Development of a Continuous Fluorescence Assay for Rhinovirus 14 3C Protease Using Synthetic Peptides

Abstract

SummaryRhinovirus 3C protease is an attractive target For therapeutic intervention owing to its important role in virion maturation and inFectivity. In order to Facilitate the identification of potential 3C protease inhibitors, we have developed a continuous Fluorescence assay using 5-[(2-aminoethyl}amino]naphthalene-l-sulphonic acid (Edans) as a Fluorescent donor and 4-(4-dimethylaminophenylazo}benzoic acid (Dabcyl) as a quenching acceptor. Several F1uorogenic peptide substrates For 3C protease were synthesized by both solution chemistry and solid phase peptide synthesis. One of the synthetic Edans/ Dabcyl substrates, with an amino acid sequence derived From the 2C/3A site of the virus polyprotein, yielded a 24-Fold increase in Fluorescence intensity after 3C cleavage. Data regarding substrate cleavage kinetics, assay sensitivity and optimization are presented. The application ofthisassay to the evaluation of 3C protease inhibitors is also shown.

Human Rhinovirus 3C Protease as a Potential Target for the Development of Antiviral Agents

“…This compound reversibly inhibited HRV14 3Cpro with a Ki of 0.47 μM and had mod-erate antiviral activity in cell culture with an EC 50 of 3.4 μM and a very low level of cytotoxicity [44]. Several of them displayed low micromolar antiviral activity in cell culture assay with relative broad spectrum [39]. These inhibitors contain different isosteric replacements for the P1 carboxamide side chain and reversibly inhibit 3Cpro with Ki values ranging from 5-640 nM [39].…”

“…The availability of active recombinant HRV 3Cpro enzymes permitted biochemical characterization and structure determination of this important viral enzyme. Alternatively, small peptides, modified with various fluorescent or chromogenic moieties, have been developed for HRV 3Cpro [20,[38][39][40]. These assays employ either viral polyprotein fragments or small peptides derived from the 3Cpro native cleavage sites as substrates [17][18][19][20].…”

“…To avoid the problems such as side chain cyclization during synthesis as seen with the P1 glutamine, a dipeptide aldehyde (compound 2.0), containing a P1 methionine sulfone to replace the glutamine residue, was then described [44]. These inhibitors contain different isosteric replacements for the P1 carboxamide side chain and reversibly inhibit 3Cpro with Ki values ranging from 5-640 nM [39]. In addition, a series of tripeptide aldehyde inhibitors of HRV 3Cpro has been disclosed.…”

“…In addition, a series of tripeptide aldehyde inhibitors of HRV 3Cpro has been disclosed. For example, one of the most potent inhibitor (compound 3.0) had a Ki of 6 nM for HRV14 3Cpro and moderate EC 50 of 2.4 μM against HRV14 [39]. Several of them displayed low micromolar antiviral activity in cell culture assay with relative broad spectrum [39].…”

Identification and Characterization of a Monofunctional Glycosyltransferase from Staphylococcus aureus

“…The protein band, visualized by Coomassie blue staining, was cut from the filter and used for sequence analysis. Electrospray ionization mass spectrometry analysis was conducted using a PESciex API III triple-stage quadrupole mass spectrometer equipped with a pneumatically assisted electrospray (IonSpray) interface as described previously (23).…”