Evaluation of ATM Kinase inhibitor KU-55933 as potential anti-Toxoplasma gondii agent

Abstract

Toxoplasma gondii is an apicomplexan protozoan parasite with a complex life cycle composed of multiple stages that infect mammals and birds. Tachyzoites rapidly replicate within host cells to produce acute infection during which the parasite disseminates to tissues and organs. Highly replicative cells are subject to Double Strand Breaks (DSBs) by replication fork collapse and ATM, a member of the phosphatidylinositol 3-kinase (PI3K) family, is a key factor that initiates DNA repair and activates cell cycle checkpoints. Here we demonstrate that the treatment of intracellular tachyzoites with the PI3K inhibitor caffeine or ATM kinase-inhibitor KU-55933 affects parasite replication rate in a dose-dependent manner. KU-55933 affects intracellular tachyzoite growth and induces G1-phase arrest. Addition of KU-55933 to extracellular tachyzoites also leads to a significant reduction of tachyzoite replication upon infection of host cells. ATM kinase phosphorylates H2A.X (γH2AX) to promote DSB damage repair. The level of γH2AX increases in tachyzoites treated with camptothecin (CPT), a drug that generates fork collapse, but this increase was not observed when co-administered with KU-55933. These findings support that KU-55933 is affecting the Toxoplasma ATM-like kinase (TgATM). The combination of KU-55933 and other DNA damaging agents such as methyl methane sulfonate (MMS) and CPT produce a synergic effect, suggesting that TgATM kinase inhibition sensitizes the parasite to damaged DNA. By contrast, hydroxyurea (HU) did not further inhibit tachyzoite replication when combined with KU-55933.