Binding Sites Of Human Serum Albumin Research Articles

In vitro spectroscopic studies of 9-amino-5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazine chloride with main carrier plasma proteins

Current methods of cancer treatment, particularly chemotherapy, are associated with harmful side effects. For this reason, it is significant to study new substances with anticancer potential with the highest possible efficacy and the lowest possible side effects. The aim of the study was the spectroscopic analysis of the interaction between 9-amino-5-alkyl-12(H)-quino[3,4-b][1,4]benzothiazine chloride (Salt3) and main carrier proteins, such as human serum albumin (HSA), α1 acid glycoprotein (AGP), human γ globulin (HGG) and controlled normal serum (CNS).The association constants (Ka [mol·L-1]) and the number of binding site classes (n) for the binding of Salt3 with studied carrier proteins and controlled normal serum were calculated using the Klotz equation. To study HSA and AGP high affinity binding sites, the fluorescent markers were used. Spectral parameter A and the second derivative of differential absorption spectra were used to assess environmental changes around aromatic amino acids residues. The changes in HSA and AGP secondary structure in the complexes with Salt3 were evaluated using the analysis using circular dichroism.Salt3 slightly binds to HSA, AGP, HGG molecules and CNS. In addition, Salt3 affects the tertiary structure of the studied proteins, while it does not damage the secondary structure of the main carrier proteins responsible for Salt3 distribution in the bloodstream.Because Salt3 binds weakly to model carrier proteins and normal control serum, it can lead to both strong therapeutic and toxic effects. Considering these preliminary spectroscopic studies, additional tests as well as expanding research to include other techniques seem justified.

Transfer of ANS-Like Drugs from Micellar Drug Delivery Systems to Albumin Is Highly Favorable and Protected from Competition with Surfactant by "Reserved" Binding Sites.

Micellar drug delivery systems (MDDS) for the intravenous administration of poorly soluble drugs have great advantages over alternative formulations in terms of the safety of their excipients, storage stability, and straightforward production. A classic example is mixed micelles of glycocholate (GC) and lecithin, both endogenous substances in human blood. What limits the use of MDDS is the complexity of the transitions after injection. In particular, as the MDDS disintegrate partially or completely after injection, the drug has to be transferred safely to endogenous carriers in the blood, such as human serum albumin (HSA). If this transfer is compromised, the drug might precipitate─a process that needs to be excluded under all circumstances. The key question of this paper is whether the high local concentration of GC at the moment and site of MDDS dissolution might transiently saturate HSA binding sites and, hence, endanger quick drug transfer. To address this question, we have used a new approach, which is time-resolved fluorescence spectroscopy of the single tryptophan in HSA, Trp-214, to characterize the competitive binding of GC and the drug substitute anilinonaphthalenesulfonate (ANS) to HSA. Time-resolved fluorescence of Trp-214 showed important advantages over established methods for tackling this problem. ANS has been the standard "model drug" to study albumin binding for decades, given its structural similarity to the class of naphthalene-containing acidic drugs and the fact that it is displaced from HSA by numerous drugs (which presumably bind to the same sites). Our complex global fit uses the critical approximation that the average lifetimes behave similarly to a single lifetime, but the resulting errors are found to be moderate and the results provide a convincing explanation of the, at first glance, counterintuitive behavior. Accordingly, and largely in line with the literature, we observed two types of sites binding ANS at HSA: 3 type A, rather peripheral, and 2 type B, likely more central sites. The latter quench Trp-214 by Förster Resonance Energy Transfer (FRET) with a rate constant of ≈0.4 ns-1 per ANS. Adding millimolar concentrations of GC displaces ANS from the A sites but not from B sites. At incomplete ANS saturation, this causes a GC-induced translocation of ANS from A to the more FRET-active B sites. This leads to the apparent paradox that the partial displacement of ANS from HSA increases its quenching effect on Trp-214. The most important conclusion is that (ANS-like) drugs cannot be displaced from the type-B sites, and consequently, drug transfer to these sites is not impaired by competitive binding of GC in the vicinity of a dissolving micelle. The second conclusion is that for unbound GC above the CMC (9 mM), ANS equilibrates between HSA and GC micelles but with a strong preference for free sites on HSA. That means that even persisting micelles would lose their cargo readily once exposed to HSA. For all MDDS sharing this property, targeted drug delivery approaches involving them as the nanocarrier would be pointless.

