RÉSUMÉ
Neuroblastoma is the most prevalent and aggressive solid tumor that develops extracranially in children between the ages of 0-14 years, which accounts for 8-10% of all childhood malignancies and â¼15% of pediatric cancer-related mortality. The polycomb repressive complex 2 (PRC2) protein, EZH2, is overexpressed in neuroblastoma and mediates histone H3 methylation at lysine 27 (K27) positions through its methyl transferase activity and is a potential epigenetic silencer of many tumor suppressor genes in cancer. Phosphorylation of EZH2 decreases its stability and leads to proteasomal degradation. The 4-oxo-N-(4-hydroxyphenyl) retinamide (4O4HPR) promotes EZH2 degradation via activation of PKC-δ, but its limited solubility and physiological instability limit its application. In the current study, the encapsulation of 4O4HPR in Human Serum Albumin Nanoparticles (HSANPs) enhanced the solubility and physiological stability of the nanoformulation, leading to improved therapeutic efficacy through G2-M cell cycle arrest, depolarization of mitochondrial membrane potential, generation of reactive oxygen species and caspase 3 mediated apoptosis activation. The molecular mechanistic approach of 4O4HPR loaded HSANPs has activated caspase 3, which further cleaves PKC-δ into two fragments wherein the cleaved fragment of PKC-δ possesses the kinase activity that phosphorylates EZH2 and decreases the protein stability leading to its further ubiquitination in SH-SY5Y cells. Co-immunoprecipitation experiments revealed the direct interaction between PKC-δ and EZH2 phosphorylation, followed by ubiquitination. Moreover, 4O4HPR loaded HSANPs demonstrated improved in vivo biodistribution, greater dispersibility, and biocompatibility and exhibited enhanced protein instability and degradation of EZH2 in the neuroblastoma xenograft mouse model.
Sujet(s)
Protéine-2 homologue de l'activateur de Zeste , Nanoparticules , Neuroblastome , Protéine-2 homologue de l'activateur de Zeste/métabolisme , Protéine-2 homologue de l'activateur de Zeste/composition chimique , Humains , Neuroblastome/anatomopathologie , Neuroblastome/métabolisme , Neuroblastome/traitement médicamenteux , Animaux , Lignée cellulaire tumorale , Nanoparticules/composition chimique , Souris , Sérum-albumine humaine/composition chimique , Sérum-albumine humaine/métabolisme , Apoptose/effets des médicaments et des substances chimiques , Fenrétinide/composition chimique , Fenrétinide/pharmacologie , Espèces réactives de l'oxygène/métabolisme , Souris nude , Protéolyse/effets des médicaments et des substances chimiques , Potentiel de membrane mitochondriale/effets des médicaments et des substances chimiques , Tests d'activité antitumorale sur modèle de xénogreffe , Antinéoplasiques/composition chimique , Antinéoplasiques/pharmacologie , Caspase-3/métabolismeRÉSUMÉ
The significant health risks of nanoplastics (NPs) and cadmium (Cd) are currently attracting a great deal of attention and research. At present, the effects and mechanisms of NPs and Cd on human serum albumin (HSA), a key functional protein in the organism on transportation, remain unknown. Here, the differences in the effects and mechanisms of action of Cd alone and composite systems (NPsCd) were explored by enzyme activity assay, multi-spectroscopy analysis and molecular docking. The results showed that HSA activity was inhibited and decreased to 80 % and 69.55 % (Cd = 30 mg/L) by Cd alone and NPs-Cd exposure, respectively. Exposure to Cd induced backbone disruption and protein defolding of HSA, and secondary structure disruption was manifested by the reduction of α-helix. Cd exposure also induces fluorescence sensitization of HSA. Notably, the addition of NPs further exacerbated the effects associated with Cd exposure, which was consistent with the changes in HSA activity. Thus, the above conformational changes may be responsible for inducing the loss of enzyme activity. Moreover, it was determined by RLS spectroscopy that NPs-Cd bound to HSA in the form of protein crowns. Molecular docking has further shown that Cd binds to the surface of Sudlow site II of HSA, suggesting that Cd impairs the function of HSA by affecting the protein structure. More importantly, the addition of NPs further exacerbated the disruption of the protein structure by the adherent binding of HSA on the surface of the plastic particles, which induced a greater change in the enzyme activity. This study provides useful perspectives for investigating the impact of composite pollution on HSA of human functional proteins.
