Study on the binding of chlorogenic acid to pepsin by spectral and molecular docking

The interaction of pepsin with chlorogenic acid (CHA) was investigated using fluorescence, UV/vis spectroscopy and molecular modeling methods. Stern–Volmer analysis indicated that the fluorescence quenching of pepsin by CHA resulted from a static mechanism, and the binding constant was 1.1846 × 10⁵ and 1.1587 × 10⁵ L/mol at 288 and 310 K, respectively. The distance between donor (pepsin) and acceptor (CHA) was calculated to be 2.39 nm and the number of binding sites for CHA binding on pepsin was ~ 1. The results of synchronous fluorescence and three‐dimensional fluorescence showed that binding of CHA to pepsin could induce conformational changes in pepsin. Molecular docking experiments found that CHA bonded with pepsin in the area of the hydrophobic cavity with Van der Waals' forces or hydrogen bonding interaction, which were consistent with the results obtained from the thermodynamic parameter analysis. Furthermore, the binding of CHA can inhibit pepsin activity in vitro. Copyright © 2013 John Wiley & Sons, Ltd.     

Investigation and comparison of the binding between tolvaptan and pepsin and trypsin: Multi‐spectroscopic approaches and molecular docking

Tolvaptan (TF), a selective arginine vasopressin V2 receptor antagonist, was approved by the Food and Drug Administration in 2009. This study mainly investigated the differences between the binding of TF with pepsin and trypsin by using a series of spectroscopy and molecular modeling methods. Thermodynamic parameters and molecular docking results suggested that the binding of TF to pepsin and trypsin were both spontaneous but driven by different forces. For pepsin, the binding was driven by hydrogen bonds and van der Waals forces; but for trypsin, it was driven by electrostatic forces and hydrophobic forces. The quenching mechanism between TF and pepsin and trypsin was investigated by fluorescence experiments and time‐resolved fluorescence spectroscopy. Synchronous fluorescence and 3‐dimensional fluorescence were used to investigate the micro‐environmental and conformational changes of pepsin and trypsin after the insertion of TF. In addition, activity‐measurement results showed that both the pepsin and trypsin activities increased with increasing TF concentration, which may help to understand the possible effect of TF on the digestion and absorption of nutrients in vivo.     

Studies on the binding of pepsin with three pyrethroid insecticides by multi‐spectroscopic approaches and molecular docking

In this study, the molecular interactions between pepsin and three pyrethroid insecticides, including fenvalerate, cyhalothrin and deltamethrin, were investigated by multi‐spectroscopic and molecular docking methods under mimic physiological pH conditions. The results indicated that all of these insecticides could interact with pepsin to form insecticide–pepsin complexes. The binding constants, number of binding sites and thermodynamic parameters measured at different temperatures indicated that these three pyrethroid insecticides could spontaneously bind with pepsin mainly through electrostatic forces and hydrophobic interactions with one binding site. According to the theory of Föster's non‐radioactive energy transfer, the distance (r) between pepsin and three pyrethroid insecticides were all found to be less than 7 nm, which implied that the energy transfer occurred between pepsin and these insecticides, leading to the quenching of pepsin fluorescence. Synchronous and three‐dimensional fluorescence, CD spectra and molecular docking results indicated that all tested pyrethroid insecticides bound directly into the enzyme cavity site and the binding of insecticides into the cavity influenced the microenvironment of the pepsin activity site which resulted in the extension of peptide strands of pepsin with loss of α‐helix structures.Copyright © 2016 John Wiley & Sons, Ltd.     

Study on the mechanism of the interaction between acteoside and pepsin using spectroscopic techniques

The interaction of acteoside with pepsin has been investigated using fluorescence spectra, UV/vis absorption spectra, three‐dimensional (3D) fluorescence spectra and synchronous fluorescence spectra, along with a molecular docking method. The fluorescence experiments indicate that acteoside can quench the intrinsic fluorescence of pepsin through combined quenching at a low concentration of acteoside, and static quenching at high concentrations. Thermodynamic analysis suggests that hydrogen bonds and van der Waal's forces are the main forces between pepsin and acteoside. According to the theory of Förster's non‐radiation energy transfer, the binding distance between pepsin and acteoside was calculated to be 2.018 nm, which implies that energy transfer occurs between acteoside and pepsin. In addition, experimental results from UV/vis absorption spectra, 3D fluorescence spectra and synchronous fluorescence spectra imply that pepsin undergoes a conformation change when it interacts with acteoside. Copyright © 2015 John Wiley & Sons, Ltd.     