Unveiling the molecular interaction of hepatitis B virus inhibitor, entecavir with human serum albumin through computational, microscopic and spectroscopic approaches

Molecular docking, molecular dynamics (MD) simulation, atomic force microscopy (AFM) and multi-spectroscopic techniques were selected to unveil the molecular association between the hepatitis B virus (HBV) inhibitor, entecavir (ETR), and the major blood plasma transporter, human serum albumin (HSA). The entire docking and simulation analyses recognized ETR binding to subdomain IIA (Site I) of HSA through hydrogen bonds, hydrophobic and van der Waals forces while maintaining the complex’s stability throughout the 100 ns. A gradual lessening in the Stern-Volmer quenching constant ( K sv ) with rising temperatures registered ETR-induced quenching of HBV fluorescence as static quenching, thus advising complexation between ETR and HSA. The further advocation of this conclusion was seen from a larger value of the biomolecular quenching rate constant (( kq ) > 1010 M−1s−1), changes in the spectra (UV-Vis absorption) of HSA following ETR inclusion and ETR-induced swelling of HSA in the AFM results. The ETR appeared to bind to HSA with moderate affinity ( K a = 1.87 − 1.19 × 10 4 M−1) at 290, 300 and 310 K. Significant alterations in the protein’s secondary and tertiary structures, including changes in the protein’s Tyr/Trp microenvironment, were also detected by circular dichroism and three-dimensional fluorescence spectra when the protein was bound to ETR. The findings of the drug displacement study backed the docking results of Site I as ETR's preferred binding site in HSA. Communicated by Ramaswamy H. Sarma

Ibuprofen-Immobilized Thin Films: A Novel Approach to Improve the Clearance of Protein-Bound Uremic Toxins.

Chronic kidney disease (CKD), a pressing global health issue, affects millions and leads to end-stage renal disease (ESRD). Hemodialysis (HD) is a crucial treatment for ESRD, yet its limited efficiency in removing protein-bound uremic toxins (PBUTs) results in high morbidity and mortality rates. A high affinity of pharmaceutical drugs for human serum albumin (HSA) can be leveraged to compete effectively with PBUTs for the same HSA binding sites, thereby enabling them to be capable of displacing these toxins. One such drug is ibuprofen (IBF), known for its very high affinity for HSA and sharing the same binding site as indoxyl sulfate (IS). This study explores the development of IBF-immobilized cellulose acetate-based (CA-based) thin films. The films were created by reacting CA with IBF-modified silica precursors at varying concentrations. The presence of IBF in CA/TEOS/APTES-IBF-3 and CA/TEOS-IBF-25 films, containing 3 and 25 wt % IBF, respectively, was confirmed through 1H NMR spectra. Competitive displacement binding assays indicated that while the incorporation of 3 wt % IBF showed no significant enhancement in IS displacement, the 25 wt % IBF film increased the dialyzed IS by 1.3 when normalized to non-IBF films. Furthermore, there was a 1.2-fold decrease in the total percentage of IS, and the free percentage of IS increased 1.3 to 3.0 times. Although direct systemic infusion of IBF in HD patients achieves a 2.4 times higher removal of IS, it is impractical due to the risks it poses to ESRD patients. The IBF-immobilized films offer the advantage of localized binding, thus eliminating the need for systemic exposure. This innovative approach lays a foundation for developing more efficient HD membranes, aiming to address the challenging issue of PBUT elimination and potentially enhance the quality of life and treatment outcomes for ESRD patients.

