Interaction of Cp6-his and Cp6 with bovine serum albumin and liver microsomes: Spectroscopic and molecular docking studies

Abstract

The conjugation of photosensitizer with a molecule specific to receptors over-expressed on cancer cells is a promising approach for improving tumor selectivity in photodynamic therapy (PDT). Since chemical nature of the photosensitizer is also affected by conjugation, this can have significant influence on its transport by serum proteins and clearance via liver microsomal enzyme system. In this report, the interaction of chlorin p6-histamine conjugate (Cp6-his) and free chlorin p6 (Cp6) with bovine serum albumin (BSA) and mouse liver microsomes was assessed by measuring the quenching of intrinsic tryptophan (Trp) fluorescence of proteins. The effect of photosensitizer binding on the activity of microsomal NADPH-cytochrome P-450 and NADH-cytochrome b5 reductase was also determined. The formation of protein carbonyl and lipid peroxides was measured to assess the photodynamic damage to proteins and lipids respectively. Further, the binding interaction between Cp6-his conjugate and parent Cp6 with BSA and cytochrome P-450 was validated using in-silico molecular docking approach. Results show that as compared to free Cp6, the affinity of conjugate to bind with serum albumin is 4 orders of magnitude less, which was also corroborated with finding of docking studies. In contrast, with microsomal proteins the binding affinity of Cp6-his was observed to be lower by 2 orders of magnitude as compared to that for Cp6. Further, the inhibition of the activities of microsomal enzymes induced by Cp6 conjugate and free Cp6 was nearly same (~15-20%). Measurements on quenching of photosensitizer fluorescence by iodide ions revealed that conjugate localized slightly deeper in microsomal membrane as compared to free Cp6 consistent with the slight difference in lipophilicity of the two photosensitizers. The amount of photodynamic damage to BSA induced by Cp6-his was significantly less as compared to that for Cp6, whereas no difference was observed in damage induced to microsomal protein and lipid. These results suggest that conjugation of Cp6 to histamine affects its binding to albumin but doses not significantly alters its interaction with microsomal enzyme system.