The Compound SBI-0090799 Inhibits Zika Virus Infection by Blocking De Novo Formation of the Membranous Replication Compartment

Abstract

Zika virus (ZIKV) is a mosquito-borne pathogen classified by the World Health Organization (WHO) as a public health emergency of international concern in 2016, and it is still identified as a priority disease. Although most infected individuals are asymptomatic or show mild symptoms, a risk of neurologic complications is associated with infection in adults. Additionally, infection during pregnancy is directly linked to microcephaly and other congenital malformations. Since there are no currently available vaccines or approved therapeutics for this virus, there is a critical unmet need in developing treatments to prevent future ZIKV outbreaks. Towards this end, we performed a large-scale cell-based high-content screen of 51,520 chemical compounds to identify potential antiviral drug candidates. The compound (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) was found to inhibit replication of multiple ZIKV strains and in different cell systems. SBI-0090799 did not affect viral entry or RNA translation but suppressed RNA replication by preventing the formation of the membranous replication compartment. Selection of drug-resistant viruses identified single amino acid substitions in the N-terminal region of non-structural protein NS4A arguing this to be the likely drug target. These resistance mutations rescued viral RNA replication and restored the formation of the membranous replication compartment. This mechanism of action is similar to clinically-approved NS5A inhibitors for hepatitis C virus (HCV). Taken together, SBI-0090799 represents a promising lead candidate for the development of an antiviral treatment against ZIKV infection for the mitigation of severe complications and potential resurgent outbreaks of the virus. IMPORTANCE This study describes the elucidation of (2E)-N-benzyl-3-(4-butoxyphenyl)prop-2-enamide (SBI-0090799) as selective and potent inhibitor of Zika virus (ZIKV) replication using a high throughput screening approach. Mapping and resistance studies, supported by electron microscopy observations, indicate that the small molecule is functioning through inhibition of NS4A-mediated formation of ZIKV replication compartments in the endoplasmic reticulum (ER). Intriguingly, this defines a novel non-enzymatic target and chemical matter for the development of a new class of ZIKV antivirals. Moreover, chemical modulation affecting this non-structural protein mirrors the identification and development of hepatitis C virus (HCV) NS5A inhibitor daclatasvir and its derivatives, similarly interfering with the formation of the viral replication compartment and also targeting a protein with no enzymatic activity, which have been part of a curative strategy for HCV.

A novel zebrafish-basedin vivomodel of Zika virus infection unveils NS4A as a key viral determinant of neuropathogenesis

“…16,[75][76][77][78] Both mutagenesis and pharmacological approaches recently demonstrated that NS4A regulates the biogenesis of these replication factories. 12,17 Most notably, Riva and colleagues have shown that ZIKV NS4A function in replication organelle morphogenesis can be specifically inhibited by the compound SBI-0090799. 17 Moreover, NS4A inhibits the Akt-mTOR pathway and the growth of neurospheres.…”

“…Reverse genetics and pharmacological approaches recently demonstrated that NS4A of ZIKV and dengue virus (DENV, closely related to ZIKV) contribute to the biogenesis of ER-derived replication organelles which host the vRNA synthesis process. 12,17 In addition, the NS4A-NS4B precursor is also believed to play specific roles in flavivirus life cycle. 18 Interestingly, DENV NS4B is the target of a several highly potent antivirals, including two currently in phase 2 clinical trials.…”

The in vitro and in vivo antiviral effects of aloperine against Zika virus infection

“…To determine at which stage the drug displayed inhibitory efficiency, a time-of-addition assay was developed as previously described. 32 Vero cells were seeded in a 12-well plate overnight and infected with ZIKV at an MOI = 0.1. Then the culture medium was removed after 1 h of infection with ZIKV viral inoculum.…”

Structures and Dynamics of Dengue Virus Nonstructural Membrane Proteins

“…It has been noted that residues of the N-terminal region of NS4A are indispensable for NS4A stability. Point mutations of some conserved residues within this region affect membrane proliferation [101,110]. Such observed effects might be due to alterations in membrane binding after mutation.…”