Skip to main content
NCBI home page
Science Advances logoLink to Science Advances
. 2024 Feb 23;10(8):eadk9004. doi: 10.1126/sciadv.adk9004

An orally active entry inhibitor of influenza A viruses protects mice and synergizes with oseltamivir and baloxavir marboxil

Irina Gaisina 1,2,*,†, Ping Li 3,†, Ruikun Du 4,†, Qinghua Cui 4,†, Meiyue Dong 4, Chengcheng Zhang 3, Balaji Manicassamy 5, Michael Caffrey 6, Terry Moore 1,7, Laura Cooper 8,*, Lijun Rong 2,8,*
PMCID: PMC10889430  PMID: 38394202

Abstract

Seasonal or pandemic illness caused by influenza A viruses (IAVs) is a major public health concern due to the high morbidity and notable mortality. Although there are several approved drugs targeting different mechanisms, the emergence of drug resistance calls for new drug candidates that can be used alone or in combinations. Small-molecule IAV entry inhibitor, ING-1466, binds to hemagglutinin (HA) and blocks HA-mediated viral infection. Here, we show that this inhibitor demonstrates preventive and therapeutic effects in a mouse model of IAV with substantial improvement in the survival rate. When administered orally it elicits a therapeutic effect in mice, even after the well-established infection. Moreover, the combination of ING-1466 with oseltamivir phosphate or baloxavir marboxil enhances the therapeutic effect in a synergistic manner. Overall, ING-1466 has excellent oral bioavailability and in vitro absorption, distribution, metabolism, excretion, and toxicity profile, suggesting that it can be developed for monotherapy or combination therapy for the treatment of IAV infections.

ING-1466, an influenza A virus entry inhibitor, is potent and synergistic with oseltamivir and baloxivir marboxil in mice.

INTRODUCTION

Influenza viruses contain a single-stranded, negative-sense, segmented viral RNA genome that belongs to the Orthomyxoviridae family (1). There are four genera within the Orthomyxoviridae family: influenzavirus A, B, C, and D (2). As a human respiratory pathogen, the influenza A virus (IAV) and influenza B virus cause seasonal epidemics of influenza, (3) which result in 290,000 to 650,000 influenza-associated respiratory deaths a year globally (World Health Organization). Four global pandemics (1918, 1957, 1968, and 2009) were caused by antigenic shifts due to the reassortment of IAV segmented genomes, which posed serious threats to public health (4, 5). Furthermore, influenza A is found in many domestic species, such as swine and poultry, allowing these animals to serve as reservoirs for pandemic influenza viruses. Because of the risks associated with these viruses, treatments are needed to mitigate their impact on human health.

Vaccines and therapeutic drugs are the two options used against influenza infections. Active immunization is the principal prophylactic for controlling influenza infections (6–8). Vaccine efficacy is also limited in most common seasonally circulating strains. According to the Centers for Disease Control and Prevention findings, the seasonal influenza vaccine is typically 40 to 60% effective at reducing the risks associated with influenza illness. Despite vaccination, some individuals, such as immunocompromised people, can still get severe influenza infections. Furthermore, existing vaccines have limited efficacy in the early phase of a pandemic, warranting a need for small-molecule therapeutics (9, 10).

Three approved classes of anti-influenza small-molecule antiviral drugs have been clinically used: viral matrix protein 2 (M2) inhibitors amantadine and rimantadine; neuraminidase inhibitors oseltamivir phosphate (OSP), zanamivir, peramivir, and laninamivir; polymerase inhibitors baloxavir marboxil (BXM) targeting the acidic protein endonuclease and viral RNA-dependent RNA polymerase inhibitor favipiravir, which prevents the inclusion of nucleotides for viral RNA replication, leading to an increased mutation frequency, and lethal mutagenesis (11–16). However, most current seasonal H1N1 and H3N2 strains are resistant to the M2 inhibitors and OSP (17–20). BXM is a first-in-class medication with Food and Drug Administration (FDA) approval to treat both influenza A and B viral infections (21). However, viral variants harboring the I38T substitution render BXM less effective in clinical practice (22, 23). Therefore, there is an urgent need for additional therapeutic agents with different mechanisms of action that can effectively treat influenza viruses, either alone or in combination with other drugs.

Hemagglutinin (HA) is essential for viral attachment and membrane fusion, which makes it an attractive therapeutic target (24, 25). HA inhibitors have the ability to block the initial step in the viral life cycle, leading to viral neutralization. Small molecules have the ability to bind to the HA protein and inhibit HA-mediated fusion. Previously, our group screened the Chembridge Small Molecule Library of 19,200 compounds and identified 2,6-dichloro-N-(1-isopropyl-piperidin-4-yl)benzamide (designated as CBS1117) as the initial hit; subsequently, our medicinal chemistry and hit-to-lead optimization efforts produced the improved inhibitor ING-1466 (fig. S1, compound 16 from previous study) (24–26). This lead inhibitor is potent and selective against group 1 IAVs. Furthermore, this compound showed synergistic activity when combined with OSP in vitro against influenza H1N1 A/Puerto Rico/8/1934 (PR8) (24). ING-1466 is predicted to bind directly to the stalk region of the group 1 HA like other previously identified influenza entry inhibitors (27, 28). In this study, we report on the in vivo efficacy of the HA-targeting IAV infection inhibitor ING-1466 on infections induced in mice by H1N1. We assessed the oral efficacy of ING-1466 and its synergistic effect with OSP and BXM in a mouse model. Our results highlight the preventive and therapeutic efficacy of ING-1466, and the potential benefits for combination therapy of drugs with different mechanisms of action.

RESULTS

To progress as therapeutic agents, potential antiviral agents need to be assessed for their ability to provide protection in an infected and susceptible animal model (29, 30). An important first step in the evaluation of a candidate drug is the determination of the therapeutic index, which is first determined in an in vitro setting, followed by in vivo evaluation. Assessment of toxicity allows for dose-range–finding studies, which is a necessary step toward the determination of in vivo efficacy. ING-1466 has promising in vitro efficacy with a median effective concentration (EC50) value of 0.18 μM against IAV H1N1 (PR8-NS1-Gluc); moreover, at a concentration of 1 μM, it caused a 90% reduction in viral titers when tested against wild-type (WT) H1N1 (A/Puerto Rico/8/1934) (24). ING-1466 has an EC50 value of 0.013 μM against pdm2009 H1N1 (A/Brownsville/39H/2009), but it is less effective against a tissue culture adapted H1N1 PR8 virus (EC50 3.10 μM) that has a V115M mutation in the HA2 (ATCC VR-1469) (fig. S2). Noteworthy, this compound showed no cytotoxicity at 100 μM in both A549 and HepG2 cells, which provided a selectivity index (SI = CC50/EC50) of >100.

