Introduction
Influenza is a highly contagious respiratory tract infection that threatens the lives of many people all over the world [
1]. Influenza viruses (IVs) possess a segmented RNA genome of negative sense [
2]. They belong to the Orthomyxoviridae family and divide into four types: influenza A viruses (IAVs), influenza B viruses (IBVs), influenza C viruses (ICVs) and influenza D viruses (IDVs) [
3]. Due to high genetic dynamism, the continuous tendency to antigenic drift/shift, efficient viral transmission, the rapid emergence of drug resistance and limited efficacy of currently available medications, the members of IAV genus are considered major life-threatening respiratory pathogens as it is responsible for all documented influenza pandemics [
4,
5]. Remarkably, the estimated number of deaths caused by the influenza A/H1N1 virus during the “Spanish influenza” pandemic from 1918 to 1920 (> 50 million deaths) exceeded those caused by coronavirus disease 2019 (COVID-19) (6.9 million deaths) [
6,
7]. Another member of the IAV genus, the avian influenza A/H5N1virus, is characterized by an extremely high mortality rate in poultry and human populations [
8]; hence it is considered a highly pathogenic avian influenza virus (HPAIV) [
9,
10].
Vaccination and antiviral therapy are the main strategies used worldwide to control influenza infection in humans. However, the effectiveness of available vaccines to control seasonal influenza infection is relatively low due to different factors such as the suitability of the vaccine to the viral strain, transmission across species and sudden genetic mutations to the vaccine strain [
11‐
13]. Moreover, the antiviral resistance and the emergence of mutant viral strains have reduced the efficacy of the known FDA-approved synthetic antiviral agents such as M2-channel blockers and neuraminidase inhibitors (NAIs) [
14‐
16]. Therefore, antiviral research should be prioritized to discover new alternatives for the prophylaxis and control of IAV infections. Phytochemicals derived from plants and natural resources are considered one of the safest and most efficacious treatment options for controlling viral infections including influenza [
17‐
21]. The largest known class of phytochemicals are called alkaloids which were documented for the first time, over 4 thousand years ago [
22]. These basic compounds consist of one or more nitrogen atoms bound to a heterocyclic nucleus [
23]. Higher plants especially those belonging to Ranunculaceae, Leguminosae, Papaveraceae, Menispermaceae, and Loganiaceae families are considered main reservoirs for alkaloids [
24]. Alkaloids can serve as therapeutic options for the treatment of a variety of diseases and exhibit promising biological activities [
25,
26].
Pharmacoinformatics have been forefront of the drug design and development research. They have decreased the expense, time, and labor of drug discovery [
27]. Hence, computational chemistry methods were applied to estimate various pharmacodynamic and pharmacokinetic parameters that relate the chemical structure of compounds to their activity and to characterize the interaction of compounds with biological targets [
28‐
30].
Herein, the anti-influenza activities of some alkaloid compounds were investigated against two different subtypes of IAVs with varied host ranges: the avian influenza A/H5N1 and the seasonal influenza A/H1N1 viruses to help in stockpiling of antiviral medications to be ready for any future pandemic situation. In addition, a molecular docking approach was applied to investigate the binding patterns of the active compounds against the prospective biological targets (influenza H1N1 neuraminidase, influenza H5N1 neuraminidase, and influenza M2).
Discussion
Influenza pandemics and seasonal epidemics threaten the public health of human and animal populations [
68]. Annually, seasonal influenza epidemics result in about 3–5 million cases of severe illness, and about 290,000 to 650,000 respiratory deaths [
69]. Seasonal influenza vaccines and limited options of anti-influenza medications are currently available, however, their effectiveness has always been debated due to the emergence of resistance to antivirals and relatively low and unpredictable efficiency of the seasonal influenza vaccines compared to other vaccines [
70]. The classical M
2 ion channel blockers (rimantadine and amantadine) and neuraminidase inhibitors (oseltamivir and zanamivir) are two classes of antiviral medications that have been authorized by the FDA organization for the treatment of influenza [
71,
72]. However, misuse of these therapies and the continuous genetic and antigenic drift of IAVs during replication in different host species has resulted in drug-resistant and/or reassortant strains [
73], with high risk to public health [
73‐
75]. Consequently, the global fear of future influenza pandemics urges the need to innovate broad-spectrum anti-influenza medications that are not subtype- or strain-specific [
76]. Phytomedicine is one of the most effective ways to cure various ailments [
21]. In this regard, we aimed in this study to determine the anti-influenza potential of naturally available nitrogenous alkaloids against two distinct IAV subtypes, influenza A/H5N1 and A/H1N1.
Notably, non-cytotoxic concentrations of the tropane alkaloid, atropine sulphate, exerted anti-influenza effects against the avian A/H5N1 virus and the seasonal A/H1N1 virus with high selectivity indices through direct virucidal action against the avian virus subtype of H5N1 subtype. However, previous studies regarding the anti-influenza activities of atropine sulphate are rare. Prior investigations on atropine revealed its in vitro antiviral activities against herpes simplex virus type-1 (HSV-1) and parainfluenza type-3 (PI-3) with therapeutic doses between 0.05 and 0.8 µg/ml [
39].
