Abstract
Febuxostat is a nonpurine selective xanthine oxidase inhibitor used chronically, often in combination with other medications, to treat hyperuricemia and gout. Its high plasma protein binding necessitates a thorough understanding of its interactions with human serum albumin (HSA) in order to assess potential protein-binding-mediated drug interactions. In this study, the interaction between febuxostat and HSA was investigated using equilibrium dialysis and circular dichroism (CD) spectroscopy. Equilibrium dialysis indicated that approximately six febuxostat molecules bind to one HSA molecule. Febuxostat induced Cotton effects in the presence of HSA, and the febuxostat concentration-dependent change in CD spectra supported the involvement of multiple binding regions with distinct microenvironments on HSA. Febuxostat caused only minor displacement of site-specific ligands, suggesting a limited impact on protein-binding-mediated drug interactions. Additionally, while fatty acids and pH did not significantly affect the free fraction of febuxostat, myristate and pH markedly altered the induced CD spectra, indicating sensitivity to conformational and microenvironmental changes in HSA. These findings further demonstrate that the febuxostat–HSA-induced CD signal provides a practical probe for monitoring albumin conformational dynamics.
Introduction
Febuxostat is widely used as a nonpurine xanthine oxidase inhibitor to treat hyperuricemia and gout (Figure ). , As patients receiving febuxostat often require long-term therapy and may be comedicated with various drugs for comorbidities, , it is important to understand its interactions with plasma proteins. This understanding can help predict pharmacokinetic behavior and potential drug–drug interactions. Febuxostat has a high plasma protein binding ratio of approximately 99%, predominantly binding to human serum albumin (HSA).
1.
Chemical structure of febuxostat.
HSA is the most abundant plasma protein and plays a pivotal role in the transport and distribution of numerous endogenous compounds and drugs. HSA possesses multiple ligand-binding regions within its three-domain structure (domains I–III, each divided into subdomains A and B). It is widely recognized that many drugs bind with high affinity to two primary sites: Sudlow’s site I and site II, which are located in subdomains IIA and IIIA, respectively. ,− Additionally, subdomain IB has been identified as another major drug-binding region. − The binding of drugs to these regions can significantly affect their free fractions and pharmacological effects, potentially leading to competitive or allosteric interactions with coadministered drugs or endogenous ligands. Importantly, the ligand-binding properties of HSA are dynamic and are influenced by both physiological and pathological factors. Despite its extensive binding to HSA, febuxostat has been reported not to significantly affect the HSA binding of several coadministered drugs, such as warfarin, ibuprofen, and digitoxin.
In the present study, we conducted a detailed characterization of the interaction between febuxostat and HSA. The binding parameters and binding stoichiometry were determined by equilibrium dialysis, and the binding mode was evaluated by circular dichroism (CD) spectroscopy. Additionally, we examined the effects of febuxostat on the binding of representative site-specific ligands for subdomains IB, IIA, and IIIA to assess potential binding-based interactions. Moreover, we investigated the influence of fatty acids (octanoate and myristate) and pH on the febuxostat–HSA interaction to assess the viability of using the CD signal from the febuxostat–HSA system as a probe for monitoring conformational changes in HSA. Finally, we conducted computational molecular docking to elucidate the binding sites of febuxostat to HSA.
Results
Analysis of the Binding of Febuxostat to HSA by Equilibrium Dialysis
The binding curve, expressed as the molar ratio of bound febuxostat to HSA (r), increased with the free concentration (C f) and approached an r value of approximately 6 at higher C f (Figure ). The data were well fitted to a multisite binding model assuming comparable association constants (eq , m = 1), yielding an apparent binding stoichiometry of n 1 = 6.30 and an association constant of K 1 = 5.36 × 104 M–1 (Table ). These results indicate that approximately six molecules of febuxostat can bind to one HSA molecule.
2.
Binding of febuxostat to HSA at pH 7.4 and 25 °C. The concentration of HSA was 40 μM and that of febuxostat was 20–640 μM. Solid line represents the theoretical curve assuming binding to the primary sites (eq , m = 1).
