Binding of Nile Blue (NB) with calf thymus DNA has been studied using molecular modeling, spectroscopic, and thermodynamic techniques. Our study revealed that NB binds to the DNA helix by two types of modes (groove binding and intercalation) simultaneously. The thermodynamic study showed that the overall binding free energy is a combination of several negative and positive free energy changes. The binding was favored by negative enthalpy and positive entropy changes (due to the release of water from the DNA helix). The docking study validated all experimental evidence and showed that NB binds to a DNA minor groove at low concentrations and switches to intercalation mode at higher concentrations.

Background Cushing’s disease, the most common cause of Cushing’s syndrome, is driven by pituitary tumors (corticotropinoma) and characterized by the overexpression of CRH R1 and V1b receptors. Accurate detection of these tumors remains challenging. This study aims to develop and evaluate a corticotropinoma specific radiopharmaceutical, [⁶⁸Ga]Ga-DOTA-mDesmo, that targets the V1b receptor for both anatomical and functional identification of corticotropinomas. Methods Molecular docking was used to validate the binding affinity of [⁶⁸Ga]Ga-DOTA-mDesmo to the V1b receptor. Radiolabeling was optimized with gallium-68, followed by quality controls and physicochemical characterization. ACTH-release assay was performed using primary-cultured corticotropinoma cells. Receptor specificity was confirmed via radioimmunoassay using recombinant human V1b receptor. Ex vivo biodistribution studies were performed in healthy male Wistar rats at 30-, 60-, and 120-min post-injection (8 ± 1.1 MBq). Results Here we show that [⁶⁸Ga]Ga-DOTA-mDesmo binds effectively to the V1b receptor, with a binding energy of −13.98 kcal/mol and the key interacting residues of V1b are GLN301, SER304, ASN309, and ASP314. Radiolabeling achieves high yield (~96%) and purity (>99%), with human serum stability for up to 4 h. In vitro studies confirm that DOTA-mDesmo acts as an agonist in corticotropinoma cells. Excess cold DOTA-mDesmo results in a 50% blocking in binding. Biodistribution in rats indicates renal clearance, with high %ID/g in the kidneys (7.37 ± 0.58) and urinary bladder (4.32 ± 0.48), and negligible uptake in the pituitary gland, our organ of interest. Conclusions These finding support [⁶⁸Ga]Ga-DOTA-mDesmo as a promising radiotracer for non-invasive, receptor-targeted PET/CT imaging of corticotropinomas in patients with Cushing’s disease.