Development of Novel Anti-influenza Thiazolides with Relatively Broad-Spectrum Antiviral Potentials

Seasonal and pandemic influenza causes 650,000 deaths annually in the world. The emergence of drug resistance to specific anti-influenza virus drugs such as oseltamivir and baloxavir marboxil highlights the urgency of novel anti-influenza chemical entity discovery. In this study, we report a series of novel thiazolides derived from an FDA-approved drug, nitazoxanide, with antiviral activity against influenza and a broad range of viruses. The preferred candidates 4a and 4d showed significantly enhanced anti-influenza virus potentials, with 10-fold improvement compared to results with nitazoxanide, and were effective against a variety of influenza virus subtypes including oseltamivir-resistant strains.

ABSTRACT

Seasonal and pandemic influenza causes 650,000 deaths annually in the world. The emergence of drug resistance to specific anti-influenza virus drugs such as oseltamivir and baloxavir marboxil highlights the urgency of novel anti-influenza chemical entity discovery. In this study, we report a series of novel thiazolides derived from an FDA-approved drug, nitazoxanide, with antiviral activity against influenza and a broad range of viruses. The preferred candidates 4a and 4d showed significantly enhanced anti-influenza virus potentials, with 10-fold improvement compared to results with nitazoxanide, and were effective against a variety of influenza virus subtypes including oseltamivir-resistant strains. Notably, the combination using compounds 4a/4d and oseltamivir carboxylate or zanamivir displayed synergistic antiviral effects against oseltamivir-resistant strains. Mode-of-action analysis demonstrated that compounds 4a/4d acted at the late phase of the viral infection cycle through inhibiting viral RNA transcription and replication. Further experiments showed that treatment with compounds 4a/4d significantly inhibited influenza virus infection in human lung organoids, suggesting the druggability of the novel thiazolides. In-depth transcriptome analysis revealed a series of upregulated cellular genes that may contribute to the antiviral activities of 4a/4d. Together, the results of our study indicated the direction to optimize nitazoxanide as an anti-influenza drug and discovered two candidates with novel structures, compounds 4a/4d, that have relatively broad-spectrum antiviral potentials.

INTRODUCTION

Seasonal influenza causes a heavy burden to public health annually, and pandemic influenza remains one of the most concerned medical issues in the world (1, 2). A vaccine is the most effective means to prevent influenza, and antiviral therapy is essential for clinical treatment of influenza. Currently, three types of influenza vaccines are clinically available: trivalent/quadrivalent inactivated vaccines, live attenuated vaccines, and the recombinant vaccine Flublok (3). Antiviral drugs, including the neuraminidase (NA) inhibitor (NAI) oseltamivir, the M2 ion channel inhibitor amantadine, and the polymerase acidic protein inhibitor baloxavir marboxil play important roles in combating influenza. As a novel cap-dependent endonuclease inhibitor, baloxavir marboxil has been approved in Japan and the United States for the treatment of influenza virus infection by the U.S. Food and Drug Administration (FDA) (4). Notably, a single oral dose could achieve the cure of acute uncomplicated influenza. However, the emergence of drug resistance is rendering current drugs ineffective. The Centers for Disease Control and Prevention (CDC) recommended discontinuing use of M2 channel blockers (amantadine and rimantadine) (5). The emergence of oseltamivir-resistant and baloxavir-resistant strains is also worth notice (6, 7).

Nitazoxanide (NTZ) is an FDA-approved antiprotozoal thiazolide drug whose broad-spectrum antiviral potential has been highlighted in recent years (8). NTZ is effective against arboviruses including Zika virus (9), Japanese encephalitis virus (10), and chikungunya virus (11). Studies have suggested the possible mechanisms underlying the antiviral activity of NTZ: it induces transcription of the antiviral phosphatase GADD34 and enhances the activities of several antiviral factors including RIG-I-like receptor, interferon regulatory factor 3 (IRF3), interferon, and mitochondrial antiviral signaling protein (12). For inhibition of important hepatitis viruses, NTZ was reported effective against hepatitis B virus (HBV) and hepatitis C virus (HCV) as well (13, 14). A clinical trial revealed that a combination of nitazoxanide, peginterferon alfa-2a, and ribavirin increased the percentages of patients with rapid and sustained virologic responses. Thiazolides were found to elicit host antiviral innate immunity and reduce HIV replication (15). NTZ exhibited in vitro activity against Middle East respiratory syndrome coronavirus (MERS-CoV) and other coronaviruses by inhibiting expression of the viral N protein (16). For treating viral diarrhea, clinical studies suggested that NTZ may play important roles in managing adult viral gastroenteritis caused by norovirus (17) and viral diarrhea in children caused by rotaviruses (18). In addition, NTZ was reported to have an antiviral effect against rubella virus and vaccinia virus (19, 20).

Researchers have repurposed NTZ as a new-generation anti-influenza drug. NTZ inhibits influenza virus replication by targeting the maturation of viral hemagglutinin (HA) (21). Moreover, NTZ showed potent antiviral activity for drug-resistant strains of seasonal influenza A (H3N2) viruses (IAVs) carrying NAI resistance-associated NA substitution (22). NTZ was shown to inhibit replication of influenza A(H1N1)pdm09 and B viruses in vitro (23). Against avian influenza A (H5N9) and canine influenza A (H3N8) viruses, NTZ was reported to inhibit these nonhuman influenza viruses potently (24, 25). A clinical trial focusing on the treatment of influenza using NTZ showed that treatment with 600 mg of nitazoxanide twice daily for 5 consecutive days was associated with a reduction of the duration of symptoms in patients with acute uncomplicated influenza (26). However, as revealed by a recent clinical study, NTZ failed to meet clinical endpoints in the treatment of respiratory illness, and improvement was needed to enhance the antiviral efficacy of this drug (27). In this study, we made structural modifications to tizoxanide, the bioactive form of NTZ, to generate a series of novel thiazolides. We attempted to obtain the next-generation antiviral thiazolide candidates with enhanced anti-influenza potentials. The candidates 4a and 4d exhibited significantly enhanced anti-influenza potentials compared with the effect of tizoxanide and had significant synergistic antiviral effects when used together with other first-line anti-influenza drugs such as oseltamivir and zanamivir. Mechanism-of-action analysis revealed that 4a/4d modulated intracellular events of influenza virus infection, and the upregulated cellular genes obtained through in-depth transcriptome analysis provided a clue to the specific antiviral target of 4a/4d. Antiviral effects of 4a/4d were validated using a lung organoid infection system, suggesting the therapeutic potential of these preferred compounds.