1. Binding Parameters Obtained by Equilibrium Dialysis for the Binding of Febuxostat to HSA at pH 7.4 and 25 °C .
| parameters | values |
|---|---|
| K 1 (×104 M–1) | 5.36 ± 0.44 |
| n 1 | 6.30 ± 0.12 |
Data are presented as the mean ± standard deviation (N = 3).
Measurement of CD Spectra of the Febuxostat–HSA System
The binding of febuxostat to HSA induced positive and negative Cotton effects around 335 and 245 nm, respectively (Figure ). The intensity of the Cotton effect at 335 nm increased with the febuxostat-to-HSA ratio up to 4, but decreased when the ratio exceeded 4. In addition, the increase in the intensity of the Cotton effect at 245 nm appeared to level off at a febuxostat-to-HSA ratio of approximately 6. Weak Cotton effects were also detectable in the 260–310 nm region at a febuxostat-to-HSA ratio of 0.5, consisting of small negative and positive peaks in the 280–310 nm and 260–280 nm regions, respectively. Although these signals became more pronounced as the febuxostat-to-HSA ratio increased, the spectral shapes in this region no longer changed proportionally; instead, they became increasingly complex when the ratio exceeded 4. These concentration-dependent changes in the Cotton effects support the involvement of multiple binding sites of febuxostat on HSA.
3.
CD spectra of the febuxostat–HSA system at pH 7.4 and 25 °C. The concentration of HSA was 10 μM, and the febuxostat-to-HSA concentration ratios were 0.5 (black), 1 (green), 2 (red), 4 (blue), and 6 (brown).
Binding of Fusidic Acid, Warfarin, and Ibuprofen to HSA in the Presence of Febuxostat
The effects of febuxostat on the binding of fusidic acid (a drug primarily binding to subdomain IB), warfarin (a drug primarily binding to subdomain IIA ,, ), and ibuprofen (a drug primarily binding to subdomain IIIA ,, ) to HSA were examined (Figure ). The free fractions of fusidic acid and ibuprofen showed slight increases with the febuxostat-to-HSA ratio in the ranges of 2–6 and 4–6, respectively. In contrast, the free fraction of warfarin slightly and gradually increased over the entire febuxostat-to-HSA ratio range investigated. Overall, the observed changes in the free fraction of these drugs by febuxostat were small.
4.
Effects of febuxostat on the free fraction of fusidic acid (A), warfarin (B), ibuprofen (C) in HSA solution at pH 7.4 and 25 °C. The following concentrations were used: HSA 40 μM, fusidic acid, warfarin, and ibuprofen 20 μM. Values are expressed as the mean ± SD (N = 3). **p < 0.01 in comparison with control (febuxostat-to-HSA ratio = 0).
Effect of Fatty Acids and pH on the Interactions between Febuxostat and HSA
The effects of octanoate and myristate, representing medium-chain and long-chain fatty acids, respectively, on the interactions between febuxostat and HSA were examined using the free fraction and CD spectra as indicators (Figure ). Only a slight increase in the free fraction was observed upon addition of myristate, whereas no significant change occurred upon addition of octanoate or with variations in pH (Figure ). The binding curve between febuxostat and HSA in the presence of myristate revealed a decreased apparent binding stoichiometry and, in contrast, an increased association constant compared with those observed in the absence of myristate (Supplementary Figure 4 and Supplementary Table 1).
5.
Effects of octanoate (A) and myristate (B) on the free fraction of febuxostat in HSA solution at pH 7.4 and 25 °C. The following concentrations were used: HSA 10 μM, febuxostat 60 μM. Values are expressed as the mean ± SD (N = 3). *p < 0.05 in comparison with control (octanoate- or myristate-to-HSA ratio = 0).
6.
Effects of pH on the free fraction of febuxostat in HSA solution at 25 °C. The following concentrations were used: HSA 10 μM, febuxostat 60 μM. Values are expressed as the mean ± SD (N = 3).
