Chapter 4 Biological assay of inhibitor candidates to dengue virus type 2 and dengue virus type 3
4.3 In vivo plaque formation assay
To assess whether those individual tetracycline derivatives could indeed affect the DV propagation as predicted, different concentrations of the compounds were added separately to cultures of BHK-21 cells, followed by the addition of DV2 PL046 strain or DV3 H87 strain at a fixed number of plaque formation units (PFUs). The reduction in the number of plaques reflected the portion of the virion infection that was inhibited by the presence of the particular compound. Therefore, using the number of PFUs from the culture plates added the solvent of the compound (0 μM) as 100%, the relative percentage of the PFUs from the culture plates with compounds was calculated (PFU %).
4.3.1 Tetracycline
The concentrations of tetracycline in the culture were 0 μM, 10 μM, 50 μM, 100 μM, 300 μM, 500 μM, 700 μM, and 1000 μM. Using the number of PFUs from the culture plates added 0 μM tetracycline as 100%, the relative percentage of the PFUs was calculated as PFU %. The PFU % of the culture plates with DV2 PL046 strain were 106.44% (10 μM), 121.55% (50 μM), 119.45% (100 μM), 85.55% (300 μM), 40.52% (500 μM), 23.47% (700 μM), and 12.86% (1000 μM), yielding the estimated IC50 value of 457.89 μM (Fig 4.2).
The PFU % of the culture plates with DV3 H87 strain were 95.61% (10 μM), 100.46%
(50 μM), 100.46% (100 μM), 54.79% (300 μM), 26.49% (500 μM), 27.99% (700 μM), and 11.33% (1000 μM), yielding the estimated IC50 value of 333.85 μM (Fig. 4.3).
4.3.2 Doxycycline
The concentrations of doxycycline in the culture were 0 μM, 10 μM, 50 μM, 100 μM, 200 μM, 300 μM, 500 μM, and 700 μM. Using the number of PFUs from the culture plates added 0 μM doxycycline as 100%, the relative percentage of the PFUs was calculated as PFU %.
The PFU % of the culture plates with DV2 PL046 strain were 126.82% (10 μM), 45.20% (50 μM), 14.83% (100 μM), 5.90% (200 μM), 1.69% (300 μM), 0.57% (500 μM), and 0% (700 μM), yielding the estimated IC50 value of 47.64 μM (Fig. 4.4).
The PFU % of the culture plates with DV3 H87 strain were 116.25% (10 μM), 156.28%
(50 μM), 84.48% (100 μM), 48.94% (200 μM), 25.48% (300μM), 16.40% (500 μM), and 6.58% (700 μM), yielding the estimated IC50 value of 197.02μM (Fig. 4.5).
4.3.3 Chlortetracycline
The concentrations of chlortetracycline in the culture were 0 μM, 10 μM, 50 M, 100 μM, 200 μM, 300 μM, 500 μM, and 700 μM. Using the number of PFUs from the culture plates added 0 μM chlortetracycline as 100%, the relative percentage of the PFUs was calculated as
PFU %. The PFU % of the culture plates with DV2 PL046 strain were 101.13% (10 μM), 48.91% (50 μM), 27.81% (100 μM), 13.34% (200 μM), 6.94% (300 μM), 6.39% (500 μM), and 2.16% (700 μM), yielding the estimated IC50 value of 49.17 μM (Fig. 4.6).
The PFU % of the culture plates with DV3 H87 strain were 98.67% (10 μM), 60.11% (50 μM), 59.52% (100 μM), 34.60% (200 μM), 17.81% (300 μM), 12.04% (500 μM), and 3.95%
(700 μM), yielding the estimated IC50 value of 138.20 μM (Fig. 4.7).
4.3.4 Rolitetracycline
The concentrations of rolitetracycline in the culture were 0 μM, 10 μM, 50 μM, 100 μM, 200 μM, 300 μM, 500 μM, and 700 μM. Using the number of PFUs from the culture plates added 0 μM rolitetracycline as 100%, the relative percentage of the PFUs was calculated as PFU %. The PFU % of the culture plates with DV2 PL046 strain were 128.38% (10 μM), 171.93% (50 μM), 160.02% (100 μM), 54.41% (200 μM), 26.14% (300 μM), 5.26% (500 μM), and 0.92% (700 μM), yielding the estimated IC50 value of 215.60 μM (Fig. 4.8).
The PFU % of the culture plates with DV3 H87 strain were 100.99% (10 μM), 119.13%
(50 μM), 110.74% (100 μM), 68.95% (300 μM), 40.28% (500 μM), and 13.34% (700 μM), yielding the estimated IC50 value of 432.19 μM (Fig. 4.9).
