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Open Access Dissertation
Doctor of Philosophy (PhD)
Year Degree Awarded
Jeanne A. Hardy
Michael J. Knapp
Biochemistry, Biophysics, and Structural Biology
Dengue Fever is a global problem with a worldwide effectiveness that put 2.5 Billion people under the risk, infect 50 million people and causes 30000-50000 people death each year. DHF was first recognized in the 1950s during the dengue epidemics in the Philippines and Thailand. By 1970 nine countries had experienced epidemic DHF and now, the number has increased more than fourfold and continues to rise. Today emerging DHF cases are causing increased dengue epidemics in the Americas, and in Asia, where all four dengue viruses are endemic. Vaccine development against Dengue Virus has been impossible to date, due to effective vaccination to prevent DHF will require a etravalent vaccine, because epidemiologic studies have shown that preexisting heterotypic dengue antibody is a risk factor for DHF a lethal form of dengue fever. To date there are no pharmaceutical treatments for Dengue fever. DV proteome is composed of 8 proteins and dengue virus protease is one of them and it is essential for virus replication therefore it has being a potential drug target for dengue fever treatment. Active-site inhibitors of proteases have been successfully used to treat other virally transmitted diseases of global importance such as HIV and Hepatitis C, however protease active site inhibitors they are subject to development of resistance. In addition, it is often difficult to target the active site due to overlapping sequence preference with endogenous human proteases. This overlap in specificity of active-site inhibitors contributes to unwanted side effects.
A principal bottleneck is that traditional drugs are designed to bind to one protein and control the function of only that protein at the active (primary functional) site. Unfortunately, similar active sites are almost always present in related proteins, leading to lack of drug specificity and thus to many unwanted side effects. A promising alternative is to use allosteric sites. Allosteric sites are cryptic drug binding sites that are spatially distinct from the active site. They allow the protein to be locked into a unique conformation that either turns the protein ‘on’ or ‘off’. Historically it has been difficult or impossible to find allosteric sites, because mechanisms of allostery were poorly understood and tools for their identification were lacking. Our unique combination of experimental approaches has enabled us to identify new allosteric sites and new allosteric mechanisms of control in four different biomedically important proteins. Many proteins have multiple allosteric sites. Allosteric sites can often be exploited for much more specificity than active sites. We have developed and implemented a technology for discovering new allosteric sites in proteases and have applied this to NS2B-NS3pro from dengue virus type 2. In this thesis we report an allosteric site in NS2B-NS3pro that can be targeted in biomedical studies. We also have shown that the internal flexibility of NS2B-NS3pro is critical for catalytic activity.
Yildiz, Muslum, "Allosteric Regulation of Dengue Virus Type 2 Protease" (2014). Doctoral Dissertations. 30.