RxPG News : SARS

A Macaque Model of SARS

Thu, 20 Apr 2006 23:09:37 PST

( from http://www.rxpgnews.com ) The 2002 SARS outbreak that started in China spread quickly to Hong Kong, Singapore, Vietnam, and Canada. Although the 774 people it killed was a small number compared with the global death toll from other infectious diseases, the outbreak caused widespread panic because of the lack of global preparedness for what could have become a worldwide epidemic. Since then, surveillance and monitoring systems have been put into place and existing ones strengthened, but since another outbreak is always possible, researchers around the globe are still devoting much time to studying the infection. Analyzing the disease in animals to investigate the pathogenesis of the novel coronavirus that causes SARS (SARS-CoV) is crucial to developing vaccines and treatments to tackle the next epidemic.

In adults, SARS first causes flu-like symptoms, then lower respiratory tract disease, and finally severe respiratory disease. But despite having similar levels of viral replication, children tend to have milder symptoms. They do not get chills or myalgias, nor do they need help breathing, as adults tend to need toward the end of the illness.

Several animalsmice, cats, and ferretshave been tested to see whether they can support replication of SARS-CoV, and otherscivets and wild batshave been investigated as potential viral reservoirs. In studies on nonhuman primates, the focus has been to document histopathological disease rather than to look for more advanced symptoms such as radiographic evidence of pulmonary disease, as happens in humans.

In a new study, Jason Paragas, James Lawler, and colleagues now describe what happened when they infected eight macaques with the SARS-CoV Urbani strain; four in the nasal cavities and bronchus; two in the nasal cavities and conjunctiva; and two intravenously.

Although all animals had evidence of viral replication and produced neutralizing antibodies, none of the animals developed fever, and only those in the first two groups had mild-to-moderate symptoms (decreased activity and feeding, and slightly labored breathing). By contrast, the animals that had been intravenously infected showed no clinical symptoms. When tested for the presence of the virus, all animals had viral DNA in nasal swabs and urine samplesirrespective of how they had been infected. Paragas and colleagues also took chest radiographs of six of the animalsnever before done in any SARS-CoV study on nonhuman primates. Three nonhuman primates showed signs of pneumonia by radiographs.

More interesting findings came from the fact that some animals were infected with wild-type virus, and others with a recombinant infectious clone. All developed similar disease, indicating that it was just the SARS-CoV that was responsible for disease, and that no coinfection was required, as has been suggested by some workers. In addition, six animals that were reinfected with SARS-CoV 13 weeks after the first infection were immuneimportantly, two of these had initially had the recombinant virus, which means that the molecular clone could induce protection against the wild-type form.

Paragas and colleagues' work differs from previous studies of SARS-CoV in nonhuman primates. Some researchers found more severe clinical disease; others, no overt disease at all. Tests on African green monkeys showed that one monkey had fever on the third day after infection. These differences could have been because of the strain, the dose, or the route of infection.

Ultimately, disease in nonhuman primates is far milder than that in adult humans. What is interesting is that it is similar to SARS-CoV infection in human children. The researchers suggest that the key to the difference in disease severity could lie in the fact that adult humans with SARS-CoV have far higher levels of inflammatory cytokines than do children, or, as this research suggests, nonhuman primates.

Researchers probe papain-like-protease (PLpro) enzyme that may lead to new SARS drugs

Wed, 05 Apr 2006 14:13:37 PST

( from http://www.rxpgnews.com ) Researchers at the University of Illinois at Chicago and two other institutions have unraveled the structure of an important new drug target from the virus that causes SARS, severe acute respiratory syndrome.

"By unlocking the three-dimensional structure of this enzyme -- known as papain-like-protease (PLpro) -- we now have a molecular road map to design new drugs that could potentially treat SARS-infected patients, or perhaps patients suffering from other SARS-related illnesses such as the common cold, bronchitis or pneumonia," said Andrew Mesecar, associate professor of pharmaceutical biotechnology in the UIC College of Pharmacy. "We are attempting to use the same approach that has been accomplished in designing effective drugs against HIV protease, which has led to the development of new drugs to fight the AIDS virus."

The research is published in the March 27 issue of the Proceedings of the National Academy of Sciences.

Mesecar said that the knowledge gained from this new atomic structure -- the first-ever reported on this class of proteases -- has the potential to go beyond the treatment of patients infected with the coronavirus that causes SARS.

Coronaviruses, which produce upper respiratory tract infections, were discovered in the late 1960s. The viruses are responsible for 10 percent to 30 percent of all common colds. Recently, two new coronaviruses -- NL63 and HKU1 -- were found to cause many cases of severe pneumonia in children and the elderly throughout the world, he said.

