Screening and identification of bioactive compounds from citrus against non-structural protein 3 protease of hepatitis C virus genotype 3a by fluorescence resonance energy transfer assay and mass spectrometry

Figure 1 Expression and purification of full-length His6-NS3 and non-structural protein 4a-non-structural protein 3 protease domain. A: Analysis of the full-length His₆–NS3 protein (68.3 kDa) expressed in Escherichia coli BL21 (DE3) using pET11a-His₆-NS3. Lane M: SeeBlue^TM Pre-Stained Protein Marker (LC5625); Lane UI: Uninduced BL21 (DE3) cells and Lane I: Induced BL21 (DE3) cells; B: Analysis of soluble and insoluble fractions of the His₆–NS3 protein expressed in E. coli BL21-CodonPlus (DE3)-RIL cells using pET11a-His₆-NS3. Lane M: Invitrogen Cat 1891868 See Blue® Plus2 Pre-Stained ladder; Lane UI: Uninduced cells; Lane I: Induced cells; Lane T: Total cell lysate of induced cells; Lane S: Soluble fraction of lysed cells; C: Analysis of soluble and insoluble fractions of the His₇–non-structural protein 4a-non-structural protein 3 (NS4A-NS3) protease domain expressed in E. coli BL21 (DE3) cells using pET11a-His₇-NS4A-NS3. Lane M: SeeBlue^TM Pre-Stained Protein Marker (LC5625); Lane UI: Uninduced cells; Lane I: Induced cells; Lane T: Total cell lysate of induced cells; Lane S: Soluble fraction of lysed cells; D: Analysis of the Ni-NTA His₆–NS3 protein by gel filtration, presence of proteins in peak 1, 2 and 3 is analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis; and E: Analysis of the native NS4–NS3 protease domain by gel filtration, presence of proteins in peak 1 and 2 is analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Figure 2 Inhibition of non-structural protein 4a-non-structural protein 3 protease domain by commercial inhibitors. A: VX-950; B: Danoprevir; C: Asunaprevir; and D: BILN 2061 and determination of inhibition constant (IC₅₀).

Figure 3 Percentage inhibition pattern and inhibition constant calculation of non-structural protein 4a-non-structural protein 3 protease domain by different citrus fruit extracts inhibitors. A: Grapefruit mesocarp extract; B: Orange extract; C: Bitter orange extract; D: Mandarin extract; and E: Lemon extract. IC₅₀: Inhibition constant.

Figure 4 Electrospray ionization mass spectrometry analysis of grapefruit mesocarp extract on negative ion mode.

Figure 5 Proposed fragmentation of hesperidin based on quasi-electrospray ionization -MSⁿ (up to MS³) spectra in negative ion mode.

Figure 6 Inhibition studies of hepatitis C virus non-structural protein 4a-non-structural protein 3 protease domain by hesperidin. A: Hepatitis C virus (HCV) non-structural protein 4a-non-structural protein 3 (NS4A-NS3) protease domain model built by Swiss Model in the blue color cartoon. The active site residues are represented in yellow color for clarification; B: Docking of Hesperidin inside the receptor-binding site of HCV NS4A-NS3 protease domain (HIS 57, ASP 81 and SER 139). Hesperidin is represented in orange color. Blue ribbon represents protease domain of HCV NS3; and C: Inhibition constant (IC₅₀) calculation of HCV NS4A-NS3 protease domain by hesperidin.