Thiosemicarbazide Derivatives Targeting Human TopoIIα and IDO-1 as Small-Molecule Drug Candidates for Breast Cancer Treatment

**Background:** Breast cancer became the most frequently diagnosed cancer in 2020, with nearly 2.3 million new cases. Despite treatment advances, chemotherapy efficacy is limited by low selectivity and drug resistance. Topoisomerase IIα and IDO-1 are both upregulated in breast cancer and represent promising therapeutic targets. The authors previously identified thiosemicarbazide derivatives 1–3 as first-in-class dual inhibitors of human topoIIα and IDO-1, showing up to 90-fold higher cytotoxicity than etoposide. This study investigates their mechanisms of action in breast cancer cells and evaluates ADME-Tox properties.

**Methods:** MCF-7 (ER/PR-positive) and MDA-MB-231 (triple-negative) breast cancer cells, along with normal MCF-10A breast epithelial cells, were used. Cytotoxicity was assessed by MTT assays. Apoptosis was measured by annexin V-FITC/PI staining via flow cytometry. Caspase-8 and caspase-9 activation was evaluated using FLICA assays with and without the pan-caspase inhibitor Z-VAD-FMK (100 µM). Autophagy was detected using an Autophagy Assay (Red kit). Cell cycle analysis was performed by PI staining and flow cytometry. ABC transporter activity (MDR1, MRP1/2, BCRP) was measured using an MDR Assay kit with specific inhibitors (verapamil, MK-571, noviobiocin), expressed as multidrug resistance activity factor (MAF). Hemolytic activity was tested on human RBCs at 10 µg/mL. CYP3A4 and CYP2D6 activities were evaluated using Vivid™ screening kits with ketoconazole and quinidine as positive controls.

**Key Results:** Compounds 1–3 showed IC50 values against MCF-10A normal cells of 25.31 ± 2.27, 42.74 ± 3.16, and 40.45 ± 4.59 µg/mL, respectively. Selectivity indices ranged from 2.57 to 5.30 for MCF-7 and 2.75 to 5.60 for MDA-MB-231. Compound 3 induced apoptosis in nearly 68% of MCF-7 cells (IC50 = 7.67 µg/mL), while compound 2 caused the greatest increase in apoptotic MDA-MB-231 cells to 70% (IC50 = 7.64 µg/mL). Etoposide (20 µg/mL) resulted in only about 8.5% apoptotic cells. Both caspase-8 and caspase-9 were significantly activated by compounds 1–3 in a dose-dependent manner, even after pan-caspase inhibitor pretreatment, indicating involvement of both extrinsic and intrinsic pathways. Compound 1 induced autophagy in 11.5 ± 1.2% of MCF-7 cells and 5.5 ± 0.9% of MDA-MB-231 cells at 20 µg/mL, versus ~1% in controls. All three compounds caused S-phase cell cycle arrest: in MCF-7 cells at 20 µg/mL, S-phase increased from 19.2% (control) to 35.9% (1), 40.7% (2), and 49.1% (3). Compounds 2 and 3 strongly inhibited ABC transporters in MCF-7 cells even at 10 µg/mL (MAF values <20). Compound 2 showed the highest inhibitory potency against MDR1, MRP1/2, and BCRP in MDA-MB-231 cells (at 10 µg/mL: MAF_MDR1 = 13.3, MAF_MRP = 7.0, MAF_BCRP = 4.9). None of the compounds significantly affected CYP3A4 or CYP2D6 activity at 10 µg/mL. Hemolysis ranged from 0.86 ± 0.18% to 1.08 ± 0.26%, not significantly different from untreated RBCs (0.81 ± 0.11%).

**Clinical Implications:** These thiosemicarbazide derivatives represent promising dual-targeting small-molecule candidates for breast cancer treatment. Their selective cytotoxicity toward cancer cells over normal cells, combined with inhibition of ABC transporters (suggesting lower risk of drug resistance), activation of both apoptotic pathways, and favorable ADME-Tox profiles (no CYP3A4/CYP2D6 inhibition, no hemolytic activity), support further preclinical development. The differential effects on ER/PR-positive versus triple-negative cells suggest potential for treating multiple breast cancer subtypes. However, the autophagy findings require further investigation, and immune-mediated hemolytic effects could only be assessed in vivo.