Limits...
Discovering novel anti-HCV compounds with inhibitory activities toward HCV NS3/4A protease.

Yu Y, Jing JF, Tong XK, He PL, Li YC, Hu YH, Tang W, Zuo JP - Acta Pharmacol. Sin. (2014)

Bottom Line: Among them, two representative compounds HZ-1157 and LZ-110618-6 inhibited HCV NS3/4A protease with IC50 values of 1.0 and 0.68 μmol/L, respectively.Furthermore, HZ-1157 and LZ-110618-6 inhibited HCV infection in vitro with IC50 values of 0.82 and 0.11 μmol/L, respectively.Some 2,4-diaminoquinazoline derivatives and carboxamide analogues have been identified as novel anti-HCV compounds.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

ABSTRACT

Aim: To discover novel hepatitis C virus (HCV) inhibitors and elucidate the mechanism of action of the active compounds.

Methods: HCV subgenomic replicon-based luciferase reporter cell line was used to screen 1200 synthetic compounds with novel structures. Huh7.5.1 cell line stably transfected with HCV NS3/4A protease reporter was established to investigate the anti-HCV mechanism of the active compounds. The active compounds were further examined in an in vitro HCV infection assay to confirm their anti-HCV activity.

Results: After two-round screening in the anti-HCV replicon assay, some 2,4-diaminoquinazoline derivatives and carboxamide analogues were found to possess anti-HCV replicon activities (the IC50 values were less than 5 μmol/L). Among them, two representative compounds HZ-1157 and LZ-110618-6 inhibited HCV NS3/4A protease with IC50 values of 1.0 and 0.68 μmol/L, respectively. Furthermore, HZ-1157 and LZ-110618-6 inhibited HCV infection in vitro with IC50 values of 0.82 and 0.11 μmol/L, respectively.

Conclusion: Some 2,4-diaminoquinazoline derivatives and carboxamide analogues have been identified as novel anti-HCV compounds.

Show MeSH

Related in: MedlinePlus

HCV NS3/4A expression of the pSelect-NS3/4A-Seap construct. Cells were lysed 72 h after transient transfection for western blotting. For the western blot, 15 μg total protein samples were loaded, and the NS3/4A fusion protein was detected with an anti-NS3 antibody. Con-1, infectious HCV virus (J399EM) infected Huh7.5.1 cell lysate; Huh7.5.1, normal Huh7.5.1 cell lysate; Vector, pSELECT-zeo-Seap plasmid (Invivogen). The representative result is from two independent experiments with the same results.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4125721&req=5

fig3: HCV NS3/4A expression of the pSelect-NS3/4A-Seap construct. Cells were lysed 72 h after transient transfection for western blotting. For the western blot, 15 μg total protein samples were loaded, and the NS3/4A fusion protein was detected with an anti-NS3 antibody. Con-1, infectious HCV virus (J399EM) infected Huh7.5.1 cell lysate; Huh7.5.1, normal Huh7.5.1 cell lysate; Vector, pSELECT-zeo-Seap plasmid (Invivogen). The representative result is from two independent experiments with the same results.

Mentions: To identify whether these anti-HCV active compounds act against the HCV NS3/4A protease, a HCV NS3/4A protease reporter plasmid was constructed. The construct expresses a HCV NS3/4A-Seap fusion protein linked by a natural NS3/4A cleavage site (Figure 1). Successful self-cleavage by the NS3/4A protein releases the Seap protein, which is then secreted into the culture supernatant due to the exposed N-terminal signal peptide. In a mutated plasmid, pSelect-NS3/4A(mut)Seap, the serine in the catalytic triad was changed to alanine, making the mutated NS3 non-functional. The pSelect-NS3/4A-Seap plasmid and its mutated version were transiently transfected into Huh7.5.1 cells to validate their function by Western blot. As expected, a band of 70 kDa, corresponding to the NS3/4A fusion protein, was detected in cells transfected with the pSelect-NS3/4A-Seap plasmid (Figure 3, lane 4). Also as expected, a band of 130 kDa was detected in cells transfected with the pSelect-NS3/4A(mut)-Seap plasmid, corresponding to the NS3/4A-Seap fusion peptide with the Seap protein still attached (Figure 3, lane 3). This result indicated that the cleavage of NS3/4A-Seap fusion protein and the subsequent Seap secretion were fully dependent on HCV NS3 protease activity. Telaprevir (VX-950), a known inhibitor of the HCV NS3/4A serine protease22, was able to decrease the Seap level in the supernatant by 74% at 10 μmol/L (Figure 4A). The free form of NS3 also decreased by western blot analysis (Figure 4B).