Octanoate caused a slight decrease in the intensity of the Cotton effects at 336 and 245 nm and generated a weak positive Cotton effect around 265 nm (Figure A). In contrast, myristate and pH markedly altered the CD spectra (Figures B and ). Since myristate itself generates a negative Cotton effect in the 260–295 nm region at higher myristate-to-HSA ratios (4 or 6) (Supplementary Figure 1), spectral changes observed in this region may include contributions from the myristate–HSA system.
7.
Effects of octanoate (A) and myristate (B) on the CD spectra of febuxostat–HSA systems at pH 7.4 and 25 °C. The concentrations of HSA and febuxostat were 10 and 60 μM, respectively. The octanoate- or myristate-to-HSA ratios were 0 (black), 0.5 (green), 1 (red), 2 (blue), 4 (brown), and 6 (light blue).
8.
Effects of pH on the CD spectra of febuxostat–HSA systems at 25 °C. The concentrations of HSA and febuxostat were 10 and 60 μM, respectively. pH values were 6.5 (black), 7.4 (green), and 8.2 (red).
Interactions between HSA and oleate, another long-chain fatty acid, were also examined. Changes in the free fraction of febuxostat at various oleate concentrations, as well as the induced CD spectra of the oleate–HSA system, measured in the presence or absence of febuxostat, were similar to those observed for the myristate–HSA system (Supplementary Figures 2 and 3).
Taken together, the results obtained in CD spectroscopy indicated that, despite possible contributions from the myristate–HSA system in the 260–295 nm region, the overall evidence supports the interpretation that long-chain fatty acids and pH modify the local environment of the febuxostat-binding regions in HSA.
Molecular Docking of Febuxostat to HSA
Because the binding parameters between febuxostat and HSA indicated that the molar ratio of febuxostat bound to HSA was approximately six (Table ), molecular docking simulations were performed to predict the most probable binding sites for six febuxostat molecules on HSA. The docking results suggested that febuxostat molecules bind widely across all domains of HSA (Figure and Table ). Among the six docked molecules, two molecules (designated as C and E in Figure ) were located within the pockets of the drug-binding sites, subdomain IIA and subdomain IIIA, which primarily bind warfarin and ibuprofen, respectively. In contrast, none of the docked febuxostat molecules showed interactions with residues in subdomain IB that are known to contribute to the binding of fusidic acid.
9.
Structural illustration of the predicted binding poses of six molecules of febuxostat, designated as A–F, on HSA obtained by molecular docking. The three domains of HSA are shown in different colors, and each subdomain is distinguished by light and dark shades.
2. Docking Scores of HSA–Febuxostat Complexes.
| molecules | GBVI/WSA dG score (kcal/mol) |
|---|---|
| A | –6.825 |
| B | –7.955 |
| C | –8.440 |
| D | –7.020 |
| E | –7.433 |
| F | –7.064 |
Discussion
In the present study, we demonstrated that approximately six molecules of febuxostat bind to one HSA molecule with comparable affinity (n 1 = 6.30, K 1 = 5.36 × 104 M–1) using equilibrium dialysis (Table ). These results suggest that febuxostat binds to multiple regions on HSA. Similar multisite binding behavior has also been reported for benzbromarone, another antihyperuricemic agent that binds to both primary and secondary sites on HSA. Under physiological conditions, the total plasma concentration of HSA (600 μM) is typically much higher than the maximum concentration (C max) of febuxostat (5.75 μM) following a 40 mg dose. Therefore, it is expected that most febuxostat will be bound to plasma proteins under these conditions. Calculations using eqs and predict that 99.5% of febuxostat is bound to HSA under these clinically relevant conditions, which agrees well with the reported high plasma protein binding of febuxostat (approximately 99%).