"NL63 and HKU1 infections are severe and often lead to hospitalization," Mesecar said. "It is believed these viruses have been around a long time, but only recently have we developed the technology to identify and detect them. The SARS outbreak and quick response of the scientific community has led to the rapid development of such technology."

SARS was first reported in Asia in early 2003. Over the next several months the illness spread to more than 29 countries in North and South America, Europe and Asia before it was contained. It begins with a high fever, headache and body aches. About 10 percent to 20 percent of patients have diarrhea, and after two to seven days, a dry cough may develop. Most patients develop pneumonia. The infection spreads by close personal contact, often through coughing or sneezing.

According to the World Health Organization, 8,098 people worldwide were diagnosed with SARS during the 2003 outbreak; 774 died. There were 29 cases reported in the United States, with no fatalities.

The papain-like-protease enzyme is essential for viral replication and infection of all of the coronaviruses involved in upper respiratory infections. Eliminating the enzyme should stop the infection, Mesecar said.

During the UIC study, graduate student Kiira Ratia, a member of Mesecar's research team, used X-ray crystallography, a technique that involves bombarding a crystalline form of the enzyme with an intense beam of X-rays that are bent by atoms in the molecules to unlock the details of the molecular structure. The X-ray studies were conducted at Argonne National Laboratory's Advanced Photon Source.

As the X-rays leave the crystal, a unique pattern is created on an ultra-high resolution charge-coupled device camera, a sensor for recording images often used in digital photography and astronomy. The images were then interpreted by computer to reconstruct the positions of all the component atoms.

"We have already discovered compounds that can bind to these pockets and inhibit the activity of this enzyme," Mesecar said. "We have made remarkable progress in a short period of time in generating lead drug-like compounds against the enzyme."

Benzotriazole Esters Based Enzyme inhibitors block replication of SARS virus

Tue, 28 Mar 2006 20:31:37 PST

( from http://www.rxpgnews.com ) The study was conducted by researchers from Scripps Research; the Genomics Research Center, Academia Sinica, Taiwan; and the National Taiwan University. It is being published today in the journal Chemistry and Biology (Vol. 13, No. 3).

Chi-Huey Wong is currently the Ernest W. Hahn Chair in Chemistry at the Skaggs Institute of Chemical Biology and directs the Scripps Research lab heading the study. He said the new finding is an important step in developing a possible drug treatment against SARS.

"We have been working on the problem of SARS since the epidemic started in 2003," Wong said. "This new class of inhibitors represents the most potent SARS virus protease inhibitors known today."

The path to today's research finding has taken several years. In 2002, Severe Acute Respiratory Syndrome (SARS) emerged in rural China and eventually spread to 32 countries, according to the World Health Organization. SARS is caused by a ring-shaped virus, known as a coronavirus. The SARS coronavirus is suspected of originating in animal populations before migrating to humans. Hardest-hit were six Asian nations. By the time the epidemic had been controlled in 2003, the disease infected more than 8,000 people, causing 800 deaths. There is no current effective treatment or vaccine.

Researchers have known since 2003 that a site on the virus is responsible for mediating proteases that allow the virus to replicate. Since then researchers have been testing protease inhibitors to lock up this site, known as SARS 3CLpro, and effectively stop the virus from infecting additional cells in the body.

In 2004, Wong's lab discovered that Lopinavir, a protease inhibitor of HIV also known as TL3, also served as weak inhibitor of the SARS 3CLpro site (PNAS, 101, 10012-10017). Since then, members of Wong's group further studied Lopinavir and are preparing it for clinical trials against SARS.

Researchers in Wong's lab at Scripps Research and in Taiwan have been looking at other Liponavir-related compounds for similar blocking effects. During these experiments, they found that a group of catalyzing agents used to help promote chemical reactions in the laboratory were actually more powerful in blocking the SARS protease than either the Lopinavir or any of the target compounds.

These organic compounds are called benzotriazole esters. The esters entered the SARS protease site, formed an intermediary compound, then inactivated the SARS enzyme. The findings were confirmed using mass spectrometry analysis of the enzyme intermediary.

"These benzotriazole esters are relatively stable and act as suicide inhibitors," Wong said. "They block the enzyme, are transformed through a co-valent bond, and are unable to get out."

Wong said the findings published today provide better insight into the mode of action of the enzyme, which may lead to development of a drug against SARS. The findings were made by using rapid drug discovery techniques developed in the Wong lab to screen large numbers of weak enzyme inhibitors, and then attaching additional compounds to look for stronger reactions.

Research Associate Chung-Yi Wu, a member of the Wong lab, is the paper's lead author. He said the finding was unexpected.

"We wanted to improve Liponavir activity," Wu said. "But we found this very surprising and serendipitous result."