Compound ING-1466 has favorable absorption, distribution, metabolism, excretion, and toxicity properties

Preliminary pharmacokinetic (PK) studies provide insight into drug exposure, and this information assists in the design of dose levels and schedules for in vivo antiviral efficacy studies. Encouraged by the potent in vitro activity of ING-1466, we had previously conducted early PK studies in BALB/c mice (24) and based the design of our experiments on the data obtained (all in vitro and in vivo PK studies were performed by Pharmaron Inc.). Previously, we showed that the PK analysis of the plasma and liver levels of ING-1466 following intraperitoneal administration or oral (PO) gavage indicated that the maximal levels are rapidly reached, with Tmax ranging from 15 to 60 min (24). The Cmax level in the plasma with the 10 mg/kg intraperitoneal dose was found to be 1140 ng/ml or 3.12 μM. We determined that ING-1466 is systemically available following both intraperitoneal and PO administration (fig. S3). Another important finding is that, during the PK evaluations and subsequent acute toxicity studies, no adverse clinical signs were observed in the mice that received 50 mg/kg of ING-1466. All mice that received 100 mg/kg exhibited slight hypoactivity, and the mice that received 200 and 400 mg/kg exhibited hypoactivity and ataxia, or even body distortion. All adverse clinical signs were relieved in 4 hours. No bodyweight loss or deaths were observed in any of the treatment groups (fig. S4).

In vitro PK studies include absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies of drug candidates. The ADMET properties of ING-1466, are summarized in Table 1. Inhibitor ING-1466 showed notable metabolic stability as indicated by the high percentage of compound remaining after 60 min of incubation in human plasma, liver microsomes, and hepatocytes. It displayed median inhibitory concentration (IC50) values >50.0 μM against cytochrome P450 (CYP) CYP1A2, CYP2C9, CYP2C19, and CYP3A4, the most common CYP isoforms in drug metabolism, suggesting that this compound will not drastically alter the metabolism of other xenobiotics or endogenous compounds that are substrates for the most common CYP isoforms. CYP2D6, which contributes to 2% of the overall hepatic CYP450 isoforms, had a lower IC50 = 6.04 μM (dextromethorphan substrate). Inhibition of CYP2D6 could cause potential drug-drug interactions; therefore, it will be advisable to further examine inhibitory effect of ING-1466 on CYP2D6, using different test substrates. CYP2D6 Inhibition is not an insurmountable issue, as many FDA-approved drugs, such as fluoxetine, cause inhibition of CYP2D6 (31, 32).

Table 1. Summary ADMET properties of ING-1466.

ADMET characteristics Observed activity
Solubility (vehicle) 292 μM (PBS, pH = 7.4); 306 μM (FaSSGF); 265 μM (FeSSIF); 327 μM (FaSSIF)*
Permeability PAMPA −LogPe = 4.92, with 94.4% recovery†
Human plasma protein binding 21.3% bound, 87.4% recovery
Plasma stability HP: 109% remaining‡ t1/2 = 2936§ MP: 90.13% remaining‡; t1/2 = 744§
Liver microsomal stability HLM: 94.4% remaining‡; t1/2 = 544; Clint = 2.55§ MLM: 97.1% remaining‡, t1/2 = 680; Clint = 2.04§
Human hepatocytes stability 98.4% remaining║
CYP P450 inhibition IC50 > 50 μM (CYP1A2, CYP2C9, CYP2C19, and CYP3A4); CYP2D6 IC50 = 6.04 μM
Cytotoxicity CC50 > 100 μM (HepG2 and A549)
Kv11.1 (hERG) inhibition IC50 = 7.51 μM¶
hNav1.5 inhibition IC50 = 22.4 μM
Cav1.2 inhibition 1.83% inhibition @ 30.0 μM
Ames Negative
Open in a new tab

*FaSSGF, fasted state simulated gastric fluid; FeSSIF, fed state simulated intestinal fluid; FaSSIF, fasted state simulated intestinal fluid.

†Compounds that have a −Log Pe < 6 are classified as having high permeability.

‡After 60 min of incubation with mouse plasma (MP), human plasma (HP), mouse liver microsomes (MLM), or human liver microsomes (HLM) at 2 μM final concentration.

§t1/2 in minutes; Clint = intrinsic clearance (μl/min per milligram protein).

║After 60 min of incubation at 2 μM final concentration.

¶Considered as moderate in the range: 1 μM < IC50 < 10 μM.

The potential inhibitory effect of ING-1466 on the human Ether-à-go-go related gene (hERG) that encodes a potassium ion channel Kv11.1 evaluated in transfected HEK293 cells by using a manual patch-clamp assay. On the basis of these results, this compound ranked as a moderate inhibitor with an IC50 value of 7.51 μM. When compared to the plasma Cmax of the drug delivered at the 50 mg/kg dose in BALB/c mice (24), the hERG inhibition raised the concern of potential cardiac toxicity from drug-induced fatal arrhythmias. To further evaluate the ability of ING14–66 to induce drug cardiac toxicity, we screened two additional voltage-gated ion channels, Nav1.5 and Cav1.2, which are essential for the development of arrhythmias (33, 34). ING-1466 is not a potent inhibitor of either Nav1.5 (IC50 = 22.4 μM) or Cav1.2 (IC50 > 30.0 μM).

The mutagenic potential of ING-1466 was evaluated by measuring its ability to induce reverse mutations at the His locus of Salmonella typhimurium (TA98, TA100, TA1535, and TA1537) and Escherichia coli WP2 uvrA (pKM101) with or without metabolic activation by the liver S9 fraction. No effects were seen in the Ames test, which further supports the favorable outcome of the earlier reverse mutation study. Kinetic solubility of ING-1466 in different media and a parallel artificial membrane permeability assay (PAMPA) permeability both fell into the range ideal for oral absorption. Because of the favorable early PK and toxicity studies results, we progressed to experiments to determine the activity of ING-1466 in an H1N1 mouse model.