Interaction between azo dye Acid Red 14 and pepsin by multispectral methods and docking studies

The interaction of synthetic azo dye Acid Red 14 with pepsin was studied by fluorescence spectroscopy, UV–vis spectroscopy, circular dichroism and molecular docking. Results from the fluorescence spectroscopy show that Acid Red 14 has a strong capability to quench the intrinsic fluorescence of pepsin with static quenching. Binding constant, number of the binding sites and thermodynamic parameters were measured at different temperatures. The result indicates that Acid Red 14 interact with pepsin spontaneously by hydrogen bonding and van der Waals interactions. Three‐dimensional fluorescence spectra and circular dichroism spectra reveal that Acid Red 14 could slightly change the structure of pepsin. The hydrogen bond is formed between Acid Red 14 and Tyr‐189 and Thr‐218 residues of pepsin. Furthermore, the binding between Acid Red 14 and pepsin inhibits pepsin activity. The study can provide a way to analyze the biological safety of Acid Red 14 on digestive proteases or other proteins.     

Multi-spectroscopic studies on the interaction between traditional Chinese herb,helicid with pepsin

Study on the binding properties of helicid by pepsin systematically using multi-spectroscopic techniques and molecular docking method, and these interactions comprise biological recognition at molecular level and backbone of biological significance in medicine concerned with the uses, effects, and modes of action of drugs. We investigated the mechanism of interaction between helicid and pepsin by using various spectroscopic techniques viz., fluorescence spectra, UV–Vis absorption spectra, circular dichroism (CD), 3D spectra, synchronous fluorescence spectra and molecular docking methods. The quenching mechanism associated with the helicid–pepsin interaction was determined by performing fluorescence measurements at different temperatures. From the experimental results show that helicid quenched the fluorescence intensity of pepsin via a combination of static and dynamic quenching process. The binding constants (K_a) at three temperatures (288, 298, and 308 K) were 7.940?×?10⁷, 2.082?×?10⁵ and 3.199?×?10⁵ L mol^?1, respectively, and the number of binding sites (n) were 1.44, 1.14, and 1.18, respectively. The n value is close to unity, which means that there is only one independent class of binding site on pepsin for helicid. Thermodynamic parameters at 298 K were calculated as follows: ΔH^o (??83.85 kJ mol^?1), ΔG^o (??33.279 kJ mol^?1), and ΔS^o (??169.72 J K^?1 mol^?1). Based on thermodynamic analysis, the interaction of helicid with pepsin is driven by enthalpy, and Van der Waals’ forces and hydrogen bonds are the main forces between helicid and pepsin. A molecular docking study further confirmed the binding mode obtained by the experimental studies. The conformational changes in the structure of pepsin was confirmed by 3D fluorescence spectra and circular dichroism.     

Spectroscopic and molecular docking studies on the interaction of antiviral drug nevirapine with calf thymus DNA

The interaction of calf thymus DNA with nevirapine at physiological pH was studied by using absorption, circular dichroism, viscosity, differential pulse voltammetry, fluorescence techniques, salt effect studies and computational methods. The drug binds to ct-DNA in a groove binding mode, as shown by slight variation in the viscosity of ct-DNA. Furthermore, competitive fluorimetric studies with Hoechst 33258 indicate that nevirapine binds to DNA via groove binding. Moreover, the structure of nevirapine was optimized by DFT calculations and was used for the molecular docking calculations. The molecular docking results suggested that nevirapine prefers to bind on the minor groove of ct-DNA.     