Sujet(s)
Cadmium , Simulation de docking moléculaire , Sérum-albumine humaine , Cadmium/toxicité , Humains , Sérum-albumine humaine/composition chimique , Sérum-albumine humaine/métabolisme , Liaison aux protéinesRÉSUMÉ
BACKGROUND Neonatal hypoxic-ischemic encephalopathy (HIE) is a significant cause of perinatal and postnatal morbidity and mortality worldwide. Catalase (CAT) activity detection is used to determine levels of inflammation and oxidative stress. Glutathione (GSH) is the most critical non-enzymatic endogenous antioxidant. Lipid peroxidation levels marked after hypoxia can be detected based on the level of malondialdehyde (MDA). Ischemia-modified albumin (IMA) is considered a biomarker for cardiac ischemia and is known to increase in the liver, brain, and kidney in states of insufficient oxygenation. We aimed to explain the results and relations between the oxidant and antioxidants to detail oxidant-antioxidant balance and cellular mechanisms. MATERIAL AND METHODS Serum levels of IMA and MDA, as an oxidative stress marker, and CAT and GSH, as antioxidant enzymes, were measured in first blood samples of 59 neonates diagnosed with HIE, with pH <7, base excess >12, and APGAR scores. RESULTS Neonates who were ≥37 weeks of gestation and had hypoxia were included. Compared with healthy newborns (n=32), CAT was statistically significantly lower in the hypoxia group (P=0.0001), while MDA serum levels were significantly higher in neonates with hypoxia (P=0.01). There was no difference between hypoxic and healthy neonates in GSH and IMA measurements (P=0.054, P=0.19 respectively). CONCLUSIONS HIE pathophysiology involves oxidative stress and mitochondrial energy production failure. Explaining the pathways between oxidant-antioxidant balance and cell death, which explains the pathophysiology of HIE, is essential to develop treatment strategies that will minimize the effects of oxygen deprivation on other body organs, especially the brain.
Sujet(s)
Antioxydants , Marqueurs biologiques , Hypoxie-ischémie du cerveau , Malonaldéhyde , Stress oxydatif , Humains , Nouveau-né , Hypoxie-ischémie du cerveau/métabolisme , Hypoxie-ischémie du cerveau/sang , Hypoxie-ischémie du cerveau/physiopathologie , Marqueurs biologiques/sang , Marqueurs biologiques/métabolisme , Antioxydants/métabolisme , Femelle , Mâle , Malonaldéhyde/sang , Malonaldéhyde/métabolisme , Glutathion/sang , Glutathion/métabolisme , Sérum-albumine humaine/métabolisme , Catalase/sang , Catalase/métabolisme , Peroxydation lipidiqueRÉSUMÉ
The competition among drugs for binding to plasma proteins is regarded as a pharmacokinetic drug interaction. Competition between antitumor agents and other drugs for plasma protein binding can alter the free concentration of the drug, potentially impacting its efficacy and increasing the risk of toxic side effects. Through a range of spectroscopic techniques, this study examined the interaction between limonin and human serum albumin (HSA) in the context of berberine (Ber) and curcumin (Cur) under physiological conditions to clarify the binding mechanisms of binary and ternary systems at the molecular level. As demonstrated by fluorescence quenching experiments, Static quenching was identified as the mechanism of interaction between HSA and limonin. The results of site competition experiments indicated that the binding site between limonin and HSA was site I, a result further supported by molecular docking simulations. Through the use of thermodynamic data calculations, it was determined that limonin forms a stable complex with HSA by establishing hydrogen bonds and van der Waals forces. Circular dichroism (CD) spectroscopy, three-dimensional (3D) fluorescence spectroscopy, and synchronous fluorescence spectroscopy (SFS) employed to validate the notion that limonin perturbed the microenvironment of amino acids and induced conformational changes in HSA. What's more, the presence of Ber or Cur was found to have further modified the alterations observed in the interaction between the original HSA-limonin binary system. In vitro cellular experiments showed that interaction with HSA reduced the antitumor activity of limonin. In contrast, adding Ber or Cur increased the inhibition rate of tumor cells. The coexistence of both Ber and Cur significantly diminished limonin's binding affinity to HSA. The current investigation enhances comprehension regarding the binding characteristics and interaction mechanisms involving limonin, Ber, Cur, and HSA. It explores the potential of HSA as a versatile drug carrier and furnishes theoretical underpinnings for co-administrative strategies.
