Title: Identifying inhibitors of Schistosoma mansoni cyclic nucleotide phosphodiesterases as new lead compounds against schistosomiasis
Funder: The Academy of Medical Sciences

Abstract: Schistosomiasis is considered the second devastating neglected tropical parasitic disease, caused by parasitic worms of genus Schistosoma, especially in poor societies with unclean water and inadequate sanitation. The disease has different stages and is endemic in areas of the tropic,subtropics including Egypt. Despite the innumerable studies on schistosomiasis, many questions have not been answered yet. The transformation of the different stages of the parasite is associated with many transcriptional changes which is associated with many physiological, transcriptional and biochemical changes. Among others, Cyclic AMP has been shown to induce gene transcription through the activation of cAMP-dependent protein kinase (PKA), which subsequently phosphorylates CREB. Increased levels of cAMP appear to be signaling through a PKA as evidenced by a more rapid transformation of miracidia in the presence of PKA inhibitors compared to controls. On the other hand, Phosphodiesterases (PDEs) are among the most exciting new drug targets in human parasites. In the previous visit several SmPDEs were cloned into the expression vectors for functional complementation in the standardised T. brucei complementation/expression system. This can now be used to identify potent inhibitors that will also be tested against Schistosomes in vitro. Taken together, the main aims of the present work: 1.To measure SmPDE activity in extracts of T. brucei strains expressing the Schistosoma enzymes. This is much more straightforward than purifying overexpressed His-tagged enzyme on a nickel column, as the thus-purified enzymes are poorly soluble and poorly stable. The complementing T. brucei lines have had their main phosphodiesterase genes deleted and survive exclusively from the high expression of the single Schistosoma PDE. The resulting activity of cAMP and/or cGMP hydrolysis should therefore be specific to that enzyme. Initially, the Km value for each cyclic nucleotide will be established. 2.The complementation lines will next be used for inhibitor studies. As the strains are designed to be dependent on only the SmPDE, inhibition is lethal. Thus, using our standard high throughput drug screening protocols with the panel of single SmPDE strains, it is possible to quickly build a comparative inhibition profile for the inhibitor series, which will yield potency in the form of EC50 values as well as data on inhibitor selectivity of one SmPDE over another. As many of these inhibitors have already been tested against Schistosoma mansoni in vitro (and some also in vivo) at the Theodor Bilharz Research Institute in Cairo, this will allow the development of hypotheses as to which SmPDE(s) are the best drug targets. These enzymes will then be prioritised for production as purified catalytic domains, as described, and high-throughput inhibitor screening in the next stage of the research. Given the above information, the proposed work proposes providing new insight into a critical part of Schistosoma biochemistry and exploit this for the development of new drugs against the parasite using Schistosoma mansoni phosphodiesterases (enzymes of the cAMP signalling pathway). We hope to provide novel targets for combating this neglected disease.


Research activity codes: 5.1 PHARMACEUTICALS;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION