6′-β-Fluoro-Homoaristeromycin and 6′-Fluoro-Homoneplanocin A Are Potent Inhibitors of Chikungunya Virus Replication through Their Direct Effect on Viral Nonstructural Protein 1

Abstract

Alphaviruses are arthropod-borne, positive-stranded RNA viruses capable of causing severe disease with high morbidity. Chikungunya virus (CHIKV) is an alphavirus that causes a febrile illness which can progress into chronic arthralgia. The current lack of vaccines and specific treatment for CHIKV infection underscores the need to develop new therapeutic interventions. To discover new antiviral agents, we performed a compound screen in cell culture-based infection models and identified two carbocyclic adenosine analogues, 6=-␤-fluoro-homoaristeromycin (FHA) and 6=-fluoro-homoneplanocin A (FHNA), that displayed potent activity against CHIKV and Semliki Forest virus (SFV) with 50% effective concentrations in the nanomolar range at nontoxic concentrations. The compounds, designed as inhibitors of the host enzyme S-adenosylhomocysteine (SAH) hydrolase, impeded postentry steps in CHIKV and SFV replication. Selection of FHNA-resistant mutants and reverse genetics studies demonstrated that the combination of mutations G230R and K299E in CHIKV nonstructural protein 1 (nsP1) conferred resistance to the compounds. Enzymatic assays with purified wild-type (wt) SFV nsP1 suggested that an oxidized (3=-keto) form, rather than FHNA itself, directly inhibited the MTase activity, while a mutant protein with the K231R and K299E substitutions was insensitive to the compound. Both wt nsP1 and the resistant mutant were equally sensitive to the inhibitory effect of SAH. Our combined data suggest that FHA and FHNA inhibit CHIKV and SFV replication by directly targeting the MTase activity of nsP1, rather than through an indirect effect on host SAH hydrolase. The high potency and selectivity of these novel alphavirus mRNA capping inhibitors warrant further preclinical investigation of these compounds.

Suramin inhibits SARS-CoV-2 infection in cell culture by interfering with early steps of the replication cycle

“…The clinical isolate SARS-CoV-2/Leiden-002 was isolated from a nasopharyngeal sample at LUMC and its sequence and characterization will be described elsewhere (manuscript in preparation). SARS-CoV-2/Leiden-002 was passaged twice in Vero E6 cells and virus titers were determined by plaque assay as described before (17). Working stocks yielded titers of 5 × 10 6 plaque forming units (PFU)/ml.…”

“…Intracellular RNA was collected by lysing the cells in 150 µl Tripure reagent. Analysis of viral progeny in supernatant from Calu-3 cells was performed by plaque assay on Vero E6 cells (17). Potential cytotoxicity of the compound was tested in parallel on uninfected cells using the MTS assay (Promega) as described for the CPE reduction assay.…”

“…These were used as inoculum to infect the Vero E6 cells in 6-well clusters. After 1 h at 37°C, the inoculum was removed and cells were incubated in Avicel-containing overlay medium without suramin for 3 days, after which they were fixed with 3.7% formaldehyde, stained with crystal violet and plaques were counted (17).…”

Suramin Inhibits Chikungunya Virus Replication by Interacting with Virions and Blocking the Early Steps of Infection

“…In the case of CHIKV and other alphaviruses (SFV, ONNV), evolutionary pressures have maintained both variants (stop and arginine codon) as it offers a fitness advantage when switching between vertebrate and invertebrate hosts [35][36][37]. The combination of the R171Q and opal524R mutations was also found independently during the selection of CHIKV variants that were resistant to an unrelated compound [38], and they merely appear to reflect adaptation to repeated passaging in mammalian cells (under stress).…”

Small-Molecule Inhibitors of Chikungunya Virus: Mechanisms of Action and Antiviral Drug Resistance

“…The mutations G230R and K299E in nsP1 were identified by resistance selection and reverse genetics to confer resistance to both FHA and FHNA (59). Difluoromethylornithine (DFMO), an inhibitor of on October 30, 2020 by guest http://aac.asm.org/ Downloaded from ornithine decarboxylase 1, was shown to have a broad-spectrum antiviral effect on a variety of RNA viruses, including CHIKV (60).…”