Dataset Information

Clinical and veterinary trypanocidal benzoxaboroles target CPSF3.

ABSTRACT: 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 Trypanosoma brucei 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.

SUBMITTER: Wall RJ

PROVIDER: S-EPMC6156652 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Clinical and veterinary trypanocidal benzoxaboroles target CPSF3.

Wall Richard J RJ Rico Eva E Lukac Iva I Zuccotto Fabio F Elg Sara S Gilbert Ian H IH Freund Yvonne Y Alley M R K MRK Field Mark C MC Wyllie Susan S Horn David D

Proceedings of the National Academy of Sciences of the United States of America 20180905 38

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-c ...[more]

PMID: 30185555

Similar Datasets

Project description:Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism. T. brucei assay is reported in the current study <a href='https://www.ebi.ac.uk/metabolights/MTBLS1474' rel='noopener noreferrer' target='_blank'>MTBLS1474</a>.T. congolense assay is reported in <a href='https://www.ebi.ac.uk/metabolights/MTBLS1309' rel='noopener noreferrer' target='_blank'>MTBLS1309</a>. Linked cross omic data sets:Genome data associated with this study are available in the European Nucleotide Archive (ENA): accession number <a href='https://www.ebi.ac.uk/ena/browser/view/PRJEB34627' rel='noopener noreferrer' target='_blank'>PRJEB34627</a>.

Project description:Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism. T. congolense assay is reported in the current study <a href='https://www.ebi.ac.uk/metabolights/MTBLS1309' rel='noopener noreferrer' target='_blank'>MTBLS1309</a>.T. brucei assay is reported in <a href='https://www.ebi.ac.uk/metabolights/MTBLS1474' rel='noopener noreferrer' target='_blank'>MTBLS1474</a>. Linked cross omic data sets:Genome data associated with this study are available in the European Nucleotide Archive (ENA): accession number <a href='https://www.ebi.ac.uk/ena/browser/view/PRJEB34627' rel='noopener noreferrer' target='_blank'>PRJEB34627</a>.

Dataset Information

Clinical and veterinary trypanocidal benzoxaboroles target CPSF3.

Publications

Clinical and veterinary trypanocidal benzoxaboroles target CPSF3.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets