Hemin-Binding Site Competition and Hemin Cytotoxicity Evaluation: Extension of "Discovery of a novel miconazole analogue as a dual apo-IDO1 inhibitor and ABCB1 modulator for cancer immunotherapy and chemosensitizing"
Description
This repository contains a graphical figure for a functional Hemin-Binding Site Competition Assay and an LDH Cytotoxicity Assay. The functional assay assesses how the addition of exogenous hemin modulates the suppression of kynurenine (Kyn) production by our Apo-IDO1 inhibitor (i.e., 1g), confirming a competitive mechanism of action. The LDH cytotoxicity assay serves as an experimental control, demonstrating that the concentrations of hemin used in the competition study do not compromise cell viability. These experiments provide further validation of the molecular hypothesis reported in our paper in the European Journal of Pharmaceutical Sciences (DOI: 10.1016/j.ejps.2026.107605).
Files
Steps to reproduce
Hemin Competition Assay (Hemin-Binding Site Competition Assay): SK-OV-3 and HeLa cells were seeded in 96-well plates at a density of 2.5 × 10⁴ cells per well and cultured for 24 h in medium supplemented with 100 μM L-tryptophan. The next day, cells were treated with IFN-γ at 25 ng/mL for 24 h. On the third day, the medium was replaced with either standard culture medium or hemin-supplemented medium containing 1g at the indicated concentrations, followed by an additional 24 h of incubation. The supernatant was collected, and kynurenine (Kyn) levels were measured using an absorbance assay. The percentage of Kyn secretion was calculated relative to its respective untreated control (cells in standard medium or hemin-supplemented medium without 1g). Each experiment was repeated at least three times. All media used during the compound treatment phase were supplemented with 1% FBS.
Institutions
- National Cheng Kung UniversityTaiwan, Tainan City
- Hacettepe UniversityAnkara, Ankara