Sudlow's Site Research Articles

A cobalt coordination complex binds on a unique binding site between domain-I and domain-III of serum albumin

Serum albumin binding influences physiological effects and pharmacokinetics of drugs. Major binding sites in serum albumin are sudlow sites I and II, located on domains IIA and IIIA, respectively. However, groove between domain I and III as a binding site is not well studied. In this work, we studied the binding of a cobalt coordination complex (DCoP) with bovine serum albumin (BSA) using multi-spectroscopic methods and molecular docking. DCoP strongly interacts with BSA with association constant of ∼7 × 105M−1. The binding was found to significantly alter protein's conformation and unfolding pathway. Results of this study shows that DCoP binds at the groove formed between domains III and I. We conclude that the groove is a rare binding site for large molecules and coordination complexes. The knowledge of unique binding site of serum albumin enlightens the comprehensive role of protein as a carrier biomacromolecule.

Generation of affinity maps for thiazolidinediones with human serum albumin using affinity microcolumns. II. Effects of advanced glycation end products on multisite drug binding

Multisite protein interactions by the thiazolidinedione-class drugs pioglitazone and rosiglitazone were examined by using high-performance affinity microcolumns that contained normal human serum albumin (HSA) vs HSA that had been modified to form advanced glycation end products by glyoxal (Go) or methylglyoxal (MGo). The results were used to generate an affinity map for these drugs at several key regions on HSA. Strong binding (∼105 M−1) by these drugs was seen at both Sudlow sites I and II. About a 50 % decrease in the affinities at Sudlow site II was observed for pioglitazone for Go-modified HSA, while either a 47 % decrease or 1.6-fold increase in affinity was seen for MGo-modified HSA, depending on the extent of modification. The binding affinity for rosiglitazone at Sudlow site II had a 40–83 % decrease for Go-modified HSA and either a non-significant change or 1.4-fold increase for MGo-modified HSA. At Sudlow site I, pioglitazone gave a 41 % decrease in affinity for either Go or MGo-modified HSA, and for rosiglitazone up to a 55 % decrease or 1.3-fold increase in affinity was noted. Positive allosteric effects were seen by these drugs with the tamoxifen site of HSA, and neither drug had any notable binding at the digitoxin site for the normal or modified forms of HSA. Rosiglitazone also had weak interactions at a site in subdomain IB, which increased in affinity by up to 5.0-fold with the Go- or MGo-modified HSA. This study illustrated how affinity microcolumns can be used to provide a detailed analysis of solute-protein systems that involve complex interactions. The data obtained should also be valuable in providing a better understanding of how drug interactions with HSA and other proteins can be altered by modifications of these binding agents in diseases such as diabetes.

Ligand-human serum albumin analysis: the near-UV CD and UV-Vis spectroscopic studies.

Spectroscopic methods offer many new opportunities to study protein-ligand interactions. The aim of this study was to evaluate the possibility of using near-UV CD as well as UV-Vis spectroscopic techniques to study the interaction between human serum albumin (HSA) and markers of Sudlow's site I (warfarin, phenylbutazone) and II (ketoprofen, ibuprofen), as well as prednisolone and indapamide. In order to perform the planned measurements, near-UV CD spectropolarimetry and UV-Vis spectrophotometry have been used. It has been demonstrated that both techniques allow for rapid evaluation of non-covalent interactions between HSA and ligand, as well as identification of the HSA aromatic amino acid residues involved in this process. The near-UV CD spectroscopic data were more valuable than the analysis based on the second derivative of differential UV-Vis absorption spectra, especially for ligands with a non-specified binding site and low affinity towards HSA, such as prednisolone. The combination of both techniques makes it possible for comprehensive analysis of the interaction between HSA and ligands.