Sujet(s)
Antinéoplasiques , Berbérine , Curcumine , Limonines , Simulation de docking moléculaire , Liaison aux protéines , Sérum-albumine humaine , Spectrométrie de fluorescence , Humains , Berbérine/pharmacologie , Berbérine/composition chimique , Berbérine/métabolisme , Curcumine/pharmacologie , Curcumine/métabolisme , Curcumine/composition chimique , Limonines/pharmacologie , Limonines/composition chimique , Limonines/métabolisme , Antinéoplasiques/pharmacologie , Antinéoplasiques/composition chimique , Antinéoplasiques/métabolisme , Sérum-albumine humaine/métabolisme , Sérum-albumine humaine/composition chimique , Sites de fixation , Thermodynamique , Lignée cellulaire tumorale , Dichroïsme circulaireRÉSUMÉ
BACKGROUND: The yeast Komagataella phaffii is widely used for manufacturing recombinant proteins, but secreted titers of recombinant proteins could be improved by genetic engineering. In this study, we hypothesized that cellular resources could be redirected from production of endogenous proteins to production of recombinant proteins by deleting unneeded endogenous proteins. In non-model microorganisms such as K. phaffii, however, genetic engineering is limited by lack gene annotation and knowledge of gene essentiality. RESULTS: We identified a set of endogenous secreted proteins in K. phaffii by mass spectrometry and signal peptide prediction. Our efforts to disrupt these genes were hindered by limited annotation of essential genes. To predict essential genes, therefore, we designed, transformed, and sequenced a pooled library of guide RNAs for CRISPR-Cas9-mediated knockout of all endogenous secreted proteins. We then used predicted gene essentiality to guide iterative disruptions of up to 11 non-essential genes. Engineered strains exhibited a ~20× increase in the production of human serum albumin and a twofold increase in the production of a monoclonal antibody. CONCLUSIONS: We demonstrated that disruption of as few as six genes can increase production of recombinant proteins. Further reduction of the endogenous proteome of K. phaffii may further improve strain performance. The pooled library of secretome-targeted guides for CRISPR-Cas9 and knowledge of gene essentiality reported here will facilitate future efforts to engineer K. phaffii for production of other recombinant proteins and enzymes.
Sujet(s)
Systèmes CRISPR-Cas , Protéines recombinantes , Saccharomycetales , Saccharomycetales/génétique , Saccharomycetales/métabolisme , Protéines recombinantes/génétique , Protéines recombinantes/biosynthèse , Protéines recombinantes/métabolisme , Humains , Techniques de knock-out de gènes/méthodes , Protéines fongiques/génétique , Protéines fongiques/métabolisme , Protéome/métabolisme , Anticorps monoclonaux/biosynthèse , Sérum-albumine humaine/génétique , Sérum-albumine humaine/métabolismeRÉSUMÉ
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.
Sujet(s)
Anti-inflammatoires non stéroïdiens , Kétoprofène , Kétorolac , Liaison aux protéines , Sérum-albumine humaine , Humains , Kétoprofène/composition chimique , Kétoprofène/métabolisme , Kétoprofène/pharmacocinétique , Anti-inflammatoires non stéroïdiens/composition chimique , Anti-inflammatoires non stéroïdiens/métabolisme , Anti-inflammatoires non stéroïdiens/pharmacocinétique , Kétorolac/composition chimique , Kétorolac/métabolisme , Kétorolac/pharmacocinétique , Sérum-albumine humaine/composition chimique , Sérum-albumine humaine/métabolisme , Dichroïsme circulaire , Thermodynamique , Spectrométrie de fluorescence , Sites de fixationRÉSUMÉ
Human serum albumin (HSA) is the most abundant plasma protein, which functions to transport a large range of ligands within the circulation. These interactions have important implications for human health and disease. The primary binding site for CuII ions on HSA is known to be the so-called amino-terminal CuII and NiII binding (ATCUN) motif. However, the number and identity of secondary binding sites is currently not understood. In this study, we harnessed a suite of contemporary electron paramagnetic resonance (EPR) spectroscopy methods to investigate recombinantly produced constructs of HSA bearing single-histidine knockouts, with the aim to characterise its endogenous CuII ion binding sites.
