Chief Investigator:

Dr Nicholas Eyre

Funding Amount:

$71,386

Recipient:

The University of Adelaide

Overview:

The recent outbreak of Zika virus (ZIKV) in South America and its association with birth defects has been declared by the World Health Organization (WHO) as a “public health emergency of international concern”. Accordingly, there is an urgent need for improved diagnostics, vaccines and antiviral therapies to combat the spread and impact of ZIKV. This project will identify inhibitors of the ZIKV NS2B/3 protease that may be developed as future antiviral therapies to treat ZIKV infection.

Research Outcomes:

Researchers:

Nicholas Eyre, Michael Beard

Research Completed:

2018

Research Findings:

The recent explosive outbreak of Zika virus (ZIKV) in Brazil in 2015 revealed the unexpected ability of the virus to be transmitted from infected pregnant women to their unborn babies. This infection of foetuses can cause severe neurodevelopmental defects, including microcephaly, in which the newborn baby’s head is smaller than expected due to abnormal brain development. Given that ZIKV can be readily transmitted via the bite of an infected mosquito and, unexpectedly, via sexual contact, there is an urgent need for the development of safe and effective vaccines and antiviral therapies to limit the spread and impact of ZIKV infection. In this study we used an advanced robotic screening facility, ‘CellScreen SA’, at Flinders University to screen a library of approximately 3,000 approved and well-characterised drugs for compounds that inhibit ZIKV infection of cultured cells and may therefore be explored as novel antiviral therapies to treat ZIKV infection. This screening identified 15 drugs that significantly inhibited ZIKV infection and could be explored as future antiviral therapies. Amongst these drugs were molecules that are synthetic mimics of the female sex hormone estrogen. In subsequent experiments we demonstrated that these synthetic estrogens can inhibit infections ZIKV and related viruses dengue virus and West Nile virus at low doses in several relevant cell culture models of viral infection. Interestingly, these antiviral effects appear to be independent of the well-characterised hormonal activity of these drugs and act to inhibit early events in viral infection. Taken together, our studies indicate that the antiviral activity of synthetic estrogens and chemically related compounds warrants further investigation towards their application as antiviral therapies to limit the impact of ZIKV infection and infections with related flaviviruses.

Key Outcomes:

Zika virus (ZIKV) is a mosquito-borne flavivirus that is closely related to other significant human flavivirus pathogens such as dengue virus (DENV), West Nile virus (WNV) and yellow fever virus (YFV). The virus was originally identified in Uganda in 1947 and, until recently, was thought to only cause mild febrile illness. This changed when large outbreaks in Micronesia in 2007 and in French Polynesia in 2013 lead to the association of ZIKV infection with the autoimmune disease Guillain-Barre syndrome. Moreover, the large and explosive ZIKV outbreak in Brazil and the Americas in 2014-2015 lead to the definitive identification of ZIKV as a causative agent in the development of serious neurodevelopmental disorders in unborn foetuses, frequently culminating in microcephaly and foetal demise. Furthermore, demonstration that ZIKV can be transmitted vertically from mother to foetus and via sexual contact added to concern that the impact of ZIKV could extend beyond South America and the seasonal nature of outbreaks that is typically associated with mosquito-borne viral epidemics. The explosiveness of the South American outbreak, the association of infection with foetal microcephaly and the lack of availability of a protective vaccine or safe and effective antiviral drugs lead the World Health Organization (WHO) to declare this ZIKV outbreak as ‘a public health emergency of international concern’. Accordingly, development of safe and effective antiviral drugs to limit the spread and impact of ZIKV infection is a major international research priority.

In this project we employed high-throughput automated microscopy to screen a library of ~2,900 approved drugs and well-characterised pharmacologically active compounds for small molecules that inhibit ZIKV infection in cultured cells. This approach has many advantages over classical approaches to develop new antiviral drugs that are typically employed in the pharmaceutical industry. Specifically, as the library of compounds that we screened primarily contains drugs that have already been approved for treatment of other conditions, any compounds that are found to display antiviral activity can potentially rapidly progress through pre-clinical and clinical testing as their properties are already well known and their safety in humans has already been demonstrated. Interestingly, this screening identified several synthetic estrogens, including Quinestrol and Raloxifene hydrochloride, as potent antiviral inhibitors of ZIKV infection. These drugs, which are currently used in hormone replacement therapies and in treatment of certain breast cancers, were found to inhibit infection with ZIKV and related flaviviruses DENV and WNV in several different cultured cell lines at low (micromolar) concentrations. While these drugs are known to bind and modulate the activity of human estrogen receptors, we found that the cell lines that we used to demonstrate the antiviral activity of these drugs lack detectable expression of estrogen receptors. This indicates that the antiviral properties of these drugs are independent of their ability to modulate estrogen receptor activity and instead may involve unanticipated effects on the viral replication cycle. Further validation studies revealed that these drugs predominantly disrupt early events in viral infection such as virus attachment and uptake or initial establishment of viral replication in infected cells.

In summary this project has identified 15 approved drugs that inhibit ZIKV infection, with subsequent validation studies revealing that the synthetic estrogens Quinestrol and Raloxifene hydrochloride potently inhibit ZIKV infection by disrupting early events in viral infection in a manner that is independent of estrogen receptor activation that is typically associated with the activity of these drugs. Further studies to modify the chemical properties of these drugs to improve their antiviral activity and reduce their estrogenic effects may identify safe and effective antiviral therapies to combat the spread and impact of ZIKV and related flaviviruses.

Research Papers:

Manuscript in preparation:

Eyre NS, Kirby EK, Anfiteatro D, Bracho G, Aloia AL, Beard MR. Identification of estrogen receptor modulators as inhibitors of flavivirus infection. Manuscript in Preparation for submission to ‘ACS Infectious Diseases’

(The support of the Channel 7 Children’s Research Foundation will appropriately be acknowledged in the ‘Acknowledgements’ section of this publication)

Conference presentations:

Functional High Throughput Technologies Australia (FHTTA) 2018 Meeting, October 24^th-25^th, Canberra Australia. Invited Oral Presentation: ‘Identification of Novel FDA-Approved Inhibitors of Zika Virus Infection. Delivered by Dr. Amanda Aloia (CellScreen SA [CeSSA], Flinders University, SA) on behalf of the research team.

(The support of CRF was appropriately acknowledged in an ‘Acknowledgements’ slide at the end of this presentation and the abstract for this talk can be found in the attachment: FHTTA 2018 Program and Abstract Booklet)

Positive-Strand RNA Viruses Keystone Symposium 2019, June 9^th-13^th, Killarney, Ireland. Abstract Submitted: ‘Identification of estrogen receptor modulators as inhibitors of flavivirus infection’

(The support of the Channel 7 Children’s Research Foundation will appropriately be acknowledged in the ‘Acknowledgements’ section of this publication)

Related Publications:

fhtta_2018_program_and_abstract_booklet

Future Outcomes:

The results of this project cannot be directly commercialised given that the drugs that we have screened for antiviral effects have already been well-characterised in pharmaceutical industry and academic research studies and are already protected by third party intellectual property. However, in future studies it may be possible to develop compounds that are chemically related to the antiviral drugs that we identified and display improved antiviral properties and reduced estrogenic effects. This possibility may be explored in future projects and associated funding applications.

Remarks:

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