Subdomain IIA Of Human Serum Albumin Research Articles

Probing the binding of two 19-nortestosterone derivatives to human serum albumin: insights into the interactions of steroid hormone drugs with functional biomacromolecule.

Norethindrone acetate (NETA) is a fatty acid ester of norethindrone (NET) that can convert to its more active parent compound NET when orally administered. To study the interactions of NETA and NET with human serum albumin (HSA), we applied fluorescence spectroscopy, circular dichroism (CD), and molecular docking. The effects of metal ions on the HSA-NETA/NET system were also explored. Fluorescence data showed that the quenching mechanism of HSA by NETA and NET was consistent with a static model and that the binding constant of NETA was higher than that of NET. Thermodynamic parameters indicated that hydrogen bonds and van der Waals forces were the main forces maintaining the stability of the HSA-NETA/NET complex. Molecular modeling studies revealed that NETA and NET were bound within subdomain IIA of HSA, in accordance with the site probe results. Synchronous fluorescence spectroscopy, CD, and three-dimensional fluorescence spectroscopy further confirmed that the binding of NETA/NET to HSA changed the secondary structure of the protein. All other metal ions, except for Ca(2+) , decreased the K value of the HSA-NETA/NET system with enhancement of the maximum effectiveness of NETA/NET. Three commercially available steroid hormone drugs influenced the binding ability of NETA on HSA to different extents. This study provides novel insights into the interactions between HSA and NETA/NET, as well as a solid foundation for future research on drug pharmacokinetics and pharmacodynamics. Copyright © 2016 John Wiley & Sons, Ltd.

Exploring binding properties of sertraline with human serum albumin: Combination of spectroscopic and molecular modeling studies

Human serum albumin (HSA)-drug binding is an important factor to determine half life and bioavailability of drugs. In the present research, the interaction of sertraline (SER) to HSA was investigated using combination of spectroscopic and molecular modeling techniques. Changes in the UV–Vis, CD and FT-IR spectra as well as a significant degree of tryptophan fluorescence quenching were observed upon SER-HSA interaction. Data obtained by spectroscopic methods along with the computational studies suggest that SER binds to residues located in subdomain IIA of HSA. Analysis of spectroscopic data represented the formation of 1:1 complex, significant binding affinity, negative values of entropy and enthalpy changes and the essential role of hydrophobic interactions in binding of SER to HSA. The binding models were demonstrated in the aspects of SER's conformation, active site interactions, important amino acids and hydrogen bonding. Computational mapping of the possible binding site of SER confirmed that the ligand to be bound in a large hydrophobic cavity of HSA. In accordance with experimental data, computational analyses indicated that SER binding does not alter the secondary structure of the protein. The results not only lead to a better understanding of interaction between SER and HSA but also provide useful data about the influence of SER on the protein conformation.

Developing Anticancer Copper(II) Pro-drugs Based on the Nature of Cancer Cells and the Human Serum Albumin Carrier IIA Subdomain.

To synergistically enhance the selectivity and efficiency of anticancer copper drugs, we proposed and built a model to develop anticancer copper pro-drugs based on the nature of human serum albumin (HSA) IIA subdomain and cancer cells. Three copper(II) compounds of a 2-hydroxy-1-naphthaldehyde benzoyl hydrazone Schiff-base ligand in the presence pyridine, imidazole, or indazole ligands were synthesized (C1-C3). The structures of three HSA complexes revealed that the Cu compounds bind to the hydrophobic cavity in the HSA IIA subdomain. Among them, the pyridine and imidazole ligands of C1 and C2 are replaced by Lys199, and His242 directly coordinates with Cu(II). The indazole and Br ligands of C3 are replaced by Lys199 and His242, respectively. Compared with the Cu(II) compounds alone, the HSA complexes enhance cytotoxicity in MCF-7 cells approximately 3-5-fold, but do not raise cytotoxicity levels in normal cells in vitro through selectively accumulating in cancer cells to some extent. We find that the HSA complex has a stronger capacity for cell cycle arrest in the G2/M phase of MCF-7 by targeting cyclin-dependent kinase 1 (CDK1) and down-regulating the expression of CDK1 and cyclin B1. Moreover, the HSA complex promotes MCF-7 cell apoptosis possibly through the intrinsic reactive oxygen species (ROS) mediated mitochondrial pathway, accompanied by the regulation of Bcl-2 family proteins.

