virology – Page 3 – Drug Discovery Opinion

Candidate Drug for Henipavirus Infection

Two viruses from the Henipavirus genus, Nipah (NiV) and Hendra (HeV), are recently emerged zoonitic (transmissible from animals to humans) paramyxoviruses that cause encephalitis in humans. HeV, previously known as equine morbillivirus, emerged as the causative agent of an outbreak of fatal respiratory disease in horses and man in Australia in 1994. NiV emerged in 1998/1999 in Malaysia and Singapore causing fatal encephalitis in humans. Human fatality rates can be as high as 75%.
Henipavirus structure
chloroquine structure Researchers at Weill Medical College of Cornell University, Australian Animal Health Laboratory, University of Tennessee Health Science Center and Rockefeller University have now developed a high-throughput assay that is able to identify inhibitors that target several stages of the viral life cycle. Their initial screen showed that chloroquine, approved for malaria treatment, inhibited infection with live HeV and NiV at a concentration of 1µM in vitro (IC₅₀=2µM), lower than the plasma concentrations present in humans receiving chloroquine treatment for malaria.

The scientists speculate that the mechanism of action of chloroquine is likely to be inhibition of cathepsin L, a host enzyme essential for processing of the viral fusion glycoprotein and maturation of newly budding virions. In the absence of this processing step, virions are not infectious. Chloroquine has previously been shown to suppress the activity of cathepsin L.

The authors of the current study, published online ahead of print in the Journal of Virology, suggest that the established safety profile and broad experience with chloroquine in humans should provide an option for treating individuals infected by these deadly viruses.

Myriad Acquires Novel HIV Drug

Myriad Pharmaceuticals recently announced the acquisition, from Panacos Pharmaceuticals, of bevirimat, a novel HIV-1 maturation inhibitor.

Bevirimat is a derivative of betulinic acid, a triterpenoid isolated from the leaves of the Chinese herb, Syzygium claviflorum, which was found to have potent inhibitory activity against HIV-1. Bevirimat shows potent in vitro activity against a broad range of HIV-1 strains, including isolates that are resistant to drugs currently approved for the treatment of HIV-1: protease inhibitor-resistant HIV-1 strains appear to be especially sensitive to bevirimat. Like HIV-1 protease inhibitors, bevirimat interferes with proteolytic processing of the newly translated viral polyprotein, Gag. One of the last steps in viral maturation is cleavage at the capsid-SP1 junction, and bevirimat is believed to prevent cleavage by binding to the Gag polyprotein at this site. Release of SP1 is essential for proper capsid condensation and function: preventing release of SP1 results in non-infectious virions containing abnormal, unstable cores. Bevirimat-resistant strains of HIV-1 can be generated in vitro, but arise more slowly in strains resistant to protease inhibitors than in wild-type strains. Mutations conferring resistance to bevirimat occur at or near the capsid-SP1 cleavage site.

bevirimat structure Clinical studies have shown that bevirimat is well tolerated and have demonstrated significant and clinically relevant reductions in viral load in a subset of patients. Studies have suggested that clinical resistance to bevirimat does not develop rapidly, possibly because of selective pressure to maintain the highly conserved capsid-SP1 cleavage sequence. There may also be a greater hurdle to development of bevirimat resistance in strains of virus that are resistant to protease inhibitors, suggesting that patients with such viruses may be especially likely to benefit from treatment with maturation inhibitors such as bevirimat.

A Bad Night’s Sleep Increases Risk of Catching Cold

sleep A good night’s sleep makes a big difference to how we feel, and a new US study suggests that it could also reduce our risk of catching a cold. People who sleep for fewer than seven hours a night were found to be almost three times more likely to get a cold than those who average eight or more hours a night. Having trouble falling asleep or waking up during the night increased the risk even more.

In the study, 54 out of 153 healthy men and women (average age 37) developed cold symptoms in the 5 days after nasal administration of drops containing a rhinovirus. After 28 days, blood samples were tested for antibodies to the virus and it was found that 135 people had become infected, although not all had developed a cold.

For two weeks before being exposed to the virus, the participants were asked each day about the duration and quality of their sleep. In the five days following exposure to the virus, people who had slept for fewer than seven hours each night in the previous two weeks were almost three times more likely to report symptoms than those who had slept for eight hours or more. Broken sleep was found to be even more important: those who were awake for more than 8% of sleep time were five and a half times more likely to show symptoms than those who were awake for 2% or less of the time. Interestingly, the development of cold symptoms did not correlate with how well rested the subjects felt. After taking into account a wide variety of other factors, how long – and especially how well – individuals slept were the strongest predictors of who would develop a cold. Previous research had suggested a link between sleep deprivation and impaired immune function, but this is the first study to show that sleep disturbances can affect susceptibility to cold viruses in normal healthy people. The study is published in the January 12^th edition of Archives of Internal Medicine.

