Revisiting and Updating the Interaction between Human Serum Albumin and the Non-Steroidal Anti-Inflammatory Drugs Ketoprofen and Ketorolac.

Ketoprofen (KTF) and ketorolac (KTL) are among the most primarily used non-steroidal anti-inflammatory drugs (NSAIDs) in humans to alleviate moderate pain and to treat inflammation. Their binding affinity with albumin (the main globular protein responsible for the biodistribution of drugs in the bloodstream) was previously determined by spectroscopy without considering some conventional pitfalls. Thus, the present work updates the biophysical characterization of the interactions of HSA:KTF and HSA:KTL by 1H saturation-transfer difference nuclear magnetic resonance (1H STD-NMR), ultraviolet (UV) absorption, circular dichroism (CD), steady-state, and time-resolved fluorescence spectroscopies combined with in silico calculations. The binding of HSA:NSAIDs is spontaneous, endothermic, and entropically driven, leading to a conformational rearrangement of HSA with a slight decrease in the α-helix content (7.1% to 7.6%). The predominance of the static quenching mechanism (ground-state association) was identified. Thus, both Stern-Volmer quenching constant (KSV) and binding constant (Kb) values enabled the determination of the binding affinity. In this sense, the KSV and Kb values were found in the order of 104 M-1 at human body temperature, indicating moderate binding affinity with differences in the range of 0.7- and 3.4-fold between KTF and KTL, which agree with the previously reported experimental pharmacokinetic profile. According to 1H STD-NMR data combined with in silico calculations, the aromatic groups in relation to the aliphatic moiety of the drugs interact preferentially with HSA into subdomain IIIA (site II) and are stabilized by interactions via hydrogen bonding and hydrophobic forces. In general, the data obtained in this study have been revised and updated in comparison to those previously reported by other authors who did not account for inner filter corrections, spectral backgrounds, or the identification of the primary mathematical approach for determining the binding affinity of HSA:KTF and HSA:KTL.

Crystal Structure, Theoretical Analysis, and Protein/DNA Binding Activity of Iron(III) Complex Containing Differently Protonated Pyridoxal-S-Methyl-Isothiosemicarbazone Ligands.

Pyridoxal-S-methyl-isothiosemicarbazone (PLITSC) is a member of an important group of ligands characterized by different complexation modes to various transition metals. In this contribution, a new complex containing two differently protonated PLITSC ligands ([Fe(PLITSC-H)(PLITSC)]SO4)∙2.5H2O was obtained. The crystal structure was solved by the X-ray analysis and used further for the optimization at B3LYP/6-311++G(d,p)(H,C,N,O,S)/def2-TZVP(Fe) level of theory. Changes in the interaction strength and bond distance due to protonation were observed upon examination by the Quantum Theory of Atoms in Molecules. The protein binding affinity of [Fe(PLITSC-H)(PLITSC)]SO4 towards transport proteins (Bovine Serum Albumin (BSA) and Human Serum Albumin (HSA)) was investigated by the spectrofluorimetric titration and molecular docking. The interactions with the active pocket containing fluorescent amino acids were examined in detail, which explained the fluorescence quenching. The interactions between complex and DNA were followed by the ethidium-bromide displacement titration and molecular docking. The binding along the minor groove was the dominant process involving complex in the proximity of DNA.

The First In Vivo Study Shows That Gyrophoric Acid Changes Behavior of Healthy Laboratory Rats.

