The critical roles of the influenza vRNP for viral RNA synthesis make activities of the vRNP, such as cap-snatching and RNA polymerization, excellent antiviral targets. A recently discovered nucleotide analog preferentially utilized by viral RNA dependent RNA polymerases including influenza vRNP, is under study as a promising antiviral therapy targeting the activity of viral RNA dependent RNA polymerases [
74]. Further, the multiple essential interactions of the vRNP, such as with each other to form the RNA dependent RNA polymerase heterodimer, with host capped mRNAs to obtain primers for viral transcription, and with NP to regulate and enhance RNA replication, coupled with high conservation of these domains among influenza subtypes, make the proteins of the vRNP excellent targets for small molecule inhibitors with broad efficacy against multiple influenza A subtypes.
Ribonucleotide analogs
Favipiravir is a 6-fluoro-3-hydroxy-2-pyrazinecarboxamide molecule (also known as T-705) that upon phosphorylation becomes favipiravir-ribofuranosyl-5’-triphosphate (RTP) and inhibits many viral RNA dependent RNA polymerases [
75]. Favipiravir is effective against influenza A, influenza B, influenza C, hantaviruses, flaviviruses, noroviruses, and most recently ebola viruses [
74‐
76]. The T-705 RTP is erroneously interpreted as a purine nucleotide by the viral polymerase during RNA elongation [
75,
77]. Once incorporated into the elongating viral RNA, the analog may hinder strand extension [
77]. The antiviral activity of Favipiravir includes influenza A(H3N2), A(H1N1), A(H5N1), A(H7N9), and strains bearing resistance to both classes of the current FDA approved influenza antivirals [
74,
75,
78].
The 50% inhibitory concentration (IC
50) of favipiravir for influenza, determined by plaque reduction assay, was 0.013-0.48 μg/ml with no cytotoxic effect up to 1000 μg/ml [
74]. Human DNA polymerase α, β, or γ with 1000 μM of favipiravir showed little sign of inhibition [
79] and human RNA polymerase II had an IC
50 of 905 μM of favipiravir [
80]. Therefore, the IC
50 of host polymerases is well over 2000 times greater then the IC
50 for influenza vRNP, making favipiravir highly selective for influenza vRNP [
75]. Favipiravir for influenza therapy has finished two Phase II clinical trials in the United States and one Phase III clinical trial in Japan [
75]. Favipiravir is a favorable candidate for a broadly effective antiviral therapy targeting RNA viruses with RNA dependent RNA polymerases that preferentially incorporate favipiravir in RNA synthesis. It is not yet clear if or how quickly influenza vRNP will evolve resistance to this nucleotide analog, but based on HBV and approved nucleotide analog therapies [
81], as with any antiviral therapy, if resistance is possible, it will eventually develop, stressing the need to consistently look for novel antiviral targets and therapies.
Targeting NP
Nucleozin is a small molecule inhibitor of NP that works by promoting NP oligomerization, blocking nuclear entry through aggregation of NP molecules at the nuclear membrane [
41] and inducing vRNP aggregation during cytoplasmic trafficking [
82]. Molecular docking models identified two proposed nucleozin interaction sites at residue 289 and 309 of NP [
41]. Both residues participate to stabilize the interaction; a tyrosine at residue 289 forms aromatic ring stacking with nucleozin, while an asparagine at residue 309 shares a hydrogen bond with nucleozin. Strains found to be nucleozin resistant encoded a histidine in place of a tyrosine at residue 289 of NP [
41]. From the crystal structure of NP, residue 289 is relatively accessible to interact with nucleozin (Figure
2) [
39,
41]. The 289H NP variant likely disrupts a critical point of interaction between NP and nucleozin. Sequence analysis of the NP gene of 3,881 influenza strains revealed Y289H substitution in 527 strains. Unfortunately the A(H1N1)pdm09 strain was shown to contain this single amino acid alteration, signifying it as a nucleozin resistant strain [
41]. Nucleozin demonstrated a 50% effective concentration (EC
50) of only 0.069 μM against influenza with 50% cytotoxic concentration greater than 250 μM [
41]. Thus nucleozin still holds potential as a potent antiviral for strains of influenza housing a tyrosine at residue 289 [
41]. In addition to nucleozin, there are several other aryl piperazine amide compounds discovered in parallel that target NP and inhibit virus replication in the same manner [
41,
83‐
85]. Cianci et al. provide a detailed review into the efficacy of aryl piperazine amides, including nucleozin, as NP inhibitors [
85]. Further analysis of these compounds could lead to the synthesis of an optimized aryl piperazine amide inhibitor of viral replication, even against the circulating nucleozin resistant strains of influenza.
The essential salt bridge for NP oligomerization at residues 339 and 416 was targeted for disruption by small molecule inhibitors (Figure
2) [
42]. Peptides that mimicked the NP tail loop (residues 402–428) bound in the tail loop binding pocket and inhibited NP oligomerization, resulting in decreased viral replication by greater than fifty percent [
42]. Random virtual screening of small molecules identified four compounds (termed # 3, 7, 12, and 23) that interrupt NP oligomerization and decrease viral replication [
42]. The IC
50 of compounds 3, 7, 12, and 23 ranged from 2.4 to 118 μM in cells challenged with influenza A/WSN/33 for nine hours at a multiplicity of infection (MOI) of 0.2 [
42]. Although the targeted NP domain is highly conserved, more research will need to be carried out to ensure little to no development of resistance to the compounds. Importantly, cytotoxicity of these compounds will need to be assessed before they can be of use as antiviral therapies.