Folate-functionalized human serum albumin carrier for anticancer copper(II) complexes derived from natural plumbagin

The folate (FA)-functionalized human serum albumin (HSA) carrier (FA-HSA) is promising for improving the target and efficiency of anticancer drugs. To develop FA-HSA carrier for metal anticancer drugs, we investigated anticancer properties and mechanism of FA-HSA carrier for Cu(II) complexes derived from plumbagin. The fluorescence spectroscopy and molecular docking revealed that Cu(II) complexes bind to IIA subdomain of HSA. Compared with Cu(II) complex alone, FA-HSA-metallodrug complex enhances cytotoxicity to FA-positive cancer cells (HeLa) but do not raise cytotoxicity levels in normal cells in vitro through selectively accumulating in cancer cells to some extent; FA-HSA-metallodrug complex has a stronger capacity for cell cycle arrest in the G2/M phase of HeLa cells, and down-regulating the expression of cyclin-dependent kinase 1 (CDK1) and cyclin B1. Moreover, FA-HSA-metallodrug complex promotes HeLa cells apoptosis through intrinsic reactive oxygen species (ROS) mediated mitochondrial pathway, accompanied by the regulation of Bcl-2 family proteins.

Enhancement of the binding affinity of methylene blue to site I in human serum albumin by cupric and ferric ions.

In this work, the binding characteristics of methylene blue (MB) to human serum albumin (HSA) and the influence of Cu(2+) and Fe(3+) on the binding affinity of MB to HSA were investigated using fluorescence, absorption, circular dichroism (CD) spectroscopy and molecular modelling. The results of competitive binding experiments using the site probes ketoprofen and ibuprofen as specific markers suggested that MB was located in site I within sub-domain IIA of HSA. The molecular modelling results agreed with the results of competitive site marker experiments and the results of CD spectra indicated that the interaction between MB and HSA caused the conformational changes in HSA. The binding affinity of MB to HSA was enhanced but to a different extent in the presence of Cu(2+) and Fe(3+), respectively, which indicated that the influence of different metal ions varied. Enhancement of the binding affinity of MB to HSA in the presence of Cu(2+) is due to the formation of Cu(2+)-HSA complex leading to the conformational changes in HSA, whereas in the presence of Fe(3+), enhancement of the binding affinity is due to the greater stability of the Fe(3+)-HSA-MB complex compared with the MB-HSA complex.

Ground and excited state proton transfer of the bioactive plant flavonol robinetin in a protein environment: spectroscopic and molecular modeling studies.

We performed spectroscopic and molecular modeling studies to explore the interaction of the bioactive plant flavonol robinetin (3,7,3',4',5'-OH flavone), with the carrier protein human serum albumin (HSA). Multiparametric fluorescence sensing, exploiting the intrinsic "two color" fluorescence of robinetin (comprising excited state intramolecular proton transfer (ESIPT) and charge transfer (CT) emissions) reveals that binding to HSA significantly affects the emission and excitation profiles, with strongly blue-shifted (∼29 nm) normal fluorescence and remarkable increase in the ESIPT fluorescence anisotropy (r) and lifetime (τ). Flavonol-induced HSA (tryptophan) fluorescence quenching data yield the dynamic quenching constant (KD) as 5.42 × 10(3) M(-1) and the association constant (Ks) as 5.59 × 10(4) M(-1). Time-resolved fluorescence anisotropy decay studies show dramatic (∼170 times) increase in the rotational correlation time (τ(rot)), reflecting greatly enhanced restrictions in motion of robinetin in the protein matrix. Furthermore, prominent induced circular dichroism (ICD) bands appear, indicating that the chiral environment of HSA strongly perturbs the electronic transitions of the intrinsically achiral robinetin molecule. Molecular docking calculations suggest that robinetin binds in subdomain IIA of HSA, where specific interactions with basic residues promote ground state proton abstraction and stabilize an anionic species, which is consistent with spectroscopic observations.

Interaction of the La(III)–Morin Complex with Human Serum Albumin

Lanthanum (La) is one of the most reactive rare earth elements, whose carbonate had been approved by the FDA for marketing as a treatment drug for hyperphosphatemia. In this paper, a complex of La with the natural active compound morin was synthesized and identified. The interaction between synthesized complex (LaMO) and human serum albumin (HSA) was studied by multiple spectroscopic methods. It was found that LaMO has an efficient interaction with HSA through hydrogen bonds and van der Waals forces, with formation of the LaMO–HSA complex in its ground state. The reaction led to quenching of HSA’s fluorescence and the quenching follows a static quenching mechanism. The binding constants, reference state enthalpy change (ΔH θ), Gibbs energy change (ΔG θ) and entropy change (ΔS θ) were calculated at four different temperatures. Fluorescence probe techniques were used to identify the binding location. The results showed that LaMO competes with warfarin for Sudlow’s site I in subdomain IIA of HSA, an acknowledged site marker. Meanwhile, circular dichroism spectrum measurements revealed changes of HSA’s secondary structure in the presence of LaMO, which implies that LaMO is potentially bioactive. The study involves research about the pharmacologic mechanism of rare earth metal coordination compounds and can provide basic data for their safety evaluation.

