Antiviral activity of geldanamycin and its derivatives against influenza virus

C17 methoxyl of geldanamycin (1). Their antiviral activity was evaluated based on influenza virus propagation in embryonated chicken eggs and viral absorption by hemagglutination (HA) inhibition test. The findings indicated that these compounds inhibited viral propagation at a concentration of 12.5 μg/ml. For the viral absorption, only compounds 2 and 3 inhibited HA at a concentration of 50 μg/ml. The solubility of compounds 2 and 3 in water was 290 and 306 μM, higher than that of compound 1 about 1.91 and 2.01 times, respectively. The compounds 2 and 3 showed a moderate cytotoxic activity on LLC-MK2 and Vero cells with IC50 values of >200.00 μg/ml. These results demonstrated the invention of tryptamine-geldanamycin hybrids to prevent influenza virus infection in viral absorption and viral propagation steps.

Tryptamine was the product of the decarboxylation of tryptophan. It has been used in the past as a vasodilator, neurotransmitter, antiviral, antibacterial, antifungal, anti-inflammatory, and antioxidant agent (Kousara et al., 2017). Its modification has been conducted to be pharmacologically active compounds. Recently, tryptamine has been synthesized as a novel non nucleosidic compound against hepatitis B virus (Qu et al., 2011). It has been a useful tool for improving the solubility, biological activities, and pharmacological properties of numerous natural products (Kousara et al., 2017). According to these effects, the invention of tryptamine-geldanamycin hybrids has been designed. The C17 methoxyl group of geldanamycin molecule can permit for various nucleophiles to be introduced. Thus, geldanamycin has been a popular template for producing various types of bioactive compounds (Lin et al., 2015;Modi et al., 2011;Supko et al., 1995;Tian et al., 2004;Wrona et al., 2010). Furthermore, the other report showed that some of the 17-substituted geldanamycin derivatives contained stronger activity against hepatitis B virus than geldanamycin with higher LD 50 values than that of geldanamycin . It has been reported that influenza virus replication could be inhibited by interfering the Hsp90 function (Chase et al., 2008). However, the activity of geldanamycin and its derivatives against influenza virus has been not reported. Therefore, geldanamycin could inhibit the functions of viral proteins by interfering with the complex formation of Hsp90 and viral proteins. Inhibition of Hsp90 activity also causes the inhibition of viral protein synthesis. Furthermore, it has been reported that geldanamycin could inhibit viral replication by preventing the chaperone-mediated process in viral protein folding and functions (Li et al., 2004).
In this study, novel tryptamine-geldanamycin hybrids were synthesized, their antiviral activity against influenza virus was evaluated based on virus propagation in embryonated chicken eggs and viral absorption by hemagglutination (HA) inhibition test, and their water solubility and cytotoxicity were also determined.

Isolation and cultivation of the actinomycete
Eighteen actinomycetes were isolated from the various tissues (leaf, pseudostem, rhizome, and root) of Zingiber zerumbet (L.) Smith (Taechowisan et al., 2017). Among the 18 isolates, the isolate W14 was found to be the best producer of antibacterial substances (Taechowisan et al., 2019). This isolate was selected and identified using a polyphasic approach (Taechowisan and Lumyong, 2003;Taechowisan et al., 2019). The strain was cultured on ISP-2 agar plates at 30°C for 14 days and then was extracted with ethyl acetate. The crude extract was fractionated using lowto-high polar solvents (ethyl acetate in hexane) and purified on Thin Layer Chromatography (TLC) (Taechowisan et al., 2019). The purified compound was undertaken to investigate on Nuclear Magnetic Resonance (NMR) spectroscopy. The spectral data of this compound corresponded to be geldanamycin (C 29 H 40 N 2 O 9 ) (1).

Synthesis and solubility of geldanamycin derivatives
Geldanamycin derivatives were synthesized from geldanamycin ( Fig. 1) as described earlier (Taechowisan et al. 2019). The solubility of the novel geldanamycins in water was determined by comparing with geldanamycin.

