A gossypol derivative effectively protects against Zika and dengue virus infection without toxicity

Abstract

Background: Zika virus (ZIKV) and dengue virus (DENV) cause microcephaly and dengue hemorrhagic fever, respectively, leading to severe problems. No effective antiviral agents are approved against infections of these flaviviruses, calling for the need to develop potent therapeutics. We previously identified gossypol as an effective inhibitor against ZIKV and DENV infections, but this compound is toxic and not suitable for in vivo treatment.

Results: In this study, we showed that gossypol derivative ST087010 exhibited potent and broad-spectrum in vitro inhibitory activity against infections of at least ten ZIKV strains isolated from different hosts, time periods, and countries, as well as DENV-1-4 serotypes, and significantly reduced cytotoxicity compared to gossypol. It presented broad-spectrum in vivo protective efficacy, protecting ZIKV-infected Ifnar1^-/- mice from lethal challenge, with increased survival and reduced weight loss. Ifnar1^-/- mice treated with this gossypol derivative decreased viral titers in various tissues, including the brain and testis, after infection with ZIKV at different human isolates. Moreover, ST087010 potently blocked ZIKV vertical transmission in pregnant Ifnar1^-/- mice, preventing ZIKV-caused fetal death, and it was safe for pregnant mice and their pups. It also protected DENV-2-challenged Ifnar1^-/- mice against viral replication by reducing the viral titers in the brain, kidney, heart, and sera.

Conclusions: Overall, our data indicate the potential for further development of this gossypol derivative as an effective and safe broad-spectrum therapeutic agent to treat ZIKV and DENV diseases.

Keywords: Antiviral agent; Dengue virus; Flavivirus; In vitro inhibition; In vivo protection; Toxicity; Zika virus.

Fig. 1

Structure, anti-Zika virus (ZIKV) activity, and cytotoxicity of gossypol derivatives. The experiments were performed on Vero E6 cells, and the cytotoxicity of gossypol derivatives in this cell line is expressed as 50% cytotoxic concentration (CC₅₀). The inhibitory activity of gossypol derivatives against infection of ZIKV human strain PAN2016 (2016/Panama) is expressed as 50% inhibitory concentration (IC₅₀). IC₅₀, CC₅₀, and selectivity index (SI) values of gossypol and each of its derivatives are shown in the figure. The red shading (A) indicates that the aldehyde groups on the C8 and C8’ positions of gossypol are replaced by other groups, whereas the blue shading (B) shows that the free hydroxyl groups on the C7 and C7’ positions of gossypol core are modified. The data are presented as the mean ± standard error of the mean (s.e.m.) of duplicate wells. The experiments were repeated twice with similar results

Fig. 2

Heatmap of the IC₅₀ values of gossypol derivatives against infection of ZIKV with different strains. Inhibitory activity of “hit” gossypol derivatives against ZIKV infection is expressed as IC₅₀. Gossypol was used as a control. Human strains are highlighted in yellow, mosquito strain is in green, and rhesus strain is in red. The SI values were calculated based on the average IC₅₀ values (human strains) and the respective CC₅₀ values (i.e., CC₅₀/IC₅₀). The data are presented as the mean of duplicate wells in each experiment. The experiments were repeated twice with similar results. Magenta indicates a low IC₅₀ or CC₅₀ value whereas dark blue indicates a high IC₅₀ or CC₅₀ value

Fig. 3

Potential inhibitory mechanism of gossypol derivative ST087010 against ZIKV infection. Time-of-addition experiment was performed in Vero E6 cells, and six specific conditions are shown as follows. (1) Condition 1: pretreatment of the virus. (2) Condition 2: pretreatment of cells. (3) Condition 3: attachment. (4) Condition 4: cotreatment. (5) Condition 5: fusion. (6) Condition 6: post-entry. The data are expressed as the mean % inhibition ± s.e.m. of duplicate wells. The experiments were repeated three times with similar results

Fig. 4

Binding affinity of gossypol derivative ST087010 to ZIKV proteins. Binding of ST087010 or gossypol control to ZIKV full-length envelope (E) protein (A), domain III of E (EDIII) protein (B), or NS2B-NS3 (C) protein was assessed by ELISA. The data are expressed as the mean ± s.e.m. of 4 wells, and DMSO was used as the negative control. Surface plasmon resonance (SPR) analysis of the binding between ST087010 and ZIKV EDIII (D) or NS2B-NS3 (E) protein. Binding affinity was shown as K_D (equilibrium dissociation constant). The experiments were repeated twice with similar results

Fig. 5

Ability of gossypol derivative ST087010 to inhibit binding of ZIKV EDIII to EDIII-specific neutralizing mAbs, as well as ZIKV NS2B-NS3 protease activity. A–D Percent inhibition (% inhibition) of EDIII-mAb binding was calculated in the presence or absence of serially diluted compounds (ST087010 or gossypol control) based on the ELISA result. Four ZIKV EDIII-specific mAbs were used for testing, and IC₅₀ values were calculated. ZIKV EDI/DII-specific mAb ZKA78 (E) and DMSO were used as controls. F Ability of ST087010 in inhibition of ZIKV NS2B-NS3 protease activity. Percent inhibition (% inhibition) of protease activity was measured in the presence or absence of serially diluted compounds, and IC₅₀ values were calculated. The data are expressed as the mean ± s.e.m of 4 wells. The experiments were repeated twice with similar results

