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. 2023 Aug:216:105654.
doi: 10.1016/j.antiviral.2023.105654. Epub 2023 Jun 14.

Efficacy of an isoxazole-3-carboxamide analog of pleconaril in mouse models of Enterovirus-D68 and Coxsackie B5

Thomas R Lane  1 , Jianing Fu  2 , Barbara Sherry  3 , Bart Tarbet  4 , Brett L Hurst  4 , Olga Riabova  5 , Elena Kazakova  5 , Anna Egorova  5 , Penny Clarke  6 , J Smith Leser  6 , Joshua Frost  7 , Michael Rudy  8 , Kenneth L Tyler  9 , Thomas Klose  2 , Alexandrina S Volobueva  10 , Svetlana V Belyaevskaya  10 , Vladimir V Zarubaev  10 , Richard J Kuhn  2 , Vadim Makarov  5 , Sean Ekins  11
Affiliations

Efficacy of an isoxazole-3-carboxamide analog of pleconaril in mouse models of Enterovirus-D68 and Coxsackie B5

Thomas R Lane et al. Antiviral Res. 2023 Aug.

Abstract

Enteroviruses (EV) cause a number of life-threatening infectious diseases. EV-D68 is known to cause respiratory illness in children that can lead to acute flaccid myelitis. Coxsackievirus B5 (CVB5) is commonly associated with hand-foot-mouth disease. There is no antiviral treatment available for either. We have developed an isoxazole-3-carboxamide analog of pleconaril (11526092) which displayed potent inhibition of EV-D68 (IC50 58 nM) as well as other enteroviruses including the pleconaril-resistant Coxsackievirus B3-Woodruff (IC50 6-20 nM) and CVB5 (EC50 1 nM). Cryo-electron microscopy structures of EV-D68 in complex with 11526092 and pleconaril demonstrate destabilization of the EV-D68 MO strain VP1 loop, and a strain-dependent effect. A mouse respiratory model of EV-D68 infection, showed 3-log decreased viremia, favorable cytokine response, as well as statistically significant 1-log reduction in lung titer reduction at day 5 after treatment with 11526092. An acute flaccid myelitis neurological infection model did not show efficacy. 11526092 was tested in a mouse model of CVB5 infection and showed a 4-log TCID50 reduction in the pancreas. In summary, 11526092 represents a potent in vitro inhibitor of EV with in vivo efficacy in EV-D68 and CVB5 animal models suggesting it is worthy of further evaluation as a potential broad-spectrum antiviral therapeutic against EV.

Keywords: Antiviral; Coxsackievirus B5; Cryo-electron microscopy; EV-D68; Enteroviruses.

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Conflict of interest statement

Declaration of competing interest SE is owner, TRL is an employee at Collaborations Pharmaceuticals, Inc. SE and VM are co-inventors on a patent submitted for 11526092. All other co-authors have no competing interests.

Figures

Fig. 1.
Fig. 1.
Structures of isoxazole-3-carboxamide pleconaril derivatives
Fig. 2.
Fig. 2.
Cryo-EM structure of Enterovirus 68 (EV-D68) inhibitor 11526092 bound to the capsid Viral Protein 1 (VP1) of EV-D68 (MO strain). (A) An icosahedral asymmetric unit of the EV-D68-inhibitor 11526092 complex. VP1, VP2, VP3 and VP4 are colored in blue, green, red, and magenta, respectively. The density of inhibitor 11526092 is shown as a grey mesh. (B) A zoomed in view of the inhibitor 11526092 (grey) in the VP1 pocket (blue). (C) An enlarged view of inhibitor 11526092 fitted into the density (contour level 0.8) and the interfacing residues. (D) Comparison of the VP1s between EV-D68-inhibitor 11526092 complex (blue) and control (light purple). Large movements are highlighted by magenta dashes
Fig. 3.
Fig. 3.
EV-D68 respiratory model viremia levels in (A) whole blood and (B) lung. Infectious virus was determined for viremia and viral lung titers by assessing the infection of human rhabdomyosarcoma (RD) cells in microtiter plates. Fifty percent cell culture infectious doses (CCID50) were converted to CCID50 per gram of lung prior to statistical analysis. Ordinary ANOVA followed by a Dunnett’s T3 multiple comparisons test was performed in Graphpad Prism 9.5.0 for macOS. Asterisks represent statistical significance using the GP style, where P values of ≤0.0001, ≤0.001, ≤0.01, ≤0.05 and ≥0.05 are summarized with “****”, “***”, “**”, “*” and ns, respectively. Error bars represent the SEM.
Fig. 4.
Fig. 4.
Select cytokine and interleukins levels in lung homogenates of AG129 mice following ma-EV-D68 challenge in the AFM respiratory mouse model. A. IL-1α, B. IL-1β, C. IL-6, D. GM-CSF, E. IL-3, F. IL-5, Each cytokine/interleukin level was compared to the placebo group for statistical significance. Ordinary two-way ANOVA followed by a Dunnett’s T3 multiple comparisons test was performed in Graphpad Prism 9.5.0 for macOS. Error bars represent the SEM. For additional cytokines see Fig. S17.
Fig. 5.
Fig. 5.
Viral load in mice infected with ma-CVB5. CVB5 viral load in pancreatic tissue from CVB5-challenged mice treated with vehicle, pleconaril or 11526092 (100 mg/kg per day, twice a day, n=6/group). A virus titer in the supernatant was determined by (A) TCID50 titration (endpoint dilution) (B) and relative fold gene expression in the pancreas of CVB5-infected mice using qPCR (2−ΔΔCt) with actin B as the reference gene. Statistical significance was calculated using an ordinary one-way ANOVA followed by a Holm-Šídák's multiple comparisons test (A) or Kruskal-Wallis with Dunn’s multiple comparisons test (B) in Graphpad Prism 9.5.0 for macOS
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