The Cotton effect around 245 nm increased with the febuxostat-to-HSA ratio but tended to level off around a ratio of 6, while the signal around 335 nm increased up to a ratio of 4 and then decreased at higher ratios (Figure ). These saturating and nonmonotonic behaviors support the involvement of multiple binding regions on HSA, consistent with the binding stoichiometry inferred from equilibrium dialysis. Although the complex ratio dependence observed in the 260–310 nm region may appear to contradict the equilibrium dialysis analysis assuming comparable association constants, this behavior can be explained by the fact that induced CD reflects not only binding stoichiometry but also binding geometry and microenvironmental heterogeneity among occupied regions. This behavior is consistent with the principle that induced Cotton effects are highly sensitive to the spatial relationship between ligand chromophores and chiral centers within a protein. −
Despite slight increases in the free fractions of site-specific drugs, fusidic acid, warfarin, and ibuprofen, the magnitude of these changes remained limited across the investigated febuxostat-to-HSA ratios (Figure ). In contrast, molecular docking simulations indicated that febuxostat molecules bind to the warfarin- and ibuprofen-binding sites located on subdomains IIA and IIIA (Figure ), raising the possibility of competitive interactions with these two drugs. These seemingly inconsistent results can be interpreted by comparing the association constants of these ligands for HSA. The association constants of warfarin and ibuprofen are markedly higher than that of febuxostat, , suggesting that febuxostat does not strongly dominate the major drug-binding sites. From a pharmacological perspective, the small extent of displacement indicates that clinically significant protein-binding-mediated interactions with coadministered drugs are unlikely under therapeutic conditions. This interpretation is consistent with the limited evidence for clinically relevant interactions attributable to albumin-binding displacement. ,,
Fatty acids are abundant endogenous ligands of HSA and are also known to modulate drug binding through both direct competition and conformational changes of HSA. ,, In this study, myristate caused only a slight change in the free fraction of febuxostat, whereas octanoate showed no significant effect (Figure ). Analysis of the binding parameters revealed opposing changes in apparent binding stoichiometry and association constant upon addition of myristate compared with those observed in its absence (Table and Supplementary Table 1). These compensatory changes indicate that the global binding strength of febuxostat to HSA remains largely preserved. Meanwhile, CD spectral changes were more pronounced with myristate and oleate than with octanoate (Figure and Supplementary Figure 3). This is likely because long-chain fatty acids are known to induce conformational changes in HSA. These findings suggest that myristate can influence the microenvironment of the febuxostat-binding regions through structural rearrangements without substantially altering macroscopic binding. HSA also undergoes pH-dependent conformational transitions, including the well-characterized N–B transition, − which involves a global structural rearrangement. The changes in pH did not affect the free fraction of febuxostat but markedly altered the induced CD spectra (Figure ). The pronounced sensitivity of the febuxostat-induced CD signal to pH (Figure ) therefore reflects changes in protein conformation, although subtle changes in binding stoichiometry cannot be completely excluded. These observations on the effects of myristate and pH highlight the ability of induced CD to detect conformational dynamics around binding regions that are not evident from equilibrium binding measurements alone.
The molecular docking simulations revealed that six febuxostat molecules bind across broadly distributed regions of HSA (Figure ). Such a binding property of febuxostat suggests that it has the potential to serve as a novel induced CD probe capable of detecting subtle conformational changes in individual regions of HSA, in contrast to previous studies that have mainly focused on analyzing ligand binding modes at specific sites using induced CD. ,
Taken together, these results demonstrate that the febuxostat–HSA-induced CD signal provides a practical and experimentally simple probe for assessing conformational dynamics in the vicinity of ligand-binding regions of HSA. This approach is applicable to detecting HSA structural states modulated by endogenous fatty acids, environmental conditions such as pH, and a variety of drugs and formulations developed to bind HSA. ,
It should be noted that the specific binding site(s) responsible for individual spectral components could not be conclusively identified in the present study. Further site-mapping investigations will be required to assign the induced CD features to particular subdomains of HSA. In this regard, high-resolution structural approaches such as X-ray crystallographic analysis will be essential to map the binding modes of febuxostat on HSA definitively. In addition, we did not evaluate potential changes in the binding properties or induced CD spectra of the febuxostat–HSA system arising from interactions at the major drug-binding sites or from pH alterations. Therefore, further detailed and quantitative analyses will be required to clarify these aspects.
