Entamoeba histolytica, the agent of amebic dysentery and amebic liver abscess, is the third leading cause of death from parasitic disease world wide. Amebic cysteine proteinases appear to play an important role in the pathogenesis of invasive amebiasis, and are potential targets for the development of antiamebic drugs. The efficacy of protease inhibitors in vivo remains to be established. Currently available dipeptide inhibitors have the potential drawback of also interacting with mammalian cathepsins. We have recently observed that E. histolytica cysteine proteinases exhibit a striking binding specificity for immobilized laminin, compared to other components of the extracellular matrix, such as collagen and fibronectin, and that laminin inhibits protease activity in a fluorogenic assay with a dipeptide substrate. Dr. Stanley, one of the project leaders, has recently developed a mouse liver abscess model of experimental amebiasis. In pursuing the development of novel agents for the treatment of amebiasis, we propose investigate E. histolytica cysteine proteinases as a drug target by taking two approaches. The first is to identify the structural determinants on laminin which are involved in its interaction with amebic proteases with the eventual goal of designing inhibitors that will preferentially target the amebic proteinases over host cathepsins. Assignment of the inhibitory domain on laminin will be make by analyzing binding interaction of the amebic proteinases with defined laminin fragments and by identifying the initial site of attack by these enzymes on laminin. Efforts will be made to generate recombinant amebic cysteine proteinases utilizing yeast expression systems, to facilitate structural and molecular genetic analyses of the structural determinants on the protease which are involved in this binding interaction. Based on these structural studies, laminin-based peptide inhibitors will be designed, synthesized and studied. The second approach will be to determine the efficacy of cysteine proteinase inhibitors in vivo. We initially test a number of currently available cysteine proteinase inhibitors and eventually test athe laminin- based inhibitors in the mouse liver abscess model developed by Dr. Stanley. Immunohistochemical studies will be carried out using the Program EM core under the guidance of Dr. Russell to examine the distribution of the E. histolytica cysteine proteinases in tissue sections obtained from the study mice, in order to gain further insight in the contribution of these proteases and their interactions with the extracellular matrix to the pathogenesis of invasive amebiasis.
Showing the most recent 10 out of 21 publications
