RESUMEN
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.
Asunto(s)
Proteína BRCA1/genética , Proteína BRCA2/genética , Reparación del ADN/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/genética , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Mutaciones Letales Sintéticas/efectos de los fármacos , Regulación Alostérica , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Daño del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxirribonucleasas/antagonistas & inhibidores , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Recombinación Homóloga/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Ratones , Organoides/efectos de los fármacos , Neoplasias Ováricas/genética , Ratas , Mutaciones Letales Sintéticas/genética , Proteína 1 de Unión al Supresor Tumoral P53/deficiencia , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , ADN Polimerasa thetaRESUMEN
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.