Biophysical characterization of human serum albumin interaction with dapagliflozin: multi-spectroscopic and molecular docking study

NMR, multi-spectroscopic and molecular modeling approach to investigate the complexes between C.I. Acid Orange 7 and human serum albumin in vitro

Perfluorononanoic acid (PFNA) is the third most frequently detected in serum among all perfluoroalkyl acids (PFAAs) which is a kind of toxic emerging environmental contaminant. The influence of PFNA on the conformation and even function of human serum albumin (HSA) is still just at the beginning of research. The attempt of this paper was to completely elucidate the interaction mechanism of PFNA with HSA by means of multi-spectroscopic, molecular docking and isothermal titration calorimetry (ITC) techniques. The inner filter effect of all fluorescence data in the paper was eliminated to get accurate binding parameters. The results showed that the fluorescence of HSA was quenched by PFNA through a combined quenching procedure of dynamic and static quenching. Through site marker competitive experiments, subdomain IIA of HSA had been assigned to possess the high-affinity binding site of PFNA. Furthermore, molecular docking reconfirmed that PFNA was bound in subdomain IIA mainly through polar force, hydrophobic interaction and halogen-bond, and the calculated free energy was -26.54 kJ·mol(-1) which indicated that the PFNA molecule exhibited large binding affinity towards HSA. The thermodynamic characterizations of two different classes of binding sites by ITC displayed that the first class with a higher affinity constant was dominated by an enthalpic contribution due to electrostatic interactions and halogen-bond, whereas the second class with a lower affinity constant was preponderated by hydrophobic interaction. The three-dimensional fluorescence revealed that the conformation of HSA was changed and the hydrophobicity of the Trp and Tyr residues microenvironment increased after formation of PFNA-HSA complex. The alterations of the protein secondary structure were quantitatively calculated from circular dichroism (CD) spectroscopy with reduction of α-helix content about 14.3%, β-sheet 5.3%, β-turn 3.5%, and augment in random content from 14.4% to 37.5%. Above results revealed that the binding of PFNA with HSA can alter the secondary structure of HSA, further probably affecting HSA physiological function. The results can provide insights with the binding mechanism of PFNA with HSA and salient biophysical and biochemical clues on elucidating the transport and distribution of PFNA in vivo.

The interaction between triclosan (TCS) and human serum albumin (HSA) was investigated in order to obtain the binding mechanism, binding constant, the type of binding force, the binding distance between the donor and acceptor, and the effect of TCS on the conformation change of HSA. A HSA solution was added to the quartz cell and then titrated by successive addition of TCS. The fluorescence quenching spectra and synchronous spectra were recorded with the excitation and emission slits of the passage of band set at 10 and 20nm. Three-dimensional fluorescence spectra of HSA were recorded before and after the addition of TCS. The capillary electrophoresis was conducted with the pressure injection mode at 0.5psi for 5s, separation under 25kV, and detection at 214nm. Fluorescence data indicated the fluorescence quenching of HSA by TCS was static quenching, and the quenching constants (K ( a )) were 1.14 × 10(5), 8.75 × 10(4), 6.67 × 10(4), and 5.00 × 10(4) at 293, 298, 303, and 309K, respectively. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the interaction were calculated to be -37.9kJmol(-1) and 32.6J mol(-1)K(-1). The binding distance between TCS and tryptophan residues of HSA was obtained to be 1.81nm according to Fǒrster nonradioactive energy transfer theory. The UV-Vis absorption spectroscopy, the synchronous fluorescence spectroscopy, three-dimensional fluorescence spectroscopy, and circular dichroism spectroscopy revealed the alterations of HSA secondary structure in the presence of TCS. Finally, the interaction between TCS and HSA was further confirmed by capillary electrophoresis. TCS was bound to HSA to form the TCS-HSA complex, with the binding distance of 1.81nm. Hydrophobic interaction and hydrogen bond were dominated in the binding. TCS could change the secondary conformation of HSA. This work provides an insight into noncovalent interaction between emerging pollutants and protein, helping to elucidate the toxic mechanism of such pollutants.

