Targeting breast cancer cells with 2-indolyl-1,3,4-oxadiazole compounds by inducing apoptosis, paraptosis and autophagy

Abstract

While 2-Indolyl-1,3,4-oxadiazole derivatives are recognized for their antibacterial properties, their potential as anticancer agents remains underexplored. This study investigates the anti-breast cancer properties of a novel 2-Indolyl-1,3,4-oxadiazole compound, 5l, focusing on its ability to induce apoptosis, paraptosis, and autophagy, and targeting poly (ADP-ribose) polymerase (PARP1), a critical enzyme in DNA repair. A series of 1,3,4-oxadiazole derivatives (compounds 5a-5m) were synthesized using an optimized multi-step process, enhancing reaction efficiency and yield. In silico molecular docking was used to determine binding efficacy of these derivatives. Lead compound, 5l, underwent cytotoxicity assays against MDA-MB-231, MCF-7, BT-474, and SK-BR-3 breast cancer cell lines, as well as the non-cancerous MCF-10A cell line. Molecular docking assessed the interaction of 5l with the PARP1 active site. Frontier molecular orbital (FMO) and molecular electrostatic potential (MESP) analyses were conducted to map electron distribution and identify reactive regions within compound 5l. The effects of 5l on cellular processes such as apoptosis, autophagy, and endoplasmic reticulum (ER) integrity were evaluated using live and dead assays, Annexin V staining, ER-tracker dye staining, and acridine orange assays. Western blotting analyzed apoptosis, paraptosis, and autophagy-related genomic instability. The optimized synthesis yielded high-purity 1,3,4-oxadiazole derivatives. Compound 5l displayed significant anticancer activity, with IC50 values of 63.7 μM, 29.1 μM, 50.3 μM, and 39.8 μM for MDA-MB-231, MCF-7, BT-474, and SK-BR-3 cell lines respectively, demonstrating its cytotoxic efficacy. Molecular docking revealed that 5l binds to PARP1 active site with a binding energy of −11.7 kcal/mol, indicating a strong interaction supporting its role as a PARP1 inhibitor. Annexin V assays, ER-tracker dye staining, and Acridine orange assays were used to assess apoptosis, ER integrity, and autophagy. 5l induced upregulation of cleaved PARP and downregulation of Alix-loaded proteins, alongside increased LC3-II expression, indicating autophagy-mediated genomic instability. Compound 5l exhibits potent anti-breast cancer activity through paraptosis, apoptosis, and autophagy-mediated genomic instability and by PARP1 inhibition with typically a low IC50 values, highlighting its potential as a therapeutic agent.