In summary, we characterized the interaction between febuxostat and HSA using equilibrium dialysis and CD spectroscopy. Febuxostat binds to HSA with an apparent stoichiometry of approximately six molecules per HSA and induces characteristic Cotton effects that vary with the febuxostat-to-HSA ratio. Only minor displacement of representative site-specific ligands was observed, indicating limited influence on classical drug-binding sites and suggesting that clinically significant protein-binding-mediated drug–drug interactions are unlikely under therapeutic conditions. Notably, fatty acids and pH produced little change in the free fraction of febuxostat but markedly altered the febuxostat-induced CD spectra. This dissociation shows that induced CD is sensitive to conformational and microenvironmental modulation of HSA. These findings demonstrate that the febuxostat–HSA-induced CD signal can serve as a practical and sensitive probe for monitoring conformational dynamics in the vicinity of ligand-binding regions of HSA, and may also reflect more global structural transitions.
Methods
Materials
HSA (fraction V, fatty acid free) and warfarin were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Febuxostat, fusidic acid, and sodium myristate (myristate) were obtained from Tokyo Chemical Industry Company Ltd. (Tokyo, Japan). Ibuprofen and sodium octanoate (octanoate) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other chemicals were obtained from commercial sources and were of the highest grade. The binding experiments were carried out in 67 mM sodium phosphate buffer.
Quantitative Analysis of Ligand Bindings to HSA
The binding parameters of febuxostat to HSA were determined by equilibrium dialysis. Equilibrium dialysis experiments were carried out using 2 mL Sanko plastic dialysis cells (Fukuoka, Japan). The same volume of sample and buffer solution (1.0 mL) was added to the cell compartments, which were separated by Visking cellulose membranes, and shaken gently at 25 °C for 18 h. After reaching equilibrium, the concentrations of the ligand in the buffer (C f; unbound ligand concentration) and protein compartments (C b + C f; the sum of the bound and unbound ligand concentrations) were determined by HPLC. The concentration of bound ligand (C b) was calculated by subtracting C f from C b + C f. Data were fit to the following equation using GraphPad PRISM Version 7 (GraphPad Software, Inc., CA, U.S.A.).
| 1 |
where r is the number of moles of ligand bound per mole of protein, and P t is the concentration of the protein. Ki and ni are the association constant and the number of binding sites in the ith binding class, respectively. The value of “m” is the number of binding classes. For the binding of benzbromarone and benzarone to HSA, the analyses were performed using m = 1, since the binding sites with a single affinity were indicated in the experimental conditions (HSA; 40 μM, ligands; 20–640 μM).
Free (unbound) fractions for the bindings of febuxostat (240 μM), warfarin (20 μM), and fusidic acid (20 μM) to HSA (40 μM) were calculated using the data obtained by equilibrium dialysis as follows.
| 2 |
HPLC Conditions
The HPLC system used in this study consisted of a Hitachi model D-2000 Elite HPLC system (Hitachi Co., Tokyo, Japan). YMC-PACK ODS AM-303 (5 μm particle size, 250 × 4.6 mm I.D., YMC Co, Kyoto, Japan) was used as the stationary phase and was maintained at 40 °C. Two solvents, solvent A (50 mM sodium dihydrogen phosphate) and solvent B (50 mM sodium dihydrogen phosphate and acetonitrile (30:70, v/v)), were used as mobile phases. The following linear gradient elution of the mobile phase was programmed for febuxostat, warfarin, and ibuprofen: 0–7 min (30–100% B), 7–10 min (100% B), 10–15 min (100–30% B). Fusidic acid was detected using mobile phase B in the isocratic mode. The flow rate of the mobile phase was set constant at 1.0 mL/min. The detection wavelength was fixed at 210 nm, and the HPLC was monitored for 15 min for febuxostat, warfarin, and ibuprofen or 10 min for fusidic acid. The retention times of febuxostat, warfarin, ibuprofen, and fusidic acid in these HPLC conditions were 9.2, 10.8, 12.6, and 8.3 min, respectively.
