Clinical and veterinary trypanocidal benzoxaboroles target CPSF3

Proc Natl Acad Sci U S A. 2018 Sep 18;115(38):9616-9621. doi: 10.1073/pnas.1807915115. Epub 2018 Sep 5.

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.

Keywords: CPSF73; N-myristoyltransferase; Ysh1; drug discovery; genetic screening.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Animals
  • Benzamides / pharmacology
  • Benzamides / therapeutic use
  • Boron Compounds / pharmacology
  • Boron Compounds / therapeutic use
  • CRISPR-Cas Systems
  • Cell Nucleus / genetics
  • Cell Nucleus / metabolism
  • Cleavage And Polyadenylation Specificity Factor / antagonists & inhibitors*
  • Cleavage And Polyadenylation Specificity Factor / genetics
  • Cleavage And Polyadenylation Specificity Factor / metabolism
  • Drug Resistance / drug effects
  • Drug Resistance / genetics
  • Gene Knockdown Techniques
  • Gene Library
  • High-Throughput Screening Assays / methods
  • Humans
  • Molecular Docking Simulation
  • Protozoan Proteins / antagonists & inhibitors*
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism
  • RNA Processing, Post-Transcriptional / drug effects
  • RNA, Messenger / metabolism
  • RNA, Protozoan / metabolism
  • Trypanocidal Agents / pharmacology*
  • Trypanocidal Agents / therapeutic use
  • Trypanosoma brucei brucei / drug effects
  • Trypanosoma brucei brucei / physiology*
  • Trypanosomiasis, African / prevention & control*
  • Trypanosomiasis, African / transmission
  • Trypanosomiasis, African / veterinary
  • Valine / analogs & derivatives
  • Valine / pharmacology
  • Valine / therapeutic use

Substances

  • AN11736
  • Benzamides
  • Boron Compounds
  • Cleavage And Polyadenylation Specificity Factor
  • Protozoan Proteins
  • RNA, Messenger
  • RNA, Protozoan
  • SCYX 7158
  • Trypanocidal Agents
  • Valine