Archive for the ‘Swine Flu Research’ Category

Influenza viruses are a major cause of morbidity and mortality around the world. More recently, a swine-origin influenza A (H1N1) virus that is spreading via human-to-human transmission has become a serious public concern.

Although vaccination is the primary strategy for preventing infections, influenza antiviral drugs play an important role in a comprehensive approach to controlling illness and transmission. In addition, a search for influenza-inhibiting drugs is particularly important in the face of high rate of emergence of influenza strains resistant to several existing influenza antivirals.

Methods: We searched for novel anti-influenza inhibitors using a cell-based neutralization (inhibition of virus-induced cytopathic effect) assay.

After screening 20,800 randomly selected compounds from a library from ChemDiv, Inc ., we found that BPR1P0034 has sub-micromolar antiviral activity. The compound was resynthesized in five steps by conventional chemical techniques.

Lead optimization and a structure-activity analysis were used to improve potency. Time-of-addition assay was performed to target an event in the virus life cycle.

Results: The 50% effective inhibitory concentration (IC50) of BPR1P0034 was 0.42 +/- 0.11 uM, when measured with a plaque reduction assay.

Viral protein and RNA synthesis of A/WSN/33 (H1N1) was inhibited by BPR1P0034 and the virus-induced cytopathic effects were thus significantly reduced. BPR1P0034 exhibited broad inhibition spectrum for influenza viruses but showed no antiviral effect for enteroviruses and echovirus 9.

In a time-of-addition assay, in which the compound was added at different stages along the viral replication cycle (such as at adsorption or after adsorption), its antiviral activity was more efficient in cells treated with the test compound between 0 and 2 h, right after viral infection, implying that an early step of viral replication might be the target of the compound. These results suggest that BPR1P0034 targets the virus during viral uncoating or viral RNA importation into the nucleus.

Conclusions: To the best of our knowledge, BPR1P0034 is the first pyrazole-based anti-influenza compound ever identified and characterized from high throughput screening to show potent (sub-uM) antiviral activity.

We conclude that BPR1P0034 has potential antiviral activity, which offers an opportunity for the development of a new anti-influenza virus agent.

Author: Shin-Ru ShihTzu-Yun ChuGadarla Randheer ReddySung-Nain TsengHsiun-Ling ChenWen-Fang TangMing-sian WuJiann-Yih YehYu-Sheng ChaoJohn HsuHsing-Pang HsiehJim-Tong Horng
Credits/Source: Journal of Biomedical Science 2010, 17:13

source: 7thspace.com

An international team of scientists have made a novel discovery that might help explain how flu virus, including the currently circulating H1N1 infects human beings.

They have also identified small molecule compounds that act on several of these factors and inhibit viral replication, pointing to new ways to treat flu. Researchers have identified 295 human cell factors that influenza A strains must harness to infect a cell.

They used RNAi screening technology to selectively turn off more than 19,000 human genes to determine which human factors facilitate viral entry, uncoating, nuclear import, viral replication and other necessary functions of the virus.

“Because influenza mutates so readily, it has become a moving target for therapeutic intervention, making it difficult to treat circulating strains, including the H1N1 swine flu,” Nature quoted researcher Sumit Chanda, from Burnham Institute for Medical Research.

“As a result, there is now widespread resistance to two classes of antiviral drugs. However, by targeting more stable human host factors, we may be able to develop therapies that prevent or treat a variety of influenza A strains and are more likely to maintain their effectiveness,” Chanda added.

Each of these represents an “Achilles heel” of the virus and vastly increases the number of potential targets for new influenza antiviral drugs. The study showed that selectively impairing each of 295 cellular genes reduced viral infection, effectively illuminating the path followed by influenza viruses during the infection of a cell.

Importantly, they found that inhibiting proteins in known drug target classes, such as kinases, vATPases, and tubulin, impairs influenza growth, suggesting that small molecular weight compounds may be developed as host factor-directed antivirals.

Source: indiatimes.com

Newswise — Investigators at Burnham Institute for Medical Research (Burnham), Mount Sinai School of Medicine (Mount Sinai), the Salk Institute for Biological Studies (Salk) and the Genomics Institute of the Novartis Research Foundation (GNF) have identified 295 human cell factors that influenza A strains must harness to infect a cell, including the currently circulating swine-origin H1N1. The team also identified small molecule compounds that act on several of these factors and inhibit viral replication, pointing to new ways to treat flu. These findings were published online on December 21 in the journal Nature.

