Association of iminium and alkanolamine forms of the benzo[c]phenanthridine alkaloid chelerythrine with human serum albumin: photophysical, thermodynamic and theoretical approach

Abstract

Pharmaceutically important benzo[c]phenanthridine alkaloid chelerythrine (CHL) exists in charged iminium [CHL (I)] and neutral alkanolamine [CHL (A)] forms depending on pH. The mechanism of interaction of these two isoforms with human serum albumin (HSA) was investigated by employing various spectroscopic techniques, isothermal titration calorimetry (ITC) and molecular docking simulation. The primary objective was to corroborate the binding position of CHL within HSA as well as to investigate the comparative binding strength between CHL (I) and CHL (A). Spectroscopic investigation revealed that the alkanolamine form of CHL undergoes stronger association with HSA in comparison to its iminium counterpart. However, such a mode of binding induces some unfolding of the protein secondary and tertiary structures detectable in the circular dichroism (far-UV CD) spectra resulting in a concomitant decrease in the α-helical content of the protein. From the site selective competitive binding assay, we found that both iminium and alkanolamine forms of CHL bind at Sudlow's site I in subdomain IIA of HSA. Electrostatic interactions play the major role in CHL (I) binding whereas hydrophobic interactions are the main cause behind the stabilization of the CHL (A)–HSA complex. In addition, molecular docking specified that hydrogen bonding was also present in both cases with His 242 residue in site I. Thermodynamic parameters obtained from ITC showed that the binding of CHL (I) with HSA was favoured by a negative enthalpy change and opposed by a negative entropy change, whereas the association of CHL (A) with HSA was characterized by both positive enthalpy and entropy changes. FRET analysis showed that the binding distance between CHL (I) and CHL (A) with HSA (Trp 214) was most favourable (r = 2.32 and 2.05 nm respectively) for quenching to occur.