Spectroscopy and molecular docking study on the interaction of daidzein and genistein with pepsin

The interaction of pepsin with daidzein (Dai) or genistein (Gen) was investigated using spectroscopic techniques under simulated physiological conditions. Dai and Gen can quench the fluorescence of pepsin and the quenching mechanism was a static process. The binding site number n and apparent binding constant K were measured at different temperatures. The thermodynamic parameters ΔΗ, ΔG and ΔS were calculated. The results indicated that van der Waals forces and hydrogen bond formation played major roles in the interaction of Dai or Gen with pepsin. The binding distance between pepsin and Dai or Gen was calculated according to energy transfer theory. The results of synchronous fluorescence spectra showed that the microenvironment and conformation of pepsin were changed. UV absorption and 3D fluorescence spectra showed that the binding interaction disturbed the microenvironment of amino acid residues and induced conformational changes in pepsin. Molecular docking results showed that Dai and Gen entered into the hydrophobic cavity of pepsin and two hydrogen bonds formed between Dai or Gen and pepsin. The results demonstrated that the interaction behavior between Dai and Gen with pepsin was slightly different, which denoted that the 5‐hydroxyl group of Gen, to a certain extent, had an effect on ligand binding to proteins. Copyright © 2016 John Wiley & Sons, Ltd.     

Interaction between caffeic acid phenethyl ester and protease: monitoring by spectroscopic and molecular docking approaches

The interaction of one anticancer drug (caffeic acid phenethyl ester; CAPE) with three proteases (trypsin, pepsin and α-chymotrypsin) has been investigated with multispectral methods and molecular docking. As an active components in propolis, the findings are of great benefit to metabolism, design, and structural modification of drugs. The results show that CAPE has an obvious ability to quench the trypsin, pepsin, or α-chymotrypsin fluorescence mainly through a static quenching procedure. Trypsin has the largest binding affinity to CAPE, and α-chymotrypsin has the smallest binding affinity to CAPE. The data obtained from thermodynamic parameters and molecular docking prove that the spontaneously interaction between CAPE and each protease is mainly due to a combination of van der Waals (vdW) force and hydrogen bond (H-bond), controlled by an enthalpy-driven process. The binding force, strength, position, and the number of H-bond are further obtained from the results of molecular docking. Through ultraviolet spectroscopy, dynamic light scattering and circular dichroism experiments, the change in the protease secondary structure induced by CAPE was observed. Additionally, the addition of protease had a positive effect on the antioxidative activity of CAPE, and α-chymotrypsin has the greatest effect on the removal of 2,2-diphenyl-1-picrylhydrazyl free radicals by CAPE.     

Hydrogen bonding-assisted interaction between amitriptyline hydrochloride and hemoglobin: spectroscopic and molecular dynamics studies

Herein, we have explored the interaction between amitriptyline hydrochloride (AMT) and hemoglobin (Hb), using steady-state and time-resolved fluorescence spectroscopy, UV–visible spectroscopy, and circular dichroism spectroscopy, in combination with molecular docking and molecular dynamic (MD) simulation methods. The steady-state fluorescence reveals the static quenching mechanism in the interaction system, which was further confirmed by UV–visible and time-resolved fluorescence spectroscopy. The binding constant, number of binding sites, and thermodynamic parameters viz. ΔG, ΔH, ΔS are also considered; result confirms that the binding of the AMT with Hb is a spontaneous process, involving hydrogen bonding and van der Waals interactions with a single binding site, as also confirmed by molecular docking study. Synchronous fluorescence, CD data, and MD simulation results contribute toward understanding the effect of AMT on Hb to interpret the conformational change in Hb upon binding in aqueous solution.     

Investigation of binding between fluoroquinolones and pepsin by fluorescence spectroscopy and molecular simulation

In this paper, the interactions of pepsin with fluoroquinolones, including norfloxacin (NFX) or ofloxacin (OFX), were investigated using fluorescence spectroscopy. The effects of NFX or OFX on pepsin showed that the molecular conformation of pepsin and the microenvironment of tryptophan residues were changed under mimicked physiological conditions. Static quenching was suggested as a factor. Quenching constants and binding constants were determined and thermodynamic parameters were calculated at three temperatures (25°C, 31°C and 37°C). Molecular interaction distances (binding distance r) were obtained. Binding was enthalpy driven and the process was spontaneous. Synchronous fluorescence, three‐dimensional fluorescence spectroscopy and molecular simulation were used for analysis. Interactions were further tested using molecular modelling. Quenching and binding constants of NFX with pepsin were the highest when testing NFX/OFX/fleroxacin/gatifloxacin with pepsin combinations. NFX was the strongest quencher, and affinity of NFX for pepsin was higher than that of OFX/fleroxacin/gatifloxacin.     