ING-1466 substantially reduces virus load in mice under preventive and therapeutic treatments

In a mouse model, reporter influenza H1N1 PR8 virus, which carries a Gaussia luciferase gene (PR8-NS1-Gluc), was established for the evaluation of antiviral therapeutics (35, 36). On the basis of the previously published method, mice were intranasally infected with 1000 median tissue culture infectious dose (TCID50) of PR8-NS1-Gluc (37), and viral luciferase signal in the lungs were measured on days 2 and 4 using ex vivo imaging. Mice infected with PR8-NS1-Gluc at 1000 TCID50 showed weight loss but 100% survival after 4 days of infection. To establish the ability of ING-1466 to reduce viral luciferase signal in vivo, mice were administered the drug via intraperitoneal injection (Fig. 1A). Intraperitoneal administration of potential pharmacological agents results in faster and more complete absorption compared to oral. In addition, two treatment doses of 25 and 50 mg/kg per day were selected on the basis of the in vitro EC90 values and previously collected PK data for this compound (24). The doses were given 2 hours before infection, and all treatment groups benefitted from significantly reduced viral luciferase signal as compared to that of the vehicle control group (Fig. 1B). This indicated that ING-1466 can produce a strong inhibitory effect on infection in vivo. A substantial therapeutic effect was observed in mice for which treatment with ING-1466 started at 6 or 24 hours after infection. In these groups, the effect was very similar to that produced by a 30 mg/kg per day dose of OSP, which was used as a positive control (Fig. 1, C and D). These results suggest that when ING-1466 is administered via intraperitoneal injection, it can elicit therapeutic effects in mice, even when an infection is well established.

Fig. 1. Preventive and therapeutic effects of ING-1466.

Fig. 1.

Open in a new tab

(A) Schematic of ING-1466 preventive and therapeutic effects study using Gaussia luciferase (Gluc) ex vivo imaging. BALB/c mice were infected with 1000 TCID50 of PR8-NS1-Gluc virus and treated with ING-1466 at doses of 25 or 50 mg/kg per day by intraperitoneal injection at −2 hours (B), +6 hours (C), +24 hours (D), and the levels of luciferase in the lungs were determined. Each data point represents Mean ± SD (n = 5), and the statistical significance was calculated using one-way analysis of variance (ANOVA); ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05.

On the basis of the promising data with H1N1 PR8-NS1-Gluc, we decided to use bioluminescent live imaging (BLI) to further analyze the efficacy of ING-1466. The Gaussia luciferase gene was replaced with a firefly luciferase gene (PR8-NS1-Fluc) that can be used for BLI of mice. When mice are inoculated with a sublethal dose of 1000 TCID50 of PR8-NS1-Fluc, they accurately reflected viral replication in vivo. Mice infected with PR8-NS1-Fluc at 1000 TCID50 showed weight loss but 100% survival after 4 days of infection. Furthermore, when this system was challenged with OSP, the results accurately reflected the in vivo efficacy of OSP (37). Consequently, we used the PR8-NS1-Fluc mouse model for the determination of the in vivo efficacy of ING-1466.

To assess the potential for this compound to be used orally, mice were administered a 25 or 50 mg/kg dose of ING-1466 by the PO or intraperitoneal routes 2 hours before infection. Control drug OSP was administered via intraperitoneal injection and PO at a previously optimized dose of 30 mg/kg per day. Mice were intranasally inoculated with 1000 TCID50 of PR8-NS1-Fluc, and the live mice were imaged on days 2 and 4. The levels of the luciferase signal of treated mice were substantially reduced compared to those detected in the control groups for days 2 and 4 after infection (Fig. 2A). The mice given the drug by the PO route had a similar reduction in luciferase signal, compared to the intraperitoneal injection group. These results demonstrate that ING-1466 oral efficacy is similar to that of intraperitoneal injection when given as a prophylactic treatment (Fig. 2, B and C). Thus, ING-1466 has good oral bioavailability as predicted by the preliminary PK data.

Fig. 2. The preventive effects of ING-1466 and OSP using a bioluminescence imaging system.

Fig. 2.

Open in a new tab

(A) Schematic of ING-1466 preventive effects study using Firefly luciferase (Fluc) bioluminescence. (B) BALB/c mice were treated with 25 or 50 mg/kg per day of ING-1466 administered by intraperitoneal (IP) injection and PO, 2 hours before being intranasally inoculated with 1000 TCID50 of PR8–NS1–Fluc virus. The 30 mg/kg per day dose of oseltamivir phosphate was used as the positive control. The mice were imaged on days 2 and 4. (C) Values of photon flux for each dosage group; each data point represents Mean ± SD (n = 3). The statistical significance was calculated using one-way ANOVA; ***P ≤ 0.001.

Because of the success of prophylactic oral gavage, we used the same method to explore whether ING-1466 could produce a strong inhibitory effect when used as a therapeutic agent starting at 6 or 24 hours after infection. In addition, the control drug OSP was administered by the PO route at a dose of 30 mg/kg per day. The 50 mg/kg per day dose of ING-1466 caused a significant reduction in luciferase signal compared to the vehicle when treatment started at either 6 or 24 hours after infection on days 2 and 4 (Fig. 3A). The 25 mg/kg per day dose of ING-1466 caused a significant reduction in luciferase signal compared to the vehicle when treatment started at either 6 or 24 hours after infection on day 4 (Fig. 3, B and C). These results suggest that when ING-1466 is administered orally it can elicit a therapeutic effect in mice, even after the infection is well established.

Fig. 3. The therapeutic effects of ING-1466 and OSP using a bioluminescence imaging system.

Fig. 3.

Open in a new tab

(A) Schematic of ING-1466 therapeutic effects study using Firefly luciferase (Fluc) bioluminescence. (B) BALB/c mice were treated with 25 or 50 mg/kg per day of ING-1466 administered by PO, 6 and 24 hours after being intranasally inoculated with 1000 TCID50 of PR8–NS1–Fluc virus. The 30 mg/kg per day dose of oseltamivir phosphate was used as the positive control. The mice were imaged on days 2 and 4. (C) Values of photon flux for each dosage group; each data point represents Mean ± SD (n = 3). The statistical significance was calculated using one-way ANOVA; ***P ≤ 0.001.

ING-1466 alone or in combination with other drugs protects mice from IAV lethal infection

To evaluate the prophylactic potency of ING-1466 in a lethal infection mode, mice were inoculated with 5 × LD50 (50% lethal dose) of WT influenza A/Puerto Rico/8/1934 (PR8) virus and treated with the antiviral agent 2 hours before infection via intraperitoneal injection. Survival rates with ING-1466 treatment at 12.5, 25, and 50 mg/kg per day were 20, 20, and 60%, respectively (Fig. 4). Compared to the vehicle group, all groups treated with ING-1466 had dose-dependent prolonged survival time. This positive outcome indicates that ING-1466 given as monotherapy improves the survival rate in an otherwise lethal IAV infection.

Fig. 4. Monotherapy of ING-1466 protects mice from IAV lethal infection.

Fig. 4.