Unveiling fructose and glucose binding to human serum albumin: fluorescence measurements and docking, molecular dynamics and quantum biochemistry computations

This research examines the interaction between human serum albumin (HSA) and various sugar forms (β-D-fructofuranose (FRC), α-D-glucopyranose (GLC), Keto-D-fructose (FRO), Aldehydo-D-glucose (GLO), and modified Aldehydo-D-glucose (GLOm)) using fluorescent spectroscopy, molecular docking simulations, molecular dynamics, protein conformational clusters (EnGens), molecular fractionation with conjugate caps (MFCC) and quantum biochemistry analysis. We analyze molecular and quantum aspects, uncovering interaction energies between sugar atoms and amino acids. Total interaction energy considers protein fragmentation, energetic decomposition, and interaction energy from a bottom-up perspective. Molecular dynamics reveal that unmodified Aldehydo-D-glucose (GLO) escapes HSA binding sites, explaining gradual glycation. We pioneer studying HSA’s binding mechanism with glucose and fructose in a 1:1 ratio using long molecular dynamics simulations. Results suggest the transitional GLOm form has a higher Sudlow I site propensity than unmodified glucose, crucial for K195 glycation. FRO and GLOm interaction tendencies move toward a deeper FA7 cavity, near its center. This approach effectively elucidates small molecule binding mechanisms, consistent with previous experimental results. Communicated by Ramaswamy H. Sarma

Determination of 3- and 4-chloromethcathinone interactions with plasma proteins: study involving analytical and theoretical methods

PurposeThe purpose of this paper was to determine 3- and 4-chloromethcathinone (3- and 4-CMC) binding degree and possible binding interaction modes with human serum albumin (HSA) using analytical and theoretical methods.MethodsExperimental determination of 3- and 4-CMC binding degree with HSA was performed using gas chromatography–tandem mass spectrometry preceded by the equilibrium dialysis (ED) and ultrafiltration (UF). Nuclear magnetic resonance (NMR) spectroscopy was used to determine 3- and 4-CMC epitope-binding maps and possible binding sites in HSA. The molecular docking and molecular dynamics were employed to obtain detailed information about binding modes of 3- and 4-CMC enantiomers in HSA.ResultsAs follows from the presented data, the degree of binding of 3- and 4-CMC is at a similar level of approx. 80%. This indicates a relatively strong binding of CMC to plasma proteins. The model studies employing the NMR spectroscopy and molecular simulations indicate that both CMCs bind to HSA. The whole 3- and 4-CMC molecules are embedded in the binding sites, with aromatic moieties being in the closest contact with the HSA residues. Moreover, conducted experiments show that Sudlow site II is the main binding center for 3- and 4-CMC and Sudlow site I acts as the secondary binding site.ConclusionsAlthough many studies describe pharmacological and toxicological properties of synthetic cathinones (SC), the data taking SCs binding in plasma into consideration are scarce. To our knowledge, this is the first report presenting comprehensive experimental and theoretical characterization of 3- and 4-CMC binding with plasma proteins.

Multi-cavity molecular descriptor interconnections: Enhanced protocol for prediction of serum albumin drug binding

The role of human serum albumin (HSA) in the transport of molecules predicates its involvement in the determination of drug distribution and metabolism. Optimization of ADME properties are analogous to HSA binding thus this is imperative to the drug discovery process. Currently, various in silico predictive tools exist to complement the drug discovery process, however, the prediction of possible ligand-binding sites on HSA has posed several challenges. Herein, we present a strong and deeper-than-surface case for the prediction of HSA-ligand binding sites using multi-cavity molecular descriptors by exploiting all experimentally available and crystallized HSA-bound drugs. Unlike previously proposed models found in literature, we established an in-depth correlation between the physicochemical properties of available crystallized HSA-bound drugs and different HSA binding site characteristics to precisely predict the binding sites of investigational molecules. Molecular descriptors such as the number of hydrogen bond donors (nHD), number of heteroatoms (nHet), topological polar surface area (TPSA), molecular weight (MW), and distribution coefficient (LogD) were correlated against HSA binding site characteristics, including hydrophobicity, hydrophilicity, enclosure, exposure, contact, site volume, and donor/acceptor ratio. Molecular descriptors nHD, TPSA, LogD, nHet, and MW were found to possess the most inherent capacities providing baseline information for the prediction of serum albumin binding site. We believe that these associations may form the bedrock for establishing a solid correlation between the physicochemical properties and Albumin binding site architecture. Information presented in this report would serve as critical in provisions of rational drug designing as well as drug delivery, bioavailability, and pharmacokinetics.