Interaction between phillygenin and human serum albumin based on spectroscopic and molecular docking

Study on the interaction of the epilepsy drug, zonisamide with human serum albumin (HSA) by spectroscopic and molecular docking techniques

Coumarin is a benzopyrone which is widely used as an anti-coagulant, anti-oxidant, anti-cancer and also to cure arthritis, herpes, asthma and inflammation. Here, we studied the binding of synthesized coumarin derivatives with human serum albumin (HSA) at physiological pH 7.2 by using fluorescence spectroscopy, circular dichroism spectroscopy, molecular docking and molecular dynamics simulation studies. By addition of coumarin derivatives to HSA the maximum fluorescence intensity was reduced due to quenching of intrinsic fluorescence upon binding of coumarin derivatives to HSA. The binding constant and free energy were found to be 1.957±0.01×105 M−1, −7.175 Kcal M−1 for coumarin derivative (CD) enamide; 0.837±0.01×105 M−1, −6.685 Kcal M−1 for coumarin derivative (CD) enoate, and 0.606±0.01×105 M−1, −6.49 Kcal M−1 for coumarin derivative methylprop (CDM) enamide. The CD spectroscopy showed that the protein secondary structure was partially unfolded upon binding of coumarin derivatives. Further, the molecular docking studies showed that coumarin derivatives were binding to HSA at sub-domain IB with the hydrophobic interactions and also with hydrogen bond interactions. Additionally, the molecular dynamics simulations studies contributed in understanding the stability of protein-drug complex system in the aqueous solution and the conformational changes in HSA upon binding of coumarin derivatives. This study will provide insights into designing of the new inspired coumarin derivatives as therapeutic agents against many life threatening diseases.

Interaction of novel Aurora kinase inhibitor MK-0457 with human serum albumin: Insights into the dynamic behavior, binding mechanism, conformation and esterase activity of human serum albumin

Betulinic acid (BA) is a naturally occurring pentacyclictriterpenoid possessing anti-retroviral, anti-cancer, and anti-inflammatory properties. Here, we studied the interaction of BA with human serum albumin (HSA) by using molecular docking, and molecular dynamic simulation methods. Molecular docking studies revealed that BA can bind in the large hydrophobic cavity of drug binding site I of sub-domain IIA and IIB, mainly by the hydrophobic interactions and also by hydrogen bond interactions. In which several cyclohexyl groups of BA are interacting with Phe(206), Arg(209), Ala(210), Ala(213), Leu(327), Gly(328), Leu(331), Ala(350), and Lys(351), residues of sub-domain IIA and IIB of HSA by hydrophobic interactions. Also, hydrogen bond interactions were observed between the hydroxyl (OH) group of BA with Phe(206) and Glu(354) of HSA, with hydrogen bond distances of 0.24 nm,0.28 nm respectively. Further, specifically, the molecular dynamics study makes an important contribution in understanding the effect of the binding of BA on conformational changes of HSA and the stability of the protein-drug complex system in aqueous solution. The root mean square deviation values of atoms in the free HSA molecule were calculated from 3000 ps to 5000 ps trajectory and the results were obtained as 0.72 ± 0.036 nm and 0.81 ± 0.032 nm for free HSA and HSA-BA, respectively. The radius of gyration (Rg) values of both unliganded HSA and HSA-BA were stabilized at 2.59 ± 0.03 nm, 2.51 ± 0.01 nm, respectively. Thus, this work may play an important role in the design of new BA inspired drugs with desired HSA binding affinity.

