Benzoxaborole treatment perturbs S-adenosyl-L-methionine metabolism in Trypanosoma brucei

Steketee, Pieter C.; Vincent, Isabel M.; Achcar, Fiona; Giordani, Federica; Kim, Dong-Hyun; Creek, Darren J.; Freund, Yvonne R.; Jacobs, Robert T.; Rattigan, Kevin M.; Horn, David; Field, Mark C.; MacLeod, Annette; Barrett, Michael P.

doi:10.1371/journal.pntd.0006450

Cited by 40 publications

(42 citation statements)

References 67 publications

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“…Polyadenylation and trans-splicing of adjacent genes are coupled in trypanosomatids, however (42), and consistent with this, CPSF3 knockdown disrupted both polyadenylation and trans-splicing in T. b. brucei (49). Accordingly, inhibition of CPSF3 may explain perturbed methyl-donor metabolism after acoziborole exposure (25), which may be because of reduced mRNA methylation as a result of a splicing defect. CPSF3 also associates with the U1A snRNP splicing complex in trypanosomatids (50).…”

Section: Discussionsupporting

confidence: 61%

“…Oxaboroles are also potential antihypertensive rho-activated kinase inhibitors (22) and anti-inflammatory phosphodiesterase inhibitors for the treatment of psoriasis (23). Genomic and proteomic approaches, using oxaborole-1, a close analog of acoziborole, previously yielded lists of candidate genes implicated in the antitrypanosomal mode of action (24), whereas metabolomic analysis after acoziborole exposure indicated perturbation of S-adenosyl-L-methionine metabolism (25), but the trypanocidal targets of acoziborole and AN11736 remained unknown.…”

Section: Significancementioning

confidence: 99%

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Clinical and veterinary trypanocidal benzoxaboroles target CPSF3

Wall

Rico

Lukac

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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African trypanosomes cause lethal and neglected tropical diseases, known as sleeping sickness in humans and nagana in animals. Current therapies are limited, but fortunately, promising therapies are in advanced clinical and veterinary development, including acoziborole (AN5568 or SCYX-7158) and AN11736, respectively. These benzoxaboroles will likely be key to the World Health Organization's target of disease control by 2030. Their mode of action was previously unknown. We have developed a high-coverage overexpression library and use it here to explore drug mode of action in Initially, an inhibitor with a known target was used to select for drug resistance and to test massive parallel library screening and genome-wide mapping; this effectively identified the known target and validated the approach. Subsequently, the overexpression screening approach was used to identify the target of the benzoxaboroles, Cleavage and Polyadenylation Specificity Factor 3 (CPSF3, Tb927.4.1340). We validated the CPSF3 endonuclease as the target, using independent overexpression strains. Knockdown provided genetic validation of CPSF3 as essential, and GFP tagging confirmed the expected nuclear localization. Molecular docking and CRISPR-Cas9-based editing demonstrated how acoziborole can specifically block the active site and mRNA processing by parasite, but not host CPSF3. Thus, our findings provide both genetic and chemical validation for CPSF3 as an important drug target in trypanosomes and reveal inhibition of mRNA maturation as the mode of action of the trypanocidal benzoxaboroles. Understanding the mechanism of action of benzoxaborole-based therapies can assist development of improved therapies, as well as the prediction and monitoring of resistance, if or when it arises.

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Section: Discussionsupporting

confidence: 61%

Section: Significancementioning

confidence: 99%

Clinical and veterinary trypanocidal benzoxaboroles target CPSF3

Wall

Rico

Lukac

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

106

125

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“…This results in loss of mRNA from the cells by the normal mechanisms of mRNA turnover [ 49 ]. It has recently been shown that an accumulation of numerous metabolites, including S-adenosylmethionine, occurs in trypanosomes treated with Sinefungin, and a similar profile was identified in trypanosomes treated with acoziborole [ 50 ].…”

Section: Introductionmentioning

confidence: 99%

The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing

et al. 2018

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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.

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“…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].…”

Section: Acoziborole: Mode Of Action and Resistance Riskmentioning

confidence: 99%

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

et al. 2020

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The twentieth century ended with human African trypanosomiasis (HAT) epidemics raging across many parts of Africa. Resistance to existing drugs was emerging, and many programs aiming to contain the disease had ground to a halt, given previous success against HAT and the competing priorities associated with other medical crises ravaging the continent. A series of dedicated interventions and the introduction of innovative routes to develop drugs, involving Product Development Partnerships, has led to a dramatic turnaround in the fight against HAT caused by Trypanosoma brucei gambiense. The World Health Organization have been able to optimize the use of existing tools to monitor and intervene in the disease. A promising new oral medication for stage 1 HAT, pafuramidine maleate, ultimately failed due to unforeseen toxicity issues. However, the clinical trials for this compound demonstrated the possibility of conducting such trials in the resource-poor settings of rural Africa. The Drugs for Neglected Disease initiative (DNDi), founded in 2003, has developed the first all oral therapy for both stage 1 and stage 2 HAT in fexinidazole. DNDi has also brought forward another oral therapy, acoziborole, potentially capable of curing both stage 1 and stage 2 disease in a single dosing. In this review article, we describe the remarkable successes in combating HAT through the twenty first century, bringing the prospect of the elimination of this disease into sight.

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Benzoxaborole treatment perturbs S-adenosyl-L-methionine metabolism in Trypanosoma brucei

Cited by 40 publications

References 67 publications

Clinical and veterinary trypanocidal benzoxaboroles target CPSF3

Clinical and veterinary trypanocidal benzoxaboroles target CPSF3

The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing

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

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