Discovery of cyclosporine A and its analogs as broad-spectrum anti-influenza drugs with a high in vitro genetic barrier of drug resistance

Abstract

As the number of drug-resistant influenza viruses continues to increase, antivirals with novel mechanisms of action are urgently needed. Among the two classes of FDA-approved antiviral drugs, neuraminidase (NA) inhibitors, oseltamivir, zanamivir, and peramivir, are currently the only choice for the prevention and treatment of influenza virus infection. Due to the antigenic drift and antigenic shift, it will only be a matter of time before influenza viruses become completely resistant to these NA inhibitors. In pursuing the next generation of antiviral drugs with complementary mechanisms of action to those of the NA inhibitors, we have identified a natural product, cyclosporine A (CsA) (1), as a desired drug candidate. In this study, we discovered that CsA (1) and its analogs have broad-spectrum antiviral activity against multiple influenza A and B strains, including strains that are resistant to either NA or M2 inhibitors or both. Moreover, CsA (1) displays a high in vitro genetic barrier of drug resistance than oseltamivir carboxylate Mechanistic studies revealed that CsA (1) acts at the intermediate step of viral replication post viral fusion. Its antiviral mechanism is independent of inhibiting the isomerase activity of cyclophilin A (CypA), and CsA (1) has no effect on the viral polymerase activity The potent antiviral efficacy of CsA (1), coupled with the high in vitro genetic barrier of drug resistance and novel mechanism of action, renders CsA (1) a promising anti-influenza drug candidate for further development.

Keywords: Antiviral; Broad spectrum; CsA; Influenza virus; Resistance; Serial passage.

Figure 1

Structures of CsA (1) and TMN355 (2).

Figure 2

Plaque reduction assays of CsA (1) in inhibiting A/WSN/33 (H1N1) and clinically isolated influenza A and B viruses. The assay was carried out with MDCK cells expressing ST6Gal I gene. Plaque numbers were counted at each concentration, and the data was fit into dose-response curve with Prism 5, and the best fit EC₅₀ values were shown in Table 1. (A) Plaque assays of CsA (1) against six influenza A and two influenza B strains at 10 μM. Quantification of the plaque size by ImageJ revealed that the plaque sizes of all eight viruses were significantly reduced in the presence of 10 μM CsA (1). The plaque area ratios with and without CsA (1) are 29.5 % for A/California/07/2009 (H1N1), 15.6 % for A/Texas/04/2009 (H1N1), 24.0 % for A/Denmark/524/2009 (H1N1), 9.4 % for A/Denmark/528/2009 (H1N1), 4.1 % for A/Udorn/72 (H3N2), 0.6 % for A/Switzerland/9715293/2013 (H3N2), 7.2 % for B/Wisconsin/1/2010, and 15.3 % for B/Brisbane/60/2008. (B) CsA (1) in inhibiting A/WSN/33 (H1N1) at different concentrations; (C) CsA (1) in inhibiting A/Switzerland/9715293/2013 (H3N2) at different concentrations; (D) CsA (1) in inhibiting A/Texas/04/2009 (H1N1) at different concentrations; (E) CsA (1) in inhibiting B/Brisbane/60/2008 (Victoria) at different concentrations; (F) CsA (1) in inhibiting B/Phuket/3073/2013 (Yamagata) at different concentrations.

Figure 3

Natural CsA analogs and their antiviral efficacy against A/WSN/33 (H1N1). (A) Molecular structures of CsA (1), CsC (3), CsD (4), and CsH (5). (B) Anti-viral efficacy of CsA analogs against A/WSN/33 (H1N1). WJ332 is a known M2-S31N channel inhibitor and was used as a positive control (A/WSN/33 contains M2-S31N mutant and it is sensitive to WJ332). CsD (4) and CsH (5) displayed similar efficacy against A/WSN/33 (H1N1) as CsA (1); CsC (3) is slightly less effective than CsA (1). *, p<0.05; **, p<0.01; ***, p<0.001 in two-tail t-test when compared with No Drug condition.

Figure 4

Synthesis scheme of CsA analogs. Detailed synthesis procedures and compound characterization can be found in the Supporting Information.

Figure 5

Anti-viral efficacy of semi-synthesized CsA analogs against A/WSN/33 (H1N1) virus, and their cytotoxicity. (A) Anti-viral efficacy was evaluated with plaque reduction assay. All compounds were tested at 10 μM, and compounds (14) and (19) displayed severe cytopathic effect after 48 hours at 10 μM and no cells remained on the dishes. *, p<0.05; **, p<0.01; ***, p<0.001 in two-tail t-test when compared with No Drug condition. (B) Cytotoxicity of CsA (1) and its analogs. The cytotoxicity of compounds was evaluated using the neutral red uptake assay.

Figure 6

Summary of structure–activity relationships of CsA.

Figure 7

Anti-viral efficacy of CypA inhibitor TMN355 (2) against clinically isolated influenza A and B viruses in plaque assays. 10 μM TMN355 (2) were applied to overlay media after infection with virus. TMN355 (2) is a known CypA inhibitor.

Figure 8

Time-of-addition experiments. Cells were infected with A/WSN/33 (H1N1) virus at −2 hour time point; viruses were first incubate at 4°C for 1 hour for attachment, then at 37°C for 1 hour for virus entry. At time point 0 hour, cells were washed with PBS buffer and progeny viruses were harvested at 12 hours post infection. The titer of harvested virus was determined with plaque assay. (A) Oseltamivir carboxylate time-of addition experiment. Blue arrows show the period in which 1 μM oseltamivir carboxylate is present. (B) CsA (1) time-of-addition experiment. Red arrows show the period in which 6 μM CsA (1) is present.

Figure 9

Influenza minigenome assay. CsA (1) and CsH (5) did not inhibit A/WSN/33 (H1N1) viral polymerase activity. HEK293 cells were transfected with protein expression plasmids for influenza A/WSN/33 virus polymerase subunits PB1, PB2, PA, and nucleoprotein NP. An influenza virus-specific firefly luciferase reporter and a Renilla luciferase expression plasmid were co-transfected. Two hours after transfection, 10 μM nucleozin (21), 6 μM CsA (1), or CsH (5) were added to media. Nucleozin (21) is a known NP inhibitor and was used as a positive control. The firefly luciferase activity was normalized against Renilla luciferase activity. ns, NOT significant; *, p<0.05; **, p<0.01; ***, p<0.001 in two-tail t-test when compared with DMSO condition.