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. 2019 Aug 13;93(17):e00878-19.
doi: 10.1128/JVI.00878-19. Print 2019 Sep 1.

Novel Small Molecule Targeting the Hemagglutinin Stalk of Influenza Viruses

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Free PMC article

Novel Small Molecule Targeting the Hemagglutinin Stalk of Influenza Viruses

Jin Il Kim et al. J Virol. .
Free PMC article

Abstract

Combating influenza is one of the perennial global public health issues to be managed. Antiviral drugs are useful for the treatment of influenza in the absence of an appropriate vaccine. However, the appearance of resistant strains necessitates a constant search for new drugs. In this study, we investigated novel anti-influenza drug candidates using in vitro and in vivo assays. We identified anti-influenza hit compounds using a high-throughput screening method with a green fluorescent protein-tagged recombinant influenza virus. Through subsequent analyses of their cytotoxicity and pharmacokinetic properties, one candidate (IY7640) was selected for further evaluation. In a replication kinetics analysis, IY7640 showed greater inhibitory effects during the early phase of viral infection than the viral neuraminidase inhibitor oseltamivir. In addition, we observed that hemagglutinin (HA)-mediated membrane fusion was inhibited by IY7640 treatment, indicating that the HA stalk region, which is highly conserved across various (sub)types of influenza viruses, may be the molecular target of IY7640. In an escape mutant analysis in cells, amino acid mutations were identified at the HA stalk region of the 2009 pandemic H1N1 (pH1N1) virus. Even though the in vivo efficacy of IY7640 did not reach complete protection in a lethal challenge study in mice, these results suggest that IY7640 has potential to be developed as a new type of anti-influenza drug.IMPORTANCE Anti-influenza drugs with broad-spectrum efficacy against antigenically diverse influenza viruses can be highly useful when no vaccines are available. To develop new anti-influenza drugs, we screened a number of small molecules and identified a strong candidate, IY7640. When added at the time of or after influenza virus infection, IY7640 was observed to successfully inhibit or reduce viral replication in cells. We subsequently discovered that IY7640 targets the stalk region of the influenza HA protein, which exhibits a relatively high degree of amino acid sequence conservation across various (sub)types of influenza viruses. Furthermore, IY7640 was observed to block HA-mediated membrane fusion of H1N1, H3N2, and influenza B viruses in cells. Although it appears less effective against strains other than H1N1 subtype viruses in a challenge study in mice, we suggest that the small molecule IY7640 has potential to be optimized as a new anti-influenza drug.

Keywords: antiviral agents; hemagglutinin stalk; influenza virus; membrane fusion; small molecule.