Comparative study of the interaction mechanism of astilbin,isoastilbin, and neoastilbin with CYP3A4

The interactions of human CYP3A4 with three selected isomer flavonoids, such as astilbin, isoastilbin and neoastilbin, were clarified using spectral analysis, molecular docking, and molecular dynamics simulation. During binding with the three flavonoids, the intrinsic fluorescence of CYP3A4 was statically quenched in static mode with nonradiative energy conversion. The fluorescence and ultraviolet/visible (UV/vis) data revealed that the three flavonoids had a moderate and stronger binding affinity with CYP3A4 due to the order of the K_a1 and K_a2 values ranging from 10⁴ to 10⁵ L·mol⁻¹. In addition, astilbin had the highest affinity with CYP3A4, then isoastilbin and neoastilbin, at the three experimental temperatures. Multispectral analysis confirmed that binding of the three flavonoids resulted in clear changes in the secondary structure of CYP3A4. It was found from fluorescence, UV/vis and molecular docking analyses that these three flavonoids strongly bound to CYP3A4 by means of hydrogen bonds and van der Waals forces. The key amino acids around the binding site were also elucidated. Furthermore, the stabilities of the three CYP3A4 complexes were evaluated using molecular dynamics simulation.     

Spectroscopic,electrochemical, docking and molecular dynamics studies on the interaction of three oxovanadium (IV) Schiff base complexes with bovine serum albumin and their cytotoxicity against cancer

This study was designed to investigate the interaction of three oxovanadium (IV) Schiff base complexes with bovine serum albumin (BSA) by means of various spectroscopic and electrochemical methods along with molecular docking study and molecular dynamics simulations. Binding constants were estimated by fluorescence and UV-Vis spectroscopy. The results indicated a good affinity of the complexes for BSA in which furyl derivative had more activity. Molecular docking study showed that these complexes have the similar binding modes and located within subdomain IB in site III of BSA. The supporting of molecular docking and molecular dynamics results by experimental data, confirms the validity of the interactions data obtained by these methods. Biological activity against cancer cell showed that furyl derivative has higher activity than other complexes. Pharmaceutical analysis also showed that, these complexes potentially can be used as anticancer agents.     

Fluorescence spectroscopic analysis on interaction of fleroxacin with pepsin

The interaction between fleroxacin (FLX) and pepsin was investigated by spectrofluorimetry. The effects of FLX on pepsin showed that the microenvironment of tryptophan residues and molecular conformation of pepsin were changed based on fluorescence quenching and synchronous fluorescence spectroscopy in combination with three‐dimensional fluorescence spectroscopy. Static quenching was suggested and it was proved that the fluorescence quenching of pepsin by FLX was related to the formation of a new complex and a non‐radiation energy transfer. The quenching constants K_SV, binding constants K and binding sites n were calculated at different temperatures. The molecular interaction distance (r = 6.71) and energy transfer efficiency (E = 0.216) between pepsin and FLX were obtained according to the Forster mechanism of non‐radiation energy transfer. Hydrophobic and electrostatic interaction played a major role in FLX–pepsin association. In addition, the hydrophobic interaction and binding free energy were further tested by molecular modeling study. Copyright © 2013 John Wiley & Sons, Ltd.     