Elucidating the binding dynamics and structural impacts of Pt(II) and Pd(II) complexes on human serum albumin: A comprehensive spectroscopic and computational study

The binding of drugs to plasma proteins, such as human serum albumin (HSA), encompasses two main functions: depot and transport, which are performed by these proteins. This binding has significant pharmacodynamic and pharmacokinetic consequences. 2-(furan-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline 1,2-diamino cyclohexane platinum (Pt(II)) or palladium (Pd(II)) complexes, have a similar structure. These new synthetic complexes have desirable anticancer activity, and the binding of these complexes with HSA was investigated. Spectroscopic and thermodynamic analyses indicate that the interactions between HSA and the complexes occur spontaneously with the static-dynamic quenching mechanism. However, the interactions are primarily driven by noncovalent forces such as hydrophobic interactions and π–π stacking, as evidenced by the positive enthalpy and entropy values associated with these complexes. In the circular dichroism (CD) results, HSA had fewer structural changes with the addition of the Pt(II) than the Pd(II) complex. Docking studies were performed at three Sudlow sites, I, II, and III, to identify the complex’s binding site on HSA. The reactive sites of HSA to Pt(II) and Pd(II) complexes mainly reside within its hydrophobic cavity in domain II (site II). Furthermore, molecular dynamics simulations showed low RMSD, Rg, and binding energy values during the 400 ns for the Pt(II) complex. The results suggest that the connection between the Pt(II) complex and HSA is reversible. The Pt(II) complex was a more desirable candidate than the Pd(II) complex. The results of the experimental methods are consistent with those of dynamic simulation and molecular docking methods, indicating that HSA is more stable in the presence of the Pt(II) complex.

The study on characterization of interaction between casticin and model proteins using spectroscopic and computational methodologies

The present studies were conducted to investigate the interaction of casticin to model proteins (BSA, HSA, OVA and HEL) using fluorescence spectra, Fourier transform infrared spectra, molecular docking and molecular dynamics simulation methodologies. Our work displayed that the endogenous fluorescence intensities of BSA, HSA, OVA and HEL were quenched by various concentrations of casticin were the typical static quenching process. The binding constants (Ka) of casticin for these model proteins were in the range of 104–105 mol·L-1, with the highest affinity for HSA. On the basis of thermodynamic parameters and computational simulation studies, it was suggested that the binding force of casticin to BSA, HSA, OVA and HEL were mainly hydrogen bonds, van der Waals forces and hydrophobic interactions. The spectral results illustrated that the binding of casticin to BSA, HSA, OVA and HEL induced conformational and microenvironmental changes. Casticin could decrease the α-helix percentages of BSA, HSA and OVA, but increase that of HEL. Binding substitution studies confirmed that casticin bound to the Sudlow site I of BSA and HSA. Molecular docking predicted the binding models of casticin to BSA, HSA, OVA and HEL, followed closely by molecular dynamic simulations to assess the binding stability.

Association mechanism of bicalutamide and human serum albumin for potential clinical implications.

The binding mechanism of molecular interaction between bicalutamide and human serum albumin (HSA) in a pH7.4 phosphate buffer was studied using various spectroscopic techniques in combination with molecular modeling. Fluorescence data revealed that the fluorescence quenching of HSA by bicalutamide was a static quenching procedure. The binding constants and number of binding sites were evaluated at different temperatures. The thermodynamic parameters, ΔH and ΔS, were calculated to be 4.30 × 104J·mol-1 and 245 J·mol-1·K-1, respectively, suggesting that the binding of bicalutamide to HSA was driven mainly by hydrophobic interactions and hydrogen bonds. The displacement studies indicated neither Sudlow's site I nor II but subdomain IB as the main binding site for bicalutamide on HSA. The binding distance between bicalutamide and HSA was determined to be 3.54 nm based on the Förster theory. Analysis of circular dichroism, synchronous, and 3D fluorescence spectra demonstrated that HSA conformation was slightly altered in the presence of bicalutamide.