Profiling in-vitro release of verteporfin from VISUDYNE® liposomal formulation and investigating verteporfin binding to human serum albumin

VISUDYNE® is a liposomal formulation of verteporfin, used in the photodynamic therapy of age-related macular degeneration via intravenous administration. In this study, we developed a new in vitro method to quantify verteporfin release from VISUDYNE® under conditions that replicate in vivo conditions using human serum albumin (HSA). Verteporfin release from the liposomes was quantified using capillary electrophoresis (CE) with optical detection. Verteporfin binding to HSA was quantified by measuring HSA fluorescence that is quenched by drugs binding to specific HSA binding sites. The binding constant of verteporfin to HSA was calculated using the Stern Volmer plot and found to be 1.966 × 107 M−1 at 37 °C. Verteporfin binding to HSA involves one albumin binding site and the binding molar ratio between verteporfin and HSA is approximately 1:1. A rapid partitioning of verteporfin from VISUDYNE® onto HSA takes place within 10 min and involves the release of more than 90% of the verteporfin at physiological temperatures. This study verifies this approach of using CE to rapidly separate liposome and HSA-bound drug, thus minimizing drug release artifacts created with other methods.

Molecular docking approach on the binding stability of derivatives of phenolic acids (DPAs) with Human Serum Albumin (HSA): Hydrogen-bonding versus hydrophobic interactions or combined influences?

Molecular docking (Mol.Doc) techniques were employed to ascertain the binding affinity and energetics of hydroxy derivatives of benzoic and cinnamic acids extract from Psidium guajava L. with Human Serum Albumin (HSA). Caffeic acid (CA), Ferullic acid (FA), Sinapic acid (SA), Syringic acid (SyA) and Vanillic acid (VA) are the derivatives phenolic acids (DPA) employed in docking studies which acts as the guest molecule. Docking of various feasible conformers of DPA with HSA (host) was explored and these conformers were categorized based on the docking score which is correlated to the binding energy (BE) and the stability depends upon the molecular interactions. Among the phenolic acids, SA-HSA complex was energetically more favorable and feasible based on BE and the order of binding stability upon complex formation of various DPA-HSA follows the order SA > FA = CA > VA > SyA, though SA and SyA are structurally similar to each other, likewise FA and VA exhibit a similar structure. The stability upon complex formation is correlated to the docking of the guest molecule in the binding domains of HSA and several molecular interactions. Hydrogen-bonding (HB) interaction governs the stability of host-guest complex is established. Interestingly, the presence of multiple hydrophobic interactions (pi-pi, pi-alkyl, pi-cation or anion, pi-sigma and pi-amide) competes over HB interaction in several conformers resulting in a decrease in BE. We report that SA acts as an excellent site selective and site-specific ligand that prefers to dock in Sudlow binding site II comprising of sub domains IIIA and IIIB respectively. However, all other phenolic acids do not behave neither as site selective nor site specific ligand such that they prefer to reside both in site II and site III (non-Sudlow binding site) of HSA. We authenticate that all the DPA as well as the amino acid moieties in HSA act as HB donor as well as acceptor sites apart from several hydrophobic interactions. We further establish that all the DPA has the least probable affinity to reside in binding site I (warfarin binding site), whereas sub domain IIIA of site II is the most preferred site which is energetically most favoured among all the sub domains.

Insights into the binding of half-sandwich phosphino Ir(III) and Ru(II) complexes to deoxyribonucleic acid, albumin and apo-transferrin: Experimental and theoretical investigation