Gyrophoric acid (GA), a lichen secondary metabolite, has attracted more attention during the last years because of its potential biological effects. Until now, its effect in vivo has not yet been demonstrated. The aim of our study was to evaluate the basic physicochemical and pharmacokinetic properties of GA, which are directly associated with its biological activities. The stability of the GA in various pH was assessed by conducting repeated UV-VIS spectral measurements. Microsomal stability in rat liver microsomes was performed using Ultra-Performance LC/MS. Binding to human serum albumin (HSA) was assessed using synchronous fluorescence spectra, and molecular docking analysis was used to reveal the binding site of GA to HSA. In the in vivo experiment, 24 Sprague-Dawley rats (Velaz, Únetice, Czech Republic) were used. The animals were divided as follows. The first group (n = 6) included healthy males as control intact rats (♂INT), and the second group (n = 6) included healthy females as controls (♀INT). Groups three and four (♂GA/n = 6 and ♀GA/n = 6) consisted of animals with daily administered GA (10 mg/kg body weight) in an ethanol-water solution per os for a one-month period. We found that GA remained stable under various pH and temperature conditions. It bonded to human serum albumin with the binding constant 1.788 × 106 dm3mol-1 to reach the target tissue via this mechanism. In vivo, GA did not influence body mass gain, food, or fluid intake during the experiment. No liver toxicity was observed. However, GA increased the rearing frequency in behavioral tests (p < 0.01) and center crossings in the elevated plus-maze (p < 0.01 and p < 0.001, respectively). In addition, the time spent in the open arm was prolonged (p < 0.01 and p < 0.001, respectively). Notably, GA was able to pass through the blood-brain barrier, indicating its ability to permeate into the brain and to stimulate neurogenesis in the hilus and subgranular zone of the hippocampus. These observations highlight the potential role of GA in influencing brain function and neurogenesis.

Supported-amine-catalyzed cascade synthesis of spiro-thiazolone-tetrahydrothiophenes: assessing HSA binding activity.

We have devised a supported-amine-catalyzed efficient synthesis of spiro-thiazolone-tetrahydrothiophenes via a sulfa-Michael/aldol cascade approach. The catalyst demonstrated sustained efficacy over 21 cycles. These derivatives were found to exhibit excellent binding abilities with purified human serum albumin as indicated by both in silico and in vitro-based experiments.

Characterization of the structural and molecular interactions of Ferulic acid ethyl ester with human serum albumin and Lysozyme through multi-methods.

Ferulic acid ethyl ester (FAEE) is an essential raw material for the formulation of drugs for cardiovascular and cerebrovascular diseases and leukopenia. It is also used as a fixed aroma agent for food production due to its high pharmacological activity. In this study, the interaction of FAEE with Human serum albumin (HSA) and Lysozyme (LZM) was characterized by multi-spectrum and molecular dynamics simulations at four different temperatures. Additionally, the quenching mechanism of FAEE-HSA and FAEE-LZM were explored. Meanwhile, the binding constants, binding sites, thermodynamic parameters, molecular dynamics, molecular docking binding energy, and the influence of metal ions in the system were evaluated. The results of Synchronous fluorescence spectroscopy, UV-vis spectroscopy, CD, three-dimensional fluorescence spectrum, and resonance light scattering showed that the microenvironment of HSA and LZM and the protein conformation changed in the presence of FAEE. Furthermore, the effects of some common metal ions on the binding constants of FAEE-HSA and FAEE-LZM were investigated. Overall, the experimental results provide a theoretical basis for promoting the application of FAEE in the cosmetics, food, and pharmaceutical industries and significant guidance for food safety, drug design, and development.

Characterization and humanization of VNARs targeting human serum albumin from the whitespotted bamboo shark (Chiloscyllium plagiosum).

The Shark-derived immunoglobulin new antigen receptors (IgNARs) have gained increasing attention for their high solubility, exceptional thermal stability, and intricate sequence variation. In this study, we immunized whitespotted bamboo shark (Chiloscyllium plagiosum) to create phage display library of variable domains of IgNAR (VNARs) for screening against Human Serum Albumin (HSA), a versatile vehicle in circulation due to its long in vivo half-life. We identified two HSA-binding VNAR clones, 2G5 and 2G6, and enhanced their expression in E. coli with the FKPA chaperone. 2G6 exhibited a strong binding affinity of 13 nM with HSA and an EC50 of 1 nM. In vivo study with a murine model further provided initial validation of 2G6's ability to prolong circulation time by binding to HSA. Additionally, we employed computational molecular docking to predict the binding affinities of both 2G6 and its humanized derivative, H2G6, to HSA. Our analysis unveiled that the complementarity-determining regions (CDR1 and CDR3) are pivotal in the antigen recognition process. Therefore, our study has advanced the understanding of the potential applications of VNARs in biomedical research aimed at extending drug half-life, holding promise for future therapeutic and diagnostic progressions.