Sujet(s)
Cuivre , Sérum-albumine humaine , Spectroscopie de résonance de spin électronique , Cuivre/composition chimique , Cuivre/métabolisme , Humains , Sites de fixation , Sérum-albumine humaine/composition chimique , Sérum-albumine humaine/métabolisme , Liaison aux protéinesRÉSUMÉ
Organophosphate flame retardants (OPFRs) pose the significant risks to the environment and human health and have become a serious public health issue. Tricresyl phosphates (TCPs), a group of aryl OPFRs, exhibit neurotoxicity and endocrine disrupting toxicity. However, the binding mechanisms between TCPs and human serum albumin (HSA) remain unknown. In this study, through fluorescence and ultraviolet-visible (UV-vis) absorption spectroscopy, molecular docking and molecular dynamics (MD), tri-para-cresyl phosphate (TpCP) was selected to explore potential interactions between HSA and TCPs. The results of the fluorescence spectroscopy demonstrated that a decrease in the fluorescence intensity of HSA and a blue shift were observed with the increasing concentrations of TpCP. The binding constant (Ka) was 2.575 × 104 L/mol, 4.701 × 104 L/mol, 5.684 × 104 L/mol and 9.482 × 104 L/mol at 293 K, 298 K, 303 K, and 310 K, respectively. The fluorescence process between HSA and TpCP involved a mix of static and dynamic quenching mechanism. The gibbs free energy (ΔG0) of HSA-TpCP system was -24.452 kJ/mol, -25.907 kJ/mol, -27.363 kJ/mol, and - 29.401 kJ/mol at 293 K, 298 K, 303 K, and 310 K, respectively, suggesting that the HSA-TpCP reaction was spontaneous. The enthalpy change (ΔH0) and thermodynamic entropy change (ΔS0) of the HSA-TpCP system were 60.83 kJ/mol and 291.08 J/(mol·>k), respectively, indicating that hydrophobic force was the major driving force in the HSA-TpCP complex. Furthermore, multispectral analysis also revealed that TpCP could alter the microenvironment of tryptophan residue and the secondary structure of HSA and bind with the active site I of HSA. Molecular docking and MD simulations confirmed that TpCP could spontaneously form a stable complex with HSA, which was consistent with the fluorescence experimental results. This study provides novel insights into the mechanisms of underlying the transportation and distribution of OPFRs in humans.
Sujet(s)
Simulation de docking moléculaire , Simulation de dynamique moléculaire , Liaison aux protéines , Spectrométrie de fluorescence , Thermodynamique , Humains , Sérum-albumine humaine/composition chimique , Sérum-albumine humaine/métabolisme , Ignifuges/métabolisme , Spectrophotométrie UV , Sites de fixation , Tritolylphosphates/composition chimique , Tritolylphosphates/métabolisme , Sérumalbumine/composition chimique , Sérumalbumine/métabolisme , Liaison hydrogèneRÉSUMÉ
Folic acid receptor-targeted drug delivery system is a promising candidate for tumor-targeted delivery because its elevated expression specifically on tumor cells enables the selective delivery of cytotoxic cargo to cancerous tissue, thereby minimizing toxic side effects and increasing the therapeutic index. Pyridine bisfolate-chitosan (PyBFA@CS NPs) and folate-chitosan nanocomposite (FA@CS NPs) were synthesized with suitable particle size (256.0 ± 15.0 and 161.0 ± 5.0 nm), high stability (ζ = -27.0 ± 0.1 and -30.0 ± 0.2 mV), respectively, and satisfactory biocompatibility to target cells expressing folate receptors and try to answer the question: Is the metal center always important for activity? Since almost all pharmaceuticals work by binding to specific proteins or DNA, the in vitro binding of human serum albumin (HSA) to PyBFA@CS NPs and FA@CS NPs has been investigated and compared with PyBFA. Strong affinity to HSA is shown by quenching and binding constants in the range of 105 and 104 M-1, respectively with PyBFA@CS NPs showing the strongest. The compounds-HSA kinetic stability, affinity, and association constants were investigated using a stopped-flow method. The findings showed that all formulations bind by a static quenching mechanism that consists of two reversible steps: rapid second-order binding and a more slowly first-order isomerization reaction. The overall coordination affinity of HSA to PyBFA@CS NPs (6.6 × 106 M-1), PyBFA (4.4 × 106 M-1), and FA@CS NPs (1.3 × 106 M-1) was measured and The relative reactivity is roughly (PyBFA@CS NPs)/(PyBFA)/(FA@CS NPs) = 5/3/1. Additionally, in vitro cytotoxicity revealed that, consistent with the binding constants and coordination affinity, active-targeting formulations greatly inhibited FR-positive MCF-7 cells in compared to FRs-negative A549 cells in the following trend: PyBFA@CS NPs > PyBFA > FA@CS NPs. Furthermore, in vitro drug release of PyBFA@CS NPs was found to be stable in PBS at pH 7.4, however, the in pH 5.4 and in pH 5.4 containing 10 mM glutathione (GSH) (mimicking the tumor microenvironment) reached 43 % and 73 %, respectively indicating that the PyBFA@CS NPs system is sensitive to GSH. Folate-modified nanoparticles, PyBFA@CS NPs, are a promising therapeutic for MCF-7 therapy because they not only showed a greater affinity for HSA, but also showed higher cleavage efficiency toward the minor groove of pBR322 DNA via the hydrolytic way, as well as effective antibacterial activity that avoids the usage of extra antibiotics.â¬â¬â¬â¬â¬â¬â¬â¬â¬â¬â¬â¬ â¬â¬â¬â¬â¬â¬â¬â¬â¬â¬â¬â¬â¬â¬.