Spectroscopic and computational study of molecular interaction of Pexidartinib with homologous mammalian transport proteins

Binding of biologically important molecules with plasma proteins highly influence its availability, distribution, and metabolism, and thus has great significance in determining its therapeutic efficiency. Hence, studying its interaction with plasma proteins is prime and inevitable. Herein, we have investigated the molecular interaction of Pexidartinib, a novel, primitive and highly selective therapeutical agent against CSF-1R overexpression, with human serum albumin (HSA) and bovine serum albumin (BSA) using various spectroscopic methods, docking and simulations. The intrinsic fluorescence of the proteins considerably quenched on PEX addition accompanied by a slight blue shift. The complex formation between PEX and BSA/HSA induced some alterations in the molecular milieu of the tryptophan residues. The active binding locus was found to be within the hydrophobic cavity of Sudlow site I of both BSA and HSA. The binding dynamics suggest the interplay of hydrogen bonding and hydrophobic interactions in complex stabilisation. MD simulations provides valuable insights into the dynamic facets of the molecular recognition processes involved and governs the stability factor of protein post ligand complexation.

The hemostatic molecular mechanism of Sanguisorbae Radix's pharmacological active components based on HSA: Spectroscopic investigations, molecular docking and dynamics simulation

The interactions between human serum albumin (HSA) and the hemostatic components of the Chinese medicine Sanguisorbae Radix (SR), specifically phenolic acid compounds such as caffeic acid (CA), ferulic acid (FA) and their 1:1 mixture (1:1) were studied to investigate the molecular mechanism underlying the hemostatic effect of SR. Network pharmacology combined with the experimental and computational data revealed that HSA is one of the hemostatic targets to SR phenolic acids. SDS-PAGE and multi-spectroscopy demonstrated that the phenolic acids bind to the Sudlow site I on HSA, altering its structure and influencing its migration velocity. There is an observed synergistic effect upon the mixture of CA and FA. Quantum chemistry, molecular docking, and molecular dynamics simulations indicate that the binding of phenolic acids to HSA is stable, and variations in binding efficiency are associated with the hydrophobicity of the substituent at the C3 position of the side chain, and also, the key amino acids and functional groups for hemostasis of SR were identified, along with the active sites that contribute to the synergistic enhancement by phenolic acids.

Exploring the Binding Mechanism of 5-HT7 Specific Benzoxazolone alkyl Piperazinium Derivatives: A Comprehensive Analysis Using Spectroscopic and Computational Approaches.

Recently, the 5-HT7 receptor has achieved greater attention in research fraternity due to the involvement of neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) in several neurological disorders. Targeting this neuroreceptor, we have synthesized six compounds named as butyl-benzoxazolone substituted piperazinium derivatives (BBOP) derivatives, abbreviated as L1-L6. These compounds have been evaluated for their binding interaction with BSA through photophysical and in-silico approaches. The UV absorption of these compounds with BSA at λmax = 280nm, showed an optical density (O.D.) in the range of 0.5-0.9, i.e., 21%-53% (L1max = 1.4, L5min = 0.7385) at varied concentrations (17μM-114μM). For fluorescence studies, the Ksv value varied inversely with temperature, which confirmed the static mechanism of quenching with L1 showing maximum quenching. The parameters (ΔH, ΔS) obtained from the thermodynamic study for interaction between BSA and L1-L6 were correlated with in-silico (molecular docking) data. The in-silico docking study showed hydrophobic and the Van der Waals forces were the most significant forces. Amino acid residues ARG 217 & TRP 213 (Sudlow Site I) and LYS 116 & GLU 125 (Sudlow Site II) of BSA were primarily involved in H-bonding.Furthermore, the catalytic activity of BSA for hydrolyzingdifferent chemical entities have monitored in the presence of L1-L6 through esterase-like assay with p-NPA as a substrate, to get more insight about the interaction with catalytic residues (LYS 414, LYS 413, and TYR 411) in BSA at site II. These findings showed the potential of these 5-HT7 markers as promising ligands with appropriate drug likeliness characteristics.