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.

Extension of the circulatory half-life of recombinant ecallantide via albumin fusion without loss of anti-kallikrein activity.

Ecallantide comprises Kunitz Domain 1 of Tissue Factor Pathway Inhibitor, mutated at seven amino acid positions to inhibit plasma kallikrein (PK). It is used to treat acute hereditary angioedema (HAE). We appended hexahistidine tags to the N- or C-terminus of recombinant Ecallantide (rEcall) and expressed and purified the resulting proteins, with or without fusion to human serum albumin (HSA), using Pichia pastoris. The inhibitory constant (Ki) of rEcall-H6 or H6-rEcall for PK was not increased by albumin fusion. When 125I-labelled rEcall proteins were injected intravenously into mice, the area under the clearance curve (AUC) was significantly increased, 3.4- and 3.6-fold, for fusion proteins H6-rEcall-HSA and HSA-rEcall-H6 versus their unfused counterparts but remained 2- to 3-fold less than that of HSA-H6. The terminal half-life of H6-rEcall-HSA and HSA-H6 did not differ, although that of HSA-rEcall-H6 was significantly shorter than either other protein. Receptor Associated Protein (RAP), a Low-density lipoprotein Receptor-related Protein (LRP1) antagonist, competed H6-rEcall-HSA clearance more effectively than intravenous immunoglobulin (IVIg), a neonatal Fc receptor (FcRn) antagonist. HSA fusion decreases rEcall clearance in vivo, but LRP1-mediated clearance remains more important than FcRn-mediated recycling for rEcall fusion proteins. The properties of H6-rEcall-HSA warrant investigation in a murine model of HAE.

Molecular Insights into the Conformational and Binding Behaviors of Human Serum Albumin Induced by Surface-Active Ionic Liquids.

Extensive research has been carried out to investigate the stability and function of human serum albumin (HSA) when exposed to surface-active ionic liquids (SAILs) with different head groups (imidazolium, morpholinium, and pyridinium) and alkyl chain lengths (ranging from decyl to tetradecyl). Analysis of the protein fluorescence spectra indicates noticeable changes in the secondary structure of HSA with varying concentrations of all SAILs tested. Helicity calculations based on the Fourier transform infrared (FTIR) data show that HSA becomes more organized at the micellar concentration of SAILs, leading to an increased protein activity at this level. Small-angle neutron scattering (SANS) data confirm the formation of a bead-necklace structure between the SAILs and HSA. Atomistic molecular dynamics (MD) simulation results identify several hotspots on the protein surface for interaction with SAIL, which results in the modulation of protein conformational fluctuation and stability. Furthermore, fluorescence resonance energy transfer (FRET) experiments with the intramolecular charge transfer (ICT) probe trans-ethyl p-(dimethylamino) cinnamate (EDAC) demonstrate that higher alkyl chain lengths and SAIL concentrations result in a significantly increased energy transfer efficiency. The findings of this study provide a detailed molecular-level understanding of how the protein structure and function are affected by the presence of SAILs, with potential implications for a wide range of applications involving protein-SAIL composite systems.

Structure-activity relationship between gold nanoclusters and human serum albumin: Effects of ligand isomerization.

The interactions between gold nanoclusters (AuNCs) and proteins have been extensively investigated. Nevertheless, the structure-activity relationship between gold nanoclusters and proteins in terms of ligand isomerization remained unclear. Here, interactions between Au25NCs modified with para-, inter- and ortho-mercaptobenzoic acid (p/m/o-MBA-Au25NCs) and human serum albumin (HSA) were analyzed. The results of the multispectral approach showed that all three gold nanoclusters bound to the site I in dynamic modes to increase the stability of HSA. There were significant differences in the binding intensity, thermodynamic parameters, main driving forces, and binding ratios between these three gold nanoclusters and HSA, which might be related to the existence forms of the three ligands on the surface of AuNCs. Due to the different polarities of AuNCs themselves, the impact of three AuNCs on the microenvironment of amino acid residues in HSA was also different. It could be seen that ligand isomerization significantly affected the interactions between gold nanoclusters and proteins. This work will provide theoretical guidance for ligand selection and biological applications of metal nanoclusters.

