Synthesis and antiviral effect of novel fluoxetine analogues as enterovirus 2C inhibitors

Abstract

Enteroviruses (EV) are a group of positive-strand RNA (+RNA) viruses that include many important human pathogens (e.g. poliovirus, coxsackievirus, echovirus, numbered enteroviruses and rhinoviruses). Fluoxetine was identified in drug repurposing screens as potent inhibitor of enterovirus B and enterovirus D replication. In this paper we are reporting the synthesis and the antiviral effect of a series of fluoxetine analogues. The results obtained offer a preliminary insight into the structure-activity relationship of its chemical scaffold and confirm the importance of the chiral configuration. We identified a racemic fluoxetine analogue, 2b, which showed a similar antiviral activity compared to (S)-fluoxetine. Investigating the stereochemistry of 2b revealed that the S-enantiomer exerts potent antiviral activity and increased the antiviral spectrum compared to the racemic mixture of 2b. In line with the observed antiviral effect, the S-enantiomer displayed a dose-dependent shift in the melting temperature in thermal shift assay, indicative for direct binding to the recombinant 2C protein.

Rational design of highly potent broad-spectrum enterovirus inhibitors targeting the nonstructural protein 2C

“…The data suggested a worsening of patient condition, possibly due to its SSRI activity [26]. Additionally, our previous work showed that the antiviral activity of fluoxetine is unlikely to be decoupled from its SSRI activity [24,27]. This clearly demonstrates that more potent and safer molecules are needed for treatment of EV infections.…”

“…Subsequently, protein was expressed for 16 h at 18˚C, with shaking at 200 rpm. Further protein purification steps were performed essentially as described previously [27], with the exception that TEV protease was replaced with 3C protease (Sigma-Aldrich). The final size-exclusion chromatography step was performed at 4˚C with buffer containing 25 mM Tris (pH 8), 300 mM NaCl, and 1 mM MgCl2, using a superose 6 increase 10/ 300 column GL (GE Healthcare Life Science, Eindhoven, The Netherlands).…”

“…The final size-exclusion chromatography step was performed at 4˚C with buffer containing 25 mM Tris (pH 8), 300 mM NaCl, and 1 mM MgCl2, using a superose 6 increase 10/ 300 column GL (GE Healthcare Life Science, Eindhoven, The Netherlands). The binding of (S)-and (R)-fluoxetine and both 2b-enantiomers to WT CV-B3 2C, and the F190L variant, was monitored by the fluorescence-based thermal shift assay using a Roche LightCycler480, as described previously [27]. Each well of the thermal shift assay plates contained WT or F190L 2C protein (final concentration of 10 μM in 50 mM Tris, 300 mM NaCl, and 1 mM MgCl2 [pH 8]) and a SYPRO orange solution in concentrations recommended by the manufacturer, in a final volume of 25 μL.…”

“…We previously established that fluoxetine inhibits viral replication stereospecifically by directly binding 2C [24]. Unfortunately, the chemical moiety important for the SSRI activity is essential for the antiviral activity, and thus far these two activities could not be uncoupled [27]. This raises concerns about the therapeutic application of fluoxetine and shows that other, potent, biosafe, and broad-spectrum antiviral inhibitors are needed.…”

In vitro screening of a FDA approved chemical library reveals potential inhibitors of SARS-CoV-2 replication

“…Whereas the number of clinical trials cannot be extensively multiplied, libraries of “old” drugs can be screened in vitro in medium- to high-throughput assays to significantly reduce the number of candidate molecules. In addition, screening of approved drugs can also pave the way for medicinal chemistry programs 6 . Screenings of drugs to be repositioned against SARS-CoV, MERS-CoV or other viruses already showed their relevance for the selection of antivirals active at least in vitro 7 – 9 .…”