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.

Public Health Relevance

Trypanosomatid parasites cause devastating human diseases that affect millions of people worldwide. The drugs to treat the often lethal diseases are limited and problematic, and parasite resistance to these drugs is on the rise requiring new chemotherapeutic intervention strategies. We have recently discovered a cyclin- dependent kinase termed CRK9 that controls a parasite-specific step of gene expression required for the production of each and every protein in these parasites. Since cyclin-dependent kinases are generally considered to be excellent drug targets we propose to prepare Trypanosoma brucei CRK9 for high throughput screening of small molecule inhibitors as a first step towards developing a drug against this enzyme.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
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Rogers, Martin J
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University of Connecticut
Schools of Medicine
United States
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