Structure of Ebola Virus Protein Determined

Ebola virus, named after a river in the Democratic Republic of the Congo where it was first identified, causes a severe, often fatal haemorrhagic fever. There is no vaccine against or specific treatment for Ebola virus infection, but researchers at Iowa State University have now moved a step closer to finding a treatment. Using a combination of X-ray crystallography and nuclear magnetic resonance spectroscopy, the group has succeeded in determining the structure of a key portion of the viral protein, VP35, at a resolution of 1.4 Å. The Ebola VP35 protein forms part of the viral RNA polymerase complex, acts as a viral assembly factor, and also inhibits production of host interferon.

Binding of VP35 to dsRNA correlates with suppression of interferon activity and viral virulence, and the group hopes that the new structure of the interferon inhibitory domain will allow them to design drugs that bind to VP35 and block its function. The protein forms a unique fold with two clusters of basic residues, one of which is important for dsRNA binding and inhibition of interferon production. The study is published in the January 13^th issue of PNAS.

HIV-1 Clade is Important in Determining Progression to Dementia

HIV-1 infection is characterized by a wide variety of genetic subtypes in different geographical locations. The predominant subtype found in the western world, clade B, differs substantially from clade C, the main subtype that exists in Africa and Asia. Dementia is common amongst individuals infected with clade B HIV-1, but occurs less frequently amongst individuals infected with clade C HIV-1. Although it had been suspected that the differences in dementia rates were linked to differences in neurotoxicity between the clades, this had been difficult to prove since differences in geographical distribution, differences in host genetics, lifespan following infection or differences in access to antiretrovirals could all affect progression to dementia.

A new study using immunocompromised (SCID) mice has now confirmed the importance of HIV-1 clades in determining the incidence of HIV-associated dementia. The results demonstrate, for the first time, differences in neuropathogenesis between clade B and C HIV-1 isolates in an in vivo model. The study demonstrated that mice infected with representative clade C HIV-1 isolates performed better in cognitive tests than those infected with clade B HIV-1 isolates. Brains of mice exposed to either HIV-1 clade B or HIV-1 clade C showed similar viral loads, but those with clade B HIV-1 showed increased inflammation and neuronal damage.

Using in vitro experiments with purified HIV-1 Tat (Trans-Activator of Transcription) proteins, the team further showed that the differences in neuropathology were linked to structural differences in the Tat protein. Tat from clade C HIV-1 differs from that of other HIV-1 clades in that a highly conserved dicysteine motif (C30-C31) is replaced by a C31S polymorphism.
TAT sequence
The authors suggest that targeting HIV-1 Tat may be a way to prevent the neurological effects of AIDS, although this may be difficult to achieve in practice. In the early to mid 1990s, there was considerable interest in developing Tat antagonists but, following the unexpected failure of the selective Tat antagonist Ro 24-7429 to show antiviral activity in clinical studies, interest in the approach appears to have waned.

Bavituximab: Broad-Spectrum Antiviral Activity

Phosphotidylserine is an anionic phospholipid that is normally present on the inner surface of cell membranes. In cells infected with a wide range of enveloped viruses, however, the phosphotidylserine becomes exposed on the outside of the cell membrane. The monoclonal antibody, bavituximab, binds to the exposed phospholipid and alerts the immune system to attack the virally infected cells. Bavituximab is currently undergoing clinical trials for the treatment of hepatitis C virus infection.

New animal studies now show that bavituximab is able to protect against a broader range of viruses. Guinea pigs were infected with Pichinide virus, which is similar to human Lassa fever virus. Whilst all the untreated animals died, half of those treated with bavituximab recovered. The surviving animals also had long-term immunity to further infection with the Pichinide virus. This is the first report of an effective treatment against advanced Lassa-like fever infections in animals. Bavituximab treatment also cured mice infected with cytomegalovirus, an opportunistic infection that causes disease in immunosuppressed individuals such as transplant and AIDS patients.

Other viruses that cause phospholipids to become externalised on infected cells include influenza virus, herpes simplex virus and members of the smallpox and rabies virus families as well as hepatitis C virus and HIV.

If externalisation of phospholipids is a general feature of cells infected with enveloped viruses, treatment with antibodies such as bavituximab may prove to be a broad-spectrum antiviral treatment. Since phosphatidylserine on virus-infected cells is host-derived, resistance may also be less likely to develop than against treatments that target components of the virus.