Beta-sitosterol is a naturally occurring phytosterol that is widely used to cure atherosclerosis, diabetes, cancer, and inflammation and is also an antioxidant. Here, we studied the interaction of beta-sitosterol, isolated from the aerial roots of Ficus bengalensis, with human serum albumin (HSA) at physiological pH 7.2 by using fluorescence, circular dichroism (CD), molecular docking, and molecular dynamics simulation methods. The experimental results show that the intrinsic fluorescence of HSA is quenched by addition of beta-sitosterol through a static quenching mechanism. The binding constant of the compound to HSA, calculated from fluorescence data, was found to be K(beta-sitosterol) = 4.6 +/- 0.01 x 10(3) M(-1), which corresponds to -5.0 kcal M(-1) of free energy. Upon binding of beta-sitosterol to HSA, the protein secondary structure was partially unfolded. Specifically, the molecular dynamics study makes an important contribution to understanding the effect of the binding of beta-sitosterol on conformational changes of HSA and the stability of a protein-drug complex system in aqueous solution. Molecular docking studies revealed that the beta-sitosterol can bind in the large hydrophobic cavity of subdomain IIA, mainly by the hydrophobic interaction but also by hydrogen bond interactions between the hydroxyl (OH) group of carbon-3 of beta-sitosterol to Arg(257), Ser(287), and Ala(261) of HSA, with hydrogen bond distances of 1.9, 2.4, and 2.2 A, respectively.

The interaction of paeoniflorin with human serum albumin (HSA) was investigated using fluorescence, UV–vis absorption, circular dichroism (CD) spectra and molecular docking techniques under simulative physiological conditions. The results clarified that the fluorescence quenching of HSA by paeoniflorin was a static quenching process and energy transfer as a result of a newly formed complex (1:1). Paeoniflorin spontaneously bound to HSA in site I (subdomain IIA), which was primarily driven by hydrophobic forces and hydrogen bonds (ΔH° = − 9.98 kJ mol−1, ΔS° = 28.18 J mol−1 K−1). The binding constant was calculated to be 1.909 × 103 L mol−1 at 288 K and it decreased with the increase of the temperature. The binding distance was estimated to be 1.74 nm at 288 K, showing the occurrence of fluorescence energy transfer. The results of CD and three-dimensional fluorescence spectra showed that paeoniflorin induced the conformational changes of HSA. Meanwhile, the study of molecular docking also indicated that paeoniflorin could bind to the site I of HSA mainly by hydrophobic and hydrogen bond interactions.

Study on the interaction of tussilagone with human serum albumin (HSA) by spectroscopic and molecular docking techniques

The binding of rhodamine B (RB) to human serum albumin (HSA) in the absence and presence of Cu2+ or Fe3+ under simulated physiological conditions was studied by using various biophysical methods for the first time. The results showed that the interaction between HSA and RB could spontaneously result in the formation of HSA-RB complex (namely, static quenching mechanism) through hydrophobic interactions and hydrogen bonds irrespective of the absence or presence of metal ions. The presence of metal ions led to the reduction of binding affinity of RB to HSA compared with no metal ions, which might result from the conformational change of HSA caused by the binding of metal ions. Furthermore, the analysis of UV-vis absorption, circular dichroism, synchronous fluorescence and three-dimensional fluorescence experiments demonstrated that the addition of RB induced conformational and microenvironmental changes of HSA without and with metal ions. In short, this work will be helpful to in-depth understand the transport mechanism and biological effect of RB and the effect of metal ions on the interaction of HSA-RB in vivo.

Comparing the interaction of four structurally similar coumarins from Fraxinus Chinensis Roxb. with HSA through multi-spectroscopic and docking studies

Multi-spectroscopic and molecular docking studies for binding interaction between fluvoxamine and human serum albumin

In order to investigate the role of the tryptophan residue of human serum albumin (HSA) in drug binding, HSA oxidized with 8 molar equivalents of N-bromosuccinimide (NBS) was prepared at pH 4.1. The NBS-oxidized HSA had lost the lone tryptophan residue without change in tyrosine content, and the modified protein retained the gross polypeptide conformation of native HSA as far as could be judged from the circular dichroism (CD) spectrum. The binding ability of NBS-oxidized HSA for chlordiazepoxide was decreased by about 60% from that of HSA. However, the binding of phenylbutazone and warfarin at the most reactive site of the modified HSA was not very different from that to HSA. These results suggest that the lone tryptophan residue in HSA lies in or near the indole and benzodiazepine binding site and not in the primary binding site of warfarin and phenylbutazone.