Yanmei Huang

In vitro investigation of the interaction between the hepatitis C virus drug sofosbuvir and human serum albumin through 1H NMR, molecular docking, and spectroscopic analyses

Sofosbuvir (SOF), an oral nucleotide inhibitor of the nonstructural protein 5B RNA of the hepatitis C virus (HCV), was approved for treating chronic HCV infection by the Food and Drug Administration in 2013. Understanding drug–protein interactions is a crucial factor in determining the pharmacokinetics and pharmacodynamics of a drug. In this study, the interaction between SOF and human serum albumin (HSA) was investigated using 1H nuclear magnetic resonance (NMR) spectroscopy, the molecular docking method, fluorescence studies, Fourier transform infrared (FT-IR) spectroscopy, and circular dichroism (CD) spectroscopy. The analysis of saturation transfer difference (STD) and WaterLOGSY data indicated that SOF was bound to HSA, and the STD signals showed that the methyl and aromatic protons of the hydrophobic components of SOF have the most intimate contact with protein. The negative free energies (−7.17 kcal mol−1 and −6.18 kcal mol−1) obtained from molecular docking and fluorescence studies clearly suggested the spontaneity of the interaction of the SOF–HSA complex. STD, WaterLOGSY and fluorescence displacement experiments demonstrated that SOF was preferentially bound to site I of HSA, and this finding was supported by the docking results. In addition, synchronous and three-dimensional (3D) fluorescence, FT-IR, and CD spectrocopy provided complementary information on the micro-environmental and conformational changes of HSA with the addition of SOF. The combination of 1H NMR and conventional methods provided useful information to further elucidate the binding mechanisms of antiviral drugs with HSA.

Multispectroscopic and docking studies on the binding of chlorogenic acid isomers to human serum albumin: Effects of esteryl position on affinity

Structural differences among various dietary polyphenols affect their absorption, metabolism, and bioactivities. In this work, chlorogenic acid (CA) and its two positional isomers, neochlorogenic acid (NCA) and cryptochlorogenic acid (CCA), were investigated for their binding reactions with human serum albumin (HSA) using fluorescence, ultraviolet-visible, Fourier transform infrared and circular dichroism spectroscopies, as well as molecular docking. All three isomers were bound to HSA at Sudlows site I and affected the protein secondary structure. CCA presented the strongest ability of hydrogen-bond formation, and both CA and NCA generated more electrostatic interactions with HSA. The albumin-binding capacity of these compounds decreased in the order CCA>NCA>CA. The compound with 4-esteryl structure showed higher binding affinity and larger conformational changes to HSA than that with 3- or 5-esteryl structures. These comparative studies on structure-affinity relationship contributed to the structural modification and design of phenolic food additives or new polyphenol-like drugs.

Studies of DNA-binding properties of lafutidine as adjuvant anticancer agent to calf thymus DNA using multi-spectroscopic approaches, NMR relaxation data, molecular docking and dynamical simulation

The interactions between lafutidine (LAF) and calf thymus DNA (ctDNA) have been investigated both experimentally and theoretically. UV-vis absorption studies confirmed that LAF binds to ctDNA through non-covalent interactions. Fluorescence quenching and time-resolved fluorescence spectroscopy studies showed that the binding of LAF with ctDNA occurred through static quenching mechanism, resulting in the formation of a LAF-ctDNA complex. The binding constants (K) of the complex were found to be around 103M-1 via NMR relaxation rates and fluorescence data, and the calculated thermodynamic parameters indicated that hydrogen bonds and van der Waals forces played major roles in the binding of LAF to ctDNA. The changes in CD spectra indicated that LAF induced a slight perturbation on the base stacking and helicity of B-DNA. A comparative study of the LAF-ctDNA complex with respect to potassium iodide quenching experiments and competition displacement assays with ethidium bromide, acridine orange, and Hoechst 33258 probes suggested that LAF interacted with ctDNA by minor groove mode. Molecular docking analysis further supported the minor groove binding. Molecular dynamics simulation indicated that LAF depart from the C-G region of DNA, but it can steadily bind with the middle part of DNA composed by A-T base pairs.

Molecular mechanism of the binding of 3,4,5-tri-O-caffeoylquinic acid to human serum albumin: Saturation transfer difference NMR, multi-spectroscopy, and docking studies

