Mode of action of the anti-trypanosomal benzoxaborole AN7973

Mon9  Apr12:00pm(15 mins)
Where:
Stream 1 - Edward Llwyd 0.26 Biology Main

Authors

D Begolo8; F Giordani7; I Vincent7; M Witty5; T Rowan4; Z Bengaly2; K Gillingwater6; Y Freund1; M P Barrett7; C Clayton3
1 Anacor Pharmaceuticals, United States;  2 CIRDES, Burkina Faso;  3 DKFZ-ZMBH Alliance, Germany;  4 GALVmed, UK;  5 Global Alliance for Livestock Veterinary Medicine, UK;  6 Swiss Tropical and Public Health Institute, Switzerland;  7 Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, UK;  8 ZMBH - Heidelberg University, Germany

Discussion

In the past few years several million compounds have been tested for selective toxicity towards kinetoplastids, and thousands have shown activity. To develop such compounds, it is useful to know the mode of action. Characterization of cells that become resistant after RNAi has proven extremely useful in identifying mechanisms of uptake or intracellular activation, but not, so far, in identifying molecular targets. 

The benzoxaborole compound AN7973 was considered for development for cattle trypanosomiasis, and a similar compound, AN5568, is in phase II trials for sleeping sickness. We investigated the mode of action of AN7973 using multiple methods, including metabolome analysis and measurements of macromolecular biosynthesis.  Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by increases in partially processed tubulin mRNAs, reduced levels of mRNA, and loss of the Y-structure splicing intermediate. Methylation of the spliced leader precursor RNA was not affected, suggesting a direct effect on processing machinery. The trypanosomes also accumulated perinuclear granules typical for splicing inhibition. Prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance: while the genomes of the parasites had numerous changes, none was indicative of a primary target. 

The cleavage and polyadenylation factor CPSF3 is a known oxaborole target in Plasmodium and Toxoplasma and there was a CPSF3 gene duplication in a published line with mild resistance to a different oxaborole. We found that the EC50 for AN7973 in T. brucei was increased three-fold by ectopic expression of T. brucei CPSF3, indicating a possible role for CPSF3 as a target. 

Several other benzoxaboroles showed effects similar to those of AN7973, but metabolic changes, splicing inhibition, granule formation, and resistance after CPSF3 expression did not always correlate and there was no obvious structure-function relationship. Our results suggest that inhibition of mRNA processing, perhaps via inhibition of CPSF3, contributes to the anti-trypanosomal activity of several benzoxaboroles.    

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