virology – Drug Discovery Opinion

New (Host) Target for Treating HCV

Water droplet — Image: Flickr - Jack Newton

Hepatitis C virus (HCV) is one of the most important causes of chronic liver disease and infection can lead ultimately to cirrhosis and liver cancer. Current standard-of-care treatment – a combination of pegylated α-interferon and ribavirin – is unable to clear the virus in all patients and new antiviral agents designed to inhibit specific viral enzymes such as the protease, helicase and polymerase are being developed.

Researchers led by a team at the Gladstone Institute of Virology and Immunology (GIVI) have now identified a human enzyme that is also needed for viral infectivity, a discovery that may offer a new strategy for treatment. The enzyme, diacylglycerol acyltransferase 1 (DGAT1), is one of two DGAT enzymes that catalyse the final step in triglyceride synthesis. HCV infection is closely tied to lipid metabolism and the Gladstone team showed that infection and replication is severely impaired in liver cells that lack DGAT1 activity: either RNAi-mediated knockdown of DGAT1 or treatment with a DGAT1 inhibitor was effective in limiting production of infectious viral particles. The team went on to show that DGAT1 interacts with the viral nucleocapsid core protein and is required for the trafficking of the core protein to lipid droplets. Knockdown of the other enzyme involved in triglyceride synthesis, DGAT2, had no effect on viral replication.

DGAT1 inhibitors are already being developed as treatments for type II diabetes and obesity and the new study, which is published in Nature Medicine, suggests that they may also be useful for treating HCV infection.

Cutting off Lipid Supply Halts HCV

New research has shown that compounds that affect cellular energy status could also be used to treat hepatitis C virus (HCV) infections. Metformin, which is used to treat type II diabetes, and 5-amino-1-β-D-ribofuranosyl-1H-imidazole-4-carboxamide (AICAR), which has been shown to mimic the beneficial effects of exercise in mice, stimulate AMP-activated protein kinase (AMPK). AMPK is a key sensor of cellular energy status and regulates both lipid and glucose metabolism to maintain cellular energy balance and protect against metabolic stress. An increase in the AMP/ATP ratio initiates an AMPK-mediated switch from activities that consume ATP, such as lipid production, to activities that produce ATP, such as lipid and glucose oxidation.

Infection with viruses might be expected to activate AMPK because of the energy demands put on the cell by viral replication, but research led by scientists at the University of Leeds has shown instead that HCV switches off AMPK so that the cell continues to produce the lipids needed to provide new viral particles with a protective outer coat. When the team treated HCV-infected cells with metformin or AICAR, AMPK activity was restored and viral replication was inhibited.

The team plan to carry out a small scale clinical trial to investigate the effects of AMPK activators in HCV infection and hope that such drugs may provide much-needed new treatments for HCV.

The study is published in the Proceedings of the National Academy of Science.

Targeting Influenza A Nucleoprotein

Although the recent sporadic outbreaks of influenza A virus H5N1 and of a new variant of H1N1 in 2009 were less serious than initially feared, public health responses gave an indication of the potential for pandemic influenza A to wreak havoc amongst human populations. Timely development of vaccines should help to contain future outbreaks, but effective antiviral medicines will also be needed. Circulating strains of influenza A virus with resistance to existing neuraminidase inhibitors have already been discovered, and new molecular targets would provide additional protection in the event of a fresh outbreak.

Researchers led by a team at the University of Hong Kong have now identified a compound, nucleozin, which can aggregate the viral nucleoprotein and prevent its transport into the nucleus. The nucleoprotein plays critical roles in viral RNA replication and genome assembly, and nucleozin was shown to block replication of H1N1, H3N2, and H5N1 viruses in cell culture experiments and also to protect mice from lethal challenge with highly pathogenic avian influenza virus A H5N1.

The study, which is published in Nature Biotechnology, shows that the nucleoprotein is a viable drug target and could lead to the development of new treatments to control the impact of future influenza A outbreaks. Potential binding sites for nucleozin on the influenza nucleoprotein were also predicted using molecular docking models.

