Hijacking of Host Cell Pathways by Human Enveloped Viruses and Discovery of Novel Indirect-Acting Antiviral Agents (IAAAs)
Host cell site-1 protease (S1P)/SKI-1 as master regulator of hepatitis C virus (HCV) infection: From lipid droplet biogenesis to broad-spectrum antivirals. Inhibiting S1P activity with our protein-based inhibitor (RRLL-S) completely blocked HCV infection of hepatoma cells in a dose-dependent manner. Thus, targeting host lipid droplets (LDs) biogenesis by inhibiting S1P/SKI-1 may have far-reaching applications in the therapeutic treatment of many important Flaviviridae viruses. In the case of HCV, overstimulation of host lipid metabolism in the liver during viral infection promotes cholesterol intracellular storage in host LDs, a critical cellular event for HCV replication, assembly, and budding (see Olmstead, A. D., et al. Jean, F. (2012) PLoS Pathogens. 8(1): e1002468).
Project summary: Today’s treatment of viral diseases involves a multi-drug regimen largely based on viral enzyme inhibition and using the so-called direct-acting antivirals (DAAs). Although research related to the development of new DAAs is dramatically expanding and under investigation in clinical trials, these multidrug regimens are limited by increasing multi-class drug resistance. About half of the drugs sold worldwide are small-molecule inhibitors directed at virally encoded enzymes (e.g., proteases, helicases, polymerases), and a large volume of data now confirms resistance to the best described drugs of the current antiviral repertoire. In the case of emerging and re-emerging viruses such as hepatitis C virus (HCV) and Flaviviridae-related members [Dengue virus (DNV) and West Nile virus (WNV)], one of the key scientific challenges in developing effective antiviral drugs is their high mutation rate due to the lack of efficient proofreading or mismatched repair systems, which hinder the effectiveness of the virus-enzyme inhibitors (DAAs). To help overcome these challenges with new standards of care for treatment of HCV based on DAAs, my lab has been proposing for several years to shift the current paradigm on global antiviral strategies towards the exploration of novel host-directed strategic targets. It is now well established that the hijacking of host-cell biosynthetic pathways by human enveloped viruses is a shared molecular event essential for the viral lifecycle. The next frontier is identifying the common critical host-cell pathways that are hijacked by pathogenic human viruses in order to develop broad-spectrum host-directed antivirals with novel mechanisms of action: i.e., indirect-acting antiviral agents (IAAs). The main objective of this research program is to discover new IAA strategies for treating HCV infection that could act synergistically in combination therapy with the current and upcoming standards of care involving DAAs. Our recent work published in PLoS Pathogens (2012) supports our main research hypothesis: We demonstrated that strategic manipulation of host cell SKI-1/S1P enzymatic activity in hepatoma cells by our novel protein-based inhibitor (RRLL-S) provides a means of effectively inhibiting HCV infection. The outcome of the proposed studies will lead to new insights into IAA strategies for treating Flaviviridae infection that could act synergistically in combination therapy with the current standards of care.
Key paper: Olmstead, A. D., Knecht, W., Lazarov, I., Dixit, S. G., and Jean, F. (2012) Human subtilase SKI-1/S1P is a master regulator of the HCV lifecycle and a potential host cell target for developing indirect-acting antiviral agents. PLoS Pathogens. 8(1): e1002468. Underlined in Nature’s Science-Business exchange SciBX 5(6). Our paper is already listed as one of the most viewed PLoS Pathogens articles in the “most views, all time” section (5,185 article views as of October 27, 2012). Names of authors from my lab are underlined.
Other papers of interest from the Jean lab in the field of protein-based therapeutics: Richer M et al., Jean F et al. (2004) Proc. Natl. Acad. Sci. U.S.A 101:10560-10565; Jean F et al. (2000) Proc. Natl. Acad. Sci. U.S.A 97:2864-2869; Jean F et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95:7293-7298
Discovering Novel Direct-Acting Antiviral Agents (DAAs) Targeting Virally Encoded Proteases
Targeting viral protease-associated replication complexes for antiviral chemotherapy: From membrane-targeted activity-based probes to novel naturally occurring protease inhibitors. Left panel: We developed and applied a novel series of membrane-anchored red-shifted fluorescent protein substrates to detect West Nile virus (WNV) NS2B/NS3 protease activity in human cells. Our study is the first to provide cellular insights into the biological and enzymatic properties of NS3, a prime target for inhibitors of WNV replication [Condotta, S. et al. and Jean, F. (2010) Biological Chemistry: Cover Illustration]. Right panel: The work presented in this communication demonstrates the importance of our membrane-targeted activity-based probes in studying the enzymology of complex induced-fit viral proteases such as the ER-anchored HCV NS3/4A during viral infection. [Martin, M., Jean, F. (2006) Biological Chemistry: Cover Illustration].