CD Measurements
CD measurements were made using a Jasco model J-720A spectropolarimeter (Tokyo, Japan) with a 10 mm cell at 25 °C. Induced ellipticity is defined as the ellipticity of febuxostat-HSA mixtures minus the ellipticity of HSA alone within the same wavelength region (235–400 nm), and the values are expressed in millidegrees. No induced ellipticity was observed for febuxostat under the experimental conditions used in this study.
Molecular Docking of Febuxostat to HSA
Molecular docking of febuxostat to HSA was performed using commercially available software (Molecular Operating Environment version 2024.0601; Chemical Computing Group, Montreal, QC, Canada). The X-ray structure of HSA for docking was obtained from the RCSB Protein Data Bank (PDB ID 4G03). Potential energy was calculated using the AMBER10:EHT force field. Prior to docking simulations, structural errors were corrected, and energy minimization of protonated atoms was performed according to the manufacturer’s instructions.
To search potential ligand-binding sites on HSA, the Site Finder module was employed, yielding 28 putative binding sites. Docking simulations between febuxostat and each predicted site were performed using the Triangle Matcher algorithm for ligand placement, and the London dG scoring function was applied for initial ranking of binding poses.
The top-scoring poses were subsequently refined and rescored using the GBVI/WSA dG scoring function under an induced fit model. Among the docking poses, those overlapping with febuxostat were excluded, except for the pose with the lowest docking score. Finally, the six docking poses with the lowest GBVI/WSA dG scores were selected as the most probable binding mode of febuxostat to HSA.
Statistical Analysis
All data are expressed as the mean ± standard deviation. Statistical significance of mean differences was evaluated using the Student’s t-test or Dunnett’s test for single- or multiple-comparison analyses of experimental groups, respectively. A probability value of p < 0.05 was considered to be significant.
Supplementary Material
Acknowledgments
The authors thank the members of the Faculty of Pharmaceutical Sciences and DDS Research Institute, Sojo University, for their support of this work.
The data supporting the findings of this study are available within the article and its Supporting Information. Additional data are available from the corresponding author upon reasonable request.
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c03195.
CD spectra of octanoate–HSA and myristate–HSA systems (Supplementary Figure 1); effects of oleate on the free fraction of febuxostat in HSA solution (Supplementary Figure 2); CD spectra of the oleate–HSA system in the presence and absence of febuxostat (Supplementary Figure 3); binding of febuxostat to HSA in the presence of myristate (Supplementary Figure 4); and binding parameters of febuxostat to HSA in the presence of myristate (Supplementary Table 1) (PDF)
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
The authors declare no competing financial interest.
References
- Edwards N. L.. Febuxostat: A New Treatment for Hyperuricaemia in Gout. Rheumatology. 2009;48(Suppl. 2):ii15–ii19. doi: 10.1093/rheumatology/kep088. [DOI] [PubMed] [Google Scholar]