Open in a new tab

BALB/c mice (n = 10 per group) were intranasally inoculated with 5× LD50 of PR8 virus and treated by intraperitoneal injection with ING-1466 at doses of 12.5, 25, or 50 mg/kg per day. The body weights (A) and survival times (B) were monitored daily. Differences between groups were analyzed by log-rank test for survival. ***P ≤ 0.001; *P ≤ 0.05; compared to vehicle group.

To investigate the possibility of ING-1466 having an enhanced effect when combined with OSP, mice infected with a lethal dose of WT PR8 IAV were treated with each drug alone and in combination. The signs of virus infection, such as body weight loss and survival, were monitored daily. In mice lethally infected with IAV, treatment with OSP caused substantial protection in a dose-dependent manner. Thus, survival rates of mice treated with OSP at 5, 10, and 20 mg/kg per day were 10, 50, and 90%, respectively, compared to 20% with ING-1466 at 25 mg/kg per day (Fig. 5, Table 2). The combination treatment of ING-1466 (25 mg/kg per day) with OSP given at 5 mg/kg per day increased survival up to 90%, while the combination with OSP at 10 mg/kg per day resulted in 100% survival. This strongly suggests that the drug combination protects against mortality more effectively than monotherapy alone. Recovery trends observed during virus infection indicated that ING-1466 and OSP provide a substantial synergistic antiviral effect.

Fig. 5. Combination therapy of ING-1466 with OSP protects mice from IAV lethal infection.

Fig. 5.

Open in a new tab

BALB/c mice were inoculated intranasally with 5× LD50 of PR8 virus. For antiviral treatment groups, mice were treated by intraperitoneal injection with indicated doses of ING-1466 and OSP, alone or in combination. Body weights (A) and survival times (B) of mice were monitored daily. Differences between groups were analyzed by log-rank test for survival. ***P ≤ 0.001 compared to vehicle treated group. §§P < 0.01; §§§P ≤ 0.001 compared to ING-1466 25 mg/kg–treated group. #P ≤ 0.05; ###P ≤ 0.001 compared to the same dosage of OSP groups.

Table 2. Treatment of IAV infection of mice with ING-1466 and OSP, used alone or in combination.

Differences between groups were analyzed by log-rank test for survival. ***P ≤ 0.001 compared to vehicle treatment; §§P ≤ 0.01; §§§P ≤ 0.001 compared to ING-1466 (25) group; #P ≤ 0.05; ###P ≤ 0.001 compared to the same dosage of OSP groups.

Compound 1 (mg/kg per day) Compound 2 (mg/kg per day) No. of survivors/total no. Median survival times (days)
Vehicle 0/10 6
ING-1466 (25) 2/10 *** 8.5
OSP (5) 1/10 *** 8
OSP (10) 5/10 ***
OSP (20) 9/10 ***
ING-1466 (25) OSP (5) 9/10 ***, §§, ###
ING-1466 (25) OSP (10) 10/10 ***, §§§, #
ING-1466 (25) OSP (20) 10/10 ***, §§§, #
Open in a new tab

The therapeutic effect of ING-1466 in combination with BXM was also investigated in the same model. Infected mice were treated with either ING-1466 (25 mg/kg per day), BXM (0.05, 0.1, 0.25, or 0.5 mg/kg per day), or both. Again, in the BXM-treated groups, mortality prevention was dose-dependent. Although survival rates among the mice treated with BXM alone at 0.1 and 0.25 mg/kg per day were only 10 and 20%, respectively, the combination treatment with the same doses of BXM and ING-1466 at 25 mg/kg per day provided 50 and 100% survival, respectively (Fig. 6, Table 3). Moreover, weight loss due to influenza infection was substantially halted in the groups receiving the drug combination treatment compared to the monotherapy groups. The prevention of weight loss suggests that ING-1466 enhanced the therapeutic effect of BXM in mice in a synergistic manner.

Fig. 6. Combination therapy of ING-1466 with BXM protects mice from IAV lethal infection.

Fig. 6.

Open in a new tab

BALB/c mice were inoculated intranasally with 5× LD50 of PR8 virus. For antiviral treatment groups, mice were treated by intraperitoneal injection with indicated doses of ING-1466 and baloxavir marboxil, alone or in combination. Body weights (A) and survival times (B) of mice were monitored daily. Differences between groups were analyzed by log-rank test for survival. ***P ≤ 0.001; *P ≤ 0.05; compared to vehicle treatment; §§§P ≤ 0.001; compared to ING-1466, 25 mg/kg group; ##P ≤ 0.01; ###P ≤ 0.001; compared to the same dosage of BXM groups.

Table 3. Treatment of IAV infection of mice with ING-1466 and BXM, used alone or in combination.

Differences between groups were analyzed by log-rank test for survival. ***P ≤ 0.001; *P ≤ 0.05; compared to vehicle treatment; §§§P ≤ 0.001 compared to ING-1466 (25) group; #P ≤ 0.05; ##P ≤ 0.01; ###P ≤ 0.001; compared to the same dosage of BXM groups.

Compound 1 (mg/kg per day) Compound 2 (mg/kg per day) No. of survivors/total no. Median survival times (days)
Vehicle 0/10 7
ING-1466 (25) 0/10* 7
BXM (0.05) 0/10 7
BXM (0.1) 1/10 *** 8.5
BXM (0.25) 2/10 *** 10
BXM (0.5) 10/10***
ING-1466 (25) BXM (0.05) 2/10 ***, §§§, ### 10
ING-1466 (25) BXM (0.1) 5/10 ***, §§§, ## 12
ING-1466 (25) BXM (0.25) 10/10 ***, §§§, ###
ING-1466 (25) BXM (0.5) 10/10 ***, §§§, ###
Open in a new tab

DISCUSSION

Despite the availability of annual influenza vaccination and FDA-approved therapeutic agents, treatment remains a challenge. The most effective IAV inhibitors require patients to start the antiviral treatment very soon after illness onset, and these inhibitors, through repetitive use, have developed resistance (38). ING-1466 is an entry inhibitor, which has a different mechanism of action for inhibiting influenza A, with respect to currently approved influenza antivirals. HA plays an important role in influenza virus receptor binding and membrane fusion, making it a promising drug target (39). Drug-like PK/ADMET properties and the remarkable antiviral potency of ING-1466 in cell culture against certain IAV strains supported the advancement of this compound toward in vivo efficacy studies. In this study, we demonstrated that this first-in-class IAV inhibitor that directly targets HA is efficacious against the influenza A/Puerto Rico/8/1934 virus infection in vivo. In pilot studies using PR8-NS1-Gluc, ING-1466 decreased viral luciferase signal in the lungs of infected mice. Moreover, the anti-influenza effect of ING-1466 on day 4 was similar to that of OSP, an FDA-approved prescription drug for use as a prophylactic agent and for the treatment of symptoms of influenza. Although this in vivo study was limited by the inclusion of only one strain of virus, while our previous in vitro studies indicated that ING-1466 had activity not only against influenza virus H1N1 (A/Puerto Rico/8/1934) but also against H5N1 (A/Vietnam/1203/2004) and the oseltamivir-resistant strains carrying NA/H274Y mutations (24).