Docking simulations and spectroscopy of the interactions of ellagic acid and oleuropein with human serum albumin

The primary objective of this study is to evaluate the interactions of ellagic acid (EA) and oleuropein (OLE) with human serum albumin (HSA). Plasma protein binding model prediction and molecular modeling suggest that EA binds to HSA stronger than OLE, and that both interact with the binding pocket of HSA subdomain IIA. Furthermore, fluorescence spectroscopy confirmed that EA has a stronger HSA binding affinity than OLE. Three-dimensional fluorescence spectroscopy and CD spectroscopy, analyzed using DichroWeb, showed that the binding of EA and OLE to HSA induce conformational changes.

Spectroscopic and molecular simulation studies on the interaction of di-(2-ethylhexyl) phthalate and human serum albumin.

Di-(2-ethylhexyl) phthalate (DEHP) is widely used as a plasticizer in industrial production, but may have a potential health risk. In this study, the binding characteristics of DEHP with human serum albumin (HSA) in aqueous solution at pH 7.4 were determined using UV/vis absorption, fluorescence, Fourier transform infrared (FTIR) spectroscopy and circular dichroism (CD), along with a molecular simulation technique. Analysis of the fluorescence titration data at different temperatures suggested that the fluorescence quenching mechanism of HSA by DEHP was static. The calculated thermodynamic parameters indicated that hydrophobic forces played a predominant role in formation of the DEHP-HSA complex, but hydrogen bonds could not be omitted. Site marker competitive experiments and denaturation studies showed that the binding of DEHP to HSA primarily took place in subdomain IIA of HSA, and molecular docking results further corroborated the binding sites. The synchronous fluorescence, UV/vis absorption, FTIR and CD spectra revealed that the addition of DEHP induced changes in the secondary structure of HSA. Protein surface hydrophobicity (PSH) tests indicated that DEHP binding to HSA caused an increase in the PSH. Moreover, the effects of some metal ions on the binding constant of DEHP - HSA interaction were also investigated.

Potential toxicity of amphenicol antibiotic: binding of chloramphenicol to human serum albumin.

Antibiotics are widely used in daily life but their abuse has posed a potential threat to human health. To evaluate the toxicity of chloramphenicol (CAP) at the protein level, the interaction between CAP and human serum albumin (HSA) was investigated by fluorescence, Ultraviolet-visible (UV-Vis) absorption, Fourier transform infrared (FT-IR) spectroscopy and molecular docking methods. Fluorescence data revealed that the fluorescence quenching of HSA by CAP was the result of the formation of CAP-HSA complex, and the binding constant was determined to be 3.196 × 10(4) L mol(-1) at 310 K. The thermodynamic determination indicated that the interaction was driven by enthalpy change and entropy change together, where the multiple hydrogen bonds (CAP and the residues Arg 222 and His 242 of HSA) and van der Waals forces were the dominant binding force. The site marker competition revealed that CAP bound into sub-domain IIA of HSA. The binding of CAP induced the drastic reduction in α-helix conformation and the significant enhancement in β-sheet conformation of HSA. Molecular docking study further confirmed the binding mode obtained by experimental study. This work provides a new quantitative evaluation method for antibiotics to cause the protein damage.

