Design of Light-Sensitive Triggers for Endothelial NO-Synthase Activation.

Sébastien Dilly,Anny Slama-Schwok,Eric Deprez,Linda J Roman,Nicolas Bogliotti,Juan Xie

doi:10.3390/antiox9020089

Abstract

A specific light trigger for activating endothelial Nitric Oxide-Synthase (eNOS) in real time would be of unique value to decipher cellular events associated with eNOS activation or to generate on demand cytotoxic levels of NO at specific sites for cancer research. We previously developed novel tools called nanotriggers (NT), which recognized constitutive NO-synthase, eNOS or neuronal NOS (nNOS), mainly via their 2’ phosphate group which is also present in NADPH in its binding site. Laser excitation of NT1 bound to eNOS triggered recombinant NOS activity and released NO. We recently generated new NTs carrying a 2’ or 3’ carboxylate group or two 2’ and 3’ carboxylate moieties replacing the 2’ phosphate group of NADPH. Among these new NT, only the 3’ carboxylate derivative released NO from endothelial cells upon laser activation. Here, Molecular Dynamics (MD) simulations showed that the 3’ carboxylate NT formed a folded structure with a hydrophobic hub, inducing a good stacking on FAD that likely drove efficient activation of nNOS. This NT also carried an additional small charged group which increased binding to e/nNOS; fluorescence measurements determined a 20-fold improved affinity upon binding to nNOS as compared to NT1 affinity. To gain in specificity for eNOS, we augmented a previous NT with a “hook” targeting variable residues in the NADPH site of eNOS. We discuss the potential of exploiting the chemical diversity within the NADPH site of eNOS for reversal of endothelial dysfunction in cells and for controlled generation of cytotoxic NO-derived species in cancer tissues.

Highlights

Nitric oxide is a gaseous signaling mediator involved in physiological vasodilatation and neurotransmission, generated at low (20 to 100 nM) concentration by endothelial and neuronal nitric oxide synthases, endothelial Nitric Oxide-Synthase (eNOS) and neuronal NOS (nNOS), respectively [1]
Light-activatable tools to control eNOS activity that would in real time actitation and the ability to follow eNOS in precise cellular events, such as epigenetic modifications, would be of unique value
Previous experimental data clearly emphasized the importance of electrostatic effects on their binding to eNOS by comparing the binding affinity of NT2_3, NT2_5 and NT2_7 with their diol derivative bearing no charge because both 2’ and 3’ positions were substituted by OH groups [18]

Summary

Introduction

Nitric oxide is a gaseous signaling mediator involved in physiological vasodilatation and neurotransmission, generated at low (20 to 100 nM) concentration by endothelial and neuronal nitric oxide synthases, eNOS and nNOS, respectively [1]. ENOS activity is finely regulated by post-translational modifications and local micro-environment effects, i.e., Ca2+ flux, and by interactions with other proteins such as caveoline-1, which sequesters eNOS in an inactive state at the cell membrane [8]. Only eNOS transcription enhancer AVE9488 [9,10] and drugs indirectly promoting the phosphorylation of eNOS at S1177, including statins and ACE inhibitors, have been reported to enhance eNOS activity. Light-activatable tools to control eNOS activity that would in real time actitation and the ability to follow eNOS in precise cellular events, such as epigenetic modifications, would be of unique value

Methods

Results

Conclusion