ABSTRACT
To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.
Subject(s)
BRCA1 Protein/genetics , BRCA2 Protein/genetics , DNA Repair/drug effects , DNA-Directed DNA Polymerase/genetics , Nucleic Acid Synthesis Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Synthetic Lethal Mutations/drug effects , Allosteric Regulation , Animals , Apoptosis/drug effects , Apoptosis/genetics , BRCA1 Protein/metabolism , BRCA2 Protein/metabolism , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cell Survival/drug effects , Cell Survival/radiation effects , DNA Damage/drug effects , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , Deoxyribonucleases/antagonists & inhibitors , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Female , Homologous Recombination/drug effects , Humans , Inhibitory Concentration 50 , Mice , Organoids/drug effects , Ovarian Neoplasms/genetics , Rats , Synthetic Lethal Mutations/genetics , Tumor Suppressor p53-Binding Protein 1/deficiency , Tumor Suppressor p53-Binding Protein 1/metabolism , DNA Polymerase thetaABSTRACT
The trypanocidal agents nifurtimox and benznidazole both function as prodrugs and must undergo enzyme-mediated activation, a reaction catalyzed by type I nitroreductase (NTR). In the search for new parasitic therapies, we have utilized this finding to investigate whether aziridinyl nitrobenzamide derivatives have activity against bloodstream-form Trypanosoma brucei and Trypanosoma cruzi amastigotes, parasite stages that replicate in the mammalian host. For T. cruzi drug screening, we generated trypanosomes that expressed the luciferase reporter gene and optimized a mammalian infection model in a 96-well plate format. A subset of compounds having a 5-(aziridin-1-yl)-2,4-dinitrobenzyl structure was shown to be metabolized by purified T. brucei NTR and when screened against both parasite life cycle stages displayed significant growth-inhibitory properties: the most potent compounds generated 50% inhibitory concentrations of <1 µM. The trypanocidal activity was shown to be NTR specific, since parasites overexpressing this enzyme were hypersensitive to the aziridinyl dinitrobenzyl agents. We conclude that members of the aziridinyl nitrobenzamide class of nitroheterocycles provide new lead structures that have the potential to treat trypanosomal infections.