RESULTS

Anti-influenza activity of novel thiazolides.

To improve the anti-influenza virus activity of tizoxanide, in this study we designed and synthesized a total of 15 tizoxanide derivatives according to two strategies. The in vitro anti-influenza A virus (IAV) activities of compounds 1a to 4d were evaluated in a cytopathic effect (CPE)-based cell culture model that utilizes an influenza A(H1N1)pdm09 strain [A/California/07/2009 (H1N1)pdm09, or CA/07] . The 50% effective concentration (EC₅₀) values are summarized in Fig. 1.

FIG 1.

Structure-activity relationship (SAR) analysis of tizoxanide derivatives. Structures of tizoxanide and its derivatives are shown for compounds 1a to 4d. The EC₅₀ values were defined as the concentration of inhibitor required to reduce 50% CPE in the infectivity assay. Values were determined by nonlinear regression and are presented as means ± standard deviations from three independent experiments. SAR was summarized according to the experimental inhibition data (EC₅₀).

Based on structure-activity relationship (SAR) analysis (Fig. 1), we found that the activities of derivatives decreased when the flexible side chain substituents and aliphatic ring substituents were introduced into the N atom in the amide (strategy 1). The anti-IAV activity data indicated that the larger the substituent group on the amide N atom is, the lower the activity is. Introduction of substituents on the amide N atom may not be conducive to the improvement of activity. The antiviral activities of nitazoxanide and tizoxanide are comparable, suggesting that the introduction of substituents into hydroxyl groups may be the direction for structure optimization. To verify this hypothesis, we introduced flexible side chain substituents into hydroxyl groups (strategy 2), and the antiviral activity increased with the extension of flexible side chains. Four derivatives showed 10-fold-improved activities against (H1N1)pdm09 compared with activity of tizoxanide/nitazoxanide. The EC₅₀ value of 4d was 0.16 μM, the best antiviral activity, and 4a was also very active, with an EC₅₀ value of 0.62 μM. Furthermore, the 50% cytotoxic concentration (CC₅₀) values of compounds 4a/4d against MDCK cells were 87.57 ± 5.57 μM and above 200 μM, respectively; the selection indexes (SI) were 141 and above 1,250 for 4a and 4d, respectively. The optimization position and principle were therefore outlined, and the preferred candidates 4a and 4d showed significantly enhanced anti-influenza virus potentials compared to the activity of tizoxanide. The synthetic routes of compounds 4a/4d are shown in Scheme S1 in the supplemental material.

Compounds 4a and 4d have activity against multiple influenza viruses in vitro.

To explore whether compounds 4a and 4d exert broad-spectrum anti-influenza activities, a CPE protection assay was employed to evaluate the inhibitory effects of the above compounds against representative IAV strains including H1N1, oseltamivir-resistant H1N1, H3N2, and H7N9, and influenza B virus strains. Results represented in Table 1 indicate that all strains were susceptible to 4a and 4d, suggesting that the anti-influenza efficacy is not subtype specific. The anti-influenza activities of compounds 4a/4d against A/Puerto Rico/8/1934 (H1N1) (PR/8), CA/07, and B type viruses were comparable to the activity of oseltamivir carboxylate. Moreover, compounds 4a/4d showed high antiviral efficacy against both naturally occurring NAI-resistant strains and lab-generated NAI-resistant strains, indicating that 4a and 4d are potential candidates to fight NAI resistance.

TABLE 1.

Activity of the compounds 4a/4d against multiple influenza virus strains

Influenza virus	Subtype or lineage	EC50 (μM)a
		Compound 4a	Compound 4d	Tizoxanide	Oseltamivir carboxylate
PR/8	H1N1	4.17 ± 1.58	6.25 ± 4.67	>22.22	3.58 ± 2.17
A/WSN/33	H1N1	2.94 ± 0.13	3.21 ± 0.87	10.53 ± 0.22	0.02 ± 0.01
CA/07	H1N1	0.62 ± 0.34	0.16 ± 0.05	7.41 ± 0.01	0.16 ± 0.12
ZX/1109b	H1N1	2.16 ± 0.40	1.52 ± 0.77	7.15 ± 0.51	>100
JN/15b	H1N1	1.37 ± 0.39	0.49 ± 0.04	5.33 ± 2.05	>100
PR/8-R292Kb	H1N1	1.36 ± 0.15	1.20 ± 0.05	11.35 ± 0.11	>100
GD/17SF003	H7N9	0.93 ± 0.38	0.21 ± 0.20	6.01 ± 5.77	0.12 ± 0.05
HK/68	H3N2	1.31 ± 0.09	1.28 ± 0.16	13.57 ± 1.56	0.03 ± 0.03
B/Xiamen/N912/2014	Yamagata	1.20 ± 0.02	1.14 ± 0.78	19.93 ± 1.60	3.08 ± 0.01
B/Xiamen/N843/2014	Victoria	1.60 ± 0.54	0.84 ± 0.05	9.56 ± 1.32	9.87 ± 1.68

^aCompounds were tested in three independent experiments, and the data are presented as the means ± standard deviations.

^bOseltamivir-resistant strain.