The emergence of resistance has been a major obstacle to influenza monotherapy. Thus, combination therapy with antiviral agents that are mechanistically different has been considered an effective treatment regimen against influenza infection (40). Antiviral combination therapy of two or more anti-influenza drugs with different mechanisms of action may generate synergistic efficacy (41). For example, BXM was just FDA approved in 2018 and has already shown a resistance rate of 7.9 to 9.7% with monotherapy (22). Clinical reports have linked this resistance to I38X substitutions in the polymerase acid protein (PA; I38T, I38S, and I38V). However, BXM, combined with OSP, obstructed the emergence of PA-I38X in a mouse model (42). This combination also showed synergy in improving survival duration and reducing pathological changes in the lungs of mice (43).

Furthermore, combination therapy may decrease the dosage of drugs, and this strategy is beneficial in preventing the emergence of resistance and reducing side effects (44). Oseltamivir and favipiravir (influenza polymerase inhibitor) have dose-related synergistic effects against multiple IAV strains in vitro and in vivo. Suboptimum doses of oseltamivir combined with favipiravir improved survival rate and body weight over monotherapy alone (45). However, with combinations of antivirals having the same target, such as oseltamivir and zanamivir, only additive antagonistic activities against H1N1 infection are observed (46–48). In our study, we found that ING-1466, which is an entry inhibitor, when combined with OSP or BXM, produced statistically significant improvements in survival rate. Both drugs have differentiated mechanisms of action, which is beneficial in enhancing the antiviral impact of combination therapy. The combined use of agents may contribute to prophylaxis and, more importantly, may create a more efficient treatment for future pandemics.

In addition to the antiviral efficacy of ING-1466 in lethal infection, the efficacy of ING-1466 administered by oral gavage was evaluated using a noninvasive imaging system. We found that ING-1466 is orally active in treating IAV infections, and these results agree with our previously reported PK data that predicted oral bioavailability (24). Notably, the oral route of administration is the gold standard for drug delivery in the pharmaceutical industry and is the safest, most economical, and most convenient method for treating systemic diseases (49, 50). Because both OSP and BXM are orally bioavailable drugs, we plan to repeat the ING-1466 combination with BXM or OSP using oral dosing.

It is important to note that while the potency and in vivo efficacy are promising, ING14–66 is still an investigational compound in the early stages of drug development. A major concern for this compound is the hERG/plasma Cmax ratio. However, drugs such as verapamil and ranolazine are strong inhibitors of hERG (Kv11.1) but do not induce arrhythmias (51). The lack of potent inhibition of Nav1.5 and Cav1.2 is optimistic. However, the cardiac toxicity concern needs to be further evaluated in an in vivo model. The hERG inhibition is a potential area of improvement that could benefit from additional SAR in the future.

Overall, intraperitoneal or PO dosing with ING-1466 rapidly reduced luciferase signals and potently protected mice from a lethal dose of IAV. It is remarkable that ING-1466, in combination with BXM or OSP, displayed an enhanced therapeutic effect with respect to monotherapy. The substantial oral bioavailability, monotherapeutic antiviral efficacy, and enhanced therapeutic effect in combination with other anti-influenza agents allow us to conclude that ING-1466 can provide additional therapeutic options in the mitigation of seasonal and pandemic IAVs.

MATERIALS AND METHODS

Ethics statement

The animal studies were performed under BSL-2 conditions, and study protocols for animal experiments were approved by the Institutional Animal Care and Use Committee of Shandong University of Traditional Chinese Medicine (Approval: SDUTCM20211230001).

Viruses and compounds

IAVs (PR8) and recombinant reporter virus PR8 with an NS segment carrying Gaussia luciferase PR8-NS1-Gluc or firefly luciferase PR8-NS1-Fluc were rescued and preserved at −80°C as previously described (32, 33). OSP was purchased from MedChemExpress. BXM was purchased from Cayman Chemical Inc. ING-1466 was synthesized at the University of Illinois Chicago (24). Compounds were formulated for intraperitoneal and PO drug delivery in 3% dimethyl sulfoxide (DMSO), 30% PEG400, and 57% water.

PR8-NS1-Gluc sublethal infection mouse model

Specific pathogen–free, 4- to 6-week-old BALB/c mice (Beijing Vital River Laboratory Animal Technology Co., Ltd) were used in all experiments. Female BALB/c mice were randomly divided into groups with 10 mice in each group. For infection, mice were anesthetized with isoflurane and intranasally inoculated with 1000 TCID50 of PR8-NS1-Gluc virus diluted in 30 μl of phosphate-buffered saline (PBS) and treated with the compounds at indicated time points before or after virus infection. OSP at the dose of 30 mg/kg per day was used as the positive control. All treatments were given twice daily via intraperitoneal injection for 5 days. Five mice from each group were euthanized on day 2 and the remaining five animals on day 4. The lungs were collected to measure viral luciferase signals (31).

Ex-vivo imaging

Lung homogenates were prepared in PBS and centrifuged. Viral load in supernatants was determined by the luciferase signal levels using a BioLux Gaussia Luciferase Assay Kit (NEB), as previously described (40). Briefly, 20 μl of lung homogenate (appropriate dilution can be applied) was added to 50 μl of luciferase substrate, and the relative lighting units were detected using a Sirius L Tube Luminometer (Berthold Detection Systems).

PR8-NS1-Fluc sublethal infection noninvasive imaging mouse model

Female BALB/c mice (three randomly selected mice per group) were anesthetized with isoflurane and intranasally inoculated with 1000 TCID50 of PR8–NS1–Fluc virus in 30 μl of PBS. For antiviral treatment, 25 or 50 mg/kg per day of ING-1466 was administered orally for 5 days starting at 2 hours before infection or 6 and 24 hours after infection. Noninvasive in vivo imaging was then carried out on days 2 and 4. Mice were intraperitoneally injected with firefly luciferase substrate d-Luciferin (PerkinElmer, Waltham, MA, USA) at 150 mg/kg, and imaged by Xenogen IVIS-200 10 min following substrate administration. All images were acquired and processed with the Living Image software (version 4.4) and the same bioluminescence scale was used for each image (33).