A group of cytotoxic half-sandwich iridium(III) (Ir(η5-Cp*)Cl2PPh2CH2OH (IrPOH)), (Ir(η5-Cp*)Cl2P(p-OCH3Ph)2CH2OH (IrMPOH)), and ruthenium(II) (Ru(η6-p-cymene)Cl2PPh2CH2OH (RuPOH), Ru(η6-p-cymene)Cl2P(p-OCH3Ph)2CH2OH (RuMPOH)) complexes with phosphine ligands exhibit the ability to (i) slow hydrolysis which is reversed by adding a high NaCl concentration; (ii) oxidation of NADH to NAD+; (iii) induction of cytotoxicity towards various cancer cell lines. Furthermore, we found that RuPOH and RuMPOH selectively inhibit the proliferation of skin cancer cells (WM266-4) while Ir(III) complexes were found to be moderate against prostate cancer cells (DU-145). Herein, to elucidate the cytotoxic effects, we investigated the interaction of these complexes with DNA and serum proteins by gel electrophoresis, fluorescence spectroscopy, and molecular docking studies. Fluorescence spectroscopic data (calf thymus DNA: CT-DNA titration), together with analysis of DNA fragmentation (gel electrophoresis) and molecular docking provided evidence for the multimodal interaction of Ir(III) and Ru(III) complexes with DNA with predominance of intercalation and minor groove binding. All examined complexes caused single-stranded cleavage of the sugar–phosphate backbone of plasmid DNA. The affinity of the complexes for apo-transferrin (apo-Tf) and human serum albumin (HSA) was evaluated by fluorescence emission spectroscopy to calculate the binding constants which suggested a tight and reversible binding. Moreover, ruthenium complexes can mimic the binding of iron compounds to specific biomolecules such as albumin and transferrin better than iridium complexes. In silico study indicate that complexes mostly bind to (i) apo-Tf with a preference for a single binding site and/or (ii) to dock within all the four predicted binding sites of HSA with the predominance of site I which include tryptophan residues of HSA. This class of ruthenium(II) and iridium(III) complexes has unusual features worthy of further exploration in the design of novel anticancer drugs.

Theoretical and experimental investigation of novel quinazoline derivatives: synthesis, photophysical, reactive properties, molecular docking and selective HSA biointeraction

Two new quinazoline derivatives (2a and 2b) were successfully synthesized in this work using the condensation technique in excellent yields. Using spectroscopic techniques and elemental analyses, the compounds were completely characterized. Density functional theory (DFT) computations have been used to examine the title compound’s reactive characteristics. Chemical reactivity was predicted using local reactive descriptors and molecule electrostatic potential. Additionally, Time dependent DFT (TD-DFT) simulations were used to examine the impact of solvents on the photophysical characteristics. The affinity of compounds 2a and 2b for human serum albumin (HSA) was further explored using several electronic spectroscopies. Through static mechanisms, both compounds reduce the intrinsic fluorescence of HSA. It is determined that the HSA-2b complex’s binding constant is significantly greater than the HSA-2a complex. The fluorescence spectrum measurements proved that the HSA underwent structural changes after interaction with these compounds. It was demonstrated by site marker competitive displacement studies that compounds 2a and 2b preferred to bind to site I in HSA subdomain IIA. Additionally, synchronised fluorescence spectra were utilized to analyze how HSA’s conformation changed after interacting with various substances. The molecular docking investigations of these compounds with the three critical HSA binding sites, comprising subdomains IIA, IIIA, and IB, further confirmed the experimental findings. The significant contact between the investigated compounds and HSA was supported by the docking simulations. Communicated by Ramaswamy H. Sarma

Low-dimensional compounds containing bioactive ligands. XXII. First crystal structure, cytotoxic activity and DNA and HSA binding of a zirconium(IV) complex with 8-hydroxyquinoline-2-carboxylic acid.

A new zirconium(IV) complex, diaquabis(8-hydroxyquinoline-2-carboxylato-κ3N,O2,O8)zirconium(IV) dimethylformamide disolvate, [Zr(C10H5NO3)2(H2O)2]·2C3H7NO or [Zr(QCa)2(H2O)2]·2DMF (1) (HQCaH is 8-hydroxyquinoline-2-carboxylic acid and DMF is dimethylformamide), was prepared and characterized by elemental analysis, IR spectroscopy and single-crystal X-ray structure analysis. Complex 1 is a mononuclear complex in which the ZrIV atoms sit on the twofold axis and they are octacoordinated by two N and six O atoms of two tridentate anionic QCa2- ligands, and two aqua ligands. Outside the coordination sphere are two DMF molecules bound to the complex unit by hydrogen bonds. The structure and stability of complex 1 in dimethyl sulfoxide were verified by NMR spectroscopy. The cytotoxic properties of 1 and HQCaH were studied in vitro against eight cancer cell lines, and their selectivity was tested on the BJ-5ta noncancerous cell line. Both the complex and HQCaH exhibited low activity, with IC50 > 200 µM. DNA and human serum albumin (HSA) binding studies showed that 1 binds to calf thymus (CT) DNA via intercalation and is able to bind to the tryptophan binding site of HSA (Trp-214).