A virus yield reduction assay was performed to further evaluate the inhibition of compounds 4a/4d on IAV infection. Briefly, cells were incubated with different concentrations of the compounds (2.5, 1.25, 0.625 μM) and dimethyl sulfoxide (DMSO) as a control at 37°C, and then four representative IAV strains (PR/8, A/TianJin-JinNan/15/2009 [JN/15], A/LiaoNing-ZhenXing/1109/2010 [ZX/1109], and A/Hongkong/8/68 [HK/68]) were inoculated into cells. At 24 h postinfection, the virus yields were determined by quantitative real-time PCR (qRT-PCR) for intracellular viral RNA and by plaque assay for supernatant viral particles. As shown in Fig. 2A and B, compounds 4a/4d reduced intracellular viral RNA levels in a dose-dependent manner. Both compounds significantly decreased (P < 0.05) infectious virus particle propagation in cell supernatants in a dose-dependent manner for all tested strains, as shown in Fig. 2C and D.

FIG 2.

Compounds 4a/4d inhibited virus yield in a dose-dependent manner in vitro. (A and B) MDCK cells were treated with DMSO or the indicated concentrations of compounds 4a and 4d, followed by infection with four different virus strains (PR/8, HK/68, JN/15, and ZX/1109; MOI of 0.1). Viral RNA copies in cell lysates were quantified by qRT-PCR. (C and D) MDCK cells were treated with DMSO or the indicated concentrations of compounds 4a and 4d, followed by infection with four different strains (PR/8, HK/68, JN/15, and ZX/1109; MOI of 0.1). Titers of influenza virus in supernatant were quantified by plaque assay. All data are shown as means ± standard deviations from three independent experiments. Statistical significance was calculated with one-way ANOVA. **, P < 0.01.

We next examined the effect of compounds 4a and 4d on viral protein generation during infection by fluorescence assay and Western blot assay. We generated a reporter virus strain with the NS1 gene tagged with green fluorescent protein (GFP) (PR/8-NS1-GFP) to track the dynamic activity of virus. Briefly, MDCK cells were infected with the PR/8-NS1-GFP reporter virus at a multiplicity of infection (MOI) of 0.1 in the presence of compounds 4a and 4d at the indicated concentrations in Fig. 3. Fluorescence imaging showed that generation of viral NS1 protein was strongly inhibited in the presence of compounds 4a and 4d (Fig. 3A). The Western blot experiment was carried out on A549 cells. The results revealed that the expression levels of viral HA, NA, M1, and PB1 proteins were significantly attenuated in the presence of compounds 4a and 4d (Fig. 3B and C). Consistently, the mRNA levels of viral HA, NA, M1 and PB1 were reduced (Fig. 3D). Thus, the inhibition of viral protein generation was in accordance with the reduction of the viral mRNA level.

FIG 3.

Effect of compounds 4a/4d on viral protein expression of influenza virus. (A) A549 cells were treated with compounds 4a/4d (5 and 10 μM) or DMSO, followed by PR/8-NS1-GFP reporter virus infection at an MOI of 0.1. At 2 h postinfection, cells were washed with PBS, and medium containing compounds was added. At 18 h postinfection, cells were fixed for imaging. Scale bar, 200 μm. (B and C) A549 cells were treated with compounds 4a/4d (10 μM) or DMSO, followed by PR/8 virus infection at an MOI of 0.5. At 24 h postinfection, cells were lysed for Western blot analysis with anti-influenza virus HA and NA antibodies (B) and M1 and PB1 antibodies (C). Human GAPDH was used as an internal reference protein to analyze the target proteins quantitatively. (D) Total RNA of cells isolated for qRT-PCR. The mRNA expression levels of viral genes are presented relative to the level of the control. *, P < 0.05; **, P < 0.01.

Compounds 4a/4d and NAI synergistically inhibit oseltamivir-resistant influenza virus strain in vitro.

The synergetic effects of compounds 4a/4d with NA inhibitors (oseltamivir carboxylate and zanamivir) were investigated using an 8-by-6 combinatorial design (28). Combination of the drugs led to enhanced inhibition of virus-induced CPE in MDCK cells, as reflected by the volume of the areas above the expected inhibitory effect for each drug independently (Fig. 4), as determined by MacSynergy II analysis (28). The resulting surface of merely additive interaction would appear as a horizontal plane at 0% inhibition from the expected value. The values of synergy/antagonism volumes under 25 μM²% were regarded as insignificant at 95% confidence; values between 25 μM²% and 50 μM²% were considered to indicate minor significance, values between 50 μM²% and 100 μM²% were regarded as indicating moderate significance, and values over 100 μM²% indicated strong interaction. The net volumes of synergy resulting from combined treatment of compound 4a and oseltamivir carboxylate or zanamivir were 122.2 μM²% and 372.9 μM²%, respectively, indicating a strong synergistic effect of 4a and NAIs (Fig. 4A and B). The net volumes of synergy resulting from combined treatment of compound 4d and oseltamivir carboxylate or zanamivir were 76.1 μM²% or 128.7 μM²%, respectively, indicating a moderate/strong synergistic effect of 4d (Fig. 4C and D). These results suggested that compounds 4a/4d might be potential options in fighting drug-resistant influenza virus.

FIG 4.

Synergetic antiviral effect of compounds 4a/4d in combination with oseltamivir or zanamivir in cells infected with drug-resistant influenza virus. MDCK cells were infected with the ZX/1109 strain in the presence of the indicated concentrations of compounds, and cell viability was evaluated at 3 days postinfection. The synergetic effects of compound 4a and oseltamivir carboxylate (A), compound 4a and zanamivir (B), compound 4d and oseltamivir carboxylate (C), and compound 4d and zanamivir (D) were analyzed by MacSynergy II and shown in a three-dimensional plot. The volumes above and below the planes represent synergy and antagonism, respectively.

Compounds 4a/4d inhibit the intracellular events of influenza virus infection.

Previous research showed that nitazoxanide and its active metabolite tizoxanide inhibit the replication of IAV strains by selectively blocking the maturation of the viral HA and impairing HA intracellular trafficking at a posttranslational level (21). Whether compounds 4a/4d with improved anti-influenza virus activity share the same molecular mechanisms needs to be elucidated.