Figures

FIG 1
FIG 1
Candidate chemicals selected using the rPR8-GFP virus. By a plaque reduction assay in MDCK cells (96-well plate; 4 × 104 cells/well), eight candidate chemicals were selected based on the GFP expression of oseltamivir-treated wells. A total of 100 μg of each chemical dissolved in DMSO was serially 2-fold diluted in PBS and added to overlaying agar. The multiplicity of infection of rPR8-GFP was 0.5, and GFP expression was observed at 24 h postinfection. PBS was used for mock infection.
FIG 2
FIG 2
PK evaluation and anti-influenza effects of IY7640. (A) Molecular structure of IY7640. (B) Graphic representation of IY7640 pharmacokinetics in mice. Mean values are plotted. (C) The anti-influenza effect of IY7640 in cultured cells. In MDCK cells, the inhibitory effect of IY7640 was determined against the rK09 virus compared with that of oseltamivir using a plaque reduction assay. n.d., below detection limit (10 PFU/ml).
FIG 3
FIG 3
Toxicity evaluation of IY7640. (A) Cytotoxicity test of IY7640 in MDCK cells. The number of viable cells was measured fluorometrically using the CCK-8 reagent after incubating the cells with DMSO (mock) or the designated concentrations of IY7640 (dissolved in DMSO), oseltamivir-carboxylate, or oseltamivir phosphate. The experiments were performed in duplicate. (B) Two-week repeated-oral-dose toxicity test of IY7640. Female SD rats (5 rats per group) were administered once daily with 0, 500, 1,000, or 2,000 mg of IY7640 by gastric intubation for 2 weeks, and their body weights were recorded at 1, 4, 8, 11, and 14 days posttreatment.
FIG 4
FIG 4
Molecular target of IY7640. (A) Escape mutations (L49I, M403T, and E447K) identified in the HA protein of rK09 after IY7640 treatment are adjacently located with the CR6261 epitopes in the HA structure. The colors represent the following: slate, HA1; light pink, HA2; red, escape mutations against IY7640; and orange, CR6261 epitopes. (B) Replication kinetics of escape mutant viruses were evaluated in MDCK cells. (C and D) Viral infection and replication efficiency were measured by the number of the newly generated virus particles via plaque assays (C), and the amounts of newly expressed HA were compared by Western blotting (D). (E) A hemagglutination assay using the K09 virus and 0.5% (vol/vol) tRBCs was performed in the presence of PBS (negative control), 2-fold serially diluted anti-K09 guinea pig sera (α-K09 #1 and #2; positive controls), or IY7640 (starting at 90 μM).
FIG 5
FIG 5
Structural interaction of IY7640 with H1, H3, and H5 HAs. The interaction simulation of IY7640 (green) with the CR6261 (orange) or the TBHQ (yellow) epitopes is depicted using the crystal structures of trimeric HAs (HA1 is slate-colored and HA2 is light pink) in one monomer—pH1N1 Ca04 HA of PDB ID 3UBQ in panel A, H3N2 A/Aichi/2/1968 HA of PDB ID 3ZTJ in panel B, and H5N1 A/Vietnam/1194/2004 HA of PDB ID 2IBX in panel C—with a respective enlarged image of the HA2 stalk region. The escape mutations against IY7640 are colored red. The residues are labeled according to H1, H3, or H5 numbering. The hydrogen bond interactions between IY7640 and HAs (connected by red dots) are given in angstroms: in the enlarged H1 HA (A), 2.109 and 2.468 to T318; in the enlarged H3 HA (B), 2.923 to T404 and 1.860, 1.470, and 2.016 to Y439; and in the enlarged H5 HA (C), 3.175 to H24, and 3.138 to W367. The non-hydrogen bond interactions (connected by black dots) are also shown: 3.334 to V40, 3.132 and 3.193 to W365, 3.368 to I389, and 2.767 to V396 in H1 HA (A); 2.617 to K307, 3.250 to R399, 3.104 to W437, 2.547 to L444, and 3.168 to A446 in H3 HA (B); and 3.013 to H24, 2.844 to H44, 3.003 to Q46, 2.817 to I391, 3.212 to V394, and 2.986 to T395 in H5 HA (C).
FIG 6
FIG 6
Inhibition of HA fusion activity by IY7640. (A and B) Inhibition of RBC hemolysis by IY7640. rK09 and the escape mutant (rK09/HA:M403T and rK09/HA:E447K) mixtures were preincubated with 50 μM of IY7640 in dimethyl sulfoxide (DMSO) (rK09 + IY7640) or DMSO alone (rK09 + DMSO). Then the mixtures were acidified to the designated pH and measured for the release of hemoglobin from lysed erythrocytes. (C) Inhibition of HA-mediated cell-to-cell fusion by IY7640. In Vero cells, the inhibition of HA-mediated cell-to-cell fusion was determined with IY7640 treatment (for pK09 HA, 1, 10, or 100 μM, and for pK09:M403T and pK09:E447K HAs, 10 or 100 μM) according to the pH ranges (pH 4.4 to 5.2). DMSO was used as a control.
FIG 7
FIG 7
HA susceptibility to protease and its inhibition by IY7640. MDCK cell lysates infected with the viruses were trypsinized followed by IY7640 treatment (5, 50, or 500 mM). After pH adjustment (pH 4.8 or 8.0), the cell supernatants were examined by the Western blot analysis.
FIG 8
FIG 8
Anti-influenza effects of IY7640 against pH1N1 challenge in mice. The in vivo efficacy of IY7640 was evaluated in BALB/c mice (n = 10 per group). The mice were treated twice daily (half daily dose per treatment) with IY7640 (45 mg/kg/day [IY7640-45], 90 mg/kg/day [IY7640-90] or 180 mg/kg/day [IY7640-180]) for 8 days after pH1N1 rK09 infection. Body weight changes (A) and survival rates (B) were recorded for 14 dpi. Mice treated with oseltamivir (45 mg/kg/day) were used as a therapeutic control. Mice infected with PBS (mock) or treated only with a vehicle were also used as negative controls.
FIG 9
FIG 9
Structural alignments of the CR6261 epitope (or equivalent) residues from H1, H3, and H5 HAs. (A) Alignment of the CR6261 epitope residues from H1 HA (PDB code 3UBQ [green]) and H5 HA (PDB code 2IBX [purple]). (B) The CR6261 epitope residues of H1 HA (PDB code 3UBQ [green]) and the equivalent H3 HA residues (PDB code 3ZTJ [magenta]). The alignments were performed using PyMOL. (C) Sequence alignment of the HA region comprising the CR6261 epitope or equivalent residues. Residues conserved compared with K09 HA are represented by dots. The HA residue numbers are given with H1 numbering. The residues highlighted with colors are the CR6261 epitope (orange) or equivalent residues. Shaded in green are the CR6261 epitope residues from H1 and H5 HA that interact with IY7640. Shaded in yellow are residues mutated in the K09 IY7640 escape mutants (L49I and M403T). **, the intervening sequences have been omitted. Abbreviations of the virus HAs are as follows: H1/Ca04, A/California/04/2009 (pH1N1); H5/IS06, A/chicken/IS/2006 (H5N1); H5/VN, A/Viet Nam/1203/2004 (H5N1); and H3/X-31, A/Aichi/2/1968 (H3N2).

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