HIV drugs could treat prostate cancer and CFS – if they are caused by XMRV

The retrovirus, xenotropic murine leukemia virus-related virus (XMRV), has been – controversially – linked to both prostate cancer and chronic fatigue syndrome (CFS). In an attempt to clarify the association of XMRV with disease, researchers at Emory University are developing a serum-based assay to detect neutralising antibodies to the virus which should begin to answer basic questions about how widespread the virus is, and how it is transmitted. The team found good agreement between their serum based assay and polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) tests carried out on prostate samples from cancer patients, and showed that at least some of the patients had been infected with XMRV. The study is published in the April issue of Urology.

Meanwhile, other researchers at Emory University/Atlanta Veterans Affairs Medical Center and the University of Utah have been looking for ways to treat XMRV should it turn out to have a causal role in prostate cancer or chronic fatigue syndrome. The team evaluated 45 compounds, mostly drugs approved for the treatment of HIV/AIDS, and found that four of them were able to inhibit XMRV with EC₅₀ values of < 1µM. XMRV replication was studied in both MCF-7 cells (generated from human breast cancer) and LNCaP cells (generated from human prostate cancer). The most effective compounds were two nucleoside reverse transcriptase inhibitors (zidovudine and tenofovir disoproxil fumarate) and two integrase inhibitors (raltegravir and L-000870812). Despite the lack of homology (only 14% identity) between HIV-1 integrase and XMRV integrase, raltegravir showed particularly good activity against XMRV with EC₅₀ values of 0.005µM and 0.03µM in MCF-7 and LNCaP cells respectively (cf 0.001µM for HIV-1 grown in PBMCs). The EC₉₀/EC₅₀ ratio was significantly higher for XMRV grown in MCF-7 cells than for XMRV grown in LNCaP cells or for HIV-1 grown in PBMCs (700, 15 and 9 respectively). Synergy studies were carried out in LNCaP cells: combinations of raltegravir and any of the other three compounds were found to act synergistically.

The authors hope that if XMRV is established as a cause of prostate cancer or CFS, existing HIV treatments may prove to be effective therapies for these conditions.

The study is published in PLoS ONE.

Mouse Model of Liver Disease

Although very different at a molecular level, hepatitis viruses B and C (HBV and HCV) both infect only humans and chimpanzees which means that there is a lack of suitable small animal models for studying viral lifecycles and for testing new drugs. One alternative would be to study the viruses and test new compounds in liver cells grown in vitro, but human hepatocytes are very difficult to grow and maintain in culture.

A team of researchers led by scientists at the Salk Institute has now provided a solution to the problem by generating a mouse with a liver that is almost completely ‘humanised’. The team had previously generated a mouse with a partially humanised liver but wanted to achieve more complete transformation. Around 95% of the liver cells of the new mice are human in origin and the animals are susceptible to infection by both HBV and HCV. Mice infected with HCV were shown to respond to drugs such as pegylated interferon α2a and ribavirin that are used to treat human patients. Adefovir dipivoxil, used to treat HBV patients, was found to lower viral titres in mice infected with HBV.

The mice were generated by using genetic and pharmacological pressures to lead to a growth disadvantage for mouse hepatocytes and positive selection for transplanted human hepatocytes. The mice provide a new way to study pathogens that target the human liver and to test drugs to treat human hepatitis. In the future, the mice could also be used to study other hepatotrophic pathogens such as malaria, as well as cirrhosis and liver cancer.

The study is published in the Journal of Clinical Investigation.

Enzyme Deficiency Protects from Ribavirin-Related Anaemia

Inosine Triphosphatase structure — Inosine Triphosphatase crystal structure - PDB ID=2CAR

Some 200 million people worldwide are estimated to be infected with the hepatitis C virus (HCV) which can eventually lead to cirrhosis, liver cancer, and in some cases, death. The current standard-of-care treatment for persistent infection – a combination of pegylated interferon-α and ribavirin – is able to clear the infection in around 50% of patients. In some patients, however, treatment is associated with haemolytic anaemia which may be severe enough to need dosage reduction or even discontinuation of treatment.