To investigate which stage of the influenza virus infection cycle was affected by compounds 4a/4d, we performed a time-of-addition experiment using a PR/8 reporter virus (Fig. 5A). Compounds 4a/4d inhibited influenza virus yields when they were added concurrently with IAV infection or after infection (Fig. 5B and Fig. S1) as did the control drug favipiravir, which acts at the viral genome replication stage, suggesting that 4a/4d acted at a late phase of the viral infection cycle.

FIG 5.

Compounds 4a/4d acted at the intracellular stage of virus infection. (A) Strategy of time-of-addition assay. The course of compound addition was set into five intervals, as follows: compound pretreatment (I, −2 to 0 h), cotreatment (II, 0 to 2 h), compound posttreatment (IV, 2 to 12 h), pretreatment plus cotreatment (III, −2 to 2 h), and cotreatment plus posttreatment (V, 0 to 12 h). (B) MDCK cells were treated with compounds at five time intervals, as indicated in panel A, and infected with PR/8-NS1-GFP. Viral load was detected by qRT-PCR. (C) Pseudovirus assay. MDCK cells were infected with influenza virus pseudotyped particles in the presence of compounds 4a/4d, DMSO, or NAb. Luciferase activities were measured at 24 h postinfection. (D) An HI assay was performed using influenza virus and chicken red blood cells. PBS without virus was used as a positive control, while virus alone was a negative control. (E) The inhibitory effects of compounds 4a/4d on IAV polymerase activity were tested by replicon assay. Favipiravir was set as a positive control, and oseltamivir carboxylate was set as a negative control. (F) A chemiluminescence-based NA inhibition assay was performed to test the effect of compounds 4a/4d on NA activities. Influenza virus PR/8 was used. Oseltamivir carboxylate was set as a positive control. Data represent the average of triplicate measurements and are shown as means ± standard deviations (error bars). *, P < 0.05; **, P < 0.01.

To further verify whether the compounds had an effect on virus entry, we carried out an influenza pseudovirus assay (29, 30) with three kinds of pseudotyped particles (H1N1, H5N1, and H7N9). The blockage of pseudotyped particle entry would reduce the luciferase activities. An IAV-specific HA neutralizing antibody (NAb) or DMSO was used as a positive or negative control, respectively. The results revealed that compounds 4a/4d had no effect on the entry of pseudotyped particles (Fig. 5C). This finding was further verified by the results obtained from a hemagglutination inhibition (HI) assay (31) (Fig. 5D). No inhibition of hemagglutination was observed under test concentrations, indicating that compounds 4a/4d were unable to inhibit the absorption of IAV into host cells. To address the possibility that compound 4a/4d inhibited postentry events, an influenza virus replicon assay was performed alongside the positive-control drug, favipiravir, which inhibits the RNA-dependent RNA polymerase (RdRp) of influenza virus (Fig. 5E). As expected, compounds 4a/4d inhibited replicon, with EC₅₀ values of 3.68 ± 2.19 μM and 18.87 ± 7.37 μM, respectively. We then asked if the compounds prevent NA-dependent progeny virus release, and an NA inhibition assay was performed. As shown in Fig. 5F, neither compound 4a nor 4d was able to disturb cleavage of the substrate by NA from influenza PR/8 in the test range. In contrast, oseltamivir carboxylate, as the positive control, inhibited viral NA activity with a 50% inhibitory concentration (IC₅₀) value of 2.57 ± 0.75 nM. Thus, we concluded that compounds 4a/4d acted on the virus postentry and before viral particle release rather than at virus entry and release.

Transcriptome analysis of 4a/4d.

As compounds 4a/4d inhibited virus postentry, the molecular antiviral mechanism was explored via transcriptome analysis. A549 cells were infected with PR/8 virus for 8 h at an MOI of 1 and compounds 4a/4d (10 μM) or DMSO was added to the cells simultaneously. We isolated total RNA in three independent experiments from the compound-treated cells and DMSO-treated cells and built sequencing libraries to perform deep sequencing. Total mRNA profiles were analyzed. Genes with fold changes (FC) in expression levels of more than 1.5 log₂ (upregulation) or less than −1.5 log₂ (downregulation) were defined as differentially expressed. As shown in Fig. 6A and B, a total of 618 and 446 differentially expressed genes (DEGs) were identified from compound 4a- and 4d-treated cells, respectively. We noticed that 250 DEGs overlapped between samples treated with compounds 4a and 4d (Fig. 6C). By importing data sets representing genes with differential expression obtained from transcriptome sequencing (RNA-seq) analysis for Gene Ontology (GO) enrichment, we examined the possible biological functions of DEGs and determined the component complex (CC) or biological process (BP) involved in the antiviral mechanism of compounds 4a/4d in cells. The results of GO analysis of the 10 most significant subclasses of BP, CC, and molecular function (MF) are presented in Fig. 6D according to enrichment ratios. It was revealed that the most enriched CCs mainly include the transcription AP-1 complex, transcription factor complex, and CHOP-ATF3 complex, and the most significant MFs mainly include mitogen-activated protein (MAP) kinase tyrosine/serine/threonine phosphatase activity, nuclear receptor activity, and transcription factor binding. The subclasses of the above DEGs are shown in the heat map (Fig. 6E). The known genes with antiviral potential, such as PTX3 and SERPINE1, were identified in the map. This might provide insights into the antiviral mechanism of 4a/4d and shed light on clues in the search for novel anti-influenza virus drug targets.

FIG 6.

Transcriptional profiling analysis. (A and B) Genes that showed differential expression (adjusted P value of <0.05) are shown in red (upregulation) or green (downregulation) in plots of compound 4a versus a virus control (A) and compound 4d versus a virus control (B). FPKM, fragments per kilobase per million. (C) Venn diagram illustration of the distribution of shared differentially expressed genes in cells treated with compounds 4a and 4d and infected with virus. (D) GO functional enrichment of shared differentially expressed genes in both groups. CC, cellular component; MF, molecular function; BP, biological process. (E) A heat map of shared expressed genes. The heat map represents normalized expression data on a logarithmic scale, and genes are ordered by means of hierarchical clustering.

Compounds 4a/4d inhibit influenza virus infection in human lung organoids.

