Novel indolic AMPK modulators induce vasodilatation through activation of the AMPK\u2013eNOS\u2013NO pathway

Marta Sanz-Gómez,María S Fernández-Alfonso,Elena Vega-Martín,Jose Cumella,Carolina Estarellas,Francisco Javier Luque,Laura Lagartera,Concepción Pérez,Marina Beroiz-Salaverri,Marta Gil-Ortega,Elnaz Aledavood,Ana Castro

doi:10.1038/s41598-022-07077-8

Abstract

Endothelial adenosine monophosphate-activated protein kinase (AMPK) plays a critical role in the regulation of vascular tone through stimulating nitric oxide (NO) release in endothelial cells. Since obesity leads to endothelial dysfunction and AMPK dysregulation, AMPK activation might be an important strategy to restore vascular function in cardiometabolic alterations. Here, we report the identification of a novel AMPK modulator, the indolic derivative IND6, which shows affinity for AMPKα1β1γ1, the primary AMPK isoform in human EA.Hy926 endothelial cells. IND6 shows inhibitory action of the enzymatic activity in vitro, but increases the levels of p-Thr174AMPK, p-Ser1177eNOS and p-Ser79ACC in EA.Hy926. This paradoxical finding might be explained by the ability of IND6 to act as a mixed-type inhibitor, but also to promote the enzyme activation by adopting two distinct binding modes at the ADaM site. Moreover, functional assays reveal that IND6 increased the eNOS-dependent production of NO and elicited a concentration-dependent vasodilation of endothelium-intact rat aorta due to AMPK and eNOS activation, demonstrating a functional activation of the AMPK–eNOS–NO endothelial pathway. This kinase inhibition profile, combined with the paradoxical AMPK activation in cells and arteries, suggests that these new chemical entities may constitute a valuable starting point for the development of new AMPK modulators with therapeutic potential for the treatment of vascular complications associated with obesity.

Highlights

Endothelial adenosine monophosphate-activated protein kinase (AMPK) plays a critical role in the regulation of vascular tone through stimulating nitric oxide (NO) release in endothelial cells
Hy926 Human endothelial cell line EC50 Half-maximum effective concentration EDC 1-Ethyl-3-(3-dimethylpropanopropyl)-carbodiimide Emax Pharmacological efficacy endothelial nitric oxide synthase (eNOS) Endothelial nitric oxide synthase Ki Inhibition constant Ki′ Apparent inhibition constant Ks Dissociation constant LC MS/MS Proteomic technique that consists of performing a liquid chromatography followed by two consecutive mass spectrometry analyses LKB1 Liver Kinase B1 L-NAME N(ω)-Nitro-l-arginine methyl ester Molecular Dynamics (MD) Molecular dynamics mTOR Mammalian/mechanistic target of rapamycin NA Noradrenaline NHS N-Hydroxysuccinimide NO Nitric oxide p-Acetyl CoA Carboxylase (ACC) Acetyl-CoA carboxylase phosphorylated in Ser[79] p-AMPK AMPK phosphorylated in Thr[174] p-eNOS Endothelial nitric oxide synthase phosphorylated in Ser1177 PFA Paraformaldehyde PI3K Phosphatidyl inositol 3 kinase RU Resonance units SAMS Substrate for AMP-activated kinases S.E.M
We have shown that caloric restriction in young Zucker fa/fa rats has cardiovascular benefits by reducing endothelial dysfunction through AMPK–PI3K–Akt–eNOS activation associated to a reduction in blood pressure, plasma triglyceride levels, and cardiac h ypertrophy[5]

Summary

Introduction

Endothelial adenosine monophosphate-activated protein kinase (AMPK) plays a critical role in the regulation of vascular tone through stimulating nitric oxide (NO) release in endothelial cells. Since obesity leads to endothelial dysfunction and AMPK dysregulation, AMPK activation might be an important strategy to restore vascular function in cardiometabolic alterations. Functional assays reveal that IND6 increased the eNOS-dependent production of NO and elicited a concentration-dependent vasodilation of endothelium-intact rat aorta due to AMPK and eNOS activation, demonstrating a functional activation of the AMPK–eNOS–NO endothelial pathway. This kinase inhibition profile, combined with the paradoxical AMPK activation in cells and arteries, suggests that these new chemical entities may constitute a valuable starting point for the development of new AMPK modulators with therapeutic potential for the treatment of vascular complications associated with obesity. AMPK activation might be an important strategy to restore vascular function in cardiometabolic alterations

Methods

Results

Conclusion