PR8 lethal infection mouse model

Female BALB/c mice were randomly divided into groups with 10 mice in each group. For infection, mice were anesthetized with isoflurane and challenged with 5× LD50 of PR8 virus in 30 μl of PBS. For antiviral treatment groups, mice were treated via intraperitoneal injection with indicated doses of ING-1466, OSP, and BXM alone or in combination. The compounds were administered intraperitoneally b.i.d. for 5 days starting at 2 hours before infection. The body weight and survival of mice were monitored daily. Mice showing >20% weight loss were humanely euthanized according to humane endpoints and considered to have succumbed to the infection.

ADMET assays

These studies were conducted in Pharmaron Inc. using the approved protocols. Detailed methods are in the Supplementary Materials.

Safety assessment

BALB/c mice were randomly assigned to five groups, with each group containing six mice (three male and three female). All mice were fasted for 12 hours and then dosed with ING-1466 at 50, 100, 200, or 400 mg/kg dose or vehicle only (3% DMSO, 30% PEG400, and 67% water) administered by oral gavage. The animals were observed for adverse clinical signs for 12 hours, and survival times and body weights were monitored for 14 days.

Statistical analysis

Statistical analyses were performed using GraphPad Prism version 8.0.0 software (GraphPad Software Inc., San Diego, CA). Comparisons of luminescence in the bioluminescent images of the mice and their lungs were analyzed by one-way analysis of variance (ANOVA). A comparison of the survival curves was done by the log-rank test. P values <0.05 were considered significant.

Acknowledgments

Funding: This work was supported by National Institutes of Health grant R42AI155039-02 (L.R.).

Author contributions: Conceptualization: L.R. and I.G. Methodology: R.D., P.L., Q.C., M.D., C.Z., L.C., L.R., and I.G. Investigation: I.G., L.C., R.D., P.L., Q.C., M.D., and C.Z. Supervision: L.R. and I.G. Writing—original draft: L.C., I.G., and P.L. Writing—review and editing: L.R., L.C., I.G., B.M., M.C., and T.M.

Competing interests: The authors declare the following competing financial interest(s): L.R. is the owner of Chicago BioSolutions Inc., which develops drugs. I.G. is employed by Chicago BioSolutions Inc. Filed patents pertaining to the results presented in the paper: “Inhibitors of Influenza Viral Entry” U.S. Serial No. 17/735,235 by L.R. and I.G.. The other authors declare that they have no competing interests. These materials reflect only the personal views of the authors and may not reflect the views of his/her/their employer(s).

Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.

Supplementary Materials

This PDF file includes:

Material and Methods for Supplement and ADMET Assays

Figs. S1 to S4

Tables S1 to S15

References

sciadv.adk9004_sm.pdf (686.9KB, pdf)