Fig. 6

Efficacy of gossypol derivative ST087010 in protecting Ifnar1^−/− mice from lethal ZIKV challenge. A Schematic diagram of experimental procedures. Mice were intraperitoneally (i.p.) treated with ST087010, gossypol (control), or DMSO (negative control), as shown in the figure. B The treated mice were infected with ZIKV human strain R103451 (200 PFU/mouse), followed by evaluation of survival rate (a) or weight changes (b) for 21 days. The data are expressed as the mean ± s.e.m. of 6 mice in each group. In a separate experiment, ST087010 or gossypol-treated mice were infected with ZIKV human strain PAN2016 (200 PFU/mouse). Five days post-infection (dpi), viral titers were assessed in tissues by plaque assay (C), and ZIKV or caspase-3 signals were measured in the eye (D) and testis (E) tissues by immunofluorescence staining. The data (C) are expressed as the mean ± s.e.m. of 5 mice in each group, and significant differences among mice treated with ST087010, gossypol, and DMSO in the respective tissues (heart, testis, eye, kidney, or brain) were compared and are shown as *, **, and ***. The detection limit is 25 PFU/g. ZIKV (green) and caspase-3 (red) (in D, E) were stained with anti-ZIKV and anti-active caspase-3 antibodies, respectively. Nuclei (blue) were stained with DAPI (4′,6-diamidino-2-phenylindole). Representative images of immunofluorescence staining are shown. Magnification: 63×, and scale bar: 10 μm

Fig. 7

Safety profile of gossypol derivative ST087010 in pregnant Ifnar1^−/− mice and their pups. A Weight changes of pregnant mothers at prenatal and postnatal time points. B Weight changes of pups at different postnatal time points. Alanine transaminase (ALT) (C) and creatinine (D) levels in sera of pregnant mice were measured by ALT assay and creatinine assay, respectively, before and 4 h, 1, 3, and 5 days post-last injection of ST087010 or DMSO control. The data (in A–D) are expressed as the mean ± s.e.m. of 5 mice in each group, and significant differences (*) between ST087010 (40 mg/kg) and DMSO groups at 1 day post-treatment are shown (in C, D). E Hematoxylin and eosin (H&E) staining of tissues, including the liver, spleen, kidney, and brain, from ST087010 or DMSO-treated mothers and their pups. Scale bar, 50 μm

Fig. 8

Efficacy of gossypol derivative ST087010 in protecting pregnant Ifnar1^−/− mice and their fetuses against ZIKV challenge. Pregnant mice were i.p. treated with ST087010 or DMSO (control) and infected with ZIKV human strain R116265 (10³ PFU/mouse), as described in the “Methods” section. A Detection of viral titers by plaque assay in the sera (a), placenta (b), fetal brain (c), and amniotic fluid (d) at 5 dpi. The data are expressed as the mean ± s.e.m. of 5 mice in each group, and significant differences between ST087010 and DMSO groups in the sera, placenta, fetal brain, and amniotic fluid, respectively, are shown. The detection limit is 25 PFU/ml (for sera and amniotic fluid) or 25 PFU/g (for the placenta and fetal brain). B Representative images of the morphology of mouse uteri and fetuses at 5 dpi are shown. (a) E17-19 uteri from pregnant mice challenged at E12-14. Fetal morphology (b) and fetal size (c). C ZIKV or caspase-3 signals were measured in the placenta by immunofluorescence staining. ZIKV (green) and caspase-3 (red) were stained with anti-ZIKV and anti-active caspase-3 antibodies, respectively. Nuclei (blue) were stained with DAPI. Representative images of immunofluorescence staining are shown. Magnification, 63×, and scale bar 10 μm

Fig. 9

Efficacy of gossypol derivative ST087010 in protecting Ifnar1^−/− mice from DENV-2 challenge. A Schematic diagram of experimental procedures. Mice were i.p. treated with ST087010, gossypol control, or DMSO (negative control) and infected with DENV-2 human strain V594. B Representative images of DENV-2 titers in the brain, kidney, heart, and sera analyzed by flow cytometry at 3 dpi. C6/36 cells with or without DENV-2 infection were used as positive and negative controls, respectively. The percentage of infected cells to the total number of cells is shown in each figure. C Detection of DENV-2 titers in challenged mouse tissues and sera at 3 dpi. Viral titers are expressed as infectious units/g (for the brain, kidney, or heart) or infectious units/ml (for the sera), as assessed by flow cytometry assay (see the “Methods” section) and calculated based on B. Viral titers (infectious units/ml or infectious units/g) were calculated as the formula ((% of infected cells)×(total number of cells)×(dilution factor)/(amount of inoculum added to cells)). Differences among mice treated with ST087010, gossypol, or DMSO in the respective tissues (brain, kidney, and heart) or sera were compared. *, **, and *** indicate significant differences of DENV-2 infection between ST087010 and gossypol or DMSO groups, or between gossypol and DMSO groups. The data are presented as the mean ± s.e.m of 5 mice in each group. The experiments were repeated twice with similar results