Targeting allosteric site of AKT by 5,7-dimethoxy-1,4-phenanthrenequinone suppresses neutrophilic inflammation

Po-Jen Chen; I-Ling Ko; Chia-Lin Lee; Hao-Chun Hu; Fang-Rong Chang; Yang-Chang Wu; Yann-Lii Leu; Chih-Ching Wu; Cheng-Yu Lin; Chang-Yu Pan; Yung-Fong Tsai; Tsong-Long Hwang

doi:10.1016/j.ebiom.2019.01.043

Targeting allosteric site of AKT by 5,7-dimethoxy-1,4-phenanthrenequinone suppresses neutrophilic inflammation

EBioMedicine. 2019 Feb:40:528-540. doi: 10.1016/j.ebiom.2019.01.043. Epub 2019 Jan 30.

Authors

Po-Jen Chen¹, I-Ling Ko², Chia-Lin Lee³, Hao-Chun Hu⁴, Fang-Rong Chang⁴, Yang-Chang Wu⁴, Yann-Lii Leu⁵, Chih-Ching Wu⁶, Cheng-Yu Lin², Chang-Yu Pan², Yung-Fong Tsai⁷, Tsong-Long Hwang⁸

Affiliations

¹ Department of Cosmetic Science, Providence University, Taichung 433, Taiwan; Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan.
² Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan.
³ Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung 404, Taiwan; Department of Cosmeceutics, China Medical University, Taichung 404, Taiwan.
⁴ Graduate Institute of Natural Products, College of Pharmacy and Research Center for Natural Products & Drug Development, Kaohsiung Medical University, Kaohsiung 807, Taiwan.
⁵ Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan; Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Taoyuan 333, Taiwan; Center for Traditional Chinese Medicine, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan.
⁶ Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan; Department of Otolaryngology - Head & Neck Surgery, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan.
⁷ Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan; Department of Anaesthesiology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan.
⁸ Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan; Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Taoyuan 333, Taiwan; Department of Anaesthesiology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan; Research Center for Chinese Herbal Medicine, Research Center for Food and Cosmetic Safety, Graduate Institute of Health Industry Technology, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan 333, Taiwan; Department of Chemical Engineering, Ming Chi University of Technology, New Taipei City 243, Taiwan. Electronic address: htl@mail.cgu.edu.tw.

Abstract

Background: Acute lung injury (ALI) is a severe life-threatening inflammatory disease. Neutrophil activation is a major pathogenic factor in ALI. Protein kinase B (PKB)/AKT regulates diverse cellular responses, but the significance in neutrophilic inflammation and ALI remains unknown.

Methods: Human neutrophils and neutrophil-like differentiated HL-60 (dHL-60) cells were used to examine the anti-inflammatory effects of 5,7-dimethoxy-1,4-phenanthrenequinone (CLLV-1). The therapeutic potential of CLLV-1 was determined in a mouse model of lipopolysaccharide (LPS)-induced ALI.

Findings: CLLV-1 inhibited respiratory burst, degranulation, adhesion, and chemotaxis in human neutrophils and dHL-60 cells. CLLV-1 inhibited the phosphorylation of AKT (Thr308 and Ser473), but not of ERK, JNK, or p38. Furthermore, CLLV-1 blocked AKT activity and covalently reacted with AKT Cys310 in vitro. The AKT_309-313 peptide-CLLV-1 adducts were determined by NMR or mass spectrometry assay. The alkylation agent-conjugated AKT (reduced form) level was also inhibited by CLLV-1. Significantly, CLLV-1 ameliorated LPS-induced ALI, neutrophil infiltration, and AKT activation in mice.

Interpretation: Our results identify CLLV-1 as a covalent allosteric AKT inhibitor by targeting AKT Cys310. CLLV-1 shows potent anti-inflammatory activity in human neutrophils and LPS-induced mouse ALI. Our findings provide a mechanistic framework for redox modification of AKT that may serve as a novel pharmacological target to alleviate neutrophilic inflammation.

Keywords: 5,7-dimethoxy-1,4-phenanthrenequinone; AKT; Acute lung injury; Inflammation; Neutrophil.

MeSH terms

Allosteric Site*
Anti-Inflammatory Agents / chemistry
Anti-Inflammatory Agents / pharmacology
Cell Adhesion / drug effects
Chemotaxis, Leukocyte / immunology
HL-60 Cells
Humans
Models, Molecular
Molecular Conformation
Neutrophil Infiltration / drug effects*
Neutrophils / drug effects*
Neutrophils / immunology*
Neutrophils / metabolism
Phenanthrenes / chemistry
Phenanthrenes / pharmacology*
Protein Binding
Protein Interaction Domains and Motifs / drug effects*
Proto-Oncogene Proteins c-akt / antagonists & inhibitors
Proto-Oncogene Proteins c-akt / chemistry*
Proto-Oncogene Proteins c-akt / metabolism
Quinones / chemistry
Quinones / pharmacology*
Reactive Oxygen Species
Structure-Activity Relationship

Substances

5,7-dimethoxy-1,4-phenanthrenequinone
Anti-Inflammatory Agents
Phenanthrenes
Quinones
Reactive Oxygen Species
Proto-Oncogene Proteins c-akt