As a natural dietary polyphenol, 3,4,5-tri-O-caffeoylquinic acid (3,4,5-triCQA) exhibits numerous stronger pharmacological activities than that of its analogues. Studies on interaction between 3,4,5-triCQA and protein are very helpful for understanding the mechanism of these enhanced biological functions. In this study, 1H saturation transfer difference NMR (1H STD-NMR) combined with multi-spectroscopy were used to probe the interaction of 3,4,5-triCQA with human serum albumin (HSA). Both qualitative and quantitative 1H STD-NMR indicated that 3,4,5-triCQA can specifically bind to HSA at the favored Sudlows site II with caffeoyl groups as the main recognizable moiety. Fluorescence emission spectra showed that Stern-Volmer quenching constant (KSV) decreases from 10.132×104M-1 to 9.711×104M-1 with temperature raise, indicating that 3,4,5-triCQA quenches HSA fluorescence through a static mechanism. Binding constant (Kb=5.557×105M-1) and the number of binding sites (n≈1) at 298K suggested that 3,4,5-triCQA only occupies one site in HSA with high affinity. Enthalpy (ΔH=-28.802kJ/mol) and entropy (ΔS=12.429J/mol/K) change proved the dominant role of electrostatic interaction in binding process. Multi-spectroscopic analysis also confirmed that the protein secondary structure and hydrophobicity were significantly affected. Molecular docking further verified the NMR and spectroscopic results. Overall, 3,4,5-triCQA exhibited a strong albumin affinity owing to the plural caffeoyl groups, which lead to the enhanced pharmacological activities. This study clarified the molecular mechanism of 3,4,5-triCQA in binding to HSA, and the findings are beneficial for the research on polyphenol-like drugs and antioxidants in foods or cosmetics.

Domain-specific interactions between MLN8237 and human serum albumin estimated by STD and WaterLOGSY NMR, ITC, spectroscopic, and docking techniques

Alisertib (MLN8237) is an orally administered inhibitor of Aurora A kinase. This small-molecule inhibitor is under clinical or pre-clinical phase for the treatment of advanced malignancies. The present study provides a detailed characterization of the interaction of MLN8237 with a drug transport protein called human serum albumin (HSA). STD and WaterLOGSY nuclear magnetic resonance (NMR)-binding studies were conducted first to confirm the binding of MLN8237 to HSA. In the ligand orientation assay, the binding sites of MLN8237 were validated through two site-specific spy molecules (warfarin sodium and ibuprofen, which are two known site-selective probes) by using STD and WaterLOGSY NMR competition techniques. These competition experiments demonstrate that both spy molecules do not compete with MLN8237 for the specific binding site. The AutoDock-based blind docking study recognizes the hydrophobic subdomain IB of the protein as the probable binding site for MLN8237. Thermodynamic investigations by isothermal titration calorimetry (ITC) reveal that the non-covalent interaction between MLN8237 and HSA (binding constant was approximately 105 M−1) is driven mainly by favorable entropy and unfavorable enthalpy. In addition, synchronous fluorescence, circular dichroism (CD), and 3D fluorescence spectroscopy suggest that MLN8237 may induce conformational changes in HSA.

Characterization of the interaction between acotiamide hydrochloride and human serum albumin: 1H STD NMR spectroscopy, electrochemical measurement, and docking investigations

The interaction between acotiamide hydrochloride (Z-338) and human serum albumin (HSA) was investigated by multiple spectroscopic analyses, electrochemical approaches, and computer-aided molecular docking studies. 1H nuclear magnetic resonance (NMR) spectroscopy and saturation transfer difference (STD) data indicated that Z-338 weakly interacted with HSA. STD signals showed that the benzene and five-membered rings of Z-338 were responsible for the binding efficiency. Fluorescence lifetime measurements implied that Z-338 quenched the intrinsic fluorescence of HSA with a new complex formation via static mode. Key parameters regarding this interaction were calculated from differential pulse voltammetry and fluorescence spectroscopy. Results obtained from the two methods above ascertained the static mechanism and revealed that hydrogen bonding combined with van der Waals forces played a major role in HSA–Z-338 binding. Although the displacement of probes from both sites I and II was observed from competitive STD-NMR experiments, molecular docking results suggested that Z-338 was preferentially bound to site II of HSA. This finding was supported by the esterase-like activity result. A decrease in the esterase-like activity of HSA after Z-338 binding showed that the Arg-410 and Tyr-411 of subdomain IIIA were directly involved in the binding process, which corroborated the accuracy of docking studies. Furthermore, circular dichroism spectra, Fourier transform infrared spectroscopy, and 3D fluorescence demonstrated that Z-338 slightly disturbed the microenvironment of amino residues and affected the secondary structure of HSA. Overall, this study provides valuable information to further understand the use of Z-338.

