AEE788 Inhibits Basal Body Assembly and Blocks DNA Replication in the African Trypanosome.

Abstract

Trypanosoma brucei causes human African trypanosomiasis (HAT). The pyrrolopyrimidine AEE788 (a hit for anti-HAT drug discovery) associates with three trypanosome protein kinases. Herein we delineate the effects of AEE788 on T. brucei using chemical biology strategies. AEE788 treatment inhibits DNA replication in the kinetoplast (mitochondrial nucleoid) and nucleus. In addition, AEE788 blocks duplication of the basal body and the bilobe without affecting mitosis. Thus, AEE788 prevents entry into the S-phase of the cell division cycle. To study the kinetics of early events in trypanosome division, we employed an "AEE788 block and release" protocol to stage entry into the S-phase. A time-course of DNA synthesis (nuclear and kinetoplast DNA), duplication of organelles (basal body, bilobe, kinetoplast, nucleus), and cytokinesis was obtained. Unexpected findings include the following: 1) basal body and bilobe duplication are concurrent; 2) maturation of probasal bodies, marked by TbRP2 recruitment, is coupled with nascent basal body assembly, monitored by localization of TbSAS6 at newly forming basal bodies; and 3) kinetoplast division is observed in G2 after completion of nuclear DNA synthesis. Prolonged exposure of trypanosomes to AEE788 inhibited transferrin endocytosis, altered cell morphology, and decreased cell viability. To discover putative effectors for the pleiotropic effects of AEE788, proteome-wide changes in protein phosphorylation induced by the drug were determined. Putative effectors include an SR protein kinase, bilobe proteins, TbSAS4, TbRP2, and BILBO-1. Loss of function of one or more of these effectors can, from published literature, explain the polypharmacology of AEE788 on trypanosome biology.