More Effective Mobilization of Hg2+ from Human Serum Albumin Compared to Cd2+ by L-Cysteine at Near-Physiological Conditions.

Although chronic low-level exposure to Hg2+ and Cd2+ causes human nephrotoxicity, the bioinorganic processes that deliver them to their target organs are poorly understood. Since the plasma protein human serum albumin (HSA) has distinct binding sites for these metal ions, we wanted to gain insight into these translocation processes and have employed size-exclusion chromatography coupled on-line to an inductively coupled plasma atomic emission spectrometer using phosphate-buffered saline mobile phases. When HSA 'labeled' with Hg2+ and Cd2+ (1:0.1:0.1) using 300 μM of L-methionine was analyzed, the co-elution of a single C, S, Cd, and Hg peak was observed, which implied the intact bis-metalated HSA complex. Since human plasma contains small molecular weight thiols and sulfur-containing metabolites, we analyzed the bis-metalated HSA complex with mobile phases containing 50-200 µM of L-cysteine (Cys), D,L-homocysteine (hCys), or glutathione (GSH), which provided insight into the comparative mobilization of each metal from their respective binding sites on HSA. Interestingly, 50 µM Cys, hCys, or GSH mobilized Hg2+ from its HSA binding site but only partially mobilized Cd2+ from its binding site. Since these findings were obtained at conditions simulating near-physiological conditions of plasma, they provide a feasible explanation for the higher 'mobility' of Hg2+ and its concomitant interaction with mammalian target organs compared to Cd2+. Furthermore, 50 µM Cys resulted in the co-elution of similar-sized Hg and Cd species, which provides a biomolecular explanation for the nephrotoxicity of Hg2+ and Cd2+.

Binding studies of synthetic cathinones to human serum albumin by high-performance affinity chromatography

The binding affinity to human serum albumin (HSA) of a series of fourteen synthetic cathinones, new psychoactive substances widely abused, was investigated by high-performance affinity chromatography (HPAC). Zonal elution experiments were conducted to measure the retention times of each synthetic cathinone on an HSA column, which enabled the calculation of the percentage of the drug bound. For some synthetic cathinones, enantioselectivity on HSA was found. To gather information on the HSA binding sites and better understand the chiral recognition mechanisms, enantioresolution of selected cathinones was carried out at a milligram scale through liquid chromatography (LC) with carbamate polysaccharide-based columns. This work was followed by zonal displacement chromatography using known competitors with specific binding sites on HSA, namely (S)-ibuprofen and warfarin. Competition was observed between the tested drugs and both competitors (except for pentedrone with warfarin), which is consistent with an allosteric competition involving a non-cooperative binding mechanism.

Probing the Binding Mechanism of Acylated Peptides to Human Serum Albumin

Peptides represent an increasingly important class of pharmaceutical products. During the last decade or so, acylation with fatty acids has demonstrated considerable success in prolonging the circulating half-life of therapeutic peptides by exploiting the ability of fatty acids to reversibly bind to human serum albumin (HSA), thus significantly impacting their pharmacological profiles. Employing methyl-13C-labeled oleic acid or palmitic acid as probe molecules and exploiting HSA mutants designed to probe fatty acid binding, the signals in two-dimensional (2D) nuclear magnetic resonance (NMR) spectra corresponding to high-affinity fatty acid binding sites in HSA were assigned. Subsequently, using a set of selected acylated peptides, competitive displacement experiments by 2D NMR identified a primary fatty acid binding site in HSA utilized in acylated peptide binding. These results represent an important first step toward understanding the structural basis for acylated peptides binding to HSA.

