Date Published: September 25, 2018
Publisher: Public Library of Science
Author(s): Daniela Begolo, Isabel M. Vincent, Federica Giordani, Ina Pöhner, Michael J. Witty, Timothy G. Rowan, Zakaria Bengaly, Kirsten Gillingwater, Yvonne Freund, Rebecca C. Wade, Michael P. Barrett, Christine Clayton, Margaret A. Phillips.
Kinetoplastid parasites—trypanosomes and leishmanias—infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis. In all kinetoplastids, transcription is polycistronic. Individual mRNA 5′-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate, reduced levels of mRNA, and accumulation of peri-nuclear granules. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, the results indicate that mRNA processing is a primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing, and the EC50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Molecular modeling results suggested that inhibition of CPSF3 by AN7973 is feasible. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes. Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition and resistance after CPSF3 expression did not, however, always correlate and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. It is therefore possible that the modes of action of oxaboroles that target trypanosome mRNA processing might extend beyond CPSF3 inhibition.
Kinetoplastid protists cause severe human diseases affecting millions of people. Trypanosoma cruzi causes Chagas disease in South America, and various Leishmania species cause a spectrum of diseases throughout the tropics. Salivarian trypanosomes, the subject of this study, cause sleeping sickness in humans and economically important diseases in cattle, horses and camels [1–3]. Approximately 70 million people, living in sub- Saharan Africa, are estimated to be at risk of contracting human African trypanosomosis, which is caused by Trypanosoma brucei subspecies [4, 5]. As a result of sustained international activities to control the disease [4–6], less than 3000 cases were reported in 2016 (http://www.who.int/trypanosomiasis_african/en/). Trypanosomosis in cattle, caused by infection with Trypanosoma congolense, Trypanosoma vivax and, to a lesser extent, T. brucei, is in contrast a major problem, with wide-reaching effects on human well-being: cattle are used not only as a source of milk and meat, but also for traction. Elimination of cattle trypanosomosis could create economic benefits estimated at nearly 2.5 billion US$ per year . Within Africa, trypanosomosis is transmitted by tsetse flies, but outside Africa, variants of T. brucei are transmitted venereally or by biting flies, and T. vivax can also be transmitted non cyclically by non-tsetse biting flies with massive economic losses affecting draught and milk animals from Argentina to the Philippines .
Benzoxaboroles are important drug candidates for both human and ruminant African trypanosomosis. Our results show that AN7973 inhibits mRNA processing in trypanosomes. Expression of additional CPSF3 increased the EC50 of AN7973, suggesting that AN7973 can bind to CPSF3. These results suggest that mRNA processing is an important target of AN7973, which might operate through CPSF3 inhibition. AN7973 also caused metabolite changes indicative of disturbed methylation, similar to those observed for acoziborole.