A human organoid system provided a novel platform to carry out in vitro drug evaluations under simulated in vivo conditions. Human lung organoids have certain substructures of human lungs, featuring alveolar epithelial cells, goblet cells, and basal cells. Human lung organoids could better simulate the cellular heterogeneity of human organs. Increasing evidence has suggested that organoids are superior models for research of important pathogens, such as influenza virus, Zika virus, enterovirus, etc. (32–34). To further explore the therapeutic potential of 4a and 4d, we adopted lung organoids differentiated from human embryonic stem cells (hESCs) to mimic influenza virus infection in human. After 104 days of development in three-dimensional culture, organoids were infected with influenza PR/8 and HK/68 viruses and simultaneously treated with compounds 4a/4d (10 μM), favipiravir (50 μM), or DMSO for 1 h. At 24 h postinfection, influenza virus in lung organoids was quantified by qRT-PCR (Fig. 7). The results demonstrated that compounds 4a/4d had positive antiviral efficacy comparable to that of the drug favipiravir. Moreover, in terms of inhibition of seasonal influenza H3N2 virus, 4a/4d were more potent than favipiravir.

FIG 7.

Compounds 4a/4d inhibited influenza virus infection in lung organoids. Human lung organoids were differentiated from hESCs. Organoids were inoculated with PR/8 and HK/68 and treated with compounds at the indicated concentrations. Influenza virus RNA was quantified by qRT-PCR. Data represent the average of triplicates and are shown as means ± standard deviations (error bars). *, P < 0.05; **, P < 0.01.

DISCUSSION

The history of anti-influenza drug development witnessed the coevolution of viruses and antiviral drugs. As the first generation of anti-influenza drugs, including amantadine and rimantadine, fade from history, researchers are more and more concerned about the emergence and spread of drug resistance of NAIs, which are currently the first-line drugs for influenza. Compared with virus-targeted drugs, host-targeted drugs have a low probability of drug resistance. NTZ was recently identified as a broad-spectrum antiviral drug and has been repurposed for the treatment of influenza. NTZ was reported to target host proteins and obstruct the maturation of the HA of influenza virus. However, the specific target of NTZ has not yet been identified, and this drug has various antiviral mechanisms for different viruses, which makes optimization of NTZ difficult. We first tried to enlarge the space occupied by the N atom in the amide, but the antiviral activity was not improved accordingly. On the contrary, antiviral activity decreased with enlargement of the amide N position. We therefore conclude that this position is not an ideal optimization target. Fortunately, a substantial increase in antiviral activity was observed when the space occupied by the hydroxyl group was improved. Based on this finding, we moved forward to extend this group, and the antiviral activity further increased along with elongation of side chain. The optimization position and principle were therefore outlined for influenza virus, and candidates with novel structures, 4a and 4d, were chosen for in-depth antiviral research.

We explored the antiviral effects of 4a/4d against a variety of influenza virus strains, including oseltamivir-resistant strains. Moreover, we explored the synergetic effects of 4a/4d, which highlighted their potentials as future anti-influenza virus drug candidates. The synergetic effects of compounds 4a/4d administered in combination with zanamivir or oseltamivir carboxylate were revealed using a cell model of infection with a natural oseltamivir-resistant isolate, ZX/1109. These findings provided potential strategies to combat NAI-resistant strains in the future.

Any structural modifications of a compound may change its mode of action or target. Therefore, we explored how compounds 4a/4d exert antiviral activities. Based on a series of experiments, we narrowed the action stage of 4a/4d to an intracellular period postentry and before viral release. We employed transcriptome analysis and found a series of cellular events that might contribute to the antiviral activity of 4a/4d. Notably, the subset of transcription factor AP-1 complex (P = 0.00046), including DDIT3 and FOS/FOSB, was significantly overrepresented. It was reported that influenza viral NS1 protein antagonized virus-induced activation of the stress response by downregulating AP-1-dependent gene expression (35). Here, we found that the treatment with compounds 4a/4d increased the expression of AP-1 complex, which may contribute to antiviral activity. Interestingly, it was revealed that treatment with compounds 4a/4d inhibited virus infection by modulation of cytoplasm-related genes (P = 0.001), indicating that these cytoplasm-related genes widely participated in postentry steps of influenza virus infection, such as protein translation, assembly, and transport. Significant upregulation of reported anti-influenza virus genes including pentraxin 3 (PTX3; P = 3.29 × 10⁻¹⁴), SERPINE1 (P = 1.89 × 10⁻²⁸⁴), ANKRD1 (P = 4.19 × 10⁻³⁵), PER1 (P = 2.84 × 10⁻¹⁴²), and NR4A3 (P = 1.66 × 10⁻¹³) was detected in our analysis. PTX3 was reported to mediate antiviral activity by modulating host immunity in vitro and in vivo. By recognizing the HA glycoprotein, PTX3 could play a role in virus neutralization and anti-influenza virus activity (36). SERPINE1/PAI-1 was reported to inhibit IAV spread by inhibiting glycoprotein cleavage (37). ANKRD1 was involved in the IRF3-mediated antiviral innate immune signaling pathway by enhancing induction of type I and type III interferons (38). NR4A3 is a member of the nuclear receptor subfamily. It is reported that the nuclear receptor may be involved in induction of amino acid and lipid metabolism, potentially required for recovery from influenza virus infection (39). Per-1, encoded by PER1, is a novel negative regulator of HIV-1 transcription (40). It is suggested that upregulated expression of PER1 regulated influenza virus infection through the same mechanism. The above findings suggested that compounds 4a/4d might play a role in anti-influenza virus activity by regulating intracellular transcription, the immune system, and/or lipid metabolism. These genes or biological processes may provide novel targets for future anti-influenza research.

For the host-targeted drugs such as NTZ, the evaluation model was critical for a comprehensive judgment of efficacy. The lung organoids we employed in this research were derived from hESCs, which could better simulate the infection of influenza virus in human (41, 42). The effectiveness of 4a/4d in lung organoids further strengthened our confidence that the two candidates may work in human tissue.

