Adenoviruses cause a number of acute infections, including respiratory and gastrointestinal infections, and also conjunctivitis. In patients with compromised immune systems, such as those infected with human immunodeficiency virus (HIV), an opportunistic adenovirus infection is frequently deadly. "Our new antiviral drugs are expected not only to inhibit adenovirus, but might also be effective against other organisms that use the same enzyme -- including Chlamydia, one of the most prevalent sexually transmitted diseases, and Yersinia pestis, the organism that causes the black plague," said Walter Mangel, the lead scientist on the studies.
During infection, these viruses make an enzyme called a protease, which cleaves or degrades other proteins. The protease is used by the virus to complete the maturation of newly synthesized virus particles. To explain this process, Mangel uses the example of building a cathedral around internal scaffolding. Once the cathedral is in place, the last step is to remove the scaffolding. "Similarly," says Mangel, "adenovirus particles are built with scaffolding protein inside. Once the virus particle is formed, the protease becomes activated and cleaves the scaffolding to render the virus particle infectious."
The three recent Brookhaven studies reveal that the protease is initially synthesized in an inactive form. The inactive enzyme binds to the viral DNA to become partially activated.
"Such activation of a protease by DNA has never been seen before," Mangel said.
The partially activated enzyme then cleaves out a cofactor (a protein fragment), which binds to the protease to activate it further. The fully active complex of enzyme and cofactor then moves along the viral DNA, cleaving the scaffolding proteins.
"These studies suggest that drugs that bind to the active site of the enzyme (the part involved in cleaving proteins), the cofactor binding site, or the DNA binding site should block the enzyme's action and serve as effective antiviral agents," Mangel said.
To design drugs able to bind to and block these sites, the scientists first had to characterize the molecular structures. The active site of the enzyme had been previously characterized by William McGrath, a postdoc in the lab, using an intense beam of x-rays available at Brookhaven's National Synchrotron Light Source (NSLS). The pattern of x-rays bouncing off the atoms reveals the three-dimensional molecular structure. In the current studies, Stony Brook University graduate student Mary Lynn Baniecki characterized the binding of the cofactor to the protease, identifying which parts bind to the enzyme and which parts stimulate the enzyme's activity. Both scientists then helped decipher how the protease binds to the DNA.
Based on the findings, Mangel has proposed a new form of antiviral therapy using three different drugs against these three target sites -- the active site, the cofactor binding site, and the DNA binding site -- on the same virus-coded protein. This three-pronged approach may overcome one of the biggest challenges in antiviral therapy -- the spontaneous evolution of drug-resistant strains. As Mangel explains the idea, a mutation conferring drug resistance at one site may alter the physiological functions at the other two sites, because the three sites are interdependent, thereby making drug resistance much less likely to arise.
"The adenovirus protease may be a good model system within which to test the efficacy of this form of combination therapy," Mangel said. Already, his team has developed two new drugs, one that binds reversibly and another irreversibly to the active site of the protease. These drugs will soon be tested as antiviral agents by the National Institutes of Health.
This work was funded by the U.S. Department of Energy, which supports basic research in a variety of scientific fields, and the National Institutes of Health.
The U.S. Department of Energy's Brookhaven National Laboratory (http://www.bnl.gov) conducts research in the physical, biomedical, and environmental sciences, as well as in energy technologies. Brookhaven also builds and operates major facilities available to university, industrial, and government scientists. The Laboratory is managed by Brookhaven Science Associates, a limited liability company founded by Stony Brook University and Battelle, a nonprofit applied science and technology organization.
Note to local editors: Walter Mangel lives in Shoreham, New York.
Journal
Biochemistry