How strong the interaction really are? Application of nanoITC in the analysis of the interaction between newly synthesized substances with potential anticancer activity and model carrier proteins.

Abstract

The aim of this work was to extend the existing knowledge of the interaction between newly synthetized substances with anticancer properties (5-methyl-12(H)-chino[3,4-b]-[1,4]-benzothiazine chloride (Salt1), 9-fluoro-5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazine chloride (Salt2), and 9-amino-5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazine chloride (Salt3) with model plasma carrier proteins. The thermodynamic profile of ligand-protein complexation and the contribution of bonds responsible for complex formation have been studied using calorimetry technique. The research has theoretical and experimental nature, but from a scientific point of view is novelty due to promising biological properties of Salt1, Salt2, and Salt3 and provide an important basis for further in vitro and in vivo studies. All measurements were conducted using nanoITC calorimeter (TA Instruments, New Castle, USA). The results were analyzed using Launch NanoAnalyze program (TA Instruments, New Castle, USA). Based on the obtained data, it is safe to consider the bonds within Salt1-HSA, Salt3-HSA, Salt1-AGP, Salt3-AGP, and Salt3-HGG complexes to be predominantly hydrophobic (ΔH > 0 and ΔS > 0) with K_a values: (1.95 ± 0.59)·10⁶, (34.6 ± 0.06)·10⁶, (3.34 ± 0.35)·10⁶, (0.45 ± 0.14)·10⁶, and (0.56 ± 0.09)·10⁶ (L·mol^-1), respectively. In contrast, complexes of Salt2 with proteins were stabilized by hydrogen bonds and/or van der Waals interaction (ΔH < 0 and ΔS < 0) and K_a values (25.50 ± 9.20)·10⁶, (1.37 ± 0.37)·10⁶, and (1.17 ± 0.01)·10⁶ (L·mol^-1) for HSA, AGP, and HGG, respectively, have been obtained. In turn, the reaction of Salt1-HGG complex formation was accompanied by ionic bonds (ΔH ≅ 0, ΔS > 0, and K_a = (0.64 ± 0.45)·10⁶ (L·mol^-1)). Regardless of the involvement of bonds and interaction between the ligands and proteins, the reactions occurred spontaneously (ΔG < 0). By comparing the binding parameters obtained using nanocalorimetric measurements and previously obtained spectroscopic data, due to the characteristic of complex formation, Salt2 was selected for further analysis. In addition, it was found that, despite the many advantages of the nanoITC technique, it still requires coupling with other techniques that allow analysis of the complexes formed at the molecular level and complementing spectroscopic analysis. Therefore, the use of these two techniques should be considered simultaneously.