To conclude, this research discovered the optimization principle of NTZ as an anti-influenza drug and reported a series of thiazolides with relatively broad-spectrum antiviral potential.

MATERIALS AND METHODS

Cell lines and virus infection.

Madin-Darby canine kidney (MDCK), human embryonic kidney (HEK) 293T, and human alveolar type II-like epithelial (A549) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and maintained at 37°C in 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/ml of penicillin, and 100 μg/ml of streptomycin.

Influenza A/Puerto Rico/8/1934 (H1N1) (PR/8), A/California/07/2009 (H1N1)pdm09 (CA/07), A/LiaoNing-ZhenXing/1109/2010 (H1N1; a natural oseltamivir-resistant isolate) (ZX/1109), A/TianJin-JinNan/15/2009 (H1N1; a natural oseltamivir-resistant isolate) (JN/15), PR/8-R292K mutant (H1N1; a recombinant oseltamivir-resistant strain), and low-pathogenicity A/Guangdong/17SF003/2016-NIBRG-375 (H7N9, GD/17SF003) viruses were propagated for 48 h at 37°C in the chorioallantoic cavity of 8- to 10-day-old embryonated chicken eggs. Influenza A/WSN/33 (H1N1) and A/Hongkong/8/68 (H3N2) (HK/68) and influenza B/Xiamen/N912/2014(Yamagata) and B/Xiamen/N843/2014(Victoria) viruses were propagated in MDCK cells for 3 days at 33 to 37°C in serum-free Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium (DF-12) containing 2 μg/ml tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma). Oseltamivir-resistant strains (natural isolate ZX/1109) and B/Xiamen/N912/2014 and B/Xiamen/N843/2014 were kindly provided by Yuhuan Li (Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences [CAMS]) and Yixin Chen (School of Public Health, Xiamen University), respectively. The other virus strains were stored in our lab. Virus yield was determined by plaque assay, HA titration, or 50% tissue culture infective dose (TCID₅₀) infectivity assay.

Compounds.

(i) Synthesis of ethyl(2-((5-nitrothiazol-2-yl) carbamoyl)phenyl) carbonate (4a). Tizoxanide (1.00 g, 3.77 mmol) was added to anhydrous dimethyl formamide (5 ml), anhydrous pyridine (2.5 ml), and acetonitrile (1.5 ml) at 2°C to 5°C, and then ethyl chloroformate was added drop by drop. The mixture was stirred at room temperature for 6 h, poured into water, and then extracted by ethyl acetate. The resulting precipitate was filtered and washed with water and saturated salt water in sequence, followed by drying with anhydrous sodium sulfate. The product was purified by flash chromatography on silica gel. Yield, 83.5%; melting point, 156 to 158°C; ¹H-NMR (CDCl₃, 400 MHz) δ ppm 11.73 (s, 1H), 8.23 (s, 1H), 8.11 (dd, J = 1.68, 7.84 Hz, 1H), 7.72 to 7.47 (m, 2H), 4.39 to 4.36 (m, 2H), 1.41 (t, J = 7.14 Hz, 3H); HRMS (ESI+) m/z (M+H)⁺ calculated for C₁₄H₁₁N₃O₆S: 338.0369; found: 338.0442.

(ii) Synthesis of 2-((5-nitrothiazol-2-yl)carbamoyl)phenyl butane-1-sulfonate (4d). Tizoxanide (0.50 g, 1.89 mmol) was stirred at room temperature in anhydrous tetrahydrofuran (20 ml) until dissolved completely. A mixture of triethanolamine (0.38 g, 3.78 mmol) and butanesulfonyl chloride (0.59 g, 3.77 mmol) was added to the solution and stirred at room temperature. The mixture was poured into water and extracted by ethyl acetate. The resulting precipitate was filtered and washed with water and saturated salt water in sequence, followed by drying with anhydrous sodium sulfate. The product was purified by flash chromatography on silica gel. Yield, 92.0%; melting point, 142 to 144°C; ¹H-NMR (CDCl₃, 400 MHz) δ ppm 10.81 (s, 1H), 8.25 (s, 1H), 8.01 (dd, J = 1.82, 7.70 Hz, 1H), 7.72 to 7.55 (m, 1H), 7.53 to 7.50 (m, 2H), 3.47 (t, J = 8.0 Hz, 2H), 2.04 to 2.00 (m, 2H), 1.57 to 1.51 (m, 2H), 0.97 (t, J = 8.0 Hz, 3H); HRMS (ESI+) m/z (M+H)⁺ calculated for C₁₄H₁₅N₃O₆S₂: 386.0402; found: 386.0476. Oseltamivir carboxylate, peramivir trihydrate, zanamivir, baloxavir marboxil, and favipiravir (T-705) were purchased from MedChemExpress (https://www.medchemexpress.cn/). Tizoxanide was purchased from Key Organics, Ltd. (Cornwall, UK).

CPE inhibition assay. The CPE inhibition assays with influenza viruses were performed in a 96-well plate as described previously (43). Typically, MDCK cells were seeded and grown for 24 h before infection. The growth medium was changed to virus growth medium (DF-12) containing 2 μg/ml TPCK-trypsin. The test compounds were added to cells by a 3-fold dilution (ranging from 0.005 μM to 100 μM) series in virus growth medium, and DMSO was added as a control. The cells were infected with influenza viruses at an MOI of 0.005 and suspended in DF-12 with 2 μg/ml TPCK-trypsin or were mock infected. After incubation at 37°C for 72 h, the antiviral effects of the test compounds were measured using a CellTiter-Glo cell viability assay (Promega), as described by the manufacturer. The luminescence was read by a SpectraMax M5 microplate reader (Molecular Devices). The EC₅₀ was calculated by Origin, version 8, software (OriginLab Corporation, USA).

Cytotoxicity assay. Compound toxicity was evaluated against MDCK cells using a CellTiter-Glo cell viability assay according to the manufacturer’s instructions. The test compounds were added to cells by a 2-fold dilution (from 0.78 μM to 200 μM) series in virus growth medium, and DMSO was added as a control. The luminescence for each well was read by a SpectraMax M5 microplate reader. The 50% cytotoxicity concentration (CC₅₀) was calculated by Origin, version 8, software.