Discovering novel anti-HCV compounds with inhibitory activities toward HCV NS3/4A protease.

Yu Y, Jing JF, Tong XK, He PL, Li YC, Hu YH, Tang W, Zuo JP - Acta Pharmacol. Sin. (2014)

HCV NS3/4A expression of the pSelect-NS3/4A-Seap construct. Cells were lysed 72 h after transient transfection for western blotting. For the western blot, 15 μg total protein samples were loaded, and the NS3/4A fusion protein was detected with an anti-NS3 antibody. Con-1, infectious HCV virus (J399EM) infected Huh7.5.1 cell lysate; Huh7.5.1, normal Huh7.5.1 cell lysate; Vector, pSELECT-zeo-Seap plasmid (Invivogen). The representative result is from two independent experiments with the same results.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4125721&req=5

fig3: HCV NS3/4A expression of the pSelect-NS3/4A-Seap construct. Cells were lysed 72 h after transient transfection for western blotting. For the western blot, 15 μg total protein samples were loaded, and the NS3/4A fusion protein was detected with an anti-NS3 antibody. Con-1, infectious HCV virus (J399EM) infected Huh7.5.1 cell lysate; Huh7.5.1, normal Huh7.5.1 cell lysate; Vector, pSELECT-zeo-Seap plasmid (Invivogen). The representative result is from two independent experiments with the same results.
Mentions: To identify whether these anti-HCV active compounds act against the HCV NS3/4A protease, a HCV NS3/4A protease reporter plasmid was constructed. The construct expresses a HCV NS3/4A-Seap fusion protein linked by a natural NS3/4A cleavage site (Figure 1). Successful self-cleavage by the NS3/4A protein releases the Seap protein, which is then secreted into the culture supernatant due to the exposed N-terminal signal peptide. In a mutated plasmid, pSelect-NS3/4A(mut)Seap, the serine in the catalytic triad was changed to alanine, making the mutated NS3 non-functional. The pSelect-NS3/4A-Seap plasmid and its mutated version were transiently transfected into Huh7.5.1 cells to validate their function by Western blot. As expected, a band of 70 kDa, corresponding to the NS3/4A fusion protein, was detected in cells transfected with the pSelect-NS3/4A-Seap plasmid (Figure 3, lane 4). Also as expected, a band of 130 kDa was detected in cells transfected with the pSelect-NS3/4A(mut)-Seap plasmid, corresponding to the NS3/4A-Seap fusion peptide with the Seap protein still attached (Figure 3, lane 3). This result indicated that the cleavage of NS3/4A-Seap fusion protein and the subsequent Seap secretion were fully dependent on HCV NS3 protease activity. Telaprevir (VX-950), a known inhibitor of the HCV NS3/4A serine protease22, was able to decrease the Seap level in the supernatant by 74% at 10 μmol/L (Figure 4A). The free form of NS3 also decreased by western blot analysis (Figure 4B).

Bottom Line: Among them, two representative compounds HZ-1157 and LZ-110618-6 inhibited HCV NS3/4A protease with IC50 values of 1.0 and 0.68 μmol/L, respectively.Furthermore, HZ-1157 and LZ-110618-6 inhibited HCV infection in vitro with IC50 values of 0.82 and 0.11 μmol/L, respectively.Some 2,4-diaminoquinazoline derivatives and carboxamide analogues have been identified as novel anti-HCV compounds.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

ABSTRACT

Aim: To discover novel hepatitis C virus (HCV) inhibitors and elucidate the mechanism of action of the active compounds.

Methods: HCV subgenomic replicon-based luciferase reporter cell line was used to screen 1200 synthetic compounds with novel structures. Huh7.5.1 cell line stably transfected with HCV NS3/4A protease reporter was established to investigate the anti-HCV mechanism of the active compounds. The active compounds were further examined in an in vitro HCV infection assay to confirm their anti-HCV activity.

Results: After two-round screening in the anti-HCV replicon assay, some 2,4-diaminoquinazoline derivatives and carboxamide analogues were found to possess anti-HCV replicon activities (the IC50 values were less than 5 μmol/L). Among them, two representative compounds HZ-1157 and LZ-110618-6 inhibited HCV NS3/4A protease with IC50 values of 1.0 and 0.68 μmol/L, respectively. Furthermore, HZ-1157 and LZ-110618-6 inhibited HCV infection in vitro with IC50 values of 0.82 and 0.11 μmol/L, respectively.

Conclusion: Some 2,4-diaminoquinazoline derivatives and carboxamide analogues have been identified as novel anti-HCV compounds.

Show MeSH
Related in: MedlinePlus