Biomolecular study and conjugation of two paraaminobenzoic acid derivatives with serum proteins: drug binding efficacy and protein structural analysis.

Abstract

Two aminobenzoic acid derivatives DAB-0 and DAB-1 showed distinct biological properties on murine bladder cancer (BCa) cell line MB49-I. In contrast to DAB-1, DAB-0 does not possess any anti-inflammatory activity and is less toxic. Furthermore, DAB-0 does not interfere with INFc-induced STAT1 activation and TNFa-induced IjB phosphorylation, while DAB-1 does. In order to rationalize these results, the binding efficacy of DAB-0 and DAB-1 with serum proteins such a human serum albumin (HSA), bovine serum albumin (BSA) and beta-lactoglobulin (b-LG) was investigated in aqueous solution at physiological pH. Multiple spectroscopic methods and thermodynamic analysis were used to determine the binding efficacy of DAB-0 and DAB-1 with serum proteins. Drug-protein conjugation was observed via through ionic contacts. DAB-1 forms stronger adducts than DAB-0, while b-LG shows more affinity with the order of stability b-LG>BSA>HSA. The stronger complexation of DAB-1 with serum proteins might account for its biological potential and transport in the blood. The binding efficacy ranged from 40 to 60%. Major alterations of protein secondary structures were detected upon drug complexation. Serum proteins are capable of delivering DAB-1 in vitro.Abbreviations: BSA: bovine serum albumin; DAB-0: N0-[4-(2,5-dioxo-pyrrolidin-1-yl)-benzoyl]-hydrazine carboxylic acid tert-butyl ester; DAB-1: N0-[4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-benzoyl]-hydrazine carboxylic acid tert-butyl est; FTIR: Fourier transform infrared; b-LG, beta-lactoglobulin; HAS: human serum albumin