Multi-target inhibitors of circadian clock and autophagy as a novel anticancer approach.

Autophagy inhibition is emerging as a promising anticancer strategy. We recently reported that the circadian nuclear receptor REV-ERBβ plays an unexpected role in sustaining cancer cell survival when the autophagy flux is compromised. We also identified ARN5187, 1, as a novel dual inhibitor of REV-ERBβ and autophagy, with an improved cytotoxicity against breast cancer BT-474 cells compared to chloroquine, a clinically relevant autophagy inhibitor. Here, we present the structure-activity relationship exploration around 1 that discloses the first class of dual inhibitors of REV-ERBβ and autophagy. This study led to identification of 18 and 28, which showed an improved antagonistic activity than 1 toward REV-ERBβ, and better anticancer activity against BT-474. The combination of optimal chemical and structural moieties of these analogs generated 30 (ARN16090), which inhibited both REV-ERBβ and BT-474 viability 15-fold better than 1. Furthermore, ARN16090 decreased the viability of different tumor tissue cells at concentrations from 5 to 50 times lower than chloroquine, while it did not affect the viability of normal HMEC cells. Considering the connections between circadian clock, autophagy and metabolism, we aimed to evaluate the potential synergistic activity deriving from a combination of compound 30 (ARN16090) and metabolic drugs. Two diverse inhibitors of glycolysis, 3-Bromopyruvate and Dicholoracetate, showed antagonistic effects toward our dual REV-ERB and autophagy inhibitor. With the goal of exploring the diverse cellular signaling pathways associated with a dual inhibition of REV-ERB and autophagy, we conducted a gene expression comparison between breast cancer BT-474 cells treated with ARN16090 or vehicle. This analysis revealed interesting novel therapeutic opportunities associated with our compound. Indeed, the treatment of breast cancer cell with ARN16090 resulted in the transcriptional activation of the pro-apoptotic factors such as PUMA GADD45, FAS, and p21. Experiments in isogenic colon cancer HCT116 cells having a wild-type or a mutated p53 clearly supported the p53-independent anticancer activity of ARN16090. Notably, the gene expression signature observed in ARN16090-treated cells is similar to the signature observed in cancer cells treated with DNA-damaging agents, including oxaliplatin. This observation prompted us investigating the potential synergism between ARN16090 and DNA-damaging agents adopted in cancer treatment. Indeed, synergistic anticancer activity against breast cancer cells were observe with cisplatin, doxorubicin, oxaliplatin and ectoposide. ARN16090 lacks of both intercalating and topoisomerase inhibitory activity; therefore, the transcriptional activation of DNA damage response upon ARN16090 treatment may be independent from a direct activity on DNA integrity. An additional relevant outcome of our gene expression analysis was the identification of an ARN16090-associated signature of estrogen receptor regulated genes. Accordingly, ARN16090 improved the anticancer activity of a clinically relevant ESR inhibitory drug, tamoxifen, which is used for the treatment of estrogen receptor positive breast tumors. In conclusion, we delivered a novel class of dual inhibitors toward REV-ERB and autophagy with improved in vitro anticancer activity against diverse cancer cell types compared with the single autophagy inhibitor, CQ. Furthermore, we provided evidence for the use of these innovative anticancer agents in combination with clinically relevant DNA-damaging antineoplastic drugs and/or for the treatment of chemotherapy-resistant cancer cells.