Study on the interaction of doxycycline with human serum albumin

The present study was designed to investigate the interaction of doxycycline (DC) with human serum albumin (HSA) by the inner filter effects, displacement experiments and molecular docking methods, based on classic multi-spectroscopy. With fluorescence quenching method at 298 and 310 K, the binding constants Ka, were determined to be 2. 73 X 10(5) and 0. 74X 10(5) L mol-1, respectively, and there was one binding site between DC and HSA, indicating that the binding of DC to HSA was strong, and the quenching mechanism was a static quenching. The thermodynamic parameters (enthalpy change, AH and enthropy change, delta S) were calculated to be -83. 55 kJ mol-1 and -176. 31 J mol-1 K-1 via the Vant' Hoff equation, which indicated that the interaction of DC with HSA was driven mainly by hydrogen bonding and van der Waals forces. Based on the Föster's theory of non-radiation energy transfer, the specific binding distance between Trp-214 (acceptor) and DC (donor) was 4. 98 nm, which was similar to the result confirmed by molecular docking. Through displacement experiments, sub-domain IIA of HSA was assigned to possess the high-affinity binding site of DC. Three-dimensional fluorescence spectra indicated that the binding of DC to HSA induced the conformation change of HSA and increased the disclosure of some part of hydrophobic regions that had been buried before. The results of FTIR spectroscopy showed that DC bound to HSA led to the slight unfolding of the polypeptide chain of HSA. Furthermore, the binding details between DC and HSA were further confirmed by molecular docking methods, which revealed that DC was bound at sub-domain IIA through multiple interactions, such as hydrophobic effect, polar forces and pi-pi interactions. The experimental results provide theoretical basis and reliable data for the study of the interaction between small drug molecule and human serum albumin

Comparison of Binding Characterization of Two Antiviral Drugs to Human Serum Albumin

Ribavirin and lamivudine are representatives of antiviral drugs that are widely used to treat viral infections, especially chronic liver disease. To compare binding mechanism and behavior of antiviral drugs with human serum albumin (HSA), we performed fluorescence spectroscopy and X-ray crystallography to investigate the interactions of ribavirin and lamivudine with HSA. Fluorescence spectroscopy showed ribavirin and lamivudine inhibit binding affinity each other. Our results further demonstrated that ribavirin and lamivdudine interaction with HSA could be affected by the presence of other compounds, including the non-steroidal anti-inflammatory drugs, indometacin. X-ray structures revealed that ribavirin and lamivudine bind in IIA subdomain of HSA mainly by forming hydrogen bond and hydrophobic interactions forces. The carboxamido of ribavirin forms hydrogen bonds with Arg222; Hydroxyl group (6) of ribavirin forms hydrogen bond with Arg257. Hydroxyl group (15) of lamivudine forms hydrogen bond with Arg222; amino group (4) of lamivudine forms hydrogen bond with carbonyl of Arg257. Our results reveal the key biochemical and structural characteristics of the HSA interaction with ribavirin and lamivudine, providing guidance for future development of ribavirin- and lamivudine-based compounds and a drug-HSA delivery system.

Interactions of PCBs with human serum albumin: In vitro spectroscopic study

Following absorption, polychlorinated biphenyls (PCBs) bind to albumin and are transported via blood into the target tissues. PCBs then accumulate in tissues and induce a variety of harmful chronic and developmental effects. The aim of the present study is to determine binding parameters, such as binding constant, quenching constant, and number of binding sites for three PCB congeners (PCB118, PCB126 and PCB153) in complex with human serum albumin (HSA). The binding parameters for the complexes of HSA–PCB118, HSA–PCB126, and HSA–PCB153 excited at 280nm were compared with those excited at 295nm. Quenching (static and dynamic) of HSA fluorescence was analyzed based on the Stern–Volmer method. Binding (Ka) constants were calculated according to the Scatchard method and analysis of non-linear regression was based on a two-component model with the Lavenberg–Marquardt algorithm. For all analyzed complexes, a single independent class of binding site for PCB congeners was found in HSA subdomain IIA. Tyrosine residues appear to play the most prominent role in binding of PCB126 to HSA, while tryptophan-214 played a dominant role in interactions of PCB153 with HSA. Among studied PCB congeners, PCB118 formed the most stable complexes with HSA. These results illustrate the importance of studies targeting the binding of PCBs to serum albumin as part of the strategy to understand and protect against toxicity of these environmental toxicants.

X-ray Crystallographic and Fluorometric Analysis of the Interactions of Rhein to Human Serum Albumin

To investigate the interactions between natural drugs and human serum albumin (HSA), we performed fluorescence spectroscopy and X-ray crystallography to gain insight into binding mechanism and behaviour of rhein to HSA. Our fluorescence results demonstrated that rhein strongly binds with HSA, and other compounds may affect binding affinity of rhein to different extent. Structural analysis revealed that rhein binds to the IIA subdomain of HSA. The carboxylate group of rhein forms hydrogen bonds with Arg218 and Lys199, as well as a salt bond with Arg222. Hydroxyl group (4) of rhein forms a hydrogen bond with His242, and hydroxyl group (5) of rhein forms a hydrogen bond with Arg257. Oxygen atom (7) of rhein forms a hydrogen bond with Arg222, and oxygen atom (6) of rhein forms a hydrogen bond with H₂O. Furthermore, hydroxyl group (4) of rhein also forms a hydrogen bond with H₂O. Our results reveal the biochemical and structural characteristics of the interaction between rhein and HSA, providing guidance for future development of rhein-based compounds and a drug-HSA delivery system.