Unraveling the enigma: Probing the unconventional binding site and binding mechanism of entrectinib with human serum albumin through spectroscopic, molecular docking, and dynamic simulation analyses

Entrectinib (ENB), a novel potent tyrosine kinase inhibitor, has demonstrated promising activity against specific genetic alterations in various solid tumors. Investigating the interaction of ENB with human serum albumin (HSA) provides valuable insights into its binding characteristics and pharmacological behavior. The interaction between ENB and HSA was investigated using various spectroscopic techniques, molecular docking, and simulations approaches. Fluorescence spectroscopy revealed a consistent decrease in the intrinsic fluorescence of HSA upon binding to ENB, indicating a static binding mechanism. The binding constant calculated from Stern Volmer and double-log equations (KSV) and (K) were determined to be 4.11 x104 M−1 and 4.38 x104 M−1, respectively at 298 K, indicating moderate binding affinity. Thermodynamic analysis demonstrated a spontaneous interaction (ΔG < 0) with an enthalpy change (ΔH) of −5.59 kJ mol−1 and an entropy change (ΔS) of + 70.36 J mol−1 K−1, indicating that the interaction is primarily driven by electrostatic forces with potential involvement of hydrogen bonding. Synchronous and 3D fluorescence analysis indicated no significant changes in the microenvironment surrounding Tyr and Trp residues upon ENB binding. Near and far UV spectrophotometric analyses confirmed the formation of the ENB-HSA complex and suggested no conformational changes in the protein structure. Fluorescence measurements and binding competition analysis using site markers revealed that ENB selectively binds to the HSA Sudlow sites I and III, displacing phenylbutazone at both sites. Molecular docking studies confirmed the binding of ENB to Sudlow sites I and III and identified critical residues involved in the interaction. Molecular dynamics simulations further validated the stability of the ENB-HSA complex at both sites, with more favorable binding at site III, and minimal fluctuations observed in the root mean square deviation, root mean square fluctuation, and radius of gyration. Hydrogen bond analysis and solvent accessible surface area calculations supported the strong binding and accessibility of ENB within the HSA structure. Overall, the experimental and computational results provided valuable insights into the ENB-HSA interaction, elucidating the binding mechanism and key residues involved.

Aggregation of Apo/Glycated Human Serum Albumins and Aptamer-Saturated Graphene Quantum Dot: A Simulation Study.

Human serum albumin (HSA) is a protein carrier that transports a wide range of drugs and nutrients. The amount of glycated HSA (GHSA) is used as a diabetes biomarker. To quantify the GHSA amount, the fluorescent graphene-based aptasensor has been a successful method. In aptasensors, the key mechanism is the adsorption/desorption of albumin from the aptamer-graphene complex. Recently, the graphene quantum dot (GQD) has been reported to be an aptamer sorbent. Due to its comparable size to aptamers, it is attractive enough to explore the possibility of GQD as a part of an albumin aptasensor. Therefore, molecular dynamics (MD) simulations were performed here to reveal the binding mechanism of albumin to an aptamer-GQD complex in molecular detail. GQD saturated by albumin-selective aptamers (GQDA) is studied, and GHSA and HSA are studied in comparison to understand the effect of glycation. Fast and spontaneous albumin-GQDA binding was observed. While no specific GQDA-binding site on both albumins was found, the residues used for binding were confined to domains I and III for HSA and domains II and III for GHSA. Albumins were found to bind preferably to aptamers rather than to GQD. Lysines and arginines were the main contributors to binding. We also found the dissociation of GLC from all GHSA trajectories, which highlights the role of GQDA in interfering with the ligand binding affinity in Sudlow site I. The binding of GQDA appears to impair albumin structure and function. The insights obtained here will be useful for the future design of diabetes aptasensors.

#2765 Medium chain fatty acids are potent binding competitors to improve protein-bound uremic toxin clearance in hemodialysis