Mycalamide A is an antiviral and antitumor compound isolated from
Mycale sponges but is unfortunately toxic to cells [
86]. A photo-cross-linked chemical array identified analogs of mycalamide A that possess the ability to bind NP [
87]. One compound inhibited viral replication up to 77% in a plaque assay using influenza virus (A/WSN/33) with no cytotoxic effect [
87]. Binding affinity of the analogs was greatest within the N-terminal 110 amino acids of NP [
87]. Although the mechanism of inhibition remains uncharacterized, the N-terminus of NP contains an important non-canonical nuclear localization signal (NLS) [
88,
89] and interacts with host RNA processing factors UAP56 and URH49 [
90,
91] proposed to enhance RNA replication [
92]. More research is needed on these compounds to elucidate the mechanism of inhibition and determine if or how quickly influenza NP will evolve resistance.
Naproxen is an over the counter nonsteroidal anti-inflammatory drug that inhibits NP from associating with RNA in the RNA binding groove (Figure
2) [
93]. From several molecular docking studies, residues Y148, Q149, R150, R355, R361, and F489 are believed to stabilize naproxen binding to NP [
93]. Naproxen acts selectively upon monomeric NP and was shown to protect MDCK cells and mice from a viral challenge at an MOI of 10
-2 - 10
-3 or 50–2,000 PFU respectively, with little to no cytotoxic effects [
93]. Subtypes of H1N1 and H3N2 were both susceptible to inhibition and treatment resulted in efficient protection from viral challenge with either subtype [
93]. No escape mutant viruses were produced in response to 500 μM naproxen treatment in cells after six passages [
93]. Within this experiment the mode of delivery for naproxen was intraperitoneal injection or intranasal treatment in mice that displayed an EC
50 of 40 mg/kg [
93]. Naproxen is currently used as an oral medication with a recommended dosage of 220 mg every 8 hours for pain. Naproxen is not currently used as an antiviral but could be optimized for antiviral use through further experimentation and improved drug design.
Targeting PA-PB1 interaction
Inhibitors of the PA-PB1 interaction are numerous [
94]. Compound 1 was discovered through
in silico screening [
94] using the crystal structure of PA
C-PB1
N (Figure
3) [
68]. The hydrophobic pocket of PA houses compound 1 according to molecular docking studies, and inhibition of PA-PB1 interaction was demonstrated through ELISA and immunoprecipitation of PA [
94]. Compound 1 inhibited RNA polymerase activity in a dose-dependent manner with an IC
50 of about 18 μM assessed in a minireplicon assay using a firefly luciferase reporter gene, with no significant cytotoxic effect up to concentrations of 250–1000 μM [
94]. Compound 1 inhibited viral replication in MDCK cells for several influenza A H1N1 and H3N2 strains, a swine-origin influenza virus, and an oseltamivir-resistant isolate, with IC
50 ranging from 12.2 to 22.5 μM [
94].
There are two FDA approved medications that in addition to their intended use also possess anti-IAV abilities due to their structural similarity with the N terminal domain of PB1 that interacts with the C-terminus of PA (Figure
3) [
95]. Benzbromarone is approved to treat gout and hyperuricemia by promoting the excretion of uric acid. Diclazuril is most commonly used in veterinary medicine as an anti-coccidial. Although these drugs are FDA approved, appropriate drug dosages for use against viral challenges would need to be established before they could be employed for use against influenza A. Testing for the ability of viruses to gain resistance to these drugs must also be done. Benzbromarone and diclazuril could potentially be utilized if an influenza strain arises that possesses resistance to other antivirals available [
95].
In addition, a compound derived from licorice, 18β-glycyrrhetinic acid (GHA), is a naturally occurring compound that exhibits some anti-IAV activity attributed to interaction with the C terminal domain of PA [
96]. Molecular docking studies identified GHA as a ligand to PA
C[
96]. GHA decreased polymerase activity 80%, as assessed by a primer extension assay for cRNA synthesis [
96]. These preliminary findings need to be investigated further with more informative assays including
in vitro PA-PB1 interaction inhibition study,
in vivo polymerase activity assays in tissue culture, and
in vivo infection in an animal model. There are many small molecules that mimic the N terminus of PB1 fit in the hydrophobic groove of PA to inhibit PA-PB1 interaction (Figure
3) and should be studied further for their potential as an anti-influenza A treatment.
Targeting PA endonuclease
Fullerene (C
60) is a spherical molecule of sixty carbon atoms that exhibits anti-influenza activity [
97]. Full length PA and an isolated PA endonuclease domain were tested in an
in vitro endonuclease assay in the presence of fullerene derivatives. Seven fullerene derivatives were able to inhibit the endonuclease ability of the full length PA and isolated endonuclease domain [
97]. Docking simulations reveal the fullerene skeleton fits nicely into the endonuclease domain active pocket [
97]. MDCK cells were infected with influenza A H1N1 or H3N2 mixed with 0 to 100 μM fullerene derivative and immunostained for NP at 24 hours post infection to reveal significantly less NP in cells infected with virus pre-incubated with fullerene compared to a DMSO control [
97]. Twelve derivatives of fullerene were tested and resulted in varying efficacy against influenza but showed no cytotoxic effect up to 100 μM [
97]. More investigation into the activity and expression of viral proteins in response to fullerene needs to be conducted. Importantly, treatment applied post-infection needs to be investigated. This novel compound exemplifies yet another possible antiviral target within the vRNP.