- Tomlinson B.. Febuxostat in the Management of Hyperuricemia and Chronic Gout: A Review. Ther. Clin. Risk Manag. 2008;4:1209–1220. doi: 10.2147/TCRM.S3310. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jordan A., Gresser U.. Side Effects and Interactions of the Xanthine Oxidase Inhibitor Febuxostat. Pharmaceuticals (Basel) 2018;11(2):51. doi: 10.3390/ph11020051. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kragh-Hansen U., Chuang V. T. G., Otagiri M.. Practical Aspects of the Ligand-Binding and Enzymatic Properties of Human Serum Albumin. Biol. Pharm. Bull. 2002;25(6):695–704. doi: 10.1248/bpb.25.695. [DOI] [PubMed] [Google Scholar]
- He X. M., Carter D. C.. Atomic Structure and Chemistry of Human Serum Albumin. Nature. 1992;358(6383):209–215. doi: 10.1038/358209a0. [DOI] [PubMed] [Google Scholar]
- Yamasaki K., Chuang V. T. G., Maruyama T., Otagiri M.. Albumin–Drug Interaction and Its Clinical Implication. Biochim. Biophys. Acta Gen. Subj. 2013;1830(12):5435–5443. doi: 10.1016/j.bbagen.2013.05.005. [DOI] [PubMed] [Google Scholar]
- Sudlow G., Birkett D. J., Wade D. N.. The Characterization of Two Specific Drug Binding Sites on Human Serum Albumin. Mol. Pharmacol. 1975;11(6):824–832. doi: 10.1016/S0026-895X(25)10691-3. [DOI] [PubMed] [Google Scholar]
- Sudlow G., Birkett D. J., Wade D. N.. Further Characterization of Specific Drug Binding Sites on Human Serum Albumin. Mol. Pharmacol. 1976;12(6):1052–1061. doi: 10.1016/S0026-895X(25)10855-9. [DOI] [PubMed] [Google Scholar]
- Zhu L., Yang F., Chen L., Meehan E. J., Huang M.. A New Drug Binding Subsite on Human Serum Albumin and Drug–Drug Interaction Studied by X-Ray Crystallography. J. Struct. Biol. 2008;162(1):40–49. doi: 10.1016/j.jsb.2007.12.004. [DOI] [PubMed] [Google Scholar]
- Zsila F.. Subdomain IB Is the Third Major Drug Binding Region of Human Serum Albumin: Toward the Three-Sites Model. Mol. Pharmaceutics. 2013;10(5):1668–1682. doi: 10.1021/mp400027q. [DOI] [PubMed] [Google Scholar]
- Kawai A., Yamasaki K., Otagiri M., Doi Y.. Interaction of Cephalosporins with Human Serum Albumin: A Structural Study. J. Med. Chem. 2024;67(16):14175–14183. doi: 10.1021/acs.jmedchem.4c00983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mukoyoshi M., Nishimura S., Hoshide S., Umeda S., Kanou M., Taniguchi K., Muroga H.. In Vitrodrug–Drug Interaction Studies with Febuxostat, a Novel Non-Purine Selective Inhibitor of Xanthine Oxidase: Plasma Protein Binding, Identification of Metabolic Enzymes and Cytochrome P450 Inhibition. Xenobiotica. 2008;38(5):496–510. doi: 10.1080/00498250801956350. [DOI] [PubMed] [Google Scholar]
- Zunszain P. A., Ghuman J., McDonagh A. F., Curry S.. Crystallographic Analysis of Human Serum Albumin Complexed with 4Z,15E-Bilirubin-IXα. J. Mol. Biol. 2008;381(2):394–406. doi: 10.1016/j.jmb.2008.06.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Peters, T. All about Albumin; Academic Press: San Diego, CA, 1995. [Google Scholar]
- Ghuman J., Zunszain P. A., Petitpas I., Bhattacharya A. A., Otagiri M., Curry S.. Structural Basis of the Drug-Binding Specificity of Human Serum Albumin. J. Mol. Biol. 2005;353(1):38–52. doi: 10.1016/j.jmb.2005.07.075. [DOI] [PubMed] [Google Scholar]
- Yamasaki K., Kawai A., Sakurama K., Udo N., Yoshino Y., Saito Y., Tsukigawa K., Nishi K., Otagiri M.. Interaction of Benzbromarone with Subdomains IIIA and IB/IIA on Human Serum Albumin as the Primary and Secondary Binding Regions. Mol. Pharmaceutics. 2021;18(3):1061–1070. doi: 10.1021/acs.molpharmaceut.0c01004. [DOI] [PubMed] [Google Scholar]
- Khosravan R., Grabowski B., Wu J.-T., Joseph-Ridge N., Vernillet L.. Effect of Food or Antacid on Pharmacokinetics and Pharmacodynamics of Febuxostat in Healthy Subjects. Br. J. Clin. Pharmacol. 2008;65(3):355–363. doi: 10.1111/j.1365-2125.2007.03016.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zsila F., Bikadi Z., Fitos I., Simonyi M.. Probing Protein Binding Sites by Circular Dichroism Spectroscopy. Curr. Drug Discovery Technol. 2004;1(2):133–153. doi: 10.2174/1570163043335135. [DOI] [PubMed] [Google Scholar]