4.3.5 Kanamycin
The concentrations of kanamycin in the culture were 0 μM, 10 μM, 50 μM, 100 μM, 200 μM, 300 μM, 500 μM, and 700 μM. Using the number of PFUs from the culture plates added 0 μM kanamycin as 100%, the relative percentage of the PFUs was calculated as PFU %. The PFU % of the culture plates with DV2 PL046 strain were 103.19% (10 μM), 100.06% (50 μM), 107.75% (100 μM), 111.28% (300 μM), 104.34% (500 μM), 122.10% (700 μM),and the IC50 was not determined (Fig. 4.10).
The PFU % of the culture plates with DV3 H87 strain were 88.05% (10 μM), 85.84% (50
μM), 90.48% (100 μM), 83.70% (200 μM), 91.75% (300 μM), 87.50% (500 μM), and 86.06%
(700 μM), and the IC50 was not determined (Fig. 4.11).
4.3.6 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate
The concentrations of 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate in the culture were 0 μM, 10 μM, 50 μM, 100 μM, 200 μM, 300 μM. Using the number of PFUs from the culture plates added 0 μM 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate as 100%, the relative percentage of the PFUs was calculated as PFU %. The PFU % of the culture plates with DV2 PL046 strain were 99.05% (10 μM), 93.72% (50 μM), 69.23% (100 μM), 68.59% (200 μM), and 53.68% (300 μM), and the IC50 was not determined (Fig. 4.12).
The PFU % of the culture plates with DV3 H87 strain were 88.81% (10 μM), 86.60% (50 μM), 74.41% (100 μM), 74.09% (200 μM), 68.33% (300 μM), and 63.22% (500 μM), and the IC50 was not determined (Fig. 4.13).
4.3.7 Berberine
The concentrations of Berberine in the culture were 0 μM, 10 μM, 50 μM, 10 μM, 200 μM, 300 μM. Using the number of PFUs from the culture plates added 0 μM Berberine as 100%, the relative percentage of the PFUs was calculated as PFU %. The PFU % of the culture plates with DV2 PL046 strain were 105.26% (10 μM), 132.49% (50 μM), 153.91% (100 μM), 125.94% (200 μM), and 70.13% (300 μM), and the IC50 was not determined (Fig. 4.14).
The PFU % of the culture plates with DV3 H87 strain were 94.38% (10 μM), 113.37%
(50 μM), and 95.68% (100 μM), and the IC50 was not determined (Fig. 4.15).
(II) Discussion
There were two main purposes for this study: (1) to assess whether those individual compounds, tetracycline derivatives and others, could indeed affect the DV propagation. (2) To assess whether the sequence variations between DV2 and DV3 affect the specificity of the candidate compounds.
Of the seven compounds, all tetracycline derivatives showed inhibitory effects on DV2 and DV3, compared with kanamycin, 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate, and berberine. The results also revealed that doxycycline and chlortetracycline had dramatic inhibitory effects on DV2 and DV3. For doxycycline on DV2, the IC50 value was 47.64 μM and there were only 14.83% of the PFUs remaining at 100 μM; on DV3, the IC50
value was 197.02 μM and 16.40% of the PFUs were retained at 500 μM. As for chlortetracycline on DV2, the IC50 value was 49.17 μM and there were only 13.34% of the PFUs remaining at 200 μM; on DV3, the IC50 value was 138.20 μM and 17.81% of the PFUs were retained at 300 μM. For rolitetracycline on DV2 and DV3, the IC50 values were 215.6 μM and 432.19 μM. As for tetracycline on DV2 and DV3, the IC50 values were 457.89 μM and 333.85 μM. But, previously, the result of tetracycline on DV2 showed that it had no inhibitory effect on the DV propagation carried out by Y.Y. Tu in the lab (appendix 3). This may be due to the way the tetracycline solution was made. In Tu’s study, the tetracycline might be dissolved in water. The solubility of tetracycline in alcohol is better than that in water. For kanamycin, it showed no inhibitory effect on DV2 and DV3 at concentrations ranging from 10 μM to 700 μM. For 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate and berberine on DV2 and DV3, no IC50 value was available.
9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate and berberine have cellular toxicity effects at higher concentrations (9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate from 500 μM, berberine from 200 μM), which would affect cellular morphology and cell
growth (Fig. 4.16, 4.17, 4.18), whereas the tetracycline derivatives would not. Nevertheless, in comparison with tetracycline, doxycycline, chlortetracycline, and rolitetracycline at concentrations ranged from 10 μM to 500 μM, 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate and berberine showed no significant inhibitory effect. It showed that the tetracycline derivatives, which contained the tetracyclic ring structure, had distinct inhibitory effects on DV2 and DV3 compared to kanamycin, 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate, with three connected rings structure , and berberine, with five connected rings structure (Table 8).
