Trypanosoma Brucei is a protozoan parasite and a pathogen responsible for the Human African Trypanosomiasis (HAT), or ?sleeping sickness?. HAT and other diseases caused by closely related pathogenic parasites represent a substantial global health concern, especially because there is a limited choice of effective antiparasitic drugs. Recent discovery that the dUTP pyrophosphatase (dUTPase) enzyme in T. brucei is significantly divergent from that of human or other mammalian systems has heightened the interest in this enzyme as a possible anti-parasitic drug target. dUTPase is an essential protein in most known living forms; it is required for the synthesis of deoxythymidine triphosphate (dTTP) in support of DNA replication and for limiting the incorporation of uracil into genomic DNA to reduce the detrimental DNA breaks initiated by uracil glycosylase. In the long term, the goal is to develop an effective anti-trypanosome drug with limited toxicity and high therapeutic index. The specific objective of this proposal is to identify and characterize small molecules that are cytotoxic to T. brucei by inhibition of the dUTPase activity in the following two specific aims.
In Aim1, we will take two complementary approaches to identify candidate small molecules targeting T. brucei dUPTase. As an extension of the comprehensive work into the mechanism of uracil DNA incorporation carried out in our laboratory, we designed a cell-based assay where T. brucei or human dUTPase functionally replaces the yeast protein. Using this system, we will screen for molecules differentially inhibiting the growth of yeast cells expressing T. brucei dUTPase and not the cells expressing the human enzyme. Additionally, through the use of virtual docking program, we will identify molecules binding with high affinity to T. brucei but not human dUTPase.
In Aim2, the cytotoxic effect of the candidate molecules will be further characterized in yeast and T. brucei cultures. We will also confirm that, as expected from the disruption of dUTP->dTTP metabolic pathway, the cytotoxicity is correlated with the elevated level of uracil in DNA, DNA breaks, and cell-cycle defect. The significance of the proposed research is underscored by its significant potential to provide further insight into the mechanism of anti-parasitic drugs that target the cellular dUTP/dTTP metabolism and finally a mechanistic basis for the improved efficacy of the combination chemotherapeutic regimen against HAT and other related diseases.
Currently there is a lack of effective chemotherapeutic treatment against diseases resulting from the infection of protozoan parasites such as human African Trypanosomiasis (HAT) or sleeping sickness. This proposal is highly relevant to public health because it seeks to test the efficacy of targeting dUTPase enzyme in Trypanosoma brucei (Tb), the etiological agent of HAT, based on our knowledge of how this essential enzyme functions in replication of the genome and critically prevents incorporation of uracil base into DNA. By identifying and characterizing small molecule inhibitors of Tb dUTPase, the result from the proposed research is expected to lead up to development of this enzyme as the anti-parasitic therapeutic target and pertains to the part of NIH?s mission to apply the fundamental knowledge of living system to enhance health and reduce illness.