- Simonyi, M. Probing HSA and AGP Drug-Binding Sites by Electronic Circular Dichroism. In Comprehensive Chiroptical Spectroscopy; Wiley: Hoboken, NJ, USA, 2012; pp 665–705. [Google Scholar]
- Yamasaki K., Hyodo S., Taguchi K., Nishi K., Yamaotsu N., Hirono S., Chuang V. T. G., Seo H., Maruyama T., Otagiri M.. Long Chain Fatty Acids Alter the Interactive Binding of Ligands to the Two Principal Drug Binding Sites of Human Serum Albumin. PLoS One. 2017;12(6):e0180404. doi: 10.1371/journal.pone.0180404. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ha C.-E., Petersen C. E., Park D. S., Harohalli K., Bhagavan N. V.. Investigations of the Effects of Ethanol on Warfarin Binding to Human Serum Albumin. J. Biomed. Sci. 2000;7(2):114–121. doi: 10.1007/BF02256617. [DOI] [PubMed] [Google Scholar]
- Khosravan R., Wu J.-T., Joseph-Ridge N., Vernillet L.. Pharmacokinetic Interactions of Concomitant Administration of Febuxostat and NSAIDs. J. Clin. Pharmacol. 2006;46(8):855–866. doi: 10.1177/0091270006289848. [DOI] [PubMed] [Google Scholar]
- Fujiwara S.-I., Amisaki T.. Molecular Dynamics Study of Conformational Changes in Human Serum Albumin by Binding of Fatty Acids. Proteins. 2006;64(3):730–739. doi: 10.1002/prot.21053. [DOI] [PubMed] [Google Scholar]
- Birkett D. J., Myers S. P., Sudlow G.. Effects of Fatty Acids on Two Specific Drug Binding Sites on Human Serum Albumin. Mol. Pharmacol. 1977;13(6):987–992. doi: 10.1016/S0026-895X(25)12512-1. [DOI] [PubMed] [Google Scholar]
- Wanwimolruk S., Birkett D. J.. The Effects of N-B Transition of Human Serum Albumin on the Specific Drug-Binding Sites. Biochim. Biophys. Acta. 1982;709(2):247–255. doi: 10.1016/0167-4838(82)90467-8. [DOI] [PubMed] [Google Scholar]
- Wilting J., van der Giesen W. F., Janssen L. H., Weideman M. M., Otagiri M., Perrin J. H.. The Effect of Albumin Conformation on the Binding of Warfarin to Human Serum Albumin. The Dependence of the Binding of Warfarin to Human Serum Albumin on the Hydrogen, Calcium, and Chloride Ion Concentrations as Studied by Circular Dichroism, Fluorescence, and Equilibrium Dialysis. J. Biol. Chem. 1980;255(7):3032–3037. doi: 10.1016/S0021-9258(19)85847-7. [DOI] [PubMed] [Google Scholar]
- Bos O. J. M., Labro J. F. A., Fischer M. J. E., Wilting J., Janssen L. H. M.. The Molecular Mechanism of the Neutral-to-Base Transition of Human Serum Albumin. J. Biol. Chem. 1989;264(2):953–959. doi: 10.1016/S0021-9258(19)85036-6. [DOI] [PubMed] [Google Scholar]
- Zhang Z., Zhang J., Yang T., Li S., Xu G., Liang H., Yang F.. Developing an Anticancer Platinum(II) Compound Based on the Uniqueness of Human Serum Albumin. J. Med. Chem. 2023;66(8):5669–5684. doi: 10.1021/acs.jmedchem.3c00001. [DOI] [PubMed] [Google Scholar]
- Li W., Li S., Zhang Z., Xu G., Man X., Yang F., Liang H.. Developing a Multitargeted Anticancer Palladium(II) Agent Based on the His-242 Residue in the IIA Subdomain of Human Serum Albumin. J. Med. Chem. 2023;66(13):8564–8579. doi: 10.1021/acs.jmedchem.3c00248. [DOI] [PubMed] [Google Scholar]
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Data Availability Statement
The data supporting the findings of this study are available within the article and its Supporting Information. Additional data are available from the corresponding author upon reasonable request.