REFERENCES AND NOTES

  • 1.Sethy B., Hsieh C. F., Lin T. J., Hu P. Y., Chen Y. L., Lin C. Y., Tseng S. N., Horng J. T., Hsieh P. W., Design, synthesis, and biological evaluation of itaconic acid derivatives as potential anti-influenza agents. J. Med. Chem. 62, 2390–2403 (2019). [DOI] [PubMed] [Google Scholar]
  • 2.Yassine H. M., Lee C. W., Gourapura R., Saif Y. M., Interspecies and intraspecies transmission of influenza A viruses: Viral, host and environmental factors. Anim. Health Res. Rev. 11, 53–72 (2010). [DOI] [PubMed] [Google Scholar]
  • 3.Klimov A. I., Garten R., Russell C., Barr I. G., Besselaar T. G., Daniels R., Engelhardt O. G., Grohmann G., Itamura S., Kelso A., McCauley J., Odagiri T., Smith D., Tashiro M., Xu X., Webby R., Wang D., Ye Z., Yuelong S., Zhang W., Cox N., Writing Committee of the World Health Organization Consultation on Southern Hemisphere Influenza Vaccine Composition for 2012 , WHO recommendations for the viruses to be used in the 2012 Southern Hemisphere Influenza Vaccine: Epidemiology, antigenic and genetic characteristics of influenza A(H1N1)pdm09, A(H3N2) and B influenza viruses collected from February to September 2011. Vaccine 30, 6461–6471 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Tomescu A. I., Robb N. C., Hengrung N., Fodor E., Kapanidis A. N., Single-molecule FRET reveals a corkscrew RNA structure for the polymerase-bound influenza virus promoter. Proc. Natl. Acad. Sci. U.S.A. 111, E3335–E3342 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Basu A., Antanasijevic A., Wang M., Li B., Mills D. M., Ames J. A., Nash P. J., Williams J. D., Peet N. P., Moir D. T., Prichard M. N., Keith K. A., Barnard D. L., Caffrey M., Rong L., Bowlin T. L., New small molecule entry inhibitors targeting hemagglutinin-mediated influenza a virus fusion. J. Virol. 88, 1447–1460 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Das K., Aramini J. M., Ma L. C., Krug R. M., Arnold E., Structures of influenza A proteins and insights into antiviral drug targets. Nat. Struct. Mol. Biol. 17, 530–538 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Horimoto T., Kawaoka Y., Designing vaccines for pandemic influenza. Curr. Top. Microbiol. Immunol. 333, 165–176 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Tscherne D. M., Garcia-Sastre A., Virulence determinants of pandemic influenza viruses. J. Clin. Invest. 121, 6–13 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Kemble G., Greenberg H., Novel generations of influenza vaccines. Vaccine 21, 1789–1795 (2003). [DOI] [PubMed] [Google Scholar]
  • 10.Poland G. A., The 2009-2010 influenza pandemic: Effects on pandemic and seasonal vaccine uptake and lessons learned for seasonal vaccination campaigns. Vaccine 28 (Suppl. 4), D3–D13 (2010). [DOI] [PubMed] [Google Scholar]
  • 11.Alves Galvao M. G., Rocha Crispino Santos M. A., da Cunha A. J. A., Amantadine and rimantadine for influenza A in children and the elderly. Cochrane Database Syst. Rev. 2014, CD002745 (2014). [DOI] [PubMed] [Google Scholar]
  • 12.Noshi T., Kitano M., Taniguchi K., Yamamoto A., Omoto S., Baba K., Hashimoto T., Ishida K., Kushima Y., Hattori K., Kawai M., Yoshida R., Kobayashi M., Yoshinaga T., Sato A., Okamatsu M., Sakoda Y., Kida H., Shishido T., Naito A., In vitro characterization of baloxavir acid, a first-in-class cap-dependent endonuclease inhibitor of the influenza virus polymerase PA subunit. Antiviral Res. 160, 109–117 (2018). [DOI] [PubMed] [Google Scholar]
  • 13.Loukotkova L., Basavarajappa M., Lumen A., Roberts R., Mattison D., Morris S. M., Fisher J., Beland F. A., Gamboa da Costa G., Pharmacokinetics of oseltamivir phosphate and oseltamivir carboxylate in non-pregnant and pregnant rhesus monkeys. Regul. Toxicol. Pharmacol. 112, 104569 (2020). [DOI] [PubMed] [Google Scholar]
  • 14.Toots M., Plemper R. K., Next-generation direct-acting influenza therapeutics. Transl. Res. 220, 33–42 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Furuta Y., Takahashi K., Kuno-Maekawa M., Sangawa H., Uehara S., Kozaki K., Nomura N., Egawa H., Shiraki K., Mechanism of action of T-705 against influenza virus. Antimicrob. Agents Chemother. 49, 981–986 (2005). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Baranovich T., Wong S. S., Armstrong J., Marjuki H., Webby R. J., Webster R. G., Govorkova E. A., T-705 (favipiravir) induces lethal mutagenesis in influenza A H1N1 viruses in vitro. J. Virol. 87, 3741–3751 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Pokorna J., Pachl P., Karlukova E., Hejdanek J., Rezacova P., Machara A., Hudlicky J., Konvalinka J., Kozisek M., Kinetic, thermodynamic, and structural analysis of drug resistance mutations in neuraminidase from the 2009 pandemic influenza virus. Viruses 10, (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.McKimm-Breschkin J. L., Influenza neuraminidase inhibitors: Antiviral action and mechanisms of resistance. Influenza Other Respi. Viruses 7 (Suppl. 1), 25–36 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Meijer A., Jonges M., Abbink F., Ang W., van Beek J., Beersma M., Bloembergen P., Boucher C., Claas E., Donker G., van Gageldonk-Lafeber R., Isken L., de Jong A., Kroes A., Leenders S., van der Lubben M., Mascini E., Niesters B., Oosterheert J. J., Osterhaus A., Riesmeijer R., Riezebos-Brilman A., Schutten M., Sebens F., Stelma F., Swaan C., Timen A., van 't Veen A., van der Vries E., te Wierik M., Koopmans M., Oseltamivir-resistant pandemic A(H1N1) 2009 influenza viruses detected through enhanced surveillance in the Netherlands, 2009-2010. Antiviral Res. 92, 81–89 (2011). [DOI] [PubMed] [Google Scholar]
  • 20.Jalily P. H., Duncan M. C., Fedida D., Wang J., Tietjen I., Put a cork in it: Plugging the M2 viral ion channel to sink influenza. Antiviral Res. 178, 104780 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Heo Y. A., Baloxavir: First global approval. Drugs 78, 693–697 (2018). [DOI] [PubMed] [Google Scholar]
  • 22.Omoto S., Speranzini V., Hashimoto T., Noshi T., Yamaguchi H., Kawai M., Kawaguchi K., Uehara T., Shishido T., Naito A., Cusack S., Characterization of influenza virus variants induced by treatment with the endonuclease inhibitor baloxavir marboxil. Sci. Rep. 8, 9633 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Takashita E., Kawakami C., Ogawa R., Morita H., Fujisaki S., Shirakura M., Miura H., Nakamura K., Kishida N., Kuwahara T., Ota A., Togashi H., Saito A., Mitamura K., Abe T., Ichikawa M., Yamazaki M., Watanabe S., Odagiri T., Influenza A(H3N2) virus exhibiting reduced susceptibility to baloxavir due to a polymerase acidic subunit I38T substitution detected from a hospitalised child without prior baloxavir treatment, Japan, January 2019. Euro Surveill. 24, 1900170 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Gaisina I. N., Peet N. P., Cheng H., Li P., Du R., Cui Q., Furlong K., Manicassamy B., Caffrey M., Thatcher G. R. J., Rong L., Optimization of 4-aminopiperidines as inhibitors of influenza a viral entry that are synergistic with oseltamivir. J. Med. Chem. 63, 3120–3130 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Hussein A. F. A., Cheng H., Tundup S., Antanasijevic A., Varhegyi E., Perez J., AbdulRahman E. M., Elenany M. G., Helal S., Caffrey M., Peet N., Manicassamy B., Rong L., Identification of entry inhibitors with 4-aminopiperidine scaffold targeting group 1 influenza A virus. Antiviral Res. 177, 104782 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Antanasijevic A., Durst M. A., Cheng H., Gaisina I. N., Perez J. T., Manicassamy B., Rong L., Lavie A., Caffrey M., Structure of avian influenza hemagglutinin in complex with a small molecule entry inhibitor. Life Sci Alliance 3, e202000724 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Russell R. J., Kerry P. S., Stevens D. J., Steinhauer D. A., Martin S. R., Gamblin S. J., Skehel J. J., Structure of influenza hemagglutinin in complex with an inhibitor of membrane fusion. Proc. Natl. Acad. Sci. U.S.A. 105, 17736–17741 (2008). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Kadam R. U., Wilson I. A., Structural basis of influenza virus fusion inhibition by the antiviral drug Arbidol. Proc. Natl. Acad. Sci. U.S.A. 114, 206–214 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Ryan D. M., Ticehurst J., Dempsey M. H., Penn C. R., Inhibition of influenza virus replication in mice by GG167 (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid) is consistent with extracellular activity of viral neuraminidase (sialidase). Antimicrob. Agents Chemother. 38, 2270–2275 (1994). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Sidwell R. W., Huffman J. H., Barnard D. L., Bailey K. W., Wong M. H., Morrison A., Syndergaard T., Kim C. U., Inhibition of influenza virus infections in mice by GS4104, an orally effective influenza virus neuraminidase inhibitor. Antiviral Res. 37, 107–120 (1998). [DOI] [PubMed] [Google Scholar]
