The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing

Abstract

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.

Regulation of gene expression in trypanosomatids: living with polycistronic transcription

“…The benzoxaborole acoziborole is undergoing clinical trials for sleeping sickness treatment [113], and other benzoxaboroles are under consideration for other kinetoplastid diseases [114]. The first detectable effect after treatment of T. brucei with the benzoxaborole AN7973 is an increase in mRNA precursors and a decrease in the Y-structure intermediate [3], suggesting that this compound targets mRNA processing. CPSF3 (also called CPSF73) is the enzyme that cleaves the substrate at the polyadenylation site.…”

“…Because of this, comparisons between the different models enable us to distinguish characteristics that were present in the last eukaryotic common ancestor from those that evolved later [2]. Moreover, one aspect of trypanosome gene expression—their mRNA processing—is a target of clinically useful drugs for the treatment of human and ruminant trypanosomiasis [3,4].…”

Unpacking the Pathogen Box—An Open Source Tool for Fighting Neglected Tropical Disease

“…Compounds 89 , 91 and 92 were later established to be orally efficacious leads for stage 1 and 2 HAT in murine models, with compound 92 progressing into the clinic . While the MOA is yet to be fully elucidated, several related studies have shed light on several possible MOAs . Possibly the most significant in this regard was an orthogonal proteomic and genomic study which identified 14 protein targets of 93 .…”

New Drugs for Human African Trypanosomiasis: A Twenty First Century Success Story

“…Over-expression of the gene yielded a notable loss of activity [96]. Elegant experiments using a gene over-expression library, followed by a selection of clones over-expressing genes yielding reduced sensitivity to acoziborole itself, also identified CPSF3 [97], and over-expression confirmed loss of sensitivity, pointing to CPSF3 as a target, if not the exclusive target, of these compounds in trypanosomes.…”

“…Subsequently, CPSF3 was identified as a target for benzoxaboroles in apicomplexan parasites Plasmodium [93] and Toxoplasma [94]. Based on this observation, and the fact that metabolomics experiments had revealed a profound change in the methionine metabolism [95] that might have been related to RNA processing defects, particularly given the multi-methylation of the spliced leader sequence used in trans-splicing in trypanosomes, the effect of over-expression of CPSF3 on sensitivity to the related benzoxaborole, AN7973, was tested [96].…”