A comprehensive biophysical and theoretical study on the binding of dexlansoprazole with human serum albumin

Pharmacokinetics, pharmacodynamics, and therapeutic activity of drugs are defined by the affinities between the drug and biological macromolecules through molecular recognition. The present work is on the investigation of the interaction of new generation competent proton pump inhibitor dexlansoprazole (DLP) with human serum albumin (HSA). Fluorescence and UV–visible spectroscopy studies were adopted for quantification of the interaction in terms of the quenching constant (KSV), bimolecular quenching constant (Kq), equilibrium-binding constant (Kb), the number of binding sites (n), and the thermodynamic parameters. A hypochromic effect on the addition of HSA to DLP from the absorption studies shows their interaction. The quenching and binding constants from fluorescence studies were 104 and 105 M−1 order, respectively. A higher quenching constant with increasing temperature reflects a collisional-type quenching mechanism. A higher binding constant at a higher temperature shows an increase in the rate of binding, and the negative value of the free energy change shows the spontaneity of the binding. The sign and magnitude of the change in entropy (ΔS0 = 737.12 ± 2 J mol−1 K−1) and enthalpy (ΔH0 = 193.58 ± 1.7 kJ mol−1) shows that binding is an entropy-driven process with hydrophobic force as the major driving force. Site marker competitive displacement studies using 1-Anilino-8-naphthalene sulfonate (ANS), warfarin, and ibuprofen confirmed the biding of DLP in subdomain IIA of HSA. Binding-induced structural changes were evident from the changes in the circular dichroism (CD) signatures. Molecular docking of DLP in HSA showed the position of DLP in the warfarin binding site of HSA and corroborated the experimental findings.

Detailed Experimental and In Silico Investigation of Indomethacin Binding with Human Serum Albumin Considering Primary and Secondary Binding Sites

The interaction of indomethacin with human serum albumin (HSA) has been studied here considering the primary and secondary binding sites. The Stern-Volmer plots were linear in the lower concentration range of indomethacin while a downward curvature was observed in the higher concentration range, suggesting the presence of more than one binding site for indomethacin inside HSA due to which the microenvironment of the fluorophore changed slightly and some of its fraction was not accessible to the quencher. The Stern-Volmer quenching constants (KSV) for the primary and secondary sites were calculated from the two linear portions of the Stern-Volmer plots. There was around a two-fold decrease in the quenching constants for the low-affinity site as compared to the primary binding site. The interaction takes place via a static quenching mechanism and the KSV decreases at both primary and secondary sites upon increasing the temperature. The binding constants were also evaluated, which show strong binding at the primary site and fair binding at the secondary site. The binding was thermodynamically favorable with the liberation of heat and the ordering of the system. In principle, hydrogen bonding and Van der Waals forces were involved in the binding at the primary site while the low-affinity site interacted through hydrophobic forces only. The competitive binding was also evaluated using warfarin, ibuprofen, hemin, and a warfarin + hemin combination as site markers. The binding profile remained unchanged in the presence of ibuprofen, whereas it decreased in the presence of both warfarin and hemin with a straight line in the Stern-Volmer plots. The reduction in the binding was at a maximum when both warfarin and hemin were present simultaneously with the downward curvature in the Stern-Volmer plots at higher concentrations of indomethacin. The secondary structure of HSA also changes slightly in the presence of higher concentrations of indomethacin. Molecular dynamics simulations were performed at the primary and secondary binding sites of HSA which are drug site 1 (located in the subdomain IIA of the protein) and the hemin binding site (located in subdomain IB), respectively. From the results obtained from molecular docking and MD simulation, the indomethacin molecule showed more binding affinity towards drug site 1 followed by the other two sites.

