Schistosomiasis is a prevalent and chronic helmintic disease in tropical regions. Treatment and control relies on chemotherapy with just one drug, praziquantel and this reliance is of concern should clinically relevant drug resistance emerge and spread. Therefore, to identify potential target proteins for new avenues of drug discovery we have taken a comparative chemogenomics approach utilizing the putative proteome of Schistosoma mansoni compared to the proteomes of two model organisms, the nematode, Caenorhabditis elegans and the fruitfly, Drosophila melanogaster. Using the genome comparison software Genlight, two separate in silico workflows were implemented to derive a set of parasite proteins for which gene disruption of the orthologs in both the model organisms yielded deleterious phenotypes (e.g., lethal, impairment of motility), i.e., are essential genes/proteins. Of the 67 and 68 sequences generated for each workflow, 63 were identical in both sets, leading to a final set of 72 parasite proteins. All but one of these were expressed in the relevant developmental stages of the parasite infecting humans. Subsequent in depth manual curation of the combined workflow output revealed 57 candidate proteins. Scrutiny of these for ‘druggable’ protein homologs in the literature identified 35 S. mansoni sequences, 18 of which were homologous to proteins with 3D structures including co-crystallized ligands that will allow further structure-based drug design studies. The comparative chemogenomics strategy presented generates a tractable set of S. mansoni proteins for experimental validation as drug targets against this insidious human pathogen.

A novel serine/threonine kinase, termed DIK, was cloned using the yeast two-hybrid system to screen a cDNA library from the human keratinocyte cell line HaCaT with the catalytic domain of rat protein kinase C␦ (PKC␦ cat ) cDNA as bait. The predicted 784-amino acid polypeptide with a calculated molecular mass of 86 kDa contains a catalytic kinase domain and a putative regulatory domain with ankyrin-like repeats and a nuclear localization signal. Expression of DIK at the mRNA and protein level could be demonstrated in several cell lines. The dik gene is located on chromosome 21q22.3 and possesses 8 exons and 7 introns. DIK was synthesized in an in vitro transcription/translation system and expressed as recombinant protein in bacteria, HEK, COS-7, and baculovirus-infected insect cells. In the in vitro system and in cells, but not in bacteria, various post-translationally modified forms of DIK were produced. DIK was shown to exhibit protein kinase activity toward autophosphorylation and substrate phosphorylation. The interaction of PKC␦ cat and PKC␦ with DIK was confirmed by coimmunoprecipitation of the proteins from HEK cells transiently transfected with PKC␦ cat or PKC␦ and DIK expression constructs.The members of the PKC 1 family, because of structural and enzymatic differences, can be subdivided into several groups (for reviews see Refs. 1 and 2). PKC␦, a member of the so-called nPKC subfamily, has attracted the interest of an increasing number of research groups over the last years and presumably is one of the most thoroughly studied PKC isoenzymes (for a review see Ref. 3). Like all the other PKC isoforms, PKC␦ is thought to play an individual role in various signaling pathways and to specifically affect diverse cellular processes, such as growth, differentiation, apoptosis, and tumorigenesis (4 -16). This specific action is likely to afford a sophisticated network of regulation of PKC␦ activity, subcellular localization, and substrate phosphorylation. Beside the well known regulation of enzyme activity by signal-induced second messengers, such as diacylglycerol, PKC␦ is regulated by up-and downmodulation of its expression (4, 17, 18), by phosphorylation (19 -29), and presumably by interaction with other proteins involved in signal transduction, such as other protein kinases and anchor or docking proteins (30 -38). Particularly the latter is essential for a specific subcellular localization of the enzyme and a selective phosphorylation of substrate proteins. Our knowledge of PKC␦-protein interactions, however, is rather scanty. This holds true not only for the interaction with physiologically relevant substrate proteins but also for the interaction with other proteins that might affect PKC␦ signaling.We therefore attempted to clone proteins interacting with PKC␦ by using the yeast two-hybrid system (YTHS). Here, we describe the cloning, expression, and characterization of a novel serine/threonine kinase, termed DIK (PKC-delta-interacting protein kinase), which is coimmunoprecipitated with PKC␦ and the catalyti...

A modern concept for the development of novel antiparasitic drugs is the combination of bioinformatics and chemoinformatics approaches. This covers, for example, the identification of target proteins serving as molecular points of attack for parasiticides--the idea is that, owing to some essential role, inhibition of a target protein should eradicate the parasite. To prevent toxicity problems for vertebrate host organisms, it is advantageous that these proteins show significant differences from their vertebrate counterparts. In the present work, we identified potential target proteins in parasitic nematodes (Ascaris suum, Brugia malayi, and Haemonchus contortus) and arthropods (Boophilus microplus and Rhipicephalus appendiculatus) using bioinformatic sequence comparison methods on expressed sequence tags. Interesting target proteins (e.g., S-adenosyl-l-methionine synthetase) were characterized in detail by subjecting them to in-depth bioinformatic analysis. S-Adenosyl-l-methionine synthetase was also used to elucidate chemoinformatics approaches like homology modeling and docking, which represent appropriate methods for generating valuable data for the development of new drug candidates.

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