Virus cultivation inhibition assay
Virus cultivation inhibition assay was carried out by embryonated chicken egg inoculation. About 100 µl of tested compounds (12.5, 25, and 50 µg/ml) was incubated with 100 µl of seed virus (2.86 × 10 8 virus particles/ml) at 37°C for 30 minutes, and then, 100 µl of the mixture was inoculated into each embryonated chicken egg and incubated at 37°C for 4 days. The allantoic fluid was harvested and then was tested by HA assay (Brauer and Chen, 2015). About 20 µg/ml of heparin (AppliChem, Germany) was used as a positive control.

HA inhibition (HAI) assay
HAI assay was used to evaluate the effect of the compounds in virus adsorption to target cells. The compounds (25 µl) with two-fold serial dilution with Phosphate Buffered Saline (PBS) were mixed with an equal volume of seed influenza virus (400 HAU per 50 µl). After incubation at room temperature for 30 minutes, 50 µl of the solution was mixed with an equal volume of 1% chicken erythrocyte suspension and then was incubated at 4°C for 30 minutes. The highest dilution of the compounds that prevented HA is called the HAI titer. About 20 µg/ml of heparin (AppliChem, Germany) was used as a positive control.

MTT assay for cell viability
The normal cells (LLC-MK2: rhesus monkey kidney epithelial cell line and Vero: African green monkey kidney cell line) were grown at 37°C in Dubecco's Modified Eagle Medium (DMEM) medium supplement with 10% FBS, penicillin (100 units/ml), and streptomycin sulfate (100 µg/ml) in a humidified atmosphere of 5% CO 2 . Cytotoxicity studies were performed on a 96-well plate. The cells were trypsinized and plated on a 96-well plate (2 × 10 5 per well) containing DMEM medium with 10% FBS and incubated overnight. Cells were incubated with the compounds at increasing concentrations in FBS-free medium for 24 hours. Cells were washed once, and 50 µl of FBS-free medium containing 5 mg/ ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well and incubated in 5% CO 2 at 37°C for 4 hours. The medium was replaced with 50 µl of DMSO to dissolve the formazan product. The optical density was measured at 450 nm. The half inhibitory concentration (IC 50 ) was defined as a 50% reduction of the absorbance compared with the control assay.

RESULTS
The characterization of geldanamycins was carried out by 1 H-NMR, 13 C-NMR, and mass spectral methods as follows.
Compound (1): The mass spectrum showed a [M+Na] + ion at m/z 583.2571 (molecular formula : C 29 H 40 N 2 O 9 ). The spectral data revealed this compound to be a geldanamycin, which was in agreement with those of geldanamycin (Table 1), previously described by Omura et al. (1979) and Qin and Panek (2008).
Compound (3): The mass spectrum showed a [M+Na] + ion at m/z 741.3482 (molecular formula : C 39 H 50 N 4 O 9 ). The structure was fully elucidated by 1 H-NMR, 13 C-NMR spectroscopy, DEPT-135, and 2D-NMR spectral data (Table 2), and the spectral data of this compound were compared with the spectral data of compound 1 (Table 3). The water solubility of geldanamycin (1) was found to be 151.60 µM (Table 4). In contrast, the solubility of its derivatives (2 and 3) in water was 290 and 306 µM, respectively, about 1.91 and 2.01 times higher than that of geldanamycin, respectively. These data suggest that the conjugation of a tryptamine moiety to geldanamycin at the C17 position greatly enhanced their water solubility.
The effect of geldanamycin and its derivatives on influenza virus propagation was evaluated at various concentrations in embryonated chicken eggs. The virus yields were determined by HA test. The virus propagation was obtained only in control, whereas no virus was detected in the compound treatments. In addition, the effect of geldanamycin and its derivatives on viral adsorption to chicken erythrocytes was carried out. Interestingly, as expected, the compounds 2 and 3 inhibited viral binding to the cells with HAI titer of 1:50, whereas geldanamycin could not inhibit viral binding to the cells (Table 4). These data suggested that geldanamycin and its derivatives inhibited influenza virus propagation, but tryptaminegeldanamycin hybrids could inhibit the viral adsorption (early step) of influenza virus infection. Heparin at the concentration of 20 µg/ml could completely inhibit both viral propagation and viral absorption (data not shown).
Geldanamycin and its derivatives were evaluated for cytotoxicity activity against LLC-MK2 and Vero cell lines using the MTT assay. The compounds 2 and 3 exhibited weak cytotoxicity activity toward LLC-MK2 and Vero cells with IC 50 values of >200.00 µg/ml ( Table 5). The results show that two novel geldanamycin possesses low toxicity to normal cells and can display potential application in antiviral chemoprevention and chemotherapy.

DISCUSSION
Influenza virus causes seasonal outbreaks in temperate regions, with an increase in disease and mortality rate which is a serious problem. With the expectation of exploiting the potency of Hsp90 inhibitor against influenza virus, we investigated an in vitro inhibitory activity of geldanamycin and its derivatives against influenza virus as they were promising candidates in vitro.
The toxicity and water solubility of geldanamycin have been a marked hindrance for its development for chemotherapy use. These have incentive scientists to pay attention to develop less toxic geldanamycin derivatives. In this study, compounds 2 and 3 also exhibited less cytotoxicity than geldanamycin in the normal cell lines. We also found that these compounds showed a greater increase in water solubility. It should be mentioned that the antiviral activity of geldanamycin and its derivatives appeared in influenza virus propagation, which suggests that geldanamycin and its derivatives are an option choice in terms of antiviral agents in the viral propagation step. The introduction of a tryptamine or 5′-methoxytryptamine group at the C17-position of geldanamycin could not interfere with the binding of geldanamycin derivatives to Hsp90, but it had a largely decreased toxicity and increased water solubility. As stated by the crystal structure of the geldanamycin-Hsp90 complex (Stebbins et al., 1997), the substitution in the C17 methoxyl of geldanamycin is revealed to the external cavity of the Hsp90 protein, while the difference of substituents of geldanamycin is crucial for the interaction with the Hsp90 protein. According to the report by Li et al. (2010), 17-amino-17-demethoxygeldanamycin derivatives have a great potential for antiviral activity, whereas the 19-substituted geldanamycin modification was not a possibility in terms of antiviral agents. Due to the introduction of a group, the C19-position of geldanamycin could interfere with the binding of geldanamycin derivatives to Hsp90 by the steric effects . These results led us to contemplate that Hsp90 could be a target for antiviral infection and that geldanamycin and its derivatives have a great potential for antiviral propagation by interfering with Hsp90 in the protein folding and stabilizing of virus-infected cells.
The invention of tryptamine-geldanamycin hybrids has been designed at the C17-position of geldanamycin by nucleophilic substitution reactions. These compounds inhibited not only on viral propagation but also on viral absorption. It suggested that tryptamine-geldanamycin hybrids could protect viral infection in both the steps. According to this effect, Sun et al. (2019) reported that tryptophan dendrimers could block receptor binding of enterovirus A71, and these compounds could prevent binding and internalization of the virus. Furthermore, the Chinese Academy of Science, Shanghai, has shown that tryptamine derivatives had an antiviral activity against hepatitis B virus (Qu et al., 2017). This study discovered the effects of the tryptamine-geldanamycins on inhibition of influenza virus propagation and adsorption. This study will help the researcher to uncover the structural modifications of the compounds to improve biological activities.

CONCLUSION
In summary, the antiviral activity, low toxicity, and enhanced water solubility of two new tryptamine-geldanamycins, compounds 2 and 3, were presented in this work, in comparison with geldanamycin. In particular, all of these compounds showed antiviral activity in viral propagation, whereas the tryptamine-geldanamycins not only inhibit viral propagation but also inhibit viral absorption which has low toxicity and good water solubility better than geldanamycin. These results show that the functions of Hsp90 can be inhibited by these compounds and the virus cannot be propagated. This suggests a new antiviral approach. Therefore, Hsp90 could be an excellent antiviral target, and the tryptamine-geldanamycins could be considered as a new choice for antiviral agents.