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Mol. Cells

Published online December 10, 2021

© The Korean Society for Molecular and Cellular Biology

A Novel Pyrazolo[3,4-d]pyrimidine Induces Heme Oxygenase-1 and Exerts Anti-Inflammatory and Neuroprotective Effects

Ji Ae Lee1 , Young-Won Kwon1 , Hye Ri Kim1 , Nari Shin1 , Hyo Jin Son1 , Chan Seong Cheong2 , Dong Jin Kim2,* , and Onyou Hwang1,*

1Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul 05505, Korea, 2Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology, Seoul 02792, Korea

Correspondence to : oyhwang@amc.seoul.kr (OH); djk2991@kist.re.kr (DJK)

Received: March 26, 2021; Revised: September 14, 2021; Accepted: October 15, 2021

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.

Abstract

The anti-oxidant enzyme heme oxygenase-1 (HO-1) is known to exert anti-inflammatory effects. From a library of pyrazolo[3,4-d]pyrimidines, we identified a novel compound KKC080096 that upregulated HO-1 at the mRNA and protein levels in microglial BV-2 cells. KKC080096 exhibited anti-inflammatory effects via suppressing nitric oxide, interleukin-1β (IL-1β), and iNOS production in lipopolysaccharide (LPS)-challenged cells. It inhibited the phosphorylation of IKK and MAP kinases (p38, JNK, ERK), which trigger inflammatory signaling, and whose activities are inhibited by HO-1. Further, KKC080096 upregulated anti-inflammatory marker (Arg1, YM1, CD206, IL-10, transforming growth factor-β [TGF-β]) expression. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, KKC080096 lowered microglial activation, protected the nigral dopaminergic neurons, and nigral damage-associated motor deficits. Next, we elucidated the mechanisms by which KKC080096 upregulated HO-1. KKC080096 induced the phosphorylation of AMPK and its known upstream kinases LKB1 and CaMKKbeta, and pharmacological inhibition of AMPK activity reduced the effects of KKC080096 on HO-1 expression and LPS-induced NO generation, suggesting that KKC080096-induced HO-1 upregulation involves LKB1/AMPK and CaMKKbeta/AMPK pathway activation. Further, KKC080096 caused an increase in cellular Nrf2 level, bound to Keap1 (Nrf2 inhibitor protein) with high affinity, and blocked Keap1-Nrf2 interaction. This Nrf2 activation resulted in concurrent induction of HO-1 and other Nrf2-targeted antioxidant enzymes in BV-2 and in dopaminergic CATH.a cells. These results indicate that KKC080096 is a potential therapeutic for oxidative stress- and inflammation-related neurodegenerative disorders such as Parkinson’s disease.

Keywords heme oxygenase-1, neuroinflammation, Parkinson’s disease, pyrazolo[3,4-d]pyrimidine

Article

On-line First

Mol. Cells

Published online December 10, 2021

Copyright © The Korean Society for Molecular and Cellular Biology.

A Novel Pyrazolo[3,4-d]pyrimidine Induces Heme Oxygenase-1 and Exerts Anti-Inflammatory and Neuroprotective Effects

Ji Ae Lee1 , Young-Won Kwon1 , Hye Ri Kim1 , Nari Shin1 , Hyo Jin Son1 , Chan Seong Cheong2 , Dong Jin Kim2,* , and Onyou Hwang1,*

1Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul 05505, Korea, 2Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology, Seoul 02792, Korea

Correspondence to:oyhwang@amc.seoul.kr (OH); djk2991@kist.re.kr (DJK)

Received: March 26, 2021; Revised: September 14, 2021; Accepted: October 15, 2021

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.

Abstract

The anti-oxidant enzyme heme oxygenase-1 (HO-1) is known to exert anti-inflammatory effects. From a library of pyrazolo[3,4-d]pyrimidines, we identified a novel compound KKC080096 that upregulated HO-1 at the mRNA and protein levels in microglial BV-2 cells. KKC080096 exhibited anti-inflammatory effects via suppressing nitric oxide, interleukin-1β (IL-1β), and iNOS production in lipopolysaccharide (LPS)-challenged cells. It inhibited the phosphorylation of IKK and MAP kinases (p38, JNK, ERK), which trigger inflammatory signaling, and whose activities are inhibited by HO-1. Further, KKC080096 upregulated anti-inflammatory marker (Arg1, YM1, CD206, IL-10, transforming growth factor-β [TGF-β]) expression. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, KKC080096 lowered microglial activation, protected the nigral dopaminergic neurons, and nigral damage-associated motor deficits. Next, we elucidated the mechanisms by which KKC080096 upregulated HO-1. KKC080096 induced the phosphorylation of AMPK and its known upstream kinases LKB1 and CaMKKbeta, and pharmacological inhibition of AMPK activity reduced the effects of KKC080096 on HO-1 expression and LPS-induced NO generation, suggesting that KKC080096-induced HO-1 upregulation involves LKB1/AMPK and CaMKKbeta/AMPK pathway activation. Further, KKC080096 caused an increase in cellular Nrf2 level, bound to Keap1 (Nrf2 inhibitor protein) with high affinity, and blocked Keap1-Nrf2 interaction. This Nrf2 activation resulted in concurrent induction of HO-1 and other Nrf2-targeted antioxidant enzymes in BV-2 and in dopaminergic CATH.a cells. These results indicate that KKC080096 is a potential therapeutic for oxidative stress- and inflammation-related neurodegenerative disorders such as Parkinson’s disease.

Keywords: heme oxygenase-1, neuroinflammation, Parkinson’s disease, pyrazolo[3,4-d]pyrimidine

Mol. Cells
Dec 31, 2021 Vol.44 No.12, pp. 861~919
COVER PICTURE
Structure of the fly peripheral neurons in the fly head. Flies have basic sensory organs including eyes for vision, antennae and maxillary palps for olfaction, and proboscis (magenta) for gustation which can be labelled with monoclonal antibody 22C10. The figure is a 3D reconstructed image with 30 slices of confocal sections with 3 μm interval. It shows that the proboscis is required for sensing attractive carboxylic acids such as glycolic acid, citric acid, and lactic acid (Shrestha and Lee, pp. 900-910).

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