Interaction of lafutidine in binding to human serum albumin in gastric ulcer therapy: STD-NMR, WaterLOGSY-NMR, NMR relaxation times, Tr-NOESY, molecule docking, and spectroscopic studies

In this study, lafutidine (LAF) was used as a model compound to investigate the binding mechanism between antiulcer drugs and human serum albumin (HSA) through various techniques, including STD-NMR, WaterLOGSY-NMR, (1)H NMR relaxation times, tr-NOESY, molecule docking calculation, FT-IR spectroscopy, and CD spectroscopy. The analyses of STD-NMR, which derived relative STD (%) intensities, and WaterLOGSY-NMR, determined that LAF bound to HSA. In particular, the pyridyl group of LAF was in close contact with HSA binding pocket, whereas furyl group had a secondary binding. Competitive STD-NMR and WaterLOGSY-NMR experiments, with warifarin and ibuprofen as site-selective probes, indicated that LAF preferentially bound to site II in the hydrophobic subdomains IIIA of HSA. The bound conformation of LAF at the HSA binding site was further elucidated by transferred NOE effect (tr-NOESY) experiment. Relaxation experiments provided quantitative information about the relationship between the affinity and structure of LAF. The molecule docking simulations conducted with AutoDock and the restraints derived from STD results led to three-dimensional models that were consistent with the NMR spectroscopic data. The presence of hydrophobic forces and hydrogen interactions was also determined. Additionally, FT-IR and CD spectroscopies showed that LAF induced secondary structure changes of HSA.

Novel poly(ADP-ribose) polymerase inhibitor veliparib: biophysical studies on its binding to calf thymus DNA

Veliparib (ABT-888), which can inhibit cancer growth by blocking DNA base excision repair, is one of several recently developed oral inhibitors of poly(ADP-ribose) polymerases, which are currently used in clinical trials. In this work, interaction of calf thymus DNA (ctDNA) with ABT-888 was first investigated following UV-visible absorption, nuclear magnetic resonance (NMR) spectroscopy, steady-state and time-resolved fluorescence, viscosity measurements, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy coupled with molecular docking methods. UV-visible absorption indicated that ABT-888 was indeed bound to ctDNA. Broadening and upfield shift of the proton peaks of ABT-888 in the proton NMR spectrum indicated that ABT-888 interacted with ctDNA primarily by partial intercalation. Fluorescence quenching and time-resolved fluorescence spectroscopy studies showed that binding of ABT-888 with ctDNA occurred through a static quenching mechanism, resulting in the formation of a ctDNA–ABT-888 complex. Thermodynamic calculations revealed that interaction was an enthalpy-driven process caused by hydrogen bonds and van der Waals forces. Competitive fluorescence experiments coupled with viscosity, CD, and FT-IR studies revealed that ABT-888 intercalates partially and binds to the groove, phosphate group, and deoxyribose sugar of ctDNA and also induces conformational changes. Molecular docking showed that ABT-888 preferably binds to the DNA groove. However, other types of binding, including classic intercalation and partial intercalation, cannot be ruled out.

Investigation on the Interaction of Norgestrel with Human Serum Albumin Using Spectroscopy and Molecular-Docking Method

The interaction of norgestrel with human serum albumin (HSA) was investigated by spectroscopy and molecular‐docking methods. Results of spectroscopy methods suggested that the quenching mechanism of norgestrel on HSA was static quenching and that the quenching process was spontaneous. Negative values of thermodynamic parameters (ΔG, ΔH, and ΔS) indicated that hydrogen bonding and van der Waals forces dominated the binding between norgestrel and HSA. Three‐dimensional fluorescence spectrum and circular dichroism spectrum showed that the HSA structure was slightly changed by norgestrel. Norgestrel mainly bound with Sudlow site I based on a probe study, as confirmed by molecular‐docking results. Competition among similar structures indicated that ethisterone and norethisterone affected the binding of norgestrel with HSA. CH3 in R1 had little effect on norgestrel binding with HSA. The surface hydrophobicity properties of HSA, investigated using 8‐anilino‐1‐naphthalenesulfonic acid, was changed with norgestrel addition.

Interactions of cucurbit[6,7]urils with human serum albumin and their effects on zaltoprofen transportation

Cucurbit[6,7]urils (CB[6,7]s) have been extensively investigated because of their characteristic inclusion ability. To enhance our understanding of the behaviors of CB[6,7]s in biological environments and explore their effects on drug transportation, multi-spectroscopic and molecular docking methods were applied to examine the binding mechanism of CB[6,7]s with human serum albumin (HSA) and their interference on the HSA–zaltoprofen (ZPF) binding system. 1H NMR spectra revealed the complexes that formed between CB[6,7]s and HSA, as well as between ZPF and HSA. The fluorescence of HSA was quenched by ZPF through a combined static and dynamic mechanism. The added CB[6,7]s did not significantly affect the energy transfer efficiency, but decreased the HSA–ZPF binding constant. The binding stoichiometry of ZPF with HSA was 1:1, and this value remained unchanged when CB[6,7]s were added. Circular dichroism spectroscopy, synchronous and 3D fluorescence spectroscopy demonstrated that CB[6,7]s slightly influenced the native secondary structure of HSA and its ZPF-induced unfolding. Further molecular docking suggested that CB[6,7]s preferred to bind to the HSA surface, and ZPF was primarily located in an L-shaped cavity in subdomain IB. Therefore, CB[6,7]s elicited minor damage to the structure of HSA and weak interferance with drug transportation.