Host targeting antiviral treatment strategies for Zika virus infection

Abstract

Zika virus (ZIKV) is a mosquito-borne flavivirus which caused an epidemic in the Americas between 2015 and 2016. ZIKV infection can cause neurological manifestations in humans, including congenital microcephaly in infected fetuses and Guillain-Barré syndrome. Currently, there are limited treatment options for ZIKV infection. The discovery of effective antiviral treatments for this emerging flavivirus infection is a research priority. In this thesis, two host-targeting antivirals, namely, the celecoxib derivative kinase inhibitor AR-12 (OSU-03012) and the SUMO inhibitor 2-D08, were identified as potential anti-ZIKV compounds. AR-12 is a celecoxib derivative cellular kinase inhibitor that was originally designed as anticancer drug. Interestingly, AR-12 exhibited broad-spectrum antimicrobial activities against viruses, bacteria, fungi, and parasites. In Chapter 2, we evaluated the antiviral activity of AR-12 against ZIKV and found that it inhibited the replication of both epidemic (Puerto Rico strain PRVABC59 and Guangdong strain GD) and pre-epidemic (976 Uganda strain) ZIKV strains in human neuronal (U251) and hepatic (Huh-7) cells. The half maximal inhibitory concentration (IC50) of AR-12 by viral load reduction assay using quantitative reverse transcription-polymerase chain reaction was <2 μM in these cells. Western blot and flow cytometry showed that AR-12 could inhibit ZIKV viral protein synthesis. Treatment of ZIKV-infected type I interferon receptor-deficient A129 mice with AR-12 had significantly higher survival rate (50.0% to 83.3% vs 0%, P < 0.05) and less body weight loss than mock-treated controls. Mechanistic assays revealed that AR-12 inhibited ZIKV replication by downregulating the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. In Chapter 3, we exploited in silico analysis to identify a potentially conserved SUMO-interacting motif (SIM) at the flaviviral non-structural 5 (NS5) protein. The alignment results showed that all known flaviviruses except insect-specific flaviviruses contain this SIM. Among ZIKV strains, this SIM is also highly conserved. Molecular docking predicted that the hydrophobic SIM core residues bind to the β2 strand of the human SUMO-1 protein. 2-D08 is a SUMO inhibitor which can prevent transfer of SUMO from the UBC9-SUMO thioester to the substrate. 2-D08 inhibited ZIKV replication in U251 and Huh-7 cells at high and low multiplicities of infection. The IC50 of 2-D08 against ZIKV was <8.88 μM in U251 cells and <15.63 μM in Huh-7 cells. Viral kinetics assay showed that 2-D08 could significantly inhibit ZIKV replication at up to 72 hours post-infection. Mutation of the putative SIM in ZIKV’s non-structural 5 protein resulted in the disablement of its interferon antagonism activity. Moreover, 2-D08 significantly reduced the replication of other human-pathogenic flaviviruses, including dengue virus, Japanese encephalitis virus, West Nile virus, and yellow fever virus. Overall, the novel findings in this thesis identified the PI3K/Akt inhibitor AR-12 and the SUMO inhibitor 2-D08 as potential host-targeting treatment options for ZIKV infection.