SARS Can Infect Brain Tissue

Thu, 15 Sep 2005 16:48:38 PST

( from http://www.rxpgnews.com ) SARS, a potentially fatal illness caused by a coronavirus, was first reported in Asia in February of 2003. The disease is usually transmitted by contact with coronavirus-laden droplets sprayed into the air by an infected persons coughing. Other symptoms can include high fever, headache, body aches, and pneumonia. However, some patients also exhibit central nervous system ailments. In a new study, the researchers report the case of a 39-year-old doctor who treated SARS patients in China during the 2003 outbreak and became infected himself.

He showed the usual symptoms of SARS--fever, chills, headache, muscle pain--but after hospitalization, he developed vision problems, then progressively worse central nervous system symptoms, like restlessness and delirium. A computed tomography scan indicated brain damage. He died about a month after being hospitalized, and his brain tissue was examined and found to contain the SARS coronavirus. The researchers also discovered a high level of Mig, a type of immune system regulator called a chemokine, in the mans bloodstream and brain, which may have resulted from the central nervous system infection. The researchers speculated that Mig could also have contributed to his brain damage by attracting immunological cells to the site of the viral infection in the brain, where their inflammatory effects may have done more harm than good.

There are a few possibilities for curbing Migs possible role in causing brain damage in SARS patients with central nervous system infection, according to lead author Jun Xu, PhD, of the Guangzhou Institute of Respiratory Diseases and senior author Yong Jiang, PhD, of the Key Laboratory of Functional Proteomics of Guangdong Province. There might be some ways of controlling the release of Mig, such as specific inhibitors that interfere [with] the signaling pathways involved, Dr. Jiang said. Other approaches, such as neutralizing antibodies [and] specific binding peptides, could be tried to block brain damage induced by Mig.

Four to five percent of SARS patients treated at the Guangzhou Institute of Respiratory Diseases experienced central nervous system symptoms, said Dr. Xu; therefore, physicians need to be aware of the potential for brain infection when evaluating patients with the disease. Immunosuppressive drugs should be administered carefully and on an individual basis, as they may allow amplification of the SARS coronavirus in the brain. Superinfection with other pathogens could also contribute to SARS harmful effects on the brain. Physicians should pay more attention to the prevention of brain damage if [SARS patients] are superinfected with other conditional pathogens, according to Dr. Xu and Dr. Jiang.

Cinanserin May Offer New Hope in Treating SARS

Mon, 20 Jun 2005 16:07:38 PST

( from http://www.rxpgnews.com ) Cinanserin, a drug that underwent preliminary clinical testing on humans in the 1960's, may inhibit the SARS virus say researchers from Europe and China. Their findings appear in the June 2005 issue of the Journal of Virology.

Severe acute respiratory syndrome (SARS) emerged as a highly infectious respiratory disease in 2002 and reached epidemic levels within six months. An estimated 8,096 cases were reported resulting in 774 deaths throughout 29 countries. Although the causative agent was quickly identified as a new strain of the coronavirus, an effective method of treatment has yet to be determined.

In the study researchers scanned a database of 8,000 existing drugs for those that would likely bind to the 3C-like proteinase of SARS. Cinanserin, a well-characterized serotonin antagonist, scored high in the screening and was selected for further experimentation. The antiviral activity of cinanserin was evaluated in tissue samples containing the SARS virus and revealed a strong inhibition of coronavirus replication at nontoxic drug concentrations.

"These findings demonstrate that the old drug cinanserin is an inhibitor of SARS-CoV replication, acting most likely via inhibition of the 3CL proteinase," say the researchers.

A Prototype drug for SARS

Thu, 09 Jun 2005 17:50:38 PST

( from http://www.rxpgnews.com ) A prototype drug created by researchers at the University of Illinois at Chicago shows promise in slowing replication of the virus responsible for severe acute respiratory syndrome, or SARS.

Currently, there are no effective antiviral agents or vaccines for SARS, which killed almost 800 people in an epidemic in 2002-2003.

On the basis of their success, the researchers have received an $8 million grant from the National Institute of Allergy and Infectious Diseases to develop protease inhibitors that would block key enzymes in the SARS virus and hamper its advance. Protease inhibitors, a class of drugs capable of disrupting enzymes that digest proteins, have been successfully used to thwart the human immunodeficiency virus, which causes AIDS.

"Data from SARS patients indicate that replication of the virus peaks 10 days after the onset of fever," said Michael Johnson, director of the Center for Pharmaceutical Biotechnology in the UIC College of Pharmacy and the study's principal investigator. "By administering protease inhibitors early, when feverish symptoms have started, the drugs could reduce the viral load and ameliorate the disease."

Like HIV, the SARS virus multiplies rapidly, hijacking the machinery of the cells it infects to clone itself over and over again.