Albumin levels in malaria patients: a systematic review and meta-analysis of their association with disease severity.

Albumin, a key protein in human blood plasma, has been linked to various health conditions. However, its association with malaria, particularly in assessing disease severity, remains inadequately understood. This comprehensive systematic review and meta-analysis aimed to elucidate the relationship between albumin levels and malaria severity. A comprehensive literature search was conducted across multiple databases, including Embase, Scopus, PubMed, MEDLINE, Ovid, and Google Scholar, to identify studies examining albumin levels in malaria patients. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed. Data were pooled using a random-effects model, and heterogeneity was assessed using I2 statistics. Subgroup and meta-regression analyses were performed based on publication year, study location, and Plasmodium species. A total of 37 studies were included in this review. The thematic synthesis indicated that albumin levels in malaria patients varied significantly based on geographical location. A meta-analysis of 28 studies found that albumin levels were significantly lower in malaria patients compared with non-malarial controls (P < 0.001, standardized mean differences [SMD] = -2.23, 95% CI - 3.25 to - 1.20, I2: 98%, random effects model, 28 studies). Additionally, subgroup analysis revealed variations in albumin levels based on geographical location and Plasmodium species. Regarding the association with disease severity, thematic synthesis showed that severe malaria cases generally had decreased albumin levels across various regions. However, one Brazilian study reported higher albumin levels in severe cases. A separate meta-analysis of five studies found significantly lower albumin levels in patients experiencing severe malaria relative to those with less severe forms of the disease (P < 0.001, SMD = -0.66, 95% CI - 1.07 to - 0.25), I2: 73%, random effects model, 5 studies). This study underscores the clinical significance of albumin as a potential biomarker for Plasmodium infection and the severity of malaria. The findings suggest that albumin level monitoring could be crucial in managing malaria patients, especially in assessing disease severity and tailoring treatment approaches. Additional studies are required to investigate the underlying mechanisms driving these associations and validate the clinical utility of albumin levels in malaria patient management.

Computed Tomography-Assessed Sarcopenia Predicts Mortality in Kidney Transplant Candidates.

OBJECTIVES: Sarcopenia is common in chronic kidney disease and associated with increased mortality. We investigated the prevalence of sarcopenia, defined as low muscle mass by the psoas muscle index, in endstage renal disease patients on waiting lists for kidney transplant and determined its association with prognostic nutritional index, C-reactive protein-toalbumin ratio, cardiovascular events, and mortality. MATERIALS AND METHODS: Our study included 162 patients with end-stage renal disease and 87 agematched healthy controls. We calculated nutritional status as follows: prognostic nutritional index = (10 × albumin [g/dL]) + (0.005 × total lymphocyte count (×103/µL]) and C-reactive protein-to-albumin ratio. We gathered demographic and laboratory data from medical records. RESULTS: Patients with end-stage renal disease had a mean age of 44.7 ± 14.2 years; follow-up time was 3.37 years (range, 0.35-9.60 y). Although patients with endstage renal disease versus controls had higher prevalence of sarcopenia (16.7% vs 3.4%; P = .002) and C-reactive protein-to-albumin ratio (1.47 [range, 0.12-37.10] vs 0.74 [range, 0.21-10.20]; P < .001), prognostic nutritional index was lower (40 [range, 20.4-52.2] vs 44 [range, 36.1-53.0]; P < .001). In patients with end-stage renal disease with and without sarcopenia, prognostic nutritional index (P = .005) was lower and C-reactive protein-to-albumin ratio (P = .041) was higher in those with versus those without sarcopenia. Among 67 patients on waiting lists who received kidney transplants, those without sarcopenia had better 5-year patient survival posttransplant than those with sarcopenia (P = .001). Multivariate regression analysis showed sarcopenia and low prognostic nutritional index were independentrisk factors for mortality among patients with end-stage renal disease. CONCLUSIONS: Sarcopenia was ~5 times more frequent in patients with end-stage renal disease than in healthy controls and was positively correlated with the prognostic nutritional index. Sarcopenia was an independent risk factor for mortality in patients on transplant waiting lists.

