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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.

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Related in: MedlinePlus

The inhibitory activity of telaprevir on HCV NS3/4A protease by transient transfection. (A) The Seap activity in the supernatant of cells transfected with pSelect-NS3/4A-Seap. Huh7.5.1 cells were transiently transfected with the plasmid and co-cultured with telaprevir for 72 h. Culture supernatant was collected and subjected to the Seap assay. Representative data are from three independent experiments with the same results. (B) Western blot analysis for telaprevir inhibiting NS3 cleavage. pSelect-NS3/4A-Seap-transfected cells were lysed after 72 h co-cultured with telaprevir (as above). The NS3/4A fusion peptide was detected by a specific anti-NS3 antibody; GAPDH is shown here as the control. The representative result is from two independent experiments with the same results.
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fig4: The inhibitory activity of telaprevir on HCV NS3/4A protease by transient transfection. (A) The Seap activity in the supernatant of cells transfected with pSelect-NS3/4A-Seap. Huh7.5.1 cells were transiently transfected with the plasmid and co-cultured with telaprevir for 72 h. Culture supernatant was collected and subjected to the Seap assay. Representative data are from three independent experiments with the same results. (B) Western blot analysis for telaprevir inhibiting NS3 cleavage. pSelect-NS3/4A-Seap-transfected cells were lysed after 72 h co-cultured with telaprevir (as above). The NS3/4A fusion peptide was detected by a specific anti-NS3 antibody; GAPDH is shown here as the control. 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)

The inhibitory activity of telaprevir on HCV NS3/4A protease by transient transfection. (A) The Seap activity in the supernatant of cells transfected with pSelect-NS3/4A-Seap. Huh7.5.1 cells were transiently transfected with the plasmid and co-cultured with telaprevir for 72 h. Culture supernatant was collected and subjected to the Seap assay. Representative data are from three independent experiments with the same results. (B) Western blot analysis for telaprevir inhibiting NS3 cleavage. pSelect-NS3/4A-Seap-transfected cells were lysed after 72 h co-cultured with telaprevir (as above). The NS3/4A fusion peptide was detected by a specific anti-NS3 antibody; GAPDH is shown here as the control. The representative result is from two independent experiments with the same results.
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Related In: Results  -  Collection

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

fig4: The inhibitory activity of telaprevir on HCV NS3/4A protease by transient transfection. (A) The Seap activity in the supernatant of cells transfected with pSelect-NS3/4A-Seap. Huh7.5.1 cells were transiently transfected with the plasmid and co-cultured with telaprevir for 72 h. Culture supernatant was collected and subjected to the Seap assay. Representative data are from three independent experiments with the same results. (B) Western blot analysis for telaprevir inhibiting NS3 cleavage. pSelect-NS3/4A-Seap-transfected cells were lysed after 72 h co-cultured with telaprevir (as above). The NS3/4A fusion peptide was detected by a specific anti-NS3 antibody; GAPDH is shown here as the control. 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