Likewise, our results demonstrated that pilocarpine hydrochloride exhibits a promising anti-influenza efficacy against both influenzas A viruses, subtypes H5N1 and A/H1N1, in a concentration-dependent manner with potent IC
50 values and high SI values when compared to the FDA-approved anti-influenza reference drug used in our study (zanamivir). It has been proven to counteract the influenza A/H5N1 virus via cell-free direct virucidal action. In literature, rare or no studies were reported on the antiviral effects of pilocarpine hydrochloride in medical uses, providing that it was known to be used in ophthalmic solutions to treat glaucoma for many years [
77].
Significant anti-influenza effects have also been exerted by the tropolone alkaloid, colchicine, against both IAVs subtypes and showed also to interfere with the viral replication of the avian influenza A/H5N1 virus. From the toxicological background, our findings suggested that the IC
50 values of colchicine when tested against both tested IAVs, influenza A/H5N1 and A/H1N1 virues, were 0.076 and 0.65 µg/ml, respectively and that is far below the toxic range described earlier (0.6 mg) [
78]. Concerning the anti-influenza effects of colchicine, no information was available regarding this area of study. Prior studies proved that colchicine is an antimitotic agent and this may aid in understanding the mechanism by which it affects the H5N1 viral replication [
79].
In previous investigations, papaverine has been proven to exert anti-influenza activities against different strains of IAVs and parainfluenza viruses with IC
50 values ranging between 2.02 and 36.41 µM [
43]. Nonetheless, in the primary screening stage of our study, papaverine hydrochloride showed poor anti-influenza activity against the highly pathogenic avian A/H5N1 virus. However, quinine sulphate has been described earlier to exert an antiviral effect against IAV (A/Puerto Rico/8/1934(H1N1) in mice [
47], it elucidated no anti-influenza activity against influenza A/H5N1 virus in our findings. Contextually, a previous study regarding the anti-influenza potential of L-Ephedrine against A/PR8/34 (H1N1 virus) proved its efficacy against the tested viruses with EC
50 values ranging from 5.66 to 10.96 µg/ml [
55]. Our findings proved poor anti-influenza activity of ephedrine hydrochloride against avian influenza A/H5N1 virus with an IC
50 value of 186.25 µg/ml when compared to the reference zanamivir drug. The rest of the tested alkaloids showed poor or no anti-influenza activity during the primary screening using the HPAIV A/H5N1.
In the computational studies, the three most active alkaloids (atropine, pilocarpine, and colchicine) were subjected to molecular docking investigations against three viral proteins. The first and second proteins are the neuraminidases influenza H1N1, and influenza H5N1, respectively. The neuraminidases (sialidases) bind to the sialic acid receptors in the cell surface causing the cleaving of the receptor and allowing the virus release outside the host cell to infect other cells [
80]. Accordingly, it was selected to figure out another mechanism of action for the selected compounds. Fortunately, the tested compounds showed similar binding modes to that of the reference molecules (the co-crystallized ligands) with slight variation for each tested molecule. For the binding energy, it was found that colchicine has the highest energy of binding indicating higher affinity against the two targets than atropine and pilocarpine. Additionally, atropine showed higher affinity than pilocarpine. Regarding the influenza M2 protein, it has an essential role in viral adsorption as it is responsible for the equilibration of the pH across the membrane of the virus to facilitate cell entry in addition to the entry across the Golgi membrane viral maturation [
81]. Interestingly, the in vitro and the in silico results were consistent. In the in vitro assay, colchicine expressed also a high ability to inhibit viral adsorption with a percentage of 73% at a concentration of 100 µg/ml while both atropine and pilocarpine were not able to inhibit viral adsorption. The computational studies showed that colchicine exhibited a correct binding mode against the influenza M2 protein while both atropine and pilocarpine failed to show correct binding modes.
Ultimately, this study suggests the anti-influenza efficacy of three nitrogenous alkaloids; namely atropine sulphate, pilocarpine hydrochloride and colchicine. Nevertheless, further studies must be conducted to validate the in vivo bioavailability and efficacy of the three alkaloids against influenza virus infections.
Conclusion
Since the emergence of the devastating COVID-19 pandemic, drug repurposing as an accelerated approach to identify new antiviral indications of commercially available FDA approved drugs attracted more attention and contributed actively to control the infection. Due to their various pharmacological effects, including antiviral activities, nitrogenous alkaloids are a major class of phytochemicals that are grasping the attention of many antiviral researchers. The anti-influenza efficacy of various biologically active alkaloids against avian IAV seasonal human IAV could be successfully investigated in this work. Significantly, atropine sulphate, pilocarpine hydrochloride and colchicine showed substantial anti-influenza effects against the designated strains. Additionally, atropine, pilocarpine, and colchicine showed excellent in silico potentialities to bind and inhibit the neuraminidases of both influenza H1N1, and influenza H5N1. Also, in agreement with in vitro results, only colchicine could bind correctly against proton channel M2 of IAV.
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