Identification of Clotrimazole Derivatives as Specific Inhibitors of Arenavirus Fusion

Abstract

Arenaviruses are a large family of emerging enveloped negative-strand RNA viruses that include several causative agents of viral hemorrhagic fevers. For cell entry, human-pathogenic arenaviruses use different cellular receptors and endocytic pathways that converge at the level of acidified late endosomes, where the viral envelope glycoprotein mediates membrane fusion. Inhibitors of arenavirus entry hold promise for therapeutic antiviral intervention and the identification of “druggable” targets is of high priority. Using a recombinant vesicular stomatitis virus pseudotype platform, we identified the clotrimazole-derivative TRAM-34, a highly selective antagonist of the calcium-activated potassium channel KCa3.1, as a specific entry inhibitor for arenaviruses. TRAM-34 specifically blocked entry of most arenaviruses, including hemorrhagic fever viruses, but not Lassa virus and other enveloped viruses. Anti-arenaviral activity was likewise observed with the parental compound clotrimazole and the derivative senicapoc, whereas structurally unrelated KCa3.1 inhibitors showed no antiviral effect. Deletion of KCa3.1 by CRISPR/Cas9 technology did not affect the antiarenaviral effect of TRAM-34, indicating that the observed antiviral effect of clotrimazoles was independent of the known pharmacological target. The drug affected neither virus-cell attachment, nor endocytosis, suggesting an effect on later entry steps. Employing a quantitative cell-cell fusion assay that bypasses endocytosis, we demonstrate that TRAM-34 specifically inhibits arenavirus-mediated membrane fusion. In sum, we uncover a novel antiarenaviral action of clotrimazoles that currently undergo in vivo evaluation in the context of other human diseases. Their favorable in vivo toxicity profiles and stability opens the possibility to repurpose clotrimazole derivatives for therapeutic intervention against human-pathogenic arenaviruses. IMPORTANCE Emerging human-pathogenic arenaviruses are causative agents of severe hemorrhagic fevers with high mortality and represent serious public health problems. The current lack of a licensed vaccine and the limited treatment options makes the development of novel antiarenaviral therapeutics an urgent need. Using a recombinant pseudotype platform, we uncovered that clotrimazole drugs, in particular TRAM-34, specifically inhibit cell entry of a range of arenaviruses, including important emerging human pathogens, with the exception of Lassa virus. The antiviral effect was independent of the known pharmacological drug target and involved inhibition of the unusual membrane fusion mechanism of arenaviruses. TRAM-34 and its derivatives currently undergo evaluation against a number of human diseases and show favorable toxicity profiles and high stability in vivo. Our study provides the basis for further evaluation of clotrimazole derivatives as antiviral drug candidates. Their advanced stage of drug development will facilitate repurposing for therapeutic intervention against human-pathogenic arenaviruses.

Synanthropic rodents as virus reservoirs and transmitters

“…Despite its efficacy, there are significant side effects, such as hemolytic anemia, progressive weight loss, respiratory difficulty, insomnia, and dermatitis, among others [180][181][182] . Alternative drugs with less side effects have been tested, such as favipiravir 183 and triarylmethane clotrimazole 184 . Cocktails using multiple antiviral drugs that target different steps of the viral life cycle appear to be the best strategy to limit viral multiplication with lower risk of drug resistance 185 .…”

LY6E impairs coronavirus fusion and confers immune control of viral disease

“…VSV pseudoparticles. Pseudotyping of VSV*ΔG(Fluc) was performed as previously described 32 . Briefly, 6 × 10 5 293LTV cells were seeded in a six-well plate and transfected using Lipofectamine 2000 (Invitrogen), which was complexed with DNA plasmids driving the expression of either VSV-G protein (positive control), the respective CoV spike proteins or the fluorophore mCherry (negative control).…”

Validation of a commercially available SARS-CoV-2 serological immunoassay

“…Two-fold dilutions were performed until 1:80 was reached by moving 60 ml from one well column to the following one. VSV-based SARS-CoV-2 pseudotypes (generated according to Berger, Rentsch, and Zimmer [11] and Torriani et al [12]) expressing a 19 amino acids C-terminal truncated spike protein [13] (NCBI Reference sequence: NC_045512.2) were diluted in DMEM 2% FCS in order to have MOI¼0.01 in 60 ml volume and added on top of serum dilutions (final serum dilutions obtained were from 1:10 to 1:160). The virusserum containing plate was incubated at 37 C, for 2 h. Vero E6 were then infected with 100 ml of virus-serum mixtures.…”