Super-silent FRET Sensor Enables Live Cell Imaging and Flow Cytometric Stratification of Intracellular Serine Protease Activity in Neutrophils

Thomas H Craven,Mark Bradley,Ahsan R Akram,Tashfeen Walton,Emma Scholefield,Chris Haslett,Kevin Dhaliwal,Neil Mcdonald,Nicolaos Avlonitis,Timothy S Walsh

doi:10.1038/s41598-018-31391-9

Abstract

Serine proteases are released by neutrophils to act primarily as antimicrobial proteins but excessive and unbalanced serine protease activity results in serious host tissue damage. Here the synthesis of a novel chemical sensor based on a multi-branched fluorescence quencher is reported. It is super-silent, exhibiting no fluorescence until de-quenched by the exemplar serine protease human neutrophil elastase, rapidly enters human neutrophils, and is inhibited by serine protease inhibitors. This sensor allows live imaging of intracellular serine protease activity within human neutrophils and demonstrates that the unique combination of a multivalent scaffold combined with a FRET peptide represents a novel and efficient strategy to generate super-silent sensors that permit the visualisation of intracellular proteases and may enable point of care whole blood profiling of neutrophils.

Highlights

Pharmaceutical agents as well as providing opportunities for the stratification of patients suffering from a range of acute and chronic inflammatory conditions
Multivalent fluorescent peptides have demonstrated high biocompatibilities, low toxicities and an ability to access intracellular compartments[14], and we previously reported on a lead optimised peptidic substrate incorporated into a multi-branched fluorescently labelled reporter to monitor HNE derived from human neutrophils[15]
The first step in the synthesis of the probe was the preparation of monomer (6) which was synthesised in six steps in an overall yield of 15%15

Summary

Introduction

Pharmaceutical agents as well as providing opportunities for the stratification of patients suffering from a range of acute and chronic inflammatory conditions. Multivalent fluorescent peptides have demonstrated high biocompatibilities, low toxicities and an ability to access intracellular compartments[14], and we previously reported on a lead optimised peptidic substrate incorporated into a multi-branched fluorescently labelled reporter to monitor HNE derived from human neutrophils[15]. This offered a single auto-quenching fluorophore based approach for the analysis of proteolytic activity, where amplification of signal upon substrate cleavage was possible, without needing to rely on two dyes, but demonstrated high background levels. To demonstrate proof of concept of this novel scaffold, we have used the generic AAPV peptidic substrate with potential to iterate this peptide sequence to provide additional enzyme specificity

Methods

Results

Conclusion