Investigation of oxidant-antioxidant status in patients treated with hirudotherapy: an experimental study.

Objectives: To examine the influence of hirudotherapy on parameters of oxidative stress. METHODS: The cross-sectional study was conducted from March 29 to September 29, 2021, at the Alanya Research and Training Hospital's Traditional and Complementary Medicine Application Centre, Turkey, and comprised adult volunteers of either gender. The participants were subjected to two sessions of hirudotherapy 4 weeks apart. Total antioxidant status, total oxidant status, oxidative stress index values, ischaemia-modified albumin level, paraoxonase 1, disulfide, native thiol, total thiol, and arylesterase levels were assessed at baseline and after the second hirudotherapy session. Data was analysed using SPSS 15. RESULTS: Of the 50 subjects, 30(60%) were females and 20(40%) were males. The overall mean age was 47.10±15.16 years. Oxidative stress, ischaemia-modified albumin and disulfide levels decreased, but not significantly (p>0.05). The reduction in disulfide levels was significant (p=0.021). CONCLUSIONS: Hirudotherapy, within its limitations, could reduce oxidative stress.

Protopine and Allocryptopine Interactions with Plasma Proteins.

A comprehensive study of the interactions of human serum albumin (HSA) and α-1-acid glycoprotein (AAG) with two isoquinoline alkaloids, i.e., allocryptopine (ACP) and protopine (PP), was performed. The UV-Vis spectroscopy, molecular docking, competitive binding assays, and circular dichroism (CD) spectroscopy were used for the investigations. The results showed that ACP and PP form spontaneous and stable complexes with HSA and AAG, with ACP displaying a stronger affinity towards both proteins. Molecular docking studies revealed the preferential binding of ACP and PP to specific sites within HSA, with site 2 (IIIA) being identified as the favored location for both alkaloids. This was supported by competitive binding assays using markers specific to HSA's drug binding sites. Similarly, for AAG, a decrease in fluorescence intensity upon addition of the alkaloids to AAG/quinaldine red (QR) complexes indicated the replacement of the marker by the alkaloids, with ACP showing a greater extent of replacement than PP. CD spectroscopy showed that the proteins' structures remained largely unchanged, suggesting that the formation of complexes did not significantly perturb the overall spatial configuration of these macromolecules. These findings are crucial for advancing the knowledge on the natural product-protein interactions and the future design of isoquinoline alkaloid-based therapeutics.

Mirror-Image Human Serum Albumin Domain III as a Tool for Analyzing Site II-Dependent Molecular Recognition.

Human serum albumin (HSA) as a drug carrier can significantly improve the pharmacokinetic profiles of short-lived therapeutics. Conjugation of albumin-binding moieties (ABMs) to therapeutic agents may prolong their serum half-life by promoting their association with endogenous HSA. To discover a new molecular class of ABMs from mirror-image chemical space, a preparation protocol for bioactive HSA domain III and its d-enantiomer (d-HSA domain III) was established. Structural and functional analyses suggested that the synthetic protein enantiomers exhibited mirror-image structures and stereoselective neonatal fragement crystallizable receptor (FcRn) recognition. Additionally, the ligand-binding properties of synthetic l-HSA domain III were comparable with those of site II in native HSA, as confirmed using site II-selective fluorescent probes and an esterase substrate. Synthetic d-HSA domain III is an attractive tool for analyzing the site II-dependent molecular recognition properties of HSA.

Thermal effects and drugs competition on the palmitate binding capacity of human serum albumin.