The study was carried out by scientists at the University of Texas Southwestern Medical Center and Peregrine Pharmaceuticals Inc. and is published in the Nov 23 online edition of the journal Nature Medicine.

Gardasil® also Effective in Men

Human papilloma viruses (HPV) infect the skin and mucous membranes and, although most infections cause no symptoms and are self-limiting, persistent infection can lead to warts or cancer, especially cervical cancer. HPV types are often categorized as ‘low risk’ (causing warts) or ‘high risk’ (causing cancer). Around 30-40 sub-types of the virus are sexually transmitted and the Center for Disease Control estimates that 20 million men and women are infected with HPV. Cervical cancer is estimated to kill almost 4000 women each year in the US, and 300,000 globally.

Two HPV vaccines, Gardasil® and Cervarix®, have recently been introduced to protect women against cervical cancer which is caused primarily by infection with HPV types 16 and 18. Gardasil® additionally offers protection against HPV types 6 and 11 that are most associated with warts.

Newly released results from a phase III study now show that Gardasil® prevented 90% of lesions caused by HPV types 6, 11, 16 and 18 in approximately 4000 men aged 16-26. These are the only data describing the efficacy of an HPV vaccine in preventing disease in men.

SARS Protease Inhibitor Identified

SARS virus The first cases of severe acute respiratory syndrome (SARS), which is caused by the SARS coronavirus (SARS-CoV), are thought to have occurred in Guangdong province in southern China in November 2002. The virus quickly spread around the globe, with the total number of cases exceeding eight thousand by the summer of 2003. Although this outbreak was contained, the rapid transmission and high mortality rate of SARS prompted an investigation into the viral lifecycle and a search for effective antiviral agents.

A recently published report in the journal PNAS describes the discovery of a potent inhibitor of the SARS coronavirus papain-like protease (PLpro). As well as proteolytic processing of the viral polyprotein, the protease is able to cleave ubiquitin and ISG15 conjugates, and also plays a significant role in allowing the virus to evade the immune system. Optimisation of an initial screening hit led to the identification of a non-covalent inhibitor, GRL0617, which inhibits PLpro with an IC₅₀ of 600nM and inhibits replication of SARS-CoV in Vero E6 cells with an EC₅₀ of 15µM. The X-ray crystallographic structure of GRL0617 bound to PLpro shows the compound binding within the S4-S3 subsites and inducing a loop closure that prevents catalysis at the active site.

Although SARS does not pose a current threat, the identification of GRL0617 shows that inhibition of PLpro is a viable target for the development of antivirals against SARS-CoV.

First Interferon-free Oral Combination Study for HCV

HCV virus In total, around 200million people are thought to be infected hepatitis C virus (HCV). The current standard of care treatment for (HCV) infection is a combination of pegylated interferon, an immune modulator, and ribavirin, an antiviral drug. Interferon/ribavirin therapy is effective in only around 50% of patients with the most difficult-to-treat HCV, type 1. A number of new small molecule antiviral drugs are being developed, and Roche, Pharmasset and InterMune have recently announced the first clinical trial of an interferon-free combination of an HCV NS3/4A protease inhibitor, R7227, and an HCV NS5B polymerase inhibitor prodrug, R7128. Both the protease and polymerase are essential for viral replication and the new study, which will be carried out in Australia and New Zealand, will look at safety and combined antiviral activity of the two compounds in combination.

R7128 R7227, which is being developed in collaboration with InterMune, and R7128, which is being developed in collaboration with Pharmasset, have both already successfully completed studies in combination with pegylated interferon and ribavirin.

R7227

NS1 Protein ‘Hides’ Influenza Virus RNA

The non-structural (NS1) protein of influenza A viruses plays a major role in countering host immune defence by limiting production of interferon and also limiting the antiviral effects of IFN-induced proteins. NS1 also directly modulates stages of the virus replication cycle, including viral RNA replication and viral protein synthesis. Researchers at the Baylor College of Medicine have now identified a mechanism whereby NS1 protein from the highly virulent avian influenza strain, H5N1, ‘hides’ the pieces of double-stranded RNA that would otherwise trigger an antiviral response. NS1 is comprised of two domains: an RNA binding domain and an effector domain. X-ray crystallography of full-length NS1 revealed that molecules of the NS1 protein combine to form tiny tubules which sequester viral RNA. The oligomeric organisation allows the residues involved in RNA-binding to face inwards towards a 20Å wide central tunnel. Binding sites for cellular factors, which may help to evade an immune response, were also identified on the surface of the tubules. It is not yet known whether NS1 proteins from other influenza strains also form similar tubules but, if so, it is possible that the disruption of such structures could form the basis for new treatments. The findings have been reported in the November 5th online edition of the journal, Nature.