A team led by scientists at Duke University’s Institute for Genome Sciences & Policy (IGSP) have now discovered that loss of function mutations in the gene ITPA, which encodes the enzyme inosine triphosphatase, protect against the development of anaemia. Previous studies had identified the genetic variants with enzyme deficiency and, through a genome-wide association study, the Duke team were able to show that they were also protective against anaemia caused by ribavirin. The finding may offer new treatment opportunities for HCV patients with coronary artery disease or kidney disease who are often not treated with ribavirin because of fears that anaemia could exacerbate their condition.

Inosine triphosphatase deficiency was first recognised over 30 years ago and is not thought to be clinically important. A diagnostic test that could predict deficiency, and hence reduced susceptibility to ribavirin-associated anaemia, would allow broader treatment options for HCV patients.

The study is published in the journal Nature.

Virus-like Particles Protect against Chikungunya Virus Infection.

gnarled tree — Image: Flickr - Scalespeeder

Chikungunya is a viral disease spread by mosquitoes which causes fever and severe joint pain – the name derives from a verb meaning ‘to become contorted’ and describes the appearance of sufferers bent with pain. Chikungunya is an alphavirus of the family Togaviridae and is usually spread by Aedes aegypti mosquitoes. In 2005-2006, however, a point mutation in one of the viral envelope genes facilitated transmission by Aedes albopictus (tiger mosquito), increasing the risk of outbreaks in areas where the Asian tiger mosquito is present. In the coming years, expansion of the ranges of mosquitoes and changes in insect vectors could increase the spread of Chikungunya virus and other arboviruses.

There is no cure for Chikungunya and treatment is focussed on relieving symptoms. There is also no commercially available vaccine but researchers in the US have now developed an experimental vaccine using non-infectious virus-like particles (VLPs). Selective expression of viral structural proteins produced VLPs that resemble replication-competent alphaviruses and immunization with these VLPs led to neutralizing antibodies against envelope proteins from alternative Chikungunya strains. Rhesus macaques produced high-titre neutralizing antibodies that protected against viremia after high-dose challenge. When the monkey antibodies were transferred into immunodeficient mice, they protected against subsequent lethal viral challenge, indicating a humoral mechanism of protection. VLPs could potentially be developed to offer protection from other alphaviruses such as O’nyong’nyong virus, Ross River virus and Barmah Forest virus. Virus-like particle based-vaccines against human papillomavirus and hepatitis B virus have already been approved by the Food and Drug Administration.

The study is published in the journal Nature Medicine.

Keeping out Newcomers Helps Virus Spread

Beach Huts — Image: Flickr - S John Davey

For more than 50 years, it has been supposed that the rate of spread of viruses is limited by replication kinetics in an iterative process of infection, replication and release, but scientists at Imperial College London have now challenged this view. Using live video microscopy, vaccinia virus was found to spread four times more quickly than should have been possible, based on the rate at which it can replicate. The videos showed that the virus spreads by surfing from cell to cell, using a mechanism that allows it to bounce past cells that are already infected and reach uninfected cells as quickly as possible. Soon after vaccinia infects a cell, two viral proteins, A33 and A36, are expressed at the cell surface, marking the cell as infected. When new viruses approach the infected cell, these proteins trigger the host cell to project actin ‘tails’ which physically repel approaching viruses. In this way, the viruses are propelled from cell to cell until they find one that is not already infected.

HSV-1 also spreads at a faster rate than should be possible given its replication rate and may use a similar spreading mechanism. If the ability to signal that a cell is already infected proves to be a common feature of pathogenic viruses, the discovery could eventually lead to new antiviral drugs that exploit this mechanism.

The study is published in the journal Science.