Therefore, the compounds showed inhibitory effects as long as they contained the tetracyclic ring (Table 7). The tetracyclic ring structure may be the core factor of showing inhibitory effects on the propagation of DV2 and DV3. Additionally, doxycycline and chlortetracycline revealed more obvious inhibitory effects than tetracycline and rolitetracycline, even though they all shared the core tetracyclic ring structure. The comparison of the 3D structures (Table 8) showed that the angles between the fourth ring and the third ring and the length of the side chain could confer the enhanced anti-dengue virus activity. The pharmacokinetic results of the tetracycline derivatives were showed in table 9.
The peak plasma concentrations (Cmax) of tetracycline, doxycycline, chlortetracycline, and rolitetracycline were 11.3 μM, 30 μM, 2.72 μM, and 11.4 μM, separately. When compared the Cmax (table 9) with the IC50 value of the tetracycline derivatives (table 7), it was found that doxycycline has the potential to be a therapeutic drug against dengue virus.
Zhou et al. used a hierarchical four-stage computational HTS to identify small-molecule compounds that bind to the β-OG pocket of the E protein of DV2. In biological assays, candidate compound P02 was demonstrated both to bind E protein and to have antiviral activity (Fig. 1.11). Additionally, Wang et al. performed docking on the β-OG pocket of the E protein of DV2 and found a small compound, labeled compound 6, which was identified as one of the inhibitors against DV2 (Fig. 1.12). When compared the tetracycline derivatives
with P02 and compound 6, it is found that they all have the hydrophobic properties and no similar basic structure was shared. Maybe the hydrophobic interaction is the factor to interfere the membrane fusion and conformational change of the E protein. Or there is other mechanism or reason not noticed.
Besides, in comparison with the inhibitory effects between DV2 and DV3, tetracycline showed no obvious difference while doxycycline, chlortetracycline and rolitetracycline showed more inhibitive effects on DV2 than on DV3. This suggested that the inhibitory effects may differ from E proteins of different serotype viruses. The alignment of the E protein hydrophobic pocket between DV2 and DV3 (Fig. 4.1; Table 6) showed that they were different at residues 51, 272, 274, 276, 277, and 278. Therefore, it was possible that these residues may affect compound binding and/or the subsequent membrane fusion.
All in all, all the tetracycline derivatives tested showed inhibited plaque formations on DV2 and DV3 in cell culture systems. Although the potencies varied, the compounds showed inhibitory effects as long as they contained the tetracyclic ring structure. Additionally, the angles between the fourth ring and the third ring and the length of the side chain according to the 3D structure may appear to confer the extra anti-dengue virus activity. In comparison, the inhibitory effects on DV2 were better than DV3 (Table 7). Hence, the results demonstrated that the sequence variations at residues 51, 272, 274, 276, 277, and 278 in the β-OG-binding hydrophobic pocket of E protein may affect the specificity of the tetracycline derivatives to DV E protein.
(III) Conclusion
Four tetracycline derivatives (tetracycline, doxycycline, chlortetracycline, and rolitetracycline), kanamycin, 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate, and berberine were subjected to plaque formation assay to assess whether those tetracycline derivatives could indeed affect the dengue virus propagation, as predicted. Of the seven compounds, all tetracycline derivatives showed inhibitory effects on DV2 and DV3, compared with the kanamycin, 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate, and berberine. It also revealed that doxycycline and chlortetracycline had dramatic inhibitory effects on DV2 and DV3. Although the potencies varied, the compounds showed inhibitory effects as long as they contained the tetracyclic ring structure. The tetracyclic ring structure may be the core factor of showing inhibitory effects on propagation of DV2 and DV3.
Additionally, in comparison, the inhibitory effects on DV2 were better than DV3. It demonstrated that the inhibitory effects may differ from the DV2 and DV3, with sequence variation at residues 51, 272, 274, 276, 277, and 278 in the β-OG-binding hydrophobic pocket of E protein. The sequence variations could indeed affect the specificity of the tetracycline derivatives to DV E protein.
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Table 1. Contents of the SuperScriptTM One-Step RT-PCR reaction
Components Volume/50 μl Final Concentration 2X Reaction Mix 25 μl 1X Template RNA x μl 10 pg – 1 μg Sense Primer (10 μM) 1 μl 0.2 μM Anti-sense Primer (10 μM) 1 μl 0.2 μM
RT/ PlatinumR Taq Mix 1 μl — Autoclaved distilled water to 50 μl —
Table 2. The thermal cycle program A: cDNA synthesis and
pre-denaturation