Unusual Binding of a Potential Biomarker with Human Serum Albumin

This study investigates the specific binding of a potential biomarker, [2,2'-bipyridyl]-3,3'-diol (BP(OH)2), with human serum albumin (HSA). The binding of BP(OH)2 at the two primary drug-binding sites on HSA (Sudlow's sites I and II) is explored by a competitive-binding study and monitored by considering the green-light emission from its diketo tautomer. Warfarin is used as a marker for site I and dansyl-L-proline (DP) as a competitor for site II. Steady-state and time-resolved fluorescence measurements affirm that neither of Sudlow's sites is the binding locus of BP(OH)2. To gain an idea regarding the probable binding site of BP(OH)2, we perform molecular-docking studies, which reveal a close proximity of the probe to Trp-214 in subdomain IIA of HSA. Confirmation of this contention is achieved by studying the quenching of the fluorescence of Trp-214 in the presence of BP(OH)2. Moreover, static quenching seems to be responsible for the depletion of the fluorescence of Trp-214, as manifested by the invariance of the intrinsic fluorescence lifetime of Trp-214, as a function of the concentration of BP(OH)2. Based on displacement and quenching studies, supported by molecular docking, we propose that BP(OH)2 binds in a cleft that separates subdomains IIIA and IIB, which is in close proximity to Trp-214.

Regulation of Amantadine Hydrochloride Binding with IIA Subdomain of Human Serum Albumin by Fatty Acid Chains

Human serum albumin (HSA) is a major protein component of blood plasma that has been exploited to bind and transport a wide variety of endogenous and exogenous organic compounds. Although anionic drugs readily associate with the IIA subdomain of HSA, most cationic drugs poorly associate with HSA at this subdomain. In this study, we propose to improve the association between cationic drugs and HSA by modifying HSA with fatty acid chains. For our experiments, we tested amantadine hydrochloride, a cationic drug with antiviral and antiparkinsonian effects. Our results suggest that extensive myristoylation of HSA can help stabilize the interaction between amantadine and HSA in vitro. Our X-ray crystallography data further elucidate the structural basis of this regulation. Additionally, our crystallography data suggest that anionic drugs, with a functional carboxylate group, may enhance the association between amantadine and HSA by a mechanism similar to myristoylation. Ultimately, our results provide critical structural insight into this novel association between cationic drugs and the HSA IIA subdomain, raising the tempting possibility to fully exploit the unique binding capacity of HSA's IIA subdomain to achieve simultaneous delivery of anionic and cationic drugs.

Spectroscopy of hydroxyphenyl benzazoles in solution and human serum albumin: detecting flexibility, specificity and high affinity of the warfarin drug binding site

The complex photophysics of 2-(2′-hydroxyphenyl)benzoxazole (HBO), 2-(2′-hydroxyphenyl)benzimidazole (HBI), and 2-(2′-hydroxyphenyl)benzothiazole (HBT) in different media makes them suitable as fluorescent probes to study the nature of binding sites in macromolecular systems. In this work, we investigate the spectroscopy of the three benzazole derivatives (HBXs) in different solvents and in human serum albumin (HSA) in order to understand the binding mechanism in subdomain IIA of HSA which has the ability to host a large variety of natural and pharmaceutical compounds. The three probes are found to specifically bind close to W214, the sole tryptophan residue in HSA, in a mode similar to that of the binding of the anticoagulant drug warfarin. The current results show that the structural differences between the three HBX molecules did not produce any measurable effects when binding with HSA. In particular, the change in planarity of the molecular backbone, from a perfectly planar and more rigid structure (HBO) to a twisted structure (HBI) and a flexible structure (HBT) has no effect on the mode of binding. Also, the strength of the intramolecular hydrogen bonds in HBXs (HBO > HBT > HBI) is shown not to intervene with the ability of HSA to ionize the ligands via through-space interaction with polar amino acid residues, similar to enzymatic reactions. The results emphasize the nature of HSA as a versatile and indiscriminate receptor, capable of binding a variety of ligands by adapting its binding pockets. In this regard, binding of HBXs, and other structurally similar ligands, in subdomain IIA is best described by the induced-fit model in which considerable flexibility of the binding site is necessary for molecular recognition. The results also point to the high affinity of the warfarin binding pocket (within subdomain IIA) for binding versatile molecular structures including several drugs. This affinity stems from the flexibility of the amino acids forming the binding pocket.

