RESUMO
Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV, respectively) are mosquito-borne, neuroinvasive human pathogens for which no FDA-approved therapeutic exists. Besides the biothreat posed by these viruses when aerosolized, arthropod transmission presents serious health risks to humans, as demonstrated by the 2019 outbreak of EEE disease in the United States that resulted in 38 confirmed cases, 19 deaths, and neurological effects in survivors. Here, we describe the discovery of a 2-pyrrolidinoquinazolinone scaffold, efficiently synthesized in two to five steps, whose structural optimization resulted in profound antiviral activity. The lead quinazolinone, BDGR-49, potently reduced cellular VEEV and EEEV titers by >7 log at 1 µM and exhibited suitable intravenous and oral pharmacokinetic profiles in BALB/c mice to achieve excellent brain exposure. Outstanding in vivo efficacy was observed in several lethal, subcutaneous infection mouse models using an 8-day dosing regimen. Prophylactically administered BDGR-49 at 25 mg kg-1 per day fully protected against a 10× LD50 VEEV Trinidad donkey (TrD) challenge in BALB/c mice. Similarly, we observed 70% protection when 10× LD50 EEEV FL93-939-infected C57BL/6 mice were treated prophylactically with BDGR-49 at 50 mg kg-1 per day. Last, we observed 100% therapeutic efficacy when mice, challenged with 10× LD50 VEEV TrD, were dosed at 48 hours after infection with BDGR-49 at 25 mg kg-1 per day. Mouse brain viral titers at 96 hours after infection were reduced to values near the limit of detection. Collectively, these results underscore the substantial development potential of a well-tolerated, brain-penetrant lead compound that shows promise in preventing and treating encephalitic alphavirus disease.
Assuntos
Vírus da Encefalite Equina Venezuelana , Encefalomielite Equina do Leste , Humanos , Cavalos , Animais , Camundongos , Estados Unidos , Antivirais/farmacologia , Antivirais/uso terapêutico , Camundongos Endogâmicos C57BL , EncéfaloRESUMO
Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50â=â0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.
Assuntos
Antivirais/farmacologia , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Encefalomielite Equina Venezuelana/tratamento farmacológico , Quinazolinonas/farmacologia , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Modelos Animais de Doenças , Farmacorresistência Viral/genética , Vírus da Encefalite Equina Venezuelana/genética , Encefalomielite Equina Venezuelana/virologia , Ensaios de Triagem em Larga Escala , Camundongos , Camundongos Endogâmicos C3H , Especificidade da Espécie , Relação Estrutura-Atividade , Células Vero , Ensaio de Placa Viral , Replicação Viral/efeitos dos fármacosRESUMO
Venezuelan equine encephalitis virus (VEEV) may cause encephalitis in humans, for which no FDA-approved antiviral treatment is available. Carbocyclic cytosine (carbodine) has broad-spectrum activity but toxicity has limited its utility. It was anticipated that one of the enantiomers of carbodine would show enhanced activity and reduced toxicity. The activity of the d-(-) enantiomer of carbodine [(-)-carbodine] was evaluated by infectious cell culture assay and was found to have a 50% effective concentration (EC50) of 0.2 microg/ml against the TC-83 vaccine strain of VEEV in Vero cells, while the l-(+) enantiomer had no activity. Virus titer inhibition correlated with intracellular cytidine triphosphate reduction after treatment with (-)-carbodine, as determined by HPLC analysis. Pre-treatment with 200 mg/(kgd) resulted in significant improvement in survival, virus load in the brain, weight change, and mean day-to-death in a mouse model of TC-83 VEEV disease. A single dose of (-)-carbodine resulted in a slight extension of mean time to death in mice infected with wild-type VEEV. Post-virus exposure treatment with (-)-carbodine was effective in significantly improving disease parameters in mice infected with TC-83 VEEV when treatment was initiated as late as 4 days post-virus installation (dpi). It is remarkable that (-)-carbodine is effective when initiated after the establishment of brain infection.
Assuntos
Antivirais/farmacologia , Citidina/análogos & derivados , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Encefalomielite Equina Venezuelana/tratamento farmacológico , Animais , Chlorocebus aethiops , Citidina/farmacologia , Citidina Trifosfato/metabolismo , Encefalomielite Equina Venezuelana/virologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos ICR , Células VeroRESUMO
The TC-83 vaccine strain of Venezuelan equine encephalitis virus (VEEV) causes encephalitis and death in C3H/HeN mice infected by intranasal (i.n.) instillation. Since TC-83 is exempt as a select agent, this mouse model was used in the evaluation of antiviral therapies. Virus titers in the brains of infected mice peaked on 4 dpi and persisted at high levels until death at 9.4+/-0.5 dpi. Mouse brains appeared histologically normal on 2 dpi, but developed meningoencephalitis, neuropil vacuolation, and gliosis by 8 dpi. Results from a protein cytokine array showed significant elevations over time in interleukin (IL)-1alpha, IL-1beta, IL-6, IL-12, MCP-1, IFNgamma, TNFalpha, MIP-1alpha, and RANTES in homogenized brain samples of infected mice. Immunohistochemical staining showed a colocalization of viral antigen with neuron markers. Treatment with interferon-alpha B/D or ampligen significantly improved survival, brain virus titer and cytokine levels, mean day-to-death, and weight change in infected mice. The time-course of infection and disease parameters of mice infected with TC-83 VEEV were similar in many ways to disease parameters in mice infected with other VEEV strains. Thus, infection of C3H/HeN mice with TC-83 VEEV may serve as a suitable model for the evaluation of antiviral compounds for the treatment of this viral disease.