Self-association of the anionic form of the DNA-binding anticancer drug mithramycin

The aqueous-phase self-association of mithramycin (MTR), an aureolic acid anticancer antibiotic, has been studied using different spectroscopic techniques such as absorption, fluorescence, circular dichroism, and 1H nuclear magnetic resonance spectroscopy. Results from these studies indicate self-association of the anionic antibiotic at pH 8.0 over a concentration range from micromolar to millimolar. These results could be ascribed to the following steps of self-association: M + M left arrow over right arrow M2, M2 + M left arrow over right arrow M3, and M3 + M left arrow over right arrow M4, where M, M2, M3, and M4 represent the monomer, dimer, trimer, and tetramer of mithramycin, respectively. Dynamic light scattering and isothermal titration calorimetry studies also support aggregation. In contrast, an insignificant extent of self-association is found for the neutral drug (at pH 3.5) and the [(MTR)2Mg2+] complex (at pH 8.0). Analysis of 2D NMR spectra of 1 mM MTR suggests that the sugar moieties play a role in the self-association process. Self-association of the drug might occur either via hydrophobic interaction of the sugar residues among themselves or water-mediated hydrogen bond formation between sugar residue(s). On the other hand, absence of a significant upfield shift of the aromatic protons from 100 microM to 1 mM MTR suggests against the possibility of stacking interactions between the aromatic rings as a stabilizing force for the formation of the dimer and higher oligomers.