Exploring the interaction of a potent anti-cancer drug Selumetinib with bovine serum albumin: Spectral and computational attributes.

The binding of drugs to plasma proteins determines its fate within the physiological system, hence profound understanding of its interaction within the bloodstream is important to understand its pharmacodynamics and pharmacokinetics and thereby its therapeutic potential. In this regard, our work delineates the mechanism of interaction of Selumetinib (SEL), a potent anti-cancer drug showing excellent effect against multiple solid tumors, with plasma protein bovine serum albumin (BSA), using methods such as absorption, steady-state fluorescence, time-resolved, fluorescence resonance energy transfer, Fourier transform infrared spectra (FTIR), circular dichroism (CD), synchronous and 3D-fluorescence, salt fluorescence, molecular docking and molecular dynamic simulations. The BSA fluorescence intensity was quenched with increasing concentration of SEL which indicates interactions of SEL with BSA. Stern-Volmer quenching analysis and lifetime studies indicate the involvement of dynamic quenching. However, some contributions from the static quenching mechanism could not be ruled out unambiguously. The association constant was found to be 5.34 × 105 M-1 and it has a single binding site. The Förster distance (r) indicated probable energy transmission between the BSA and SEL. The positive entropy changes and enthalpy change indicate that the main interacting forces are hydrophobic forces, also evidenced by the results of molecular modeling studies. Conformation change in protein framework was revealed from FTIR, synchronous and 3D fluorescence and CD studies. Competitive binding experiments as well as docking studies suggest that SEL attaches itself to site I (subdomain IIA) of BSA where warfarin binds. Molecular dynamic simulations indicate the stability of the SEL-BSA complex. The association energy between BSA and SEL is affected in the presence of different metals differently.

Molecular interactions and binding dynamics of Alpelisib with serum albumins: insights from multi-spectroscopic techniques and molecular docking.

Alpelisib (ALP) is a potent anti-cancer drug showing promising activity against advanced breast cancers. Hence, profound understanding of its binding dynamics within the physiological system is vital. Herein, we have investigated interaction of ALP with human serum albumin (HSA) and bovine serum albumin (BSA) using spectroscopic techniques like absorption, fluorescence, time-resolved, synchronous and 3D-fluorescence, FRET, FT-IR, CD, and molecular docking studies. The intrinsic fluorescence of both BSA and HSA quenched significantly by ALP with an appreciable red shift in its emission maxima. Stern-Volmer analysis showed increase in Ksv with temperature indicating involvement of dynamic quenching process. This was further validated by no significant change in absorption spectrum of BSA and HSA (at 280 nm) upon ALP interaction, and by results of fluorescence time-resolved lifetime studies. ALP exhibited moderately strong binding affinity with BSA (of the order 106 M-1) and HSA (of the order 105 M-1), and the major forces accountable for stabilizing the interactions are hydrophobic forces. Competitive drug binding experiments and molecular docking suggested that ALP binds to site I in subdomain IIA of BSA and HSA. The Förster distance r was found to be less than 8 nm and 0.5 Ro < r < 1.5 Ro which suggests possible energy transfer between donors BSA/HSA and acceptor ALP. Synchronous and 3D-fluoresecnce, FT-IR and CD studies indicated that ALP induces conformational changes of BSA and HSA upon interaction.Communicated by Ramaswamy H. Sarma.

Caffeine and sulfadiazine interact differently with human serum albumin: A combined fluorescence and molecular docking study.

The interaction and binding behavior of the well-known drug sulfadiazine (SDZ) and psychoactive stimulant caffeine (CAF) with human serum albumin (HSA) was monitored by in vitro fluorescence titration and molecular docking calculations under physiological condition. The quenching of protein fluorescence on addition of CAF is due to the formation of protein-drug complex in the ground state; whereas in case of SDZ, the experimental results were explained on the basis of sphere of action model. Although both these compounds bind preferentially in Sudlow's site 1 of the protein, the association constant is approximately two fold higher in case of SDZ (∼4.0×10(4)M(-1)) in comparison with CAF (∼9.3×10(2)M(-1)) and correlates well with physico-chemical properties like pKa and lipophilicity of the drugs. Temperature dependent fluorescence study reveals that both SDZ and CAF bind spontaneously with HSA. However, the binding of SDZ with the protein is mainly governed by the hydrophobic forces in contrast with that of CAF; where, the interaction is best explained in terms of electrostatic mechanism. Molecular docking calculation predicts the binding of these drugs in different location of sub-domain IIA in the protein structure.

Interaction of Sulfadiazine with Model Water Soluble Proteins: A Combined Fluorescence Spectroscopic and Molecular Modeling Approach.

The binding behavior of antibacterial drug sulfadiazine (SDZ) with water soluble globular proteins like bovine as well as human serum albumin (BSA and HSA, respectively) and lysozyme (LYS) was monitored by fluorescence titration and molecular docking calculations. The experimental data reveal that the quenching of the intrinsic protein fluorescence in presence of SDZ is due to the strong interaction in the drug binding site of the respective proteins. The Stern-Volmer plot shows positive deviation at higher quencher concentration for all the proteins and was explained in terms of a sphere of action model. The calculated fluorophore-quencher distances vary within 4 ~ 11 Å in different cases. Fluorescence experiments at different temperature indicate thermodynamically favorable binding of SDZ with the proteins with apparently strong association constant (~10(4)-10(5) M(-1)) and negative free energy of interaction within the range of -26.0 ~ -36.8 kJ mol(-1). The experimental findings are in good agreement with the respective parameters obtained from best energy ranked molecular docking calculation results of SDZ with all the three proteins.