Influenza A virus contains only enough genetic information (RNA) to produce 11 proteins and must co-opt host cellular machinery to complete its life cycle. Sumit Chanda, Ph.D., of Burnham, Megan Shaw, Ph.D., of Mount Sinai, John Young, Ph.D., of Salk, Yingyao Zhou, Ph.D., of GNF and others used RNAi screening technology to selectively turn off more than 19,000 human genes to determine which human factors facilitate viral entry, uncoating, nuclear import, viral replication and other necessary functions of the virus.

“Because influenza mutates so readily, it has become a moving target for therapeutic intervention, making it difficult to treat circulating strains, including the H1N1 swine flu,” said Dr. Chanda. “As a result, there is now widespread resistance to two classes of antiviral drugs. However, by targeting more stable human host factors, we may be able to develop therapies that prevent or treat a variety of influenza A strains and are more likely to maintain their effectiveness.”

“This study has provided us with crucial knowledge of the cellular pathways and factors the influenza virus exploits to replicate” added Dr. Shaw. “Each of these represents an ‘Achilles heel’ of the virus and vastly increases the number of potential targets for new influenza antiviral drugs.”

The team screened human A549 (lung epithelial) cells infected with a modified influenza virus against the genome-wide siRNA library. Conducting two independent screens, they confirmed that selectively impairing each of 295 cellular genes reduced viral infection, effectively illuminating the path followed by influenza viruses during the infection of a cell. Importantly, they found that inhibiting proteins in known drug target classes, such as kinases, vATPases, and tubulin, impairs influenza growth, suggesting that small molecular weight compounds may be developed as host factor-directed antivirals. Protein interactions dataset analysis confirmed 181 host cellular factors that mediate 4,266 interactions between viral or cellular proteins.

Renate Koenig, Ph.D., of Burnham and Peter Palese, Ph.D., Silke Stertz, Ph.D., and Adolfo Garcia-Sastre, Ph.D., of Mount Sinai also collaborated on this research.

“Trying to identify all the host proteins that are required for the replication of influenza viruses is a wonderful challenge and we have come closer to ‘knowing’ all the genes involved,” said Dr. Palese.

Dr. Young added, “These findings, combined with those from other RNAi screens, provide a blueprint of the cellular processes that are exploited more generally by viruses, pointing towards development of future broad-spectrum antiviral approaches.”

About Burnham Institute for Medical Research

Burnham Institute for Medical Research is dedicated to discovering the fundamental molecular causes of disease and devising the innovative therapies of tomorrow. Burnham, with operations in California and Florida, is one of the fastest-growing research institutes in the country. The institute ranks among the top four institutions nationally for NIH grant funding and among the top organizations worldwide for its research impact. For the past decade (1999-2009), Burnham ranked first worldwide in the fields of biology and biochemistry for the impact of its research publications (defined by citations per publication), according to the Institute for Scientific Information.

Burnham utilizes a unique, collaborative approach to medical research and has established major research programs in cancer, neurodegeneration, diabetes, and infectious, inflammatory, and childhood diseases. The Institute is especially known for its world-class capabilities in stem cell research and drug discovery technologies. Burnham is a nonprofit public benefit corporation. For more information, please visit www.burnham.org.

About The Mount Sinai Medical Center

The Mount Sinai Medical Center encompasses The Mount Sinai Hospital and Mount Sinai School of Medicine. The Mount Sinai Hospital is one of the nation’s oldest, largest and most-respected voluntary hospitals. Founded in 1852, Mount Sinai today is a 1,171-bed tertiary-care teaching facility that is internationally acclaimed for excellence in clinical care. Last year, nearly 50,000 people were treated at Mount Sinai as inpatients, and there were nearly 450,000 outpatient visits to the Medical Center.

Mount Sinai School of Medicine is internationally recognized as a leader in groundbreaking clinical and basic science research, as well as having an innovative approach to medical education. With a faculty of more than 3,400 in 38 clinical and basic science departments and centers, Mount Sinai ranks among the top 20 medical schools in receipt of National Institute of Health (NIH) grants. For more information, please visit www.mountsinai.org.

About the Salk Institute for Biological Studies

The Salk Institute for Biological Studies is one of the world’s preeminent basic research institutions, where internationally renowned faculty probe fundamental life science questions in a unique, collaborative, and creative environment. Focused on both discovery and mentoring future generations of researchers, Salk scientists make groundbreaking contributions to our understanding of cancer, aging, Alzheimer’s, diabetes, and cardiovascular disorders by studying neuroscience, genetics, cell and plant biology, and related disciplines.

Faculty achievements have been recognized with numerous honors, including Nobel Prizes and memberships in the National Academy of Sciences. Founded in 1960 by polio vaccine pioneer Jonas Salk, M.D., the Institute is an independent nonprofit organization and architectural landmark.