Human serum albumin (HSA) is the most abundant plasma protein of the circulatory system. It is a multidomain, multifunctional protein that, combining diverse affinities and wide specificity, binds, stores, and transports a variety of biological compounds, pharmacores, and fatty acids. HSA is finding increasing uses in drug-delivery due to its ability to carry functionalized ligands and prodrugs. All this raises the question of competition for binding sites occupancy in case of multiple ligands, which in turn influences the protein structure/dynamic/function relationship and also has an impact on the biomedical applications. In this work, the effects of interactive binding of palmitic acid (PA), warfarin (War) and ibuprofen (Ibu) on the thermal stability of HSA were studied using DSC, ATR-FTIR, and EPR. PA is a high-affinity physiological ligand, while the two drugs are widely used for their anticoagulant (War) and anti-inflammatory (Ibu) efficacy, and are exogenous compounds that accommodate in the deputed drug site DS1 and DS2, respectively overlapping with some of the fatty acid binding sites. The results indicate that HSA acquires the highest thermal stability when it is fully saturated with PA. The binding of this physiological ligand does not hamper the binding of War or Ibu to the native state of the protein. In addition, the three ligands bind simultaneously, suggesting a synergic cooperative influence due to allosteric effects. The increased thermal stability subsequent to binary and multiple ligands binding moderates protein aggregation propensity and restricts protein dynamics. The biophysics findings provide interesting features about protein stability, aggregation, and dynamics in interaction with multiple ligands and are relevant in drug-delivery.

In Search for Low-Molecular-Weight Ligands of Human Serum Albumin That Affect Its Affinity for Monomeric Amyloid ß Peptide.

An imbalance between production and excretion of amyloid ß peptide (Aß) in the brain tissues of Alzheimer's disease (AD) patients leads to Aß accumulation and the formation of noxious Aß oligomers/plaques. A promising approach to AD prevention is the reduction of free Aß levels by directed enhancement of Aß binding to its natural depot, human serum albumin (HSA). We previously demonstrated the ability of specific low-molecular-weight ligands (LMWLs) in HSA to improve its affinity for Aß. Here we develop this approach through a bioinformatic search for the clinically approved AD-related LMWLs in HSA, followed by classification of the candidates according to the predicted location of their binding sites on the HSA surface, ranking of the candidates, and selective experimental validation of their impact on HSA affinity for Aß. The top 100 candidate LMWLs were classified into five clusters. The specific representatives of the different clusters exhibit dramatically different behavior, with 3- to 13-fold changes in equilibrium dissociation constants for the HSA-Aß40 interaction: prednisone favors HSA-Aß interaction, mefenamic acid shows the opposite effect, and levothyroxine exhibits bidirectional effects. Overall, the LMWLs in HSA chosen here provide a basis for drug repurposing for AD prevention, and for the search of medications promoting AD progression.

Impact of Obesity on Hepatic Drug Clearance: What are the Influential Variables?

Drug clearance in obese subjects varies widely among different drugs and across subjects with different severity of obesity. This study investigates correlations between plasma clearance (CLp) and drug- and patient-related characteristics in obese subjects, and evaluates the systematic accuracy of common weight-based dosing methods. A physiologically-based pharmacokinetic (PBPK) modeling approach that uses recent information on obesity-related changes in physiology was used to simulate CLp for a normal-weight subject (body mass index [BMI] = 20) and subjects with various severities of obesity (BMI 25-60) for hypothetical hepatically cleared drugs with a wide range of properties. Influential variables for CLp change were investigated. For each drug and obese subject, the exponent that yields perfect allometric scaling of CLp from normal-weight subjects was assessed. Among all variables, BMI and relative changes in enzyme activity resulting from obesity proved highly correlated with obesity-related CLp changes. Drugs bound to α1-acid glycoprotein (AAG) had lower CLp changes compared to drugs bound to human serum albumin (HSA). Lower extraction ratios (ER) corresponded to higher CLp changes compared to higher ER. The allometric exponent for perfect scaling ranged from -3.84 to 3.34 illustrating that none of the scaling methods performed well in all situations. While all three dosing methods are generally systematically accurate for drugs with unchanged or up to 50% increased enzyme activity in subjects with a BMI below 30 kg/m2, in any of the other cases, information on the different drug properties and severity of obesity is required to select an appropriate dosing method for individuals with obesity.

