Synthesis and Initial Evaluation of a Novel Fluorophore for Selective FMDV 3C Protease Detection.

Samerah Malik,Alex Sinclair,Adam Le Gresley,Ali Ryan

doi:10.3390/molecules25163599

Abstract

The development and evaluation of a Boc-AL(Boc)Q(Trt)-AMC fluorophore to detect 3C Protease, produced by Foot and Mouth Disease Virus (FMDV) is reported, with a view to a potential use as a rapid screen for FMDV infected livestock The peptide-linked conjugate fluorophore is evaluated in vitro for sensitivity, specificity, stability and rapidity and shows statistically significant increases in fluorescence when exposed to physiologically relevant concentrations of 3C Protease and selectivity when compared with other common proteases likely to be located, typically in the absence of FMDV. The stability of deprotected Boc-AL(Boc)Q(Trt)-AMC is reported as a limitation of this probe.

Highlights

PCR is considered a powerful and sensitive technique, the development of portable PCR systems for FMDV detection; field testing made the limitations of the system apparent as it requires a precision thermo-cycling step that is carried out using expensive, fragile instrumentation that requires a vigorous decontamination protocol of the instrument to be followed after each site use [7]
In order to reduce the costs associated with the necessary cycling at different reaction temperatures for the portable PCR system, isothermal amplification strategies were explored in viral diagnostics
In 2000, Notomi et al developed a molecular technique based on loop mediated isothermal amplification (LAMP), a technique widely used to detect viruses [8]

Summary

Results

Compounds 1 and 2 require acid treatment to deprotect the substrate ready for biochemical testing. TTeessttiinngg uussiinngg aa rraannggee ooff ccoommmmoonn pprrootteeaasseess sshhoowwss tthhaatt tthhee ssuubbssttrraattee wwaass sseelleeccttiivveellyy cclleeaavveedd bbyy 33CCpprroo,,bbuuttnnoottbbyycchhyymmoottrryyppssiinn,,tthhrroommbbiinn,,ttrryyppssiinn aanndd tthhee TTEEVV pprrootteeaassee. Our probe for 3CPro in comparison with other proteases, suggesting the evolution of fluorescence is enzyme-linked. In solution, the deprotected detection probe was found to have a short shelf-life in comparison to a commercially available BocValProArg-AMC. This may be a limitation, owing to the need to prepare a solution thereof prior to use, the sensitivity and selectivity of substrate 2 after the removal of the trityl group is compelling and, in this manuscript, we have proved the principle of a selective fluorescent probe, which could be applied as a rapid point-of-decision-making test for FMDV in the field. The authors acknowledge that such an application would demand a stable vehicle for the probe

Synthesis

Synthesis of 1

Synthesis of 2

SDS-PAGE

Enzymatic Fluorescence Assay