About the Genomics Institute of the Novartis Research Foundation

The Genomics Institute of the Novartis Research Foundation develops and applies integrated state-of-the-art technologies in chemistry, biology, automation, and information sciences in order to pursue new approaches towards the understanding of complex biomedical problems in cancer biology, immunology, neuroscience, and metabolic as well as infectious disease. These technologies cut across the life sciences, and include genomics and proteomics tools, medicinal chemistry, cell-based ultra high throughput screening of genes or compounds, structural genomics, and forward/reverse mammalian genetics. The mission of the Institute is to exploit these technologies to identify new biological processes and understand the underlying mechanisms involved in human disease. These discoveries are being translated into human therapeutics through an internal preclinical drug discovery effort coupled with further developmental activities with Novartis. Founded in 1999, the Institute is funded through the Novartis Research Foundation.

Computer compatibility tests might help flu-fighting drugs find their groove.

A pandemic of the H1N1 swine flu virus has health officials worried that the virus could develop resistance to drugs such as Tamiflu used to treat infected people. A new computerized screening method could help find new or already existing drugs that find a flu virus’ weak spot, researchers from the University of California, San Diego reported December 6 at the annual meeting of the American Society for Cell Biology.

Researchers Daniel Dadon, Jacob Durrant and J. Andrew McCammon, all of UCSD, made a computer movie of slight structural shifts occurring in the neuraminidase 1 enzyme (the N1 in H1N1 and H5N1), a protein found in the avian and swine influenza viruses. Those changes reveal possible target areas that could allow drugs to circumvent a virus’ usual means of becoming resistant.

All influenza viruses have a neuraminidase enzyme, but the protein comes in several subtypes. Previous work had shown that the N1 subtype contains a loop that makes it more flexible than other neuraminidase subtypes, says Rommie Amaro, a computational biologist at the University of California, Irvine. “It is particularly nimble,” she says. The enzyme’s flexibility could affect the way drugs bind to it.

Enlarge

Antiviral drugs can wedge into a cavity within an active site of the N1 neuraminidase enzyme (blue) and stop the enzyme’s action. Mutations in the enzyme (colored dots) can reduce the efficiency with which antiviral drugs such as Tamiflu bind, creating drug-resistant forms of the virus. Newly discovered drugs (green) lodge in the enzyme’s active site in a different location, possibly being able to knock out viruses that have become resistant to other drugs.Daniel Dadon and Jacob Durrant, University of California, San Diego

Analyses of still frames from the simulation, which is called a relaxed complex scheme, revealed 27 different natural conformations that the N1 protein could take on under conditions it might encounter in a host cell. Some parts of the protein change shape readily and some stiffer portions are locked into place, the researchers discovered.

Drugs currently used against flu — including oseltamivir, better known as Tamiflu, peramivir and zanamivir — all insert themselves into neuraminidase at about the same location within that enzyme. When the drugs insert into that pocket, they block the enzyme’s ability to release newly made viral particles from the cell, and this blockage prevents the spread of the disease.

That location is prone to structural changes such as those revealed by the simulation, and to genetic changes that affect the amino acid building blocks that compose the protein. Many of those amino acid changes also alter the shape of the pocket, keeping the drugs from binding and thus making the flu virus resistant to the drugs.

To find drugs that could block the protein’s active site in a different way — and knock out viruses resistant to the currently used drugs — the researchers mined a library of FDA-approved drugs. The team digitally sliced up the drugs and simulated how the drug fragments might bind to all of the enzyme’s forms.

Among those fragments, the team found 15 novel compounds that could wedge into the protein’s pocket and block its action better than Tamiflu or other antiviral drugs would. A closer examination revealed that those 15 compounds share a common structure. What’s more, the compounds lodge into a part of the protein that doesn’t allow changes easily, meaning that those areas are less likely to mutate and develop drug resistance than the parts of the protein that come into contact with Tamiflu and other current flu treatments, Dadon says.

But because the computer-generated fragment molecules don’t exist in the real world, the researchers needed to see whether any existing, small molecules could work just as well. Searching four databases of drugs turned up six small molecules that had the same common structure as the digitally diced compounds. These real compounds are currently being tested by collaborators in Australia to determine whether they really do block the flu’s action.

Both of the approaches Dadon’s team followed in the new drug design scheme — examining all forms of the protein and then screening a library of fragments from approved drugs — could be easily adapted for other molecules, Amaro says. The caveat is that researchers need to have prior knowledge of an enzyme’s structure in order to develop effective drugs, she says.

Source; Sciencenews.org