Preparation of trypanosome cdc2-related kinase 9 for high throughput screening

University Of Connecticut Sch Of Med/Dnt, Farmington CT

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Abstract

DESCRIPTION (provided by applicant): The trypanosomatid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. cause the major human diseases African Sleeping Sickness, Chagas' disease, and Leishmaniasis, respectively. Since drugs for these neglected tropical diseases are limited, toxic and, in part, difficult to administer, and since parasite resistance to these drugs is on the rise, it becomes increasingly important to develop new strategies of chemotherapeutic intervention. Our research has focused on been trypanosomatid gene expression because the underlying mechanisms deviate substantially from those in the human host. For example, polycistronic transcription of protein coding genes and processing of pre-mRNA by spliced leader (SL) trans splicing are parasite-specific steps in the expression and maturation of trypanosomatid mRNAs. A key molecule being consumed in this process is the SL RNA whose 5/-terminal sequence is fused to the 5/ end of each mRNA. Thus, the viability of all trypanosomatid parasites crucially depends on strong and continuous expression of functional SL RNA which involves RNA polymerase II-mediated transcription of SL RNA genes and the formation of a highly methylated cap structure, termed cap4, at the 5/ end of the SL RNA. We serendipitously discovered that the T. brucei cyclin-dependent kinase CRK9 (cdc2-related kinase 9) controls SL RNA cap4 formation. Accordingly, CRK9 silencing blocked trans splicing and was rapidly lethal to both insect-stage and mammalian-infective trypanosomes in culture. Since cyclin-dependent kinases are considered to be excellent drug targets and since CRK9 controls this key step in trypanosomatid gene expression, we propose to prepare CRK9 for large scale drug screening. This will involve the characterization of the trypanosome CRK9 complex, validation of CRK9 as a drug target in the mouse model, high expression and reconstitution of a functional, recombinant enzyme, development of a high throughput screening assay and a small scale, pilot inhibitor screen.