Fluorescence modulation and associative behavior of lumazine in hydrophobic domain of micelles and bovine serum albumin.

The photophysical behavior of the deprotonated form of lumazine (Lum-anion) was studied in biologically relevant surfactant systems like sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB) and TritonX-100 (TX-100) and also model water soluble protein, bovine serum albumin (BSA), using steady-state and time-resolved fluorescence spectroscopy in buffer solution of pH 12.0. The association constant values were calculated from modulated fluorescence intensity of Lum-anion in different medium. The interaction of non-ionic surfactant TX-100 was found to be about 10 times greater than SDS and CTAB. However, while the driving force of binding in SDS and/or TX-100 is mainly hydrophobic in nature, electrostatic interaction with the oppositely charged micellar head group is the predominant factor in CTAB. The thermodynamic parameters like enthalpy (ΔH) and entropy (ΔS) change, etc., corresponding to the binding of Lum-anion with BSA were estimated by performing the fluorescence titration experiment at different temperatures. Thermodynamically favorable and strong binding of Lum-anion (K~10(4) M(-1)) into BSA is due to hydrophobic interaction in the ligand binding domain II. However, the binding mechanism is entirely different in presence of protein denaturing agent like urea and electrostatic interaction plays a major role under this condition.

Luminol fluorescence quenching in biomimicking environments: sequestration of fluorophore in hydrophobic domain.

The photophysical behavior of luminol (LH(2)) was studied in a variety of biologically relevant systems ranging from surfactants, cyclodextrin, and proteins using steady-state and time-resolved fluorescence spectroscopy. It was shown that, out of two possible LH(2) conformers present in solution, the sequestration of relatively less polar structure into the hydrophobic domain of biological media is the primary reason for decrease in fluorescence intensity. The efficacy of LH(2) fluorescence quenching is substantially higher in micellar subdomain of cationic surfactant and depends on the nature of the headgroup. The thermodynamic parameters like enthalpy (ΔH) and entropy (ΔS) change, etc., corresponding to the binding of LH(2) in the model water-soluble protein, bovine serum albumin (BSA), were estimated by performing the fluorescence titration experiment at different temperatures. The involvement of subdomain IA and IIA of BSA in LH(2) binding was confirmed from the ligand replacement process with bilirubin (BIL). The difference in ligand binding with structurally homologous human serum albumin (HSA) is discussed in terms of positive cooperativity among these two binding domains of BSA with a Hill coefficient (n(H)) value of 2.26 ± 0.18 and a half-maximal concentration (K(0.5)) of 5.74 ± 0.23 µM at 298 K.

Fluorescence solvatochromism in lumichrome and excited-state tautomerization: a combined experimental and DFT study.

Fluorescence solvatochromism of lumichrome (LC) was studied by steady-state and time-resolved fluorescence spectroscopy. The excited-state properties of LC do not show any correlation with solvent polarity, however, reasonably good correlation with solvent E(T)(30) parameter was observed. A quantitative estimation of contribution from different solvatochromic parameters, like solvent polarizability (π*), hydrogen bond donor (α), and hydrogen bond acceptor (ß) ability of the solvent, was made using linear free energy relationship on the basis of Kamlet-Taft equation. The analysis reveals that hydrogen bond donating ability (acidity) of the solvent is the most important parameter that characterizes the excited-state behavior of lumichrome. Quantum mechanical calculations using density functional theory (DFT) were done to study the most stable structure and excited-state tautomerization process of LC toward the formation of isoalloxazines. Charge localization in the excited state and formation of hydrogen-bonded cluster through solvent hydrogen bond donation on the N10 atom of alloxazine moiety were predicted to be the key step toward this water-catalyzed tautomerization process.

Effect of solvent hydrogen bonding on excited-state properties of luminol: a combined fluorescence and DFT study.

The effect of solvent on the photoluminescence behavior of luminol was studied by steady-state fluorescence spectroscopy. The fluorescence spectral behavior of luminol is markedly different in polar protic solvents compared to that in aprotic solvents. A quantitative estimation of the contribution from different solvatochromic parameters, like solvent polarizibility (pi*), hydrogen-bond donor (alpha), and hydrogen-bond acceptor (beta), was made using the linear free energy relationship based on the Kamlet-Taft equation. The analysis reveals that the hydrogen-bond-donating ability (acidity) of the solvent is the most important parameter to characterize the excited-state behavior of luminol. Quantum mechanical calculations using density functional theory (DFT) predict the most stable structure, out of several possible tautomeric conformers of luminol with varying degrees of hydration. In the excited state, charge localization at specific points of the luminol phthalhydrazide moiety causes the solvent to interact primarily through hydrogen-bond donation.