Project summary: The long-term goals of this research program targeted at virally encoded proteases are to increase our understanding of the virus host-cell interactions, and to discover how viral enzymatic pathways essential for the virus lifecycle can be interrupted. Our research program centers now on developing and evaluating new protease inhibitors of the Flaviviridae-encoded serine protease nonstructural (NS)3, which is essential for the viral lifecycle of hepatitis C, West Nile, and Dengue viruses. Over the last 10 years, my research in antiviral drug discovery has been punctuated by important contributions including 2 collective patents and 9 collective applications submitted to the University-Industry Liaison Office (UILO) at UBC. My team has discovered novel classes of naturally occurring direct-acting antivirals (DAAs) targeting viral proteases that are essential for the virus lifecycle of emerging and re-emerging human viruses of great concern around the world (e.g., SARS-CoV, HCV, DENV, and WNV]. One of our most exciting discoveries, novel small-molecule DAAs of the HCV NS3 protease, has been filed for a patent application and was supported by a CIHR Proof of Principle (POP) grant designed to advance discoveries towards commercializable technologies. My team has also reported the discovery of the first generation of anti-SARS agents directed at the SARS-CoV 3CL protease from marine natural products [collaborator (UBC), Dr. Andersen; Paper of the Year 2006 (Hamill P., et al. Jean F. (2006) Biological Chemistry 387: 1063-74].
Key paper: Martin, M., Condotta, S., Fenn, J., Olmstead, A., Jean, F. (2011) In-cell selectivity profiling of membrane-anchored and replicase-associated hepatitis C virus NS3-4A protease reveals a common, stringent substrate recognition profile. Biological Chemistry. 392: 927-35. The work presented in this communication demonstrates the importance of our membrane-targeted activity-based probes in studying the enzymology of complex induced-fit viral proteases such as the ER-anchored HCV NS3/4A during viral infection. The findings of our studies also demonstrate the potential of our experimental approaches based on membrane-targeted activity-based probes for antiviral drug screening directed at complex induced-fit membrane-bound viral proteases in the context of viral infection. For example, our fluorescent probes will greatly help the scientific research community in validating the specificity of novel anti-HCV small molecules targeted at HCV NS3/4A when tested against NS3-4A activity alone, in replication complexes, or within the course of HCV infection. Names of authors from my lab are underlined.
Other papers of interest from the Jean lab in the field of viral proteases as therapeutic targets: Condotta, S., et al., Jean, F. (2010) Biological Chemistry. 391: 549-59. (Cover Illustration); Martin, M. and Jean, F. (2006) Biological Chemistry. 387: 1075-80. (Cover Illustration); Hamill, P., et al., Jean, F. (2006) Biological Chemistry. 387: 1063-74 (Awarded Paper of the Year 2006: Board of Editors of Biological Chemistry); Hamill, P. and Jean, F. (2005) Biochemistry. 44:6586-96; Richer, M., et al. Jean, F. (2004) J. Biological Chemistry 279:10222-10227.
Exosomal MicroRNAs as Master Regulators of Viral Infection and Blood-Based Diagnostic Biomarkers of Human Viral Diseases
Host-cell microRNA fingerprints reveal new insights into influenza A biology. Unraveling the molecular basis of swine-origin H1N1 pandemic influenza A virus and highly pathogenic avian-origin H7N7 influenza A virus pathogenesis by microRNAome (miRNAome) analysis [see Loveday, E. K., et al., and Jean, F. (2012). Journal of Virology 86: 6109-6122]
Human exosomes – A new treasure chest for viral-disease biomarker discovery? Delivery of exosome-associated cargos molecules (e.g., miRNAs, mRNAs, polypeptides, and proteins) into a recipient host cell. Exosomes are microvesicles secreted by both normal and pathological cells and play important roles in intercellular communication. Secretory exosomes provide a rich source for discovering potential blood-based biomarkers through non-invasive blood tests (see cover image: Journal of Virology; June 2012, V. 86, Issue 12).
Project summary: As part of a Canadian Institute of Health Research (CIHR)-funded Research Team for Pandemic Preparedness (Dr. Jean, team leader), my lab has recently gathered experimental evidence of virus-specific circulatory microRNA (miRNA) expression signatures (biomarker fingerprinting) on infections with pathogenic human viruses [UBC invention disclosure (Dr. F. Jean, July 2012) and Journal of Virology (E. K. Loveday et al., F. Jean; Cover Image Vol. 86, Issue 12, 2012)]. Our discovery that the majority of the new miRNA biomarkers identified are associated with secretory microvesicles known as exosomes indicated to the team that secretory exosomes and their miRNA cargo molecules produced during viral infection could allow for the development of unique non-invasive diagnostic tests for viral-associated diseases. Circulating exosomes are detected in a wide range of biological fluids including human plasma, urine, and saliva; and they contain various subsets of cargo molecules (e.g., proteins, miRNAs, and mRNAs). Since circulating exosomes provide a rich source for discovering potential blood-based biomarkers through non-invasive blood tests, our findings also raise the possibility of identifying circulating miRNA biomarkers as novel diagnostic cellular markers in important viral-associated human diseases. Given the importance of miRNAs in viral infections (E. K. Loveday et al., F. Jean (2012), Journal of Virology) the viral strain-specific exosomal transport of miRNA cargo molecules and the rich source of secretory exosomes in biological fluids such as human serum, my laboratory is currently testing this exciting research hypothesis that exosome-associated miRNAs represent robust biomarkers for viral infectious diseases such as hepatitis C, Dengue hemorrhagic fever, and HIV-1/AIDS. With the recent breakthroughs in HCV treatments, chronic hepatitis C is expected to be curable in virtually all patients, highlighting an urgent need to develop non-invasive tests for the diagnosis of HCV-related chronic liver disease to prevent HCV-related deaths in Canada and worldwide.