Probing the combination of erlotinib hydrochloride,an anticancer drug,and human serum albumin: Spectroscopic,molecular docking,and molecular dynamic analyses

Human serum albumin (HSA) is a globular and monomeric protein in plasma that transports many drugs and compounds. Binding of some drugs to HSA can lead to changes in its stability and biological function. We investigated the binding interactions between erlotinib hydrochloride (Erlo) and HSA. Erlo is used to treat lung, pancreatic, and some other cancers. Experimental data showed that the fluorescence emission of the protein was quenched by Erlo using a static quenching mechanism. The calculation of the binding constant, K_b (1.57 × 10⁵ M⁻¹ at 300 K), confirmed the existence of a moderate binding interaction between Erlo and HSA. The interaction was enthalpy driven, spontaneous, and exothermic. The calculated thermodynamic parameters in agreement with simulation and molecular docking data showed that van der Waals and hydrogen bond forces played an important role in the interaction process. Molecular docking results indicated that Erlo has more affinity to bind to subdomain IIA (site I) of HSA. Molecular dynamics simulation analysis showed that the protein is stable in the presence of Erlo under simulation conditions.     

Synthesis,characterization, and binding assessment with human serum albumin of three bipyridine lanthanide(III) complexes

In this work, the terbium(III), dysprosium(III), and ytterbium(III) complexes containing 2, 2′-bipyridine (bpy) ligand have been synthesized and characterized using CHN elemental analysis, FT-IR, UV–Vis and ¹H-NMR techniques and their binding behavior with human serum albumin (HSA) was studied by UV–Vis, fluorescence and molecular docking examinations. The experimental data indicated that all three lanthanide complexes have high binding affinity to HSA with effective quenching of HSA fluorescence via static mechanism. The binding parameters, the type of interaction, the value of resonance energy transfer, and the binding distance between complexes and HSA were estimated from the analysis of fluorescence measurements and Förster theory. The thermodynamic parameters suggested that van der Waals interactions and hydrogen bonds play an important role in the binding mechanism. While, the energy transfer from HSA molecules to all these complexes occurs with high probability, the order of binding constants (BpyTb > BpyDy > BpyYb) represents the importance of radius of Ln³⁺ ion in the complex-HSA interaction. The results of molecular docking calculation and competitive experiments assessed site 3 of HSA, located in subdomain IB, as the most probable binding site for these ligands and also indicated the microenvironment residues around the bound mentioned complexes. The computational results kept in good agreement with experimental data.     

Multi-spectroscopic,thermodynamic and molecular docking studies to investigate the interaction of eplerenone with human serum albumin

The binding of small molecular drugs with human serum albumin (HSA) has a crucial influence on their pharmacokinetics. The binding interaction between the antihypertensive eplerenone (EPL) and HSA was investigated using multi-spectroscopic techniques for the first time. These techniques include ultraviolet-visible (UV-vis) spectroscopy, Fourier-transform infrared (FTIR), native fluorescence spectroscopy, synchronous fluorescence spectroscopy and molecular docking approach. The fluorescence spectroscopic study showed that EPL quenched HSA inherent fluorescence. The mechanism for quenching of HSA by EPL has been determined to be static in nature and confirmed by UV absorption and fluorescence spectroscopy. The modified Stern–Volmer equation was used to estimate the binding constant (K_b) as well as the number of bindings (n). The results indicated that the binding occurs at a single site (K_b = 2.238 × 10³ L mol⁻¹at 298 K). The enthalpy and entropy changes (∆H and ∆S) were 58.061 and 0.258 K J mol⁻¹, respectively, illustrating that the principal intermolecular interactions stabilizing the EPL–HSA system are hydrophobic forces. Synchronous fluorescence spectroscopy revealed that EPL binding to HSA occurred around the tyrosine (Tyr) residue and this agreed with the molecular docking study. The Förster resonance energy transfer (FRET) analysis confirmed the static quenching mechanism. The esterase enzyme activity of HSA was also evaluated showing its decrease in the presence of EPL. Furthermore, docking analysis and site-specific markers experiment revealed that EPL binds with HSA at subdomain IB (site III).     