Exploring weak ligand-protein interactions by relaxometry of long-lived spin order.

Relaxation times of nuclear spins often serve as a valuable source of information on the dynamics of various biochemical processes. Measuring relaxation as a function of the external magnetic field turned out to be extremely useful for the studies of weak ligand-protein interactions. We demonstrate that observing the relaxation of the long-lived spin order instead of longitudinal magnetization extends the capability of this approach. We studied the field-dependent relaxation of the longitudinal magnetization and the singlet order (SO) of methylene protons in alanine-glycine dipeptide and citrate in the presence of human serum albumin (HSA). As a result, SO relaxation proved to be more sensitive to ligand-protein interaction, providing higher relaxation contrast for various HSA concentrations. To assess the parameters of the binding process in more details, we utilized a simple analytical relaxation model to fit the experimental field dependences for both SO and T1 relaxation. We also tested the validity of our approach in the experiments with trimethylsilylpropanoic acid (TSP) used as a competitor in ligand binding with HSA.

Evaluating the biomolecular interaction between delamanid/formulations and human serum albumin by fluorescence, CD spectroscopy and SPR: Effects on protein conformation, kinetic and thermodynamic parameters.

Delamanid is an anti-tuberculosis drug used for the treatment of drug-resistant tuberculosis. Since delamanid has a high protein bound potential, even patients with low albumin levels should experience high and rapid delamanid clearance. However, the interaction between delamanid and albumin should be better controlled to optimize drug efficacy. This study was designed to evaluate the binding characteristics of delamanid to human serum albumin (HSA) using various methods: fluorescence spectroscopy, circular dichroism (CD), surface plasmon resonance (SPR), and molecular docking simulation. The fluorescence emission band without any shift indicated the interaction was not affected by the polarity of the fluorophore microenvironment. The reduction of fluorescence intensity at 344 nm was proportional to the increment of delamanid concentration as a fluorescence quencher. UV-absorbance measurement at the maximum wavelength (λmax, 280 nm) was evaluated using inner filter effect correction. The HSA conformation change was explained by the intermolecular energy transfer between delamanid and HSA during complex formation. The study, which was conducted at temperatures of 298 K, 303 K, and 310 K, revealed a static quenching mechanism that correlated with a decreased of bimolecular quenching rate constant (kq) and binding constant (Ka) at increased temperatures. The Ka was 1.75-3.16 × 104 M-1 with a specific binding site with stoichiometry 1:1. The negative enthalpy change, negative entropy change, and negative Gibbs free energy change demonstrated an exothermic-spontaneous reaction while van der Waals forces and hydrogen bonds played a vital role in the binding. The molecular displacement approach and molecular docking confirmed that the binding occurred mainly in subdomain IIA, which is a hydrophobic pocket of HSA, with a theoretical binding free energy of -9.33 kcal/mol. SPR exhibited a real time negative sensorgram that resulted from deviation of the reflex angle due to ligand delamanid-HSA complex forming. The binding occurred spontaneously after delamanid was presented to the HSA surface. The SPR mathematical fitting model revealed that the association rate constant (kon) was 2.62 × 108 s-1M-1 and the dissociation rate constant (koff) was 5.65 × 10-3 s-1. The complexes were performed with an association constant (KA) of 4.64 × 1010 M-1 and the dissociation constant (KD) of 2.15 × 10-11 M. The binding constant indicated high binding affinity and high stability of the complex in an equilibrium. Modified CD spectra revealed that conformation of the HSA structure was altered by the presence of delamanid during preparation of the proliposomes that led to the reduction of secondary structure stabilization. This was indicated by the percentage decrease of α-helix. These findings are beneficial to understanding delamanid-HSA binding characteristics as well as the drug administration regimen.