58º Congresso Brasileiro de Genética
Resumos do 58o Congresso Brasileiro de Genética • 11 a 14 de setembro de 2012
Rafain Palace Hotel e Convention Center • Foz do Iguaçu • PR • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
79
Screening of inhibitors against proteolytic
activity of NS3 serine proteinase (wild type
and mutants) of Hepatitis C Virus genotype 1b
Costa, ECB1; Capaccia, AM2; Ventura, GT2; Muri, EMF3; Barros, TG3; Ferreira, SB4; Teixeira, BM5; Costa, MS5;
Ferreira, VF5; Tanuri, A1; Mohana-Borges, R2
1
Instituto de Biologia (IB-UFRJ) - Rio de Janeiro, RJ; 2Instituto de Biofísica Carlos Chagas Filho (IBCCF-UFRJ) - Rio de
Janeiro, RJ; 3Faculdade de Farmácia (FF-UFF) - Niterói, RJ; 4Instituto de Química (IQ-UFRJ) - Rio de Janeiro, RJ; 5Instituto
de Química (IQ-UFF) - Niterói, RJ.
[email protected]
Keywords: Hepatitis, NS3, protease, mutants, inhibitors.
Hepatitis C virus (HCV) infection is one of the leading causes of chronic hepatitis, liver cirrhosis and hepatocellular
carcinoma. It has affected more than 170 million individuals worldwide. The current treatment with ribavirin (RBV)
and pegylated α-interferon (PEG-IFNα) is successful only on 50% of patients, and both drugs are indirect antivirals
because they do not target a specific HCV protein or RNA element. In 2011, boceprevir and telaprevir inhibitors were
approved by the FDA (Foods and Drugs Administration) as the first direct-acting antiviral agents in the treatment of
chronic hepatitis C in combination as the PEG-IFNα and RBV. Both are peptidemimetic inhibitors of the NS3/4A
protease, belonging to the class of α-ketoamid derivatives have remarkable antiviral activity against HCV genotype
1. However, even with the approval of the FDA, phenotypic and genotypic resistance have been described for HCV
mutant protease (V36A/M, T54S/A, V55A, R155K/T/Q, A156S/V/T, D168A, V170A). Currently, macrocyclic and
linear inhibitors against HCV protease are being developed as a treatment option for chronic hepatitis C in the
near future. In this study, the plasmid Con1/SG-Neo (I), containing cDNA of HCV 1b subgenomic replicon, was
used as template in the generation of the protease NS3 fused with cofactor NS4A (NS3/4Apro) constructs, and
subsequently inserted into the pET21dHisTev vector for expression and purification of the NS3/4Apro. Mediumthroughput NS3/4Apro inhibition-based screening assay (MTS) was used to identify novel molecule against protease
activity of the HCV-1b. For inhibition of proteolytic activity, we evaluated the cleavage of a peptide labeled with
5-FAM/QXL by FRET assay (fluorescence resonance energy transfer assay). We started screening of 17 compounds
tartaric acid derivates against NS3/4Apro from which two compounds were identified to suppress HCV protease
activity with calculated inhibitor effect higher than 40%. Dose-response curve of the most active compound (B05)
was compared with standard inhibitor Ac-DE-Dif-E-Cha-C, had IC50s of 76±14μM (noncompetitive inhibition)
and 0.42±0.07μM (competitive inhibition), respectively. To determine in detail the potential effect of the compound
B05 against HCV mutant protease activity, we tested mutants proteases proteins (R155K, A156V, D168A and
V170A) that confer resistance to inhibitors as boceprevir (SCH503034), telaprevir (VX-950), danoprevir (ITMN191/RG7227) and ciluprevir (BILN 2061). IC50 values from the mutant proteases and inhibition potential were
performed. Our data shown that substitution at position 168 (D168A) and 170 (V170A) conferred low-level
resistance (3.6 and 2.4-fold increase in IC50, respectively) against compound B05, whereas substitution at position
155 (R155K) and 156 (A156V) no conferred resistance. These data suggest that even though compound B05 to be
noncompetitive inhibitor, it is able to interact with important residues located near the catalytic site. In addition,
B05 exhibits potent antiviral activity against variants carrying resistance mutations to boceprevir and telaprevir.
Financial support: FAPERJ, CNPq, CAPES.