Sujet(s)
Chitosane , Acide folique , Glutathion , Nanoparticules , Pyridines , Humains , Chitosane/composition chimique , Chitosane/pharmacologie , Cellules MCF-7 , Acide folique/composition chimique , Nanoparticules/composition chimique , Pyridines/composition chimique , Pyridines/pharmacologie , Glutathion/composition chimique , Glutathion/métabolisme , Antinéoplasiques/pharmacologie , Antinéoplasiques/composition chimique , Vecteurs de médicaments/composition chimique , Survie cellulaire/effets des médicaments et des substances chimiques , Récepteurs des folates à ancre GPI/métabolisme , Systèmes de délivrance de médicaments , Sérum-albumine humaine/composition chimique , Sérum-albumine humaine/métabolisme , Libération de médicament , Prolifération cellulaire/effets des médicaments et des substances chimiques , Taille de particuleRÉSUMÉ
Human Serum Albumin (HSA), the most abundant protein in human body fluids, plays a crucial role in the transportation, absorption, metabolism, distribution, and excretion of drugs, significantly influencing their therapeutic efficacy. Despite the importance of HSA as a drug target, the available data on its interactions with external agents, such as drug-like molecules and antibodies, are limited, posing challenges for molecular modeling investigations and the development of empirical scoring functions or machine learning predictors for this target. Furthermore, the reported entries in existing databases often contain major inconsistencies due to varied experiments and conditions, raising concerns about data quality. To address these issues, a pioneering database, HSADab, was established through an extensive review of >30,000 scientific publications published between 1987 and 2023. The database encompasses over 5000 affinity data points at multiple temperatures and >130 crystal structures, including both ligand-bound and apo forms. The current HSADab resource (www.hsadab.cn) serves as a reliable foundation for validating molecular simulation protocols, such as traditional virtual screening workflows using docking, end-point, and al-chemical free energy techniques. Additionally, it provides a valuable data source for the implementation of machine learning predictors, including plasma protein binding models and plasma protein-based drug design models.
Sujet(s)
Bases de données de protéines , Sérum-albumine humaine , Humains , Sérum-albumine humaine/composition chimique , Sérum-albumine humaine/métabolisme , Apprentissage machine , Liaison aux protéines , Simulation de docking moléculaire , LigandsRÉSUMÉ
In the present study, one mononuclear Cu(II) [CuL(SCN)] (1) and one mononuclear Co(II) [CoLN3] (2) complexes, with a Schiff base ligand (HL) formed by condensation of 2-picolylamine and salicylaldehyde, have been successfully developed and structurally characterized. The square planer geometry of both complexes is fulfilled by the coordination of one deprotonated ligand and one ancillary ligand SCN-(1) or N3-(2) to the metal centre. Binding affinities of both complexes with deoxyribonucleic acid (DNA) and human serum albumin (HSA) are investigated using several biophysical and spectroscopic techniques. High values of the macromolecule-complex binding constants and other results confirm the effectiveness of both complexes towards binding with DNA and HSA. The determined values of the thermodynamic parameters support spontaneous interactions of both complexes with HSA, while fluorescence displacement and DNA melting studies establish groove-binding interactions with DNA for both complexes 1 and 2. The molecular modelling study validates the experimental findings. Both complexes are subjected to an MTT test establishing the anticancer property of complex 1 with lower risk to normal cells, confirmed by the IC50 values of the complex for HeLa cancer cells and HEK normal cells. Finally, a nuclear staining analysis reveals that the complexes have caused apoptotic cell death.