Molecular Docking approach on the effect of Site- Selective and Site-Specific Drugs on the Molecular Interactions of Human Serum Albumin (HSA) -Acridinedione dye complex

Molecular Docking (Mol.dock) of resorcinol based acridinedione dyes (ADR1 and ADR2) with a globular protein, Human Serum Albumin (HSA) were carried out. Docking studies reveal that ADR2 dye binding with HSA is energetically more stable and feasible than ADR1 dye. ADR1 dye predominantly resides in site I and III of HSA rather than binding site II wherein, ADR1 dye acts as hydrogen bonding (HB) acceptor through its carbonyl oxygen. On the contrary, ADR2 dye resides in all the binding sites of HSA such that the dye acts as the HB donor through the NH hydrogen atom and the carbonyl oxygen of the amino acid acts as the HB acceptor. The stability of dye-protein complex in the presence of several non-steroidal anti-inflammatory drugs (NSAIDs) was carried out by employing specific site selective drugs (Sudlow binding site drugs). The energetics and the bimolecular interactions of various drugs with ADR1-HSA and ADR2-HSA were generated to ascertain the influence of drug and its governance on the binding affinity of dye-protein complex. Sudlow site I binding drugs were effective in decreasing the energetics of ADR1 dye-HSA complex whereas site II binding drugs predominantly decreases the affinity of ADR2 dye with HSA. However, the dyes efficiently displaces the site specific drugs from their specific binding sites of HSA which was not observed in the case of drugs on the displacing ability over dyes situated in different domains of protein. Mol.dock studies are employed as an authentic, reliable and most effective tool to ascertain the binding stability of host–guest complex as well as to ascertain the most probable location of several competing ligands in various domains of HSA.

Exploring molecular interaction of cefpirome with human serum albumin: In vitro and in silico approaches

Cefpirome (CFP), a fourth-generation cephalosporin for parenteral administration, is highly influential against gram-negative and gram-positive bacteria. Multiple approaches, such as fluorescence, absorption, voltammetric, and molecular docking were applied to assess the binding of CFP to the primary carrier protein, human serum albumin (HSA). The CFP-triggered quenching of HSA fluorescence reported the CFP−HSA complex formation, which was accomplished by a static quenching mechanism. A moderate binding affinity (Kf = 1.78 × 104 M−1 at 298 K) was found in the CFP−HSA interaction. The complex was anticipated to be stabilized by the presence of van der Waals interactions and hydrogen bonds. Upon CFP binding, microenvironmental alterations surrounding Trp and Tyr residues of HSA were observed. To establish the location of the CFP binding site in HSA, molecular docking studies were conducted, and the results indicated that CFP attaches to site I of HSA mostly through hydrophobic interactions and hydrogen bonding. The results of fluorescence spectroscopy and molecular docking supported one another quite well.

Studies on the Interaction of Rose Bengal with the Human Serum Albumin Protein under Spectroscopic and Docking Simulations Aspects in the Characterization of Binding Sites

Rose Bengal (RB) is a xanthene dye used as a sensitizer to convert triplet (3O2) to singlet oxygen (1O2). This photophysical property makes it one of the most used dyes in photodynamic therapy. Thus, understanding its interaction with biomacromolecules can provide helpful information about its mode of action and application. The protein chosen for this study was human serum albumin (HSA), which has nine binding sites for fatty acids (FA), and at least three sites for interactions of drugs (DS). The complexation of HSA with RB caused a maximum bathochromic shift in its absorption. From this displacement and the application of the Benesi–Hildebrand model, the ligand–protein association constant (3.90 ± 0.08 × 105 M−1) was obtained. Applying the Job’s Plot method resulted in a 6:1 (ligand-protein) stoichiometry. The determination of preferred binding sites was performed by measuring the association constant in the presence of drugs for which their binding sites in HSA are already well established, such as warfarin (DS1), ibuprofen (DS2 and FA6), digitoxin (DS3), diazepam (DS2), and diflunisal (DS2 and FA6). From these studies, it was found that RB is able to bind at DS1, DS3, and FA6 sites but not at DS2. Subsequently, molecular docking studies using the 2BX8 and 2BXE crystallographic structures were performed and corroborated the experimental results. The lowest energy poses were −52.13, −58.79, and −67.55 kcal mol−1 at DS1, DS3, and FA6, respectively. Conversely, DS2 was the lower affinity binding site. In conclusion, HSA has a high affinity for RB, being able to bind up to six dye molecules.