Abstract Background and Aims Protein-bound uremic toxins (PBUTs) remain a concerning burden in patients with end-stage renal disease (ESRD) since their removal during hemodialysis is limited due to their tight binding to albumin. By increasing their circulating free fraction during HD, “binding competitors” of albumin could be used to increase their dialytic clearance. Medium chain fatty acids (MCFAs, 6 to 12 carbon length) are potent candidates for that purpose. The present study aims to investigate whether MCFAs could serve as potential binding competitors of albumin to enhance the removal of PBUTs, specifically IS and p-CS, during hemodialysis. Method Hexanoate (C6), octanoate (C8), and decanoate (C10) were purchased from Sigma-Aldrich. The binding of MCFAs to albumin was investigated in silico and using fluorescent probes: warfarin (specific of Sudlow's site I) and dansylsarcosine (specific of Sudlow's site II). Indoxyl-sulfate (IS, final concentration 212 µM) and p-cresyl sulfate (p-CS, final concentration 250 µM) were added to a 600 µM bovine serum albumin-phosphate buffered saline solution. The free fractions of IS and p-CS were assayed with or without MCFAs (1-2 mM) using ultrafiltration devices in this solutions and in patients uremic plasma. To mimic the removal of PBUTs during a HD session, batches of 2 liters of fresh bovine blood were loaded with IS and p-CS (final concentration 200 µM). A 2-hours closed-loop HD session was performed using a Fresenius 5008 CorDiax HD generator (Fresenius, Germany). A solution of 224 mM of octanoate was perfused at the rate of 150 µL/min; A solution of saline 0.9% was used as a control. One milliliter of blood was sampled every 15 minutes through the arterial sampling port and concentration of IS and p-CS were assayed by HPLC coupled with fluorescence detection. The hemolytic effect of MCFAs was evaluated in vitro by assaying the free hemoglobin concentration. Results According to the in silico prediction, the binding was predicted to be almost complete for MCFAs ≥ C8 (Fig. 1). The enhanced protein binding appeared to be inversely correlated with water solubility, which became particularly low for MCFAs ≥ C10. Octanoate and decanoate were the more prone to displace dansylsarcosine from Sudlow's site II of albumin (which is the main binding site of IS and p-CS). In vitro, the incubation with 2 mM of octanoate and decanoate increased the free fraction of PBUTs from 12% to 53% for p-CS (4.4 fold, p &amp;lt; 0.05) and from 11% to 45% for IS (4.1 fold, p &amp;lt; 0.05). Octanoate was also able to increase the free fraction of PBUTs in patients uremic plasma (1.6-fold for IS, p &amp;lt; 0.05 and 4.8 fold for p-CS, &amp;lt;0.01). The per-dialytic infusion of octanoate significantly increased the fractional removal of p-CS (from 38% to 88%, p &amp;lt; 0.001) and IS (from 36% to 91%, p &amp;lt; 0.001). No significant hemolysis was observed for concentration of MCFAs lower than 2 mmol/L. Conclusion MCFAs and especially octanoate and decanoate are serious candidates to displace the binding of PBUTs such as p-CS and IS. The per-dialytic administration of octanoate significantly increased the removal of PBUTs and could constitute a new strategy to prevent their accumulation in end-stage kidney disease patients. Due to their good safety profile, MCFAs could be better tolerated than other chemical compounds that has already been tested in a clinical setting such as ibuprofen. Further in vivo studies are however needed to carefully evaluate this potentially new therapeutic option.

Exploring the molecular basis of tucatinib interaction with human serum albumin: A spectroscopic and computational analysis

This study delves into the kinetics of interaction among anticancer drug tucatinib (TCT) and human serum albumin (HSA), a pivotal protein that transports substances in the bloodstream of humans. Employing biophysical techniques alongside in-silico approaches, the TCT-HSA complex formation is substantiated. This binding interaction is unequivocally confirmed by the inverse relationship between temperature and Stern-Volmer constant (KSV) and the hyperchromicity in UV–visible spectra. The binding constant (Kb) value (5.34 x 104 M−1) indicates a moderate affinity between TCT and HSA, with thermodynamic analysis pointing towards stabilization through hydrogen bonds and hydrophobic and van der Waals interactions. Moreover, despite causing alterations in the vicinity of Tyr and Trp amino acids, the introduction of TCT to HSA confers a substantial protective effect against the thermal denaturation of the protein. Circular dichroism unveils a decrease in the content of alpha helices upon TCT binding. At the same time, CD thermal analysis hints at a marginal increase in the melting temperature of HSA, indicating heightened stability at elevated temperatures. Molecular docking and competitive displacement assays confirm that the preferred binding site for TCT is subdomain IIA (Sudlow's site I) of HSA. Molecular dynamics simulations emphasize the stability of the TCT-HSA complex. This work sheds light on a detailed characterization of the HSA-TCT interaction, elucidating binding mechanisms and their consequential impact on HSA structure and stability.

Effects of Chrysin and Chrysin-7-sulfate on Ochratoxin A-Albumin Interactions and on the Plasma and Kidney Levels of the Mycotoxin in Rats.

The nephrotoxic mycotoxin ochratoxin A (OTA) is a common food contaminant. OTA binds to the Sudlow's Site I region of serum albumin with very high affinity, resulting in its slow elimination. The displacement of OTA from albumin may be beneficial due to the faster excretion of the mycotoxin, while it may also lead to the increased tissue uptake of OTA. Furthermore, it is challenging to displace the mycotoxin from albumin even with high-affinity Site I ligands. In this study, we tested the impacts of Site I and Heme site ligands on OTA-albumin interactions by applying fluorescence spectroscopic, ultracentrifugation, and modeling studies. Chrysin-7-sulfate (C7S) strongly displaced OTA from both human and rat albumins; therefore, the impacts of C7S (single intravenous administration) and the parent flavonoid chrysin (repeated peroral treatment) were examined on the plasma and kidney levels of OTA in rats. Chrysin barely influenced the concentrations of mycotoxin in plasma and kidneys. In the first few hours, C7S significantly decreased the plasma levels of OTA compared to the control animals; while after 24 h, only minor differences were noticed. Our study highlights the superior displacing ability of C7S vs OTA regarding human and rat albumins.

Probing berberine syringate-bovine serum albumin interactions by multi-spectroscopic, conductimetric, volumetric and molecular docking methods

In this paper, a new berberine-based salt berberine syringate ([BBR][SA]) was synthesized and [BBR][SA]-bovine serum albumin (BSA) interactions were investigated by various techniques like UV–visible absorption, fluorescence spectroscopy, Fourier transform infrared spectroscopy (FT-IR), volumetry and conductimetry. Using the experimental values of density and conductivity, the specific volume of BSA and the critical aggregation concentration of [BBR][SA] were derived. From the variation trends of specific volume for BSA with [BBR][SA] concentrations, it was observed the predominate ionic interaction/dehydration effect at small [BBR][SA] concentration and the non-polar interaction/hydration phenomenon at higher [BBR][SA] concentration for the binding of [BBR][SA] with BSA, as well as hydration shell structure changes at different temperatures in the medium with different pH values. Aggregation of [BBR][SA] is slowed by increasing concentration of BSA and temperature. The thermodynamic parameters of aggregation process have been exploited to gain more information on the interaction of [BBR][SA] with BSA. UV–vis absorption, multiple fluorescence spectroscopy (steady-state spectroscopy, synchronous fluorescence, three-dimensional fluorescence), and FT-IR have been utilized to understand the interaction mechanisms and binding strength between the two molecules. [BBR][SA] quenches the fluorescence of BSA through static quenching mechanism. The 1:1 complex between [BBR][SA] and BSA with a strong affinity was formed. The changes in microenvironment and conformation of BSA caused by [BBR][SA] were revealed by synchronous fluorescence spectra, 3D fluorescence maps and FT-IR. The binding average distance from [BBR][SA] to BSA (2.83 nm) was obtained based on the theory of Förster energy transfer. Competitive binding experiment confirms [BBR][SA] binding to Sudlow site I (sub-domain IIA) of BSA. In addition, molecular docking was generated to provide valuable information about the available binding sites of [BBR][SA] on BSA and the types of binding forces.