Sujet(s)
Antinéoplasiques , Cobalt , Complexes de coordination , Cuivre , ADN , Liaison aux protéines , Humains , ADN/composition chimique , ADN/métabolisme , Cuivre/composition chimique , Cobalt/composition chimique , Antinéoplasiques/pharmacologie , Antinéoplasiques/composition chimique , Complexes de coordination/composition chimique , Complexes de coordination/pharmacologie , Cellules HeLa , Isothiocyanates/composition chimique , Isothiocyanates/pharmacologie , Sérum-albumine humaine/composition chimique , Sérum-albumine humaine/métabolisme , Modèles moléculaires , Anions/composition chimique , Thermodynamique , Cellules HEK293 , Bases de Schiff/composition chimique , Ligands , Simulation de docking moléculaireRÉSUMÉ
Lobeline (LOB), a naturally occurring alkaloid, has a broad spectrum of pharmacological activities and therapeutic potential, including applications in central nervous system disorders, drug misuse, multidrug resistance, smoking cessation, depression, and epilepsy. LOB represents a promising compound for developing treatments in various medical fields. However, despite extensive pharmacological profiling, the biophysical interaction between the LOB and proteins remains largely unexplored. In the current article, a range of complementary photophysical and cheminformatics methodologies were applied to study the interaction mechanism between LOB and the carrier protein HSA. Steady-state fluorescence and fluorescence lifetime experiments confirmed the static-quenching mechanisms in the HSA-LOB system. "K" (binding constant) of the HSA-LOB system was determined to be 105 M-1, with a single preferable binding site in HSA. The forces governing the HSA-LOB stable complex were analyzed by thermodynamic parameters and electrostatic contribution. The research also investigated how various metal ions affect complex binding. Site-specific binding studies depict Site I as probable binding in HSA by LOB. We conducted synchronous fluorescence, 3D fluorescence, and circular dichroism studies to explore the structural alteration occurring in the microenvironment of amino acids. To understand the robustness of the HSA-LOB complex, we used theoretical approaches, including molecular docking and MD simulations, and analyzed the principal component analysis and free energy landscape. These comprehensive studies of the structural features of biomolecules in ligand binding are of paramount importance for designing targeted drugs and delivery systems.
Sujet(s)
Dichroïsme circulaire , Interactions hydrophobes et hydrophiles , Lobéline , Liaison aux protéines , Sérum-albumine humaine , Thermodynamique , Humains , Sérum-albumine humaine/composition chimique , Sérum-albumine humaine/métabolisme , Lobéline/composition chimique , Lobéline/métabolisme , Sites de fixation , Dichroïsme circulaire/méthodes , Conformation des protéines , Spectrométrie de fluorescence , Simulation de docking moléculaire , Simulation de dynamique moléculaire , Neuroprotecteurs/pharmacologie , Neuroprotecteurs/composition chimiqueRÉSUMÉ
YM201636 is the potent PIKfyve inhibitor that is being actively investigated for liver cancer efficacy. In this study, computer simulations and experiments were conducted to investigate the interaction mechanism between YM201636 and the transport protein HSA. Results indicated that YM201636 is stably bound between the subdomains IIA and IIIA of HSA, supported by site marker displacement experiments. YM201636 quenched the endogenous fluorescence of HSA by static quenching since a decrease in quenching constants was observed from 7.74 to 2.39 × 104 M-1. UV-vis and time-resolved fluorescence spectroscopy confirmed the YM201636-HSA complex formation and this binding followed a static mechanism. Thermodynamic parameters ΔG, ΔH, and ΔS obtained negative values suggesting the binding was a spontaneous process driven by Van der Waals interactions and hydrogen binding. Binding constants ranged between 5.71 and 0.33 × 104 M-1, which demonstrated a moderately strong affinity of YM201636 to HSA. CD, synchronous, and 3D fluorescence spectroscopy revealed that YM201636 showed a slight change in secondary structure. The increase of Kapp and a decrease of PSH with YM201636 addition showed that YM201636 changed the surface hydrophobicity of HSA. The research provides reasonable models helping us further understand the transportation and distribution of YM201636 when it absorbs into the blood circulatory system.
Sujet(s)
Sérum-albumine humaine , Spectrométrie de fluorescence , Humains , Sérum-albumine humaine/composition chimique , Sérum-albumine humaine/métabolisme , Thermodynamique , Phosphatidylinositol 3-kinases/métabolisme , Phosphatidylinositol 3-kinases/composition chimique , Modèles moléculaires , Inhibiteurs des phosphoinositide-3 kinases/composition chimique , Inhibiteurs des phosphoinositide-3 kinases/pharmacologie , Inhibiteurs des phosphoinositide-3 kinases/métabolisme , Pyