BioProject

African trypanosomes cause lethal human and animal diseases, known as sleeping sickness and nagana, respectively. More...

African trypanosomes cause lethal human and animal diseases, known as sleeping sickness and nagana, respectively. Current therapies are limited but, fortunately, promising new therapies are in clinical and veterinary development, including acoziborole (AN5568 or SCYX-7158) and AN11736, respectively. These benzoxaboroles are the only novel chemical entities under development for either disease and are key to the WHO target of disease elimination by 2020. Their mode-of-action was previously unknown, however. We have developed a high-coverage overexpression library to explore drug mode-of-action in Trypanosoma brucei. Inititally, an N-myristoyltransferase (NMT) inhibitor was used to select for drug-resistance and to validate a massive parallel library screening approach. This revealed both the drug-target, NMT, and a major NMT-substrate, confirming endocytosis inhibition as the mode-of-action. The approach was then used to identify the target of both benzoxaboroles, Cleavage and Polyadenylation Specificity Factor 3 (CPSF3, Tb927.4.1340). We validate the CPSF3 endonuclease as the target using independent overexpression strains. Knockdown studies provide genetic validation as an essential target and GFP-tagging reveals the expected nuclear localisation. Docking studies reveal how acoziborole blocks the active site and mRNA processing by CPSF3. Thus, we provide both genetic and chemical validation for CPSF3 as an important new drug target in trypanosomes and reveal inhibition of mRNA maturation as the mode-of-action of the oxaboroles. Understanding the mechanism of action of new oxaborole-based therapies can help to develop improved therapies and will also help predict and monitor resistance, if or when it arises. Less...