What Doesn’t Kill Them Makes Them Stronger

The concept of ‘lethal mutagenesis’ has been developed as a means of curing viral infections and has also been used to explain the action of some antiviral drugs. Although mutation is the basis for adaptation and survival, especially in the presence of antiviral drugs, most mutations are detrimental and the theory of lethal mutagenesis holds that an infecting population can be pushed to extinction by an overwhelmingly high mutation rate. Chemical mutagens have been used to increase error rates in a number of RNA viruses including HIV-1 and HCV and have been found to significantly reduce viral titres and, in some cases, achieve extinction. For example, the antiviral activity of the ribonucleoside analogue 5-azacytidine (5-AZC) against HIV-1 has been attributed primarily to an increase in mutant frequency consistent with lethal mutagenesis caused by incorporation of 5-AZC into viral DNA.

A team of researchers from the University of Texas at Austin have now raised serious concerns about the strategy of inducing lethal mutations, suggesting that it could cause viruses to become more virulent. The team predicted that growing the DNA bacteriophage T7 in the presence of a mutagen would lead to a substantial decline in viral fitness but found instead that, after 200 generations, fitness had increased despite a mutation rate two to three orders of magnitude above baseline. Although the researchers agree that extremely high mutation rates will mostly lead to viral extinction, they caution that forcing viruses to undergo rapid mutation could, if the mutation rate is not high enough, lead to well adapted ‘superviruses’.

The study is published in the journal Genetics.

Immune Response to Swine Flu May Explain Disease Severity

The pandemic swine flu (H1N1) virus has proved to be less lethal than originally feared but, although most infected individuals experience relatively mild and self-limiting symptoms, some patients with no previous underlying medical condition have died. An international team of researchers has now found a possible explanation of why some people develop severe pneumonia when infected with the H1N1 virus. The team profiled immune mediators in 20 patients with severe symptoms, 15 patients with mild symptoms and 15 healthy controls. A typical innate antiviral response with increased levels of chemokines IP-10, MCP-1 and MIP-1β and an absence of anti-H1N1 antibodies characterised the early response in all infected individuals, but elevated levels of IFN-γ and mediators that stimulate Th17 and Th1 responses were found only in hospitalised patients. Both critical and non-critical hospitalised patients had increased levels of IFN-γ, Th17 mediators (IL-8, IL-9, IL17 and IL6) and Th1 mediators (TNF-α, IL-15 and IL-12p70) compared to outpatients. All hospitalised patients showed higher levels of IL-17 and TNF-α than controls but only the non-critical patients showed significant higher levels of IL-17 and TNF-α than those with mild symptoms. Levels of IL-15, IL-12p70 increased exclusively in critical patients who also showed the highest levels of IL-6.

Around half of hospitalised patients and nearly all outpatients tested positive for virus, with all those who tested positive having similar viral loads. Significantly higher levels of IL-13 and IL-17 were found in hospitalised patients with undetectable virus. IL6 was found to show a significant inverse association with arterial blood oxygen pressure in hospitalised patients and a similar inverse relationship was found for IL-8 in the critically ill patients.

Th1 and Th17 cells form an important part of host defence against pathogens but TH17 cells have also been linked to the pathogenesis of autoimmune and inflammatory diseases. It is presently unclear whether the increase in Th1 and Th17 responses reflects a vigorous antiviral defence necessary to clear lower respiratory infection or whether the inflammatory response contributes to disease severity. Although the ability of influenza viruses to evoke an inflammatory response is well known, this is the first study to link a Th17 response to severe influenza disease in humans. The authors suggest that immunomodulatory drugs which down-modulate Th1 and Th17 responses could be used to clarify the role of these pathways in the pathogenesis of the acute respiratory symptoms shown by patients with severe H1N1 disease. “Hypercytokinemia” of specific chemokines and cytokines has previously been shown to be associated with severe and often fatal cases of human H5N1 infections.

The study is published in the journal Critical Care.