  • 31.Alfaro C. L., Lam Y. W., Simpson J., Ereshefsky L., CYP2D6 inhibition by fluoxetine, paroxetine, sertraline, and venlafaxine in a crossover study: Intraindividual variability and plasma concentration correlations. J. Clin. Pharmacol. 40, 58–66 (2000). [DOI] [PubMed] [Google Scholar]
  • 32.Foisy M. M., Yakiwchuk E. M., Hughes C. A., Induction effects of ritonavir: Implications for drug interactions. Ann. Pharmacother. 42, 1048–1059 (2008). [DOI] [PubMed] [Google Scholar]
  • 33.Kowalska M., Nowaczyk J., Nowaczyk A., KV11.1, NaV1.5, and CaV1.2 transporter proteins as antitarget for drug cardiotoxicity. Int. J. Mol. Sci. 21, 8099 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Erdemli G., Kim A. M., Ju H., Springer C., Penland R. C., Hoffmann P. K., Cardiac safety implications of hNav1.5 blockade and a framework for pre-clinical evaluation. Front. Pharmacol. 3, 6 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Li P., Cui Q., Wang L., Zhao X., Zhang Y., Manicassamy B., Yang Y., Rong L., Du R., A simple and robust approach for evaluation of antivirals using a recombinant influenza virus expressing gaussia luciferase. Viruses 10, 325 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Manicassamy B., Manicassamy S., Belicha-Villanueva A., Pisanelli G., Pulendran B., Garcia-Sastre A., Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus. Proc. Natl. Acad. Sci. U.S.A. 107, 11531–11536 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Zhao X., Lin X., Li P., Chen Z., Zhang C., Manicassamy B., Rong L., Cui Q., Du R., Expanding the tolerance of segmented Influenza A Virus genome using a balance compensation strategy. PLOS Pathog. 18, e1010756 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Uyeki T. M., Bernstein H. H., Bradley J. S., Englund J. A., File T. M., Fry A. M., Gravenstein S., Hayden F. G., Harper S. A., Hirshon J. M., Ison M. G., Johnston B. L., Knight S. L., McGeer A., Riley L. E., Wolfe C. R., Alexander P. E., Pavia A. T., Clinical practice guidelines by the infectious diseases society of america: 2018 update on diagnosis, treatment, chemoprophylaxis, and institutional outbreak management of seasonal influenzaa. Clin. Infect. Dis. 68, 895–902 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Chen Z., Cui Q., Caffrey M., Rong L., Du R., Small molecule inhibitors of influenza virus entry. Pharmaceuticals (Basel) 14, (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Dunning J., Baillie J. K., Cao B., Hayden F. G., International Severe Acute Respiratory and Emerging Infection Consortium (ISARIC) , Antiviral combinations for severe influenza. Lancet Infect. Dis. 14, 1259–1270 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Ison M. G., Finding the right combination antiviral therapy for influenza. Lancet Infect. Dis. 17, 1221–1222 (2017). [DOI] [PubMed] [Google Scholar]
  • 42.Park J. H., Kim B., Antigua K. J. C., Jeong J. H., Kim C. I., Choi W. S., Oh S., Kim C. H., Kim E. G., Choi Y. K., Baek Y. H., Song M. S., Baloxavir-oseltamivir combination therapy inhibits the emergence of resistant substitutions in influenza A virus PA gene in a mouse model. Antiviral Res. 193, 105126 (2021). [DOI] [PubMed] [Google Scholar]
  • 43.Fukao K., Noshi T., Yamamoto A., Kitano M., Ando Y., Noda T., Baba K., Matsumoto K., Higuchi N., Ikeda M., Shishido T., Naito A., Combination treatment with the cap-dependent endonuclease inhibitor baloxavir marboxil and a neuraminidase inhibitor in a mouse model of influenza A virus infection. J. Antimicrob. Chemother. 74, 654–662 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Tsiodras S., Mooney J. D., Hatzakis A., Role of combination antiviral therapy in pandemic influenza and stockpiling implications. BMJ 334, 293–294 (2007). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Smee D. F., Hurst B. L., Wong M. H., Bailey K. W., Tarbet E. B., Morrey J. D., Furuta Y., Effects of the combination of favipiravir (T-705) and oseltamivir on influenza A virus infections in mice. Antimicrob. Agents Chemother. 54, 126–133 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Smee D. F., Hurst B. L., Wong M. H., Tarbet E. B., Babu Y. S., Klumpp K., Morrey J. D., Combinations of oseltamivir and peramivir for the treatment of influenza A (H1N1) virus infections in cell culture and in mice. Antiviral Res. 88, 38–44 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Hoopes J. D., Driebe E. M., Kelley E., Engelthaler D. M., Keim P. S., Perelson A. S., Rong L., Went G. T., Nguyen J. T., Triple combination antiviral drug (TCAD) composed of amantadine, oseltamivir, and ribavirin impedes the selection of drug-resistant influenza A virus. PLOS ONE 6, e29778 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Nguyen J. T., Hoopes J. D., Le M. H., Smee D. F., Patick A. K., Faix D. J., Blair P. J., de Jong M. D., Prichard M. N., Went G. T., Triple combination of amantadine, ribavirin, and oseltamivir is highly active and synergistic against drug resistant influenza virus strains in vitro. PLOS ONE 5, e9332 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.van Dongen M. J. P., Kadam R. U., Juraszek J., Lawson E., Brandenburg B., Schmitz F., Schepens W. B. G., Stoops B., van Diepen H. A., Jongeneelen M., Tang C., Vermond J., van Eijgen-Obregoso Real A., Blokland S., Garg D., Yu W., Goutier W., Lanckacker E., Klap J. M., Peeters D. C. G., Wu J., Buyck C., Jonckers T. H. M., Roymans D., Roevens P., Vogels R., Koudstaal W., Friesen R. H. E., Raboisson P., Dhanak D., Goudsmit J., Wilson I. A., A small-molecule fusion inhibitor of influenza virus is orally active in mice. Science 363, eaar6221 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Aoki F. Y., Macleod M. D., Paggiaro P., Carewicz O., El Sawy A., Wat C., Griffiths M., Waalberg E., Ward P., IMPACT Study Group , Early administration of oral oseltamivir increases the benefits of influenza treatment. J. Antimicrob. Chemother. 51, 123–129 (2003). [DOI] [PubMed] [Google Scholar]
  • 51.Mirams G. R., Cui Y., Sher A., Fink M., Cooper J., Heath B. M., McMahon N. C., Gavaghan D. J., Noble D., Simulation of multiple ion channel block provides improved early prediction of compounds' clinical torsadogenic risk. Cardiovasc. Res. 91, 53–61 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Alqarni S., Cooper L., Galvan Achi J., Bott R., Sali V. K., Brown A., Santarsiero B. D., Krunic A., Manicassamy B., Peet N. P., Zhang P., Thatcher G. R. J., Gaisina I. N., Rong L., Moore T. W., Synthesis, optimization, and structure-activity relationships of Imidazo[1,2-a]pyrimidines as inhibitors of group 2 influenza A viruses. J. Med. Chem. 65, 14104–14120 (2022). [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Material and Methods for Supplement and ADMET Assays

Figs. S1 to S4

Tables S1 to S15

References

sciadv.adk9004_sm.pdf (686.9KB, pdf)

Articles from Science Advances are provided here courtesy of American Association for the Advancement of Science

RESOURCES