Investigations with spectroscopy, zeta potential and molecular modeling of the non-cooperative behaviour between cyclophosphamide hydrochloride and aspirin upon interaction with human serum albumin: binary and ternary systems from multi-drug therapy

The interaction between cyclophosphamide hydrochloride (CYC) and aspirin (ASA) with human serum albumin (HSA) was studied by various kind of spectroscopic, ζ potential and molecular modeling under physiological conditions. The fluorescence data showed that the binding of drugs to proteins caused strong static fluorescence quenching. The analysis of the fluorescence quenching of HSA in the binary and ternary systems displayed that ASA was affected by the complex formed between CYC and HSA. Moreover, CYC was influenced by the HSA-ASA complex. The inherent binding information, including the quenching mechanism, binding constants, number of binding sites, effective quenching constant, fraction of the initial fluorescence and thermodynamic parameters were measured by the fluorescence quenching technique at various temperatures. In addition, according to the synchronous fluorescence spectra of HSA, the results showed that the fluorescence quenching of HSA originated from the Trp and Tyr residues, and indicated a conformational change of HSA with the addition of the drugs. Far-UV CD spectra of HSA were recorded before and after the addition of ASA and CYC as binary and ternary systems. An increase in intensity of the positive CD peak of HSA was observed in the presence of the drugs. The results were interpreted by excited interactions between the aromatic residues of the HSA binding sites and the drugs bound to them. The distance r between donor and acceptor was obtained by the Forster energy according to fluorescence resonance energy transfer (FRET) and found to be 2.35 nm and 1.78 nm for CYC and ASA, respectively. This confirmed the existence of static quenching for proteins in the presence of CYC and ASA. Furthermore, docking studies pointed at a reduction of the affinity of each of the drug compounds to the protein in the presence of the other in meaningful amounts. Pre-binding of any of the said compounds forced the second to bind in a non-optimized location and orientation. The potential at the electrokinetic shear surface of the protein-drug solution were measured at several concentrations of the drugs by the ζ potential technique, which confirmed experimental and theoretical results.     

Elucidating the molecular interaction of sinigrin,a potent anticancer glucosinolate from cruciferous vegetables with bovine serum albumin: effect of methylglyoxal modification

The present study employed the spectroscopic techniques, i.e. fluorescence, and circular dichroism (CD) and the molecular docking approach to investigate the mechanism of interaction of a potent anticancer glucosinolate, sinigrin (SIN), with bovine serum albumin (BSA). SIN binding to BSA resulted in the quenching of intrinsic fluorescence, and the analysis of results revealed the presence of static quenching mechanism. Based on the results, it was evident that the interaction of SIN with BSA was mainly stabilized by hydrogen bonding. Results from CD analysis revealed that the binding of SIN does not induce significant conformational changes in BSA. Molecular docking studies showed that four hydrogen bonds stabilize the binding of SIN in the site I of BSA with a binding energy of ?6.2 kcal mol^?1. These findings will not only provide insights about the mechanism of interaction of sinigrin but also showed the effect of methylglyoxal-mediated glycation on ligand binding with BSA.     

Probing the binding mechanisms of α-tocopherol to trypsin and pepsin using isothermal titration calorimetry,spectroscopic, and molecular modeling methods

α-Tocopherol is a required nutrient for a variety of biological functions. In this study, the binding of α-tocopherol to trypsin and pepsin was investigated using isothermal titration calorimetry (ITC), steady-state and time-resolved fluorescence measurements, circular dichroism (CD) spectroscopy, and molecular modeling methods. Thermodynamic investigations reveal that α-tocopherol binds to trypsin/pepsin is synergistically driven by enthalpy and entropy. The fluorescence experimental results indicate that α-tocopherol can quench the fluorescence of trypsin/pepsin through a static quenching mechanism. The binding ability of α-tocopherol with trypsin/pepsin is in the intermediate range, and one molecule of α-tocopherol combines with one molecule of trypsin/pepsin. As shown by circular dichroism (CD) spectroscopy, α-tocopherol may induce conformational changes of trypsin/pepsin. Molecular modeling displays the specific binding site and gives information about binding forces and α-tocopherol-tryptophan (Trp)/tyrosine (Tyr) distances. In addition, the inhibition rate of α-tocopherol on trypsin and pepsin was studied. The study provides a basic data set for clarifying the binding mechanisms of α-tocopherol with trypsin and pepsin and is helpful for understanding its biological activity in vivo.