Application of molecular modelling and spectroscopic approaches for investigating the binding of tanshinone IIA to human serum albumin

The interaction of tanshinone IIA and human serum albumin (HSA) has been characterised by molecular modelling, fluorescence, Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopic methods. The results of molecular modelling suggested that tanshinone IIA located within the binding pocket of subdomain IIA of HSA is held mainly by hydrophobic forces. Fluorescence titration revealed that tanshinone IIA could quench the intrinsic fluorescence of HSA. The binding constants at three temperatures (296, 303, and 310)K are (6.42·104, 1.54·105, and 4.35·105)dm3·mol−1, respectively. In addition, the studies of CD spectroscopy and FT-IR spectroscopy showed that the binding of tanshinone IIA to HSA changed molecular conformation of HSA.

Studies of the interaction between demeclocycline and human serum albumin by multi-spectroscopic and molecular docking methods

This study was designed to examine the interaction of demeclocycline (DMCTC) with human serum albumin (HSA) by multi-spectroscopic and molecular docking methods. The inner filter effect was corrected before we calculated the binding parameters. Fluorescence and UV–vis spectroscopy revealed that DMCTC induced the fluorescence quenching of HSA though a static quenching procedure. Thermodynamic analysis by Van Hoff equation found enthalpy change (ΔH) and entropy change (ΔS) were −53.01kJmol−1 and −65.13Jmol−1K−1, respectively, which indicated hydrogen bond and van der Waals force were the predominant force in the binding process. According to fluorescence resonance energy transfer (FRET), the specific binding distances between Trp-214 (donor) and DMCTC (acceptor) were 3.18nm. Through site marker competitive experiments, subdomain IIA of HSA has been assigned to possess the high-affinity binding site of DMCTC. The three dimensional fluorescence showed that the conformation of HSA was changed after its complexation with DMCTC, and the alternations of protein secondary structure were quantitatively calculated from FT-IR with reduction of α-helices content about 4.8%, β-sheet from 30.3% to 21.6% and with increases of β-turn from 15.6% to 22.2%. Furthermore, the binding details between DMCTC and HSA were further confirmed by molecular docking studies, which revealed that DMCTC was bound at subdomain IIA through multiple interactions, such as hydrophobic effect, polar forces and π–π interactions. Moreover, the coexist metal ions such as Al3+, Fe3+, Cu2+, Cr3+ and Cd2+ can decrease the binding constants of DMCTC–HSA.

Interactive Associations of Drug–Drug and Drug–Drug–Drug with IIA Subdomain of Human Serum Albumin

Owing to advantageous biochemical and pharmacological properties of human serum albumin (HSA), HSA-based drug carrier is playing an increasing role in the clinical setting. Since the IIA subdomain of HSA is a big hydrophobic cavity, we proposed that HSA delivers multiple drugs simultaneously docking on the IIA subdomain and that drugs may influence each other's binding affinity to albumin when cobinding the HSA IIA subdomain. Therefore, we studied the interactive association of drugs with the IIA subdomain of HSA by fluorescence spectroscopy and X-ray crystallography, in order to elucidate the molecular mechanism and behavior of drugs cobinding the IIA subdomain of HSA, and develop a universal structure-based model for HSA carrier design. We solved HSA-fatty acid-cinnamic acid, HSA-fatty acid-cinnamic acid-indometacin and HSA-fatty acid-cinnamic acid-indometacin-lamivudine complex structures, respectively. HSA complex structures and fluorescence quenching of HSA revealed that different drugs can regulate binding sites, binding mode and binding affinity of each other. For example, indometacin renders cinnamic acid to make reposition, and decreases binding affinity of HSA for cinnamic acid, but increases binding affinity of itself to HSA. Lamivudine makes cinnamic acid and indometacin to bind new subsites. Cinnamic acid-indometacin enhances binding affinity of lamivudine, and cinnamic acid-lamivudine increases binding affinity of indometacin, but indometacin-lamivudine decreases binding affinity of cinnamic acid to HSA. The study provided a biochemical basis for structure-guided development of HSA delivery system.