Derived From A Platform Of Innovative Therapeutic Molecular Clusters (Tmc), Ag5 Is A Highly Potent And Differentiated Molecule That Promises To Be Efficacious In Hard-To-Treat Solid Cancers.

Abstract Bioenergetics of cancer results in an increased oxidation state and toxic by-products such as reactive oxygen species (ROS). ROS-mediated thiol oxidation of specific sensory proteins triggers the antioxidant response in order to decrease ROS levels and to trigger apoptosis when the oxidative stress cannot be overcome. This metabolic reprogramming has been implicated in refractory and relapsed disease and shaping of the microenvironment in solid cancers. Two redundant antioxidant pathways counteract ROS-mediated oxidation: the Glutathione (GSH)- and Thioredoxin (Trx)-dependent pathways. An effective way to treat tumors is to irreversibly block both pathways simultaneously. Experimental and therapeutic experience has shown that blocking only one is insufficient because one active pathway is enough for tumor survival, a reason why inhibitors of these pathways have shown limited success in the clinic. The TMC Ag5 irreversibly blocks both the GSH and Trx-dependent pathways, making it a highly active therapeutic agent. We present here for the first time a novel and innovative approach targeting tumor pathophysiology and increased REDOX state. Ag5 contains five silver atoms arranged in a specific conformation. Extensive biophysical and cell-based analyses demonstrate its catalytic activity and stability. Ag5 selectively kills high ROS cells by catalyzing the ROS-dependent oxidation of thiol groups of thioredoxins, peroxiredoxins and targets of the glutathione pathway in cells generating high ROS. As a consequence, Ag5 will preferentially kill cancer cells, but will spare normal cells due to their REDOX homeostasis. We characterized Ag5 efficacy with a range of in vitro assays and found it to be potent against a panel of established cancer cell lines derived from solid cancers with an IC50 in the low nM range. Ag5 sensitivity was correlated with high ROS levels, especially mitochondrial superoxide as measured by DHE or MitoSOX. The investigation of specific antioxidant target proteins (e.g. mitochondrial PRX3), and the use of agents that influence the REDOX state including DTT, BSO, NAC or erastin, supported the postulated mechanism of action. Furthermore, we were able to demonstrate concentration dependent cell cycle arrest, mitochondrial swelling, and induction of apoptosis by TUNEL staining, caspase activation and PARP cleavage. In addition, Ag5 was shown to be effective in reducing tumor growth in KRAS-mutated NSCLC and other relevant cell lines in vivo. Finally, Ag5 was safely administered without any clinical observations of toxicity in mouse and rat studies. In summary, Ag5 is a novel and innovative therapeutic candidate that was shown to be safe and effective in preclinical studies, and has the promise to address the unmet medical need in a range of solid cancers that currently have no effective treatment. Citation Format: Vanesa Porto, Carmen Carneiro, Erea Borrajo, Blanca Dominguez, J M. Devida, L J. Giovanetti, F G. Requejo, G Barone, Angela Turrero, David Buceta, Arturo Lopez, Anxo Vidal, Martin Treder, Fernando Dominguez. Derived from a platform of innovative therapeutic molecular clusters (TMC), Ag5 is a highly potent and differentiated molecule that promises to be efficacious in hard-to-treat solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2020.