Key paper: Loveday, E. K., Svinti, V., Diederich, S., Pasick, J., and Jean, F. (2012) Temporal and strain-specific hostmicroRNA molecular signatures associated with swine-orgin H1N1 and avian-origin H7N7 influenza A virus infection. Journal of Virology. 86: 6109-6122. (Cover Illustration Vol. 86, Issue 12). The work presented in this manuscript describes our original and exciting studies on elucidating the miRNA expression signatures induced by two important influenza A strains, low-pathogenic swine-origin influenza A virus (S-OIV) pandemic H1N1 (2009) and highly pathogenic avian-origin (A-OIV) H7N7 (2003), at multiple stages of infection. The results of our study provide the first experimental evidence that the host microRNAome is regulated in a temporal- and strain-specific manner by S- and A-OIV infections in human cells. Our results also identify novel potential exosomal miRNA biomarkers associated with pandemic S-OIV and deadly A-OIV-host infection. Featured in Microbe Magazine (July 2012) published by the American Society for Microbiology. Names of authors from my lab are underlined.
Research in the Jean Lab is Performed at UBC’s State-of-the-Art Facility
My exciting research projects are performed at the new state-of-the-art University of British Columbia Facility of Infectious Disease and Epidemic Research (UBC FINDER), which has been under my scientific directorship since January 2008. FINDER is one of the most important university-based research facilities of its kind in the world. The scientific instruments (CFI Award: $19.3M: Dr. Jean co-PI) are installed in customized laboratories within the facility [e.g., analytical/proteomic lab (1), imaging labs (2), flow cytometry lab (1), microbiology labs (3),] providing a unique national capability to apply the cutting-edge tools of genomics, proteomics, and imaging (i.e., infectomics) research to the study of emerging and re-emerging infectious diseases.
UBC FINDER website: www.finder.ubc.ca
NEXT GENERATION MOLECULAR DIAGNOSTICS FOR EMERGING VIRAL DISEASES
Project leader: DR. FRANCOIS JEAN, University of British Columbia
Funding: Canadian Networks of Centres of Excellence (NCE): IC-IMPACTS (the India-Canada Centre for Innovative Multidisciplinary Partnerships to Accelerate Community Transformation and Sustainability)
Canadian Team Members:
Dr. Leonard Foster, University of British Columbia
Dr. Mark Loeb, McMaster University
Indian Team Members
Dr. Santanu Chattopadhyay, NationWide - The Family Doctors
Dr. Navin Khanna, ICGEB
The global health burdens of emerging and re-emerging viral diseases such as West Nile fever and dengue hemorrhagic fever (DHF) are dramatically increasing. According to the World Health Organization (WHO), Canada recorded the third highest number of West Nile virus (WNV) cases in 2012; moreover, about 2.5 billion people are now at risk of dengue virus (DENV) infection worldwide with an estimated 390-million dengue cases annually, of which approximately 30% occur in India. WNV infection can lead to neuroinvasive disease and DENV infection and progress to life-threatening DHF; rapid and reliable diagnosis is therefore critical. However, current diagnostic tools are limited in their sensitivity, specificity, and multiplexing capabilities for detecting and identifying WNV and the four DENV serotypes circulating around the world. In light of these limitations, the WHO and the US Centers for Disease Control have stated that the development of early, rapid, and robust diagnostic methods for WNV and DENV should be research priorities.
In this IC-IMPACTS research program, Dr. Jean (UBC, Canada) and Dr. Chattopadhyay (NationWide, India) are leading a multidisciplinary team of distinguished investigators from Canada and India to develop and deliver a rapid, robust, and sensitive non-invasive multiplexing diagnostic test for detecting WNV and DENV in blood samples. The novel diagnostic technology will be validated and optimized using clinical samples obtained from Canadian and Indian patients who have been hospitalized with laboratory- confirmed WNV and DENV infections respectively. Upon delivery and deployment, this cutting-edge diagnostic for the detection of emerging and re-emerging viruses would have an enormous on-the-ground impact on healthcare in Canadian and Indian communities.