Plaque, HA titration, and TCID₅₀ assay.

(i) Plaque assay. Confluent MDCK cells monolayers in 12-well plates were incubated with 10-fold serial dilutions of virus for 1.5 h. The virus inoculum was then removed, and cells were overlaid with DF-12 medium containing 1% agarose, 1 μg/ml TPCK-trypsin, 100 U/ml penicillin, and 100 μg/ml streptomycin. After 72 h of incubation, cells were fixed with 4% formaldehyde for 4 h and then stained with 1% crystal violet for plaque counting.

(ii) HA titration assay. The tested virus (100 μl) was diluted in phosphate-buffered saline (PBS) with a 2-fold dilution series in U-bottomed 96-well plates, and a volume of 50 μl of virus diluent from the last well was discarded. An equal volume of preprepared chicken erythrocytes (1%, vol/vol, in PBS) was mixed into each well with the tested virus. Subsequently, the mixture was incubated for 30 min at room temperature to allow for hemagglutination to occur. The highest dilution with complete agglutination was recorded, and the reciprocal of the dilution was determined as the HA titer.

(iii) TCID₅₀ assay. Briefly, confluent MDCK cell monolayers in a 96-well plate were washed with PBS, and then the virus growth medium (DF-12) containing 2 μg/ml TPCK-trypsin was added to each well. The tested virus was serially diluted by 10-fold from 10⁰ to 10⁻⁸, and each dilution was inoculated into MDCK cells in 96-well plates. After 72 h of incubation, the 50% tissue culture infectious dose (TCID₅₀) was calculated using the method of Reed and Muench as described previously (44).

IAV replicon assay. An IAV replicon assay was assessed using a previously described method (43). The inhibition activity of test compounds was calculated by Origin, version 8, software.

NA inhibition assay. An NA-Star Influenza NA Inhibitor Resistance Detection kit (Applied Biosystems) was used to measure the inhibition of NA activity according to the manufacturer’s instructions. NA from influenza PR/8 virus was used for this assay. The IC₅₀ of the test compounds and oseltamivir carboxylate were calculated by Origin, version 8.0, software.

Entry assay of pseudotyped particle of IAV. HEK 293T cells were transfected with pNL4.3, pCAGGs H1N1-HA, and NA (or H5N1 or H7N9) at a ratio of 10:1:1, using Lipofectamine 3000, for 12 h in six-well plates. The medium was replenished with DMEM containing 0.2% bovine serum albumin (BSA), and supernatants with pseudotyped particles were collected at 60 h posttransfection. For the entry assay, MDCK cells were seeded in a 96-well plate for 18 h. The pseudotyped particles were incubated with test drugs at a concentration of 10 μM before being inoculated for 2 h on MDCK cells. The firefly luciferase activity was detected using a Bright-Glo luciferase assay system (Promega).

HI assay. An HI assay was performed as described previously (43). Test compounds were diluted in PBS with a 3-fold dilution series starting from 100 μM. Then, equal volumes of compounds and 4 hemagglutinating units (4HAU) of PR/8 virus were mixed and added into a U-bottomed 96-well plate. After a 15-min incubation at room temperature, 50 μl of freshly prepared chicken erythrocytes (1%, vol/vol, in PBS) was added to each well with multichannel pipettes. Subsequently, the mixture was incubated for 30 min at room temperature to allow for hemagglutination to occur. PBS without virus and with virus alone was used as a positive and negative control, respectively.

Quantitative real-time PCR. Total cellular RNA was isolated from cell samples infected with IAV using an RNeasy minikit (Qiagen). Absolute quantitation was performed using an ABI Step One Plus platform with a One-Step PrimeScript RT-PCR kit (TaKaRa). The details of probe sequence and primer sequences are as follows: probe, FAM-TGCAGTCCTCGCTCACTGGGCACG-BHQ1 (where FAM is 6-carboxyfluorescein and BHQ1 is Black Hole quencher 1); forward primer Inf-F, 5′-GACCRATCCTGTCACCTCTGAC-3′; reverse primer Inf-R, 5′-AGGGCATTYTGGACAAAKCGTCTA-0.3′. Relative quantitative PCR (qPCR) was performed using an ABI Step One Plus platform with a One-Step TB Green PrimeScript RT-PCR kit (TaKaRa) according to the manufacturer’s instructions. Specific primers used for qRT-PCR are as follows: for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 5′-CATGAGAAGTATGACAACAGCCT-3′ (forward) and 5′-AGTCCTTCCACGATACCAAAG T-3′ (reverse); HA, 5′-AGACCATCGGCTGTTAATGG-3′ (forward) and 5′-TTGTGTATTGGGCGTCTTGA-3′ (reverse); NA, 5′-TTCAGGCAGAATGAATGCAG-3′ (forward) and 5′-TGCGGAAAGCCTAATTGAGT-3′ (reverse); PB1, 5′-AGCGGGTATGCACAAACAGA-3′ (forward) and 5′-ATAAGTCTGGCGACCTTGGG-3′ (reverse). Data were normalized to the expression of human GAPDH. The 2^−ΔΔCT (where C_T is threshold cycle) method was used to calculate relative expression.

Time-of-addition assay. A time-of-addition assay was performed as previously described (45). Briefly, MDCK cells were seeded in a six-well plate and incubated overnight and then washed with PBS. Test compounds at a concentration of 10 μM or DMSO was added at different time intervals before or after inoculation with PR/8 reporter virus (at 0 h), as follows: phase I, −2 to 0 h; phase II, 0 to 2 h; phase III, −2 to 2 h; phase IV, 2 to 12 h; phase V, 0 to 12 h. At 12 h postinfection, the supernatants and cells were harvested. Virus titers were assessed by plaque assay and qPCR.

