase2 we identified 2,6-Dimethoxy-4-(5-Phenyl-4-Thiophen-2-yl-1H-Imidazol-2-yl)-Phenol (DPTIP), a potent (IC50 30 nM), selective, metabolically stable, and brain penetrable (AUCbrain/AUCplasma = 0.26) nSMase2 inhibitor. DPTIP dose-dependently inhibited EV release in primary astrocyte cultures. In a mouse model of brain injury conducted in GFAP-GFP mice, DPTIP potently (10 mg/kg IP) inhibited IL-1β-induced astrocyte-derived EV release (51 ± 13%; p < 0.001). This inhibition led to a reduction of cytokine upregulation in liver and attenuation of the infiltration of immune cells into the brain (80 ± 23%; p < 0.01). A structurally similar but inactive analog had no effect in vitro or in vivo.

Highlights

Neutral sphinghomyelinase 2 catalyzes the formation of ceramide, a required step in the formation and release of extracellular vesicles (EVs)[1]
Astrocyte-derived EVs (ADEVs) isolated from the plasma of AD patients contain increased amounts of complement proteins, implying that glial activation leads to the release of EVs that may play some role in regulating innate immunity[9]
Our group has shown that brain inflammation, a common theme in many neurodegenerative disorders[10], can trigger the release of EVs from astrocytes which primes the infiltration of immune cells into the brain via upregulation of cytokines in the periphery[11]

Summary

Introduction

Neutral sphinghomyelinase 2 (nSMase2) catalyzes the formation of ceramide, a required step in the formation and release of extracellular vesicles (EVs)[1]. We report on a high throughput screening (HTS) campaign of over 365,000 compounds that identified a potent inhibitor of nSMase[2] termed DPTIP, with an excellent pharmacokinetic profile including significant brain penetration, which was capable of dose-dependently blocking EV release from primary astrocytes.

Results

Conclusion