One of the first steps in that process is the production of a long chain of proteins, all of which are needed for the virus to propagate. Two enzymes, or proteases, clip the chain to release the individual proteins, the parts needed to assemble a mature virus.

These two proteases -- called 3CLpro and PLpro -- are UIC's targets for drug therapy.

"If we can block 3CLpro and probably PLpro, then we can stop the SARS virus from replicating," Johnson said.

Under the grant, Andrew Mesecar, associate professor of pharmacy, will study details of the three-dimensional structure of the two enzymes using x-ray crystallography.

His technique involves bombarding a crystalline form of the enzymes with an intense beam of x-rays generated by a multimillion-dollar instrument at the Advanced Photon Source at Argonne National Laboratory. As the x-rays are bent, or diffracted, by molecular components, they create a pattern on an ultra-high-resolution x-ray camera. Computers then mathematically interpret the pattern to reconstruct the enzyme's molecular configuration atom by atom.

The detailed structures will reveal the precise locations and chemical properties of pockets where the enzymes bind the SARS virus's long protein chain.

This information will help Arun Ghosh, professor of chemistry at UIC, build protease inhibitors, like the prototype he assembled earlier, through what is called structure-based drug design. He will create computer images of each enzyme coupled with compounds anticipated to obstruct the pockets, revealing whether and how tightly the molecules fit in the space. If the fit is poor, he can explore a library of alternative molecules that might meet the chemical and configurational requirements.

Ghosh will then assemble the molecules that appear to work, as if the chemicals were the sticks and spools of a Tinkertoy, to create a potential drug.

Crucial to the endeavor is Ghosh's strategy of targeting not the sidechains of the proteases, but their backbones. When viruses mutate, as they frequently do, thwarting the action of drugs, the mutations typically occur in these sidechains.

"By targeting the backbone, we create a drug that the virus probably will not be able to evade," Johnson said.

Susan Baker, a microbiologist at Loyola University Stritch School of Medicine collaborating in the research, will test the newly developed protease inhibitors to determine whether and how quickly they slow the enzymes' activity inside living cells.

The prototype drug developed earlier by Ghosh, an inhibitor of 3CLpro, was an improvement on a design published in the scientific literature. Shipped to the Centers for Disease Control and Prevention for testing on a live virus, the prototype proved to be 1,000 times more effective than the original compound in inhibiting 3CLpro.

However, Johnson, said, the prototype drug blocked several, but not all, of the enzyme's pockets. "The goal is to create compounds that will block all possible binding sites in the proteases to put them out of business."

"A compound that blocks all these sites, rather than just a few, and that conforms tightly to the pockets, is ultimately a better drug for human consumption because it can be prescribed in smaller doses."

Johnson emphasized that all the work being done at UIC involves isolated proteases, not live viruses. "The proteases are no more dangerous than the enzymes in your laundry detergent," he said.

Testing of the protease inhibitors on live viruses will be done at the U.S. Centers for Disease Control and Prevention.

SARS was first reported in Guangdong Province, China in November 2002 and spread rapidly to other Asian countries, North America and Europe. By July of the following year, more than 8,000 cases were reported in 26 countries on five continents, resulting in 774 deaths and enormous economic damage.

Immune Response Differs in SARS Patients

Fri, 15 Apr 2005 17:04:38 PST

( from http://www.rxpgnews.com ) Chinese investigators have revealed that the early presence of interferon-inducible protein 10 (IP-10), an immunoregulatory protein, is a prominent characteristic of the body's immune reaction to the coronavirus that causes severe acute respiratory syndrome (SARS). The disease itself differs from other non-SARS viral infections that cause no increase in IP-10. The researchers believe that levels of the protein could make a difference in both diagnosis and the development of an effective treatment for the disease.

The investigators screened 14 cytokines/chemokines (immunoregulatory proteins) in the blood of 23 patients with SARS and 25 non-SARS patients with atypical pneumonia. The SARS patients were separated into 4 groups, according the disease's course: early stage---2 days after onset of fever; progressive stage--10 to 20 days after onset; end stage--the day before death; and convalescent stage--15 to 30 days after discharge from the hospital.

They said that IP-10 was markedly elevated in the blood during the early stage of SARS, and remained at a high level during convalescence. Also, they said that IP-10 was highly expressed in both lung and lymphoid tissues.

The investigators pointed out that immunopathologic injury of host cells triggered by the immune response to virus plays a key role in the pathogenesis of virus infections. Many cytokines/chemokines released from activated immune cells not only take part in the process of antiviral immune response, but are also involved in cell damage and development of organ dysfunction. Determination of these soluble factors in the blood should aid their understanding of the immunologic processes of SARS and enable differential diagnosis of SARS from other atypical pneumonias that require quite different approaches to the management of patients.