Cysteine Proteases & Inhibitors in Nematode Development

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DESCRIPTION (provided by the applicant): Onchocerciasis, or river blindness, is a major filarial disease and is the fifth most common cause of blindness in the world. Limitations of control programs and the possible emergence of ivermectin-resistant strains suggest the need for alternative strategies for treatment and control of Onchocerciasis. Currently, few suitable targets for chemotherapy have been precisely identified in filarial and other parasitic nematodes, due in part to a lack of understanding of the basic biology and biochemistry of these parasites. We propose to explore cysteine proteases as potential targets for chemotherapy. In our previous studies we identified a cysteine protease inhibitor, onchocystatin, and a cathepsin Z-like cysteine protease, and proposed that they are essential for molting, growth and remodeling of the cuticle in larvae and adult worms, and the development of microfilariae. Recently, a cathepsin L-Iike enzyme and another member of the cystatin family were cloned, suggesting that the role of cysteine proteases and their endogenous inhibitors is more elaborate than initially thought. However, these proteins cannot be easily studied in 0. volvulus in vivo as we lack a system for observing gene expression during the development of the parasite, particularly in its adult stages and in the gravid female worms. As many of the essential genes for nematode development are conserved in free-living and parasitic nematodes, we will take advantage of the existence of homologous proteins in C. elegansto understand the cellular processes by which the 0. volvuluscysteine proteases participate in the development of the 0. volvulusparasite in humans. The integrated approach of using genetic, molecular, biochemical and anatomical studies in this proposal, combined with a well-defined organism, will result in understanding how regulation of three distinct cysteine proteases is critical for the development and survival of C. elegansand 0. volvulus. The proposed project has three specific objectives: 1. To establish the distinct physiological roles for each cathepsin Z and cathepsin L-like cysteine protease during C. elegans development and then verify that the proposed 0. volvulushomologues will perform similar functions in 0. volvulus. 2. To compare and contrast the developmental regulation and tissue specificity of 0. volvulusand C. elegans cystatins. 3. To determine the substrate specificity of each 0. volvulusand C. elegans cysteine protease and identify their specific inhibitors in vitro. We will also determine which low molecular weight inhibitors could, eventually, be tested for their in vivoeffects on adult worm survival and microfilariae development. This will provide the basis for developing effective drugs, targeting cysteine proteases, to control onchocerciasis and filariasis in the future.