Generation of affinity maps for thiazolidinediones with human serum albumin using affinity microcolumns. I. Studies of effects by glycation on multisite drug binding

Human serum albumin (HSA) is known to undergo modifications by glucose during diabetes. This process produces glycated HSA that can have altered binding to some drugs. In this study, high-performance affinity microcolumns and competition studies were used to see how glycation affects the binding by two thiazolidinedione-class drugs (i.e., pioglitazone and rosiglitazone) at specific regions of HSA. These regions included Sudlow sites I and II, the tamoxifen and digitoxin sites, and a drug-binding site located in subdomain IB. At Sudlow site II, the association equilibrium constants (or binding constants) for pioglitazone and rosiglitazone with normal HSA were 1.7 × 105 M−1 and 2.0 × 105 M−1 at pH 7.4 and 37 °C, with values that changed by up to 5.7-fold for glycated HSA. Sudlow site I of normal HSA had binding constants for pioglitazone and rosiglitazone of 3.4 × 105 M−1 and 4.6 × 105 M−1, with these values changing by up to 1.5-fold for glycated HSA. Rosiglitazone was found to also bind a second region that had a positive allosteric effect on Sudlow site I for all the tested preparations of HSA (binding affinity, 1.1–3.2 × 105 M−1; coupling constant for Sudlow site I, 1.20–1.34). Both drugs had a strong positive allosteric effect on the tamoxifen site of HSA (coupling constants, 13.7–19.9 for pioglitazone and 3.7–11.5 for rosiglitazone). Rosiglitazone also had weak interactions at a site in subdomain IB, with a binding constant of 1.4 × 103 M−1 for normal HSA and a value that was altered by up to 6.8-fold with glycated HSA. Neither of the tested drugs had any significant binding at the digitoxin site. The results were used to produce affinity maps that described binding by these thiazolidinediones with HSA and the effects of glycation on these interactions during diabetes.

Biophysical and structural characterization of tetramethrin serum protein complex and its toxicological implications.

Tetramethrin (TMT) is a commonly used insecticide and has a carcinogenic and neurodegenerative effect on humans. The binding mechanism and toxicological implications of TMT to human serum albumin (HSA) were examined in this study employing a combination of biophysical and computational methods indicating moderate binding affinity and potential hepato and renal toxicity. Fluorescence quenching experiments showed that TMT binds to HSA with a moderate affinity, and the binding process was spontaneous and predominantly enthalpy-driven. Circular dichroism spectroscopy revealed that TMT binding did not induce any significant conformational changes in HSA, resulting in no changes in its alpha-helix content. The binding site and modalities of TMT interactions with HSA as computed by molecular docking and molecular dynamics simulations revealed that it binds to Sudlow site II of HSA via hydrophobic interactions through its dimethylcyclopropane carboxylate methyl propanyl group. The structural dynamics of TMT induce proper fit into the binding site creating increased and stabilizing interactions. Additionally, molecular mechanics-Poisson Boltzmann surface area calculations also indicated that non-polar and van der Waals were found to be the major contributors to the high binding free energy of the complex. Quantum mechanics (QM) revealed the conformational energies of the binding confirmation and the degree of deviation from the global minimum energy conformation of TMT. The results of this study provide a comprehensive understanding of the binding mechanism of TMT with HSA, which is important for evaluating the toxicity of this insecticide in humans.

Effect of plasma exchange with albumin replacement on albumin functionality and organ dysfunction in acute-on-chronic liver failure