Combinational effects of compounds 4a/4d and NAIs in vitro. MDCK cells in 96-well plates were infected with the influenza virus PR/8 strain at 100 TCID₅₀/well, followed by the addition of compounds 4a/4d and NAIs in serial dilutions (for compounds 4a/4d, 0.16 to 10 μM; for zanamivir and oseltamivir carboxylate, 1.23 to 100 μM). After incubation at 37°C for 3 days, the antiviral effect was measured using a CellTiter-Glo cell viability assay (Promega). Data were analyzed using MacSynergy II software.

Western blot assay. A549 cells seeded in six-well plates were washed twice with PBS and infected with PR/8 virus at an MOI of 0.5. DF-12 medium containing 10 μM test compound or DMSO was added to the cells at the same time. After incubation for 24 h, the cells were lysed and loaded onto an SDS-PAGE gel. The proteins were then transferred to a polyvinylidene difluoride membrane. After being blocked for 1 h, the membranes were washed and incubated with anti-IAV HA, M1, PB1 (Invitrogen), NA (R&D Systems) antibodies or anti-GAPDH (Invitrogen) antibody as a control at 4°C overnight. The membranes were washed and incubated with appropriate secondary antibody at room temperature for 1 h. The proteins were detected using an electrochemiluminescence detection system.

Fluorescence assay of reporter virus. A549 cells seeded in black 96-well plates were washed twice with PBS and infected with PR/8 reporter virus at an MOI of 0.1. DF-12 medium containing test compound or a DMSO control was added to the cells at the same time. At 2 h postinfection, cells were washed with PBS, and medium containing the test compound was added. After 18 h of incubation, cell images were obtained via a Zeiss Axio Observer inverted microscope. The following filters were used: the 4′,6′-diamidino-2-phenylindole (DAPI) channel for nuclear staining and GFP channel for virus reporter.

Transcriptome sequencing. Total cellular RNA was isolated from cell samples infected with IAV using an RNeasy minikit (Qiagen). The quality and quantity of RNA were analyzed by an Agilent 2100 Bioanalyzer (Agilent Technologies) and Nanodrop 2000 (Thermo Scientific), respectively. RNA from each sample (5 μg) was used to create transcriptome libraries with an Illumina TruSeq RNA sample preparation kit (Illumina) according to the manufacturer’s guidelines. Sequencing was performed on an Illumina HiSeq 2500 platform. Q20 was used as a quality control standard to filter the raw reads. Low-quality reads were filtered while the adaptors of high-quality reads were removed, and the clean reads were aligned with the reference genome using HISAT. No more than two mismatches were allowed in the alignment.

Differential expression and GO enrichment analysis. Read count of genes was determined using a high-throughput sequencing (HT-Seq) method. Differentially expressed genes (DEGs) were identified by DESeq2 with a significance level at an adjusted P value of 0.05 and corrected by the Benjamini-Hochberg false discovery rate (FDR) (46). GO association data were downloaded from the Gene Ontology database (http://www.geneontology.org/), and enrichment analysis of the biological processes category was conducted by topGO (47). FDR correction was used to identify significantly enriched GO terms and was also used when the FDR was less than 0.05. All statistical analyses were performed using R, version 3.4.0 (www.bioconductor.org).

Lung organoid induction.

(i) hESC maintenance. Human embryonic stem cells (hESCs H9; provided by Harvard University) were maintained on a plate coated with mouse embryonic fibroblasts (MEF) and cultured in hESC maintenance medium at 37°C and 5% CO₂.

(ii) Endoderm and anterior foregut endoderm induction. The induction was performed as previously described (41, 48). Briefly, MEF were depleted by passaging onto Matrigel supplied with maintenance medium (IMDM 750 ml, Ham’s F12 250 ml, N2 5 ml, B27 10 ml, 7.5% BSA 7.5 ml, penicillin-streptomycin 1%, GultaMax 10 ml, ascorbic acid 50 μg/ml, monothioglycerol 0.4 μM) and incubated at 37°C for 24 h. After MEF depletion, primitive streak and embryoid body induction was performed in primitive streak/embryoid body formation medium (maintenance medium plus 10 μM Y-27632 and 3 ng/ml BMP4) in low-attachment 24-well plates for 12 to 16 h. The medium was replaced with endoderm induction medium (maintenance medium plus 10 μM Y-27632, 0.5 ng/ml BMP4, 2.5 ng/ml FGF2, and 100 ng/ml activin A) and incubated for 36 to 40 h. Embryoid bodies were kept supplied with fresh medium every day and maintained in a humidified 5% O₂–5% CO₂ atmosphere at 37°C. Endoderm yield was determined by the expression of CXCR4. When the endoderm yield was more than 90%, cells could be used for further induction. Embryoid bodies were digested with 0.05% EDTA-trypsin and plated on fibronectin-coated multiple-well plates on day 4. Cells were incubated in anteriorization medium 1 for 24 h followed by switching to anteriorization medium 2 for another 24 h.

(iii) Formation of LBOs and branching morphogenesis. The generation of lung bud organoids (LBO) was performed as previously described (41). At the end of anterior foregut endoderm induction (after 24 h), cells were treated with ventralization medium for 48 h, and three-dimensional clump formation was observed. The clumps, called lung bud organoids (LBOs), were suspended by gentle pipetting and removed to ultralow-attachment 24-well plates with branching medium. The LBOs were fed every other day until days 20 to 25. The LBOs from days 20 to 25 with wrinkled structures were embedded in 100% Matrigel in 24-well transwell inserts and incubated in an incubator until the Matrigel solidified. Branching medium was added to the wells, after which the transwell was inserted, and branching medium was added into the transwell insert as well. Medium was changed every other day.

Statistical analysis.

Data are presented as means ± standard deviations. Statistical significance between two groups was estimated using an unpaired Student’s test or analysis of variance (ANOVA) for comparison of three groups and performed using GraphPad Prism, version 7, software. A P value of <0.05 was considered a significant difference.

Data availability.

Transcriptome sequences have been deposited in the Sequence Read Archive database under accession number PRJNA625384.

The data that support the findings of this study are available from the corresponding author upon request.