Effective treatments for acute-on-chronic liver failure (ACLF) are a major unmet need. This proof-of-concept pilot study was aimed at evaluating the effects of plasma exchange (PE) with albumin 5% (PE-A5%) on albumin functional capacity and organ dysfunction in patients with ACLF. Ten adult patients were enrolled in a single-center phase II, prospective, open-label, non-controlled study. Six PE-A5% sessions were performed in 10 days followed by a 1-month follow-up visit. Albumin functional capacity and circulatory function were assessed, as were renal, cerebral, and liver function, and systemic inflammation. The main safety variable was the percentage of PE sessions associated with at least one procedure-related adverse event (AE). Patients with ACLF showed lower albumin binding capacity, lower antioxidant capacity, and lower levels of albumin with preserved structure compared to healthy donors (n= 19). From baseline to day 11, PE-A5% treatment increased albumin levels and improved albumin binding capacity to Sudlow site II (15.3±1.6mg/ml to 18.9±1.7mg/ml; p= 0.003), fatty acid-binding capacity (8.2±1.4μM to 3.1±1.5μM; p= 0.013) and antioxidant capacity (human mercaptalbumin 9.5±1.5mg/ml to 14.6±1.6mg/ml; p= 0.001). Native albumin levels were increased throughout day 1-11PE-A5% sessions (6.5±1.0mg/ml to 10.2±1.4mg/ml; p= 0.035). PE-A5% improved systemic hemodynamics (mean arterial pressure, heart rate, cardiac index), renal function (creatinine level, blood urea nitrogen), cerebral function (hepatic encephalopathy grade), liver parameters (transaminases, bilirubin) and inflammatory parameters (C-reactive protein, leukocyte count). All patients had at least one of the 78 AEs reported, mostly mild (product/procedure-related: 36%). Sixteen serious AEs were reported in eight patients (procedure/product-related: none). PE-A5% was a safe procedure associated with positive effects on albumin functionality, and circulatory, renal, cerebral, and liver function in patients with ACLF. Acute-on-chronic liver failure (ACLF) is a clinical condition characterized by severe systemic inflammation, organ failure, and high mortality. Plasma exchange removes patient's plasma containing pathogenic substances, replacing it with 5% albumin and fresh frozen plasma (PE-A5%). In this study, cirrhotic patients with ACLF were treated with PE-A5%, which was a safe procedure that increased binding and antioxidant capacity of patients' albumin, while improving circulatory, kidney, brain, and liver functions. These beneficial effects could impact survival in ACLF. NCT01201720. 2010-021360-15.

Revealing the mechanistic interactions of profenofos and captan pesticides with serum protein via biophysical and computational investigations

Profenofos (PF) and captan (CT) are among the most utilized organophosphorus insecticides and phthalimide fungicides, respectively. To elucidate the physicochemical and influential toxicokinetic factors, the mechanistic interactions of serum albumin and either PF or CT were carried out in the current study using a series of spectroscopy and computational analyses. Both PF and CT could bind to bovine serum albumin (BSA), a representative serum protein, with moderate binding constants in a range of 103–104 M−1. The bindings of PF and CT did not induce noticeable BSA’s structural changes. Both pesticides bound preferentially to the site I pocket of BSA, where the hydrophobic interaction was the main binding mode of PF, and the electrostatic interaction drove the binding of CT. As a result, PF and CT may not only induce direct toxicity by themselves, but also compete with therapeutic drugs and essential substances to sit in the Sudlow site I of serum albumin, which may interfere with the pharmacokinetics and equilibrium of drugs and other substances causing consequent adverse effects.

Impact of Sinapic Acid on Bovine Serum Albumin Thermal Stability.

The thermal stability of bovine serum albumin (BSA) in Tris buffer, as well as the effect of sinapic acid (SA) on protein conformation were investigated via calorimetric (differential scanning microcalorimetry-μDSC), spectroscopic (dynamic light scattering-DLS; circular dichroism-CD), and molecular docking approaches. μDSC data revealed both the denaturation (endotherm) and aggregation (exotherm) of the protein, demonstrating the dual effect of SA on protein thermal stability. With an increase in ligand concentration, (i) protein denaturation shifts to a higher temperature (indicating native form stabilization), while (ii) the aggregation process shifts to a lower temperature (indicating enhanced reactivity of the denatured form). The stabilization effect of SA on the native structure of the protein was supported by CD results. High temperature (338 K) incubation induced protein unfolding and aggregation, and increasing the concentration of SA altered the size distribution of the protein population, as DLS measurements demonstrated. Complementary information offered by molecular docking allowed for the assessment of the ligand binding within the Sudlow's site I of the protein. The deeper insight into the SA-BSA interaction offered by the present study may serve in the clarification of ligand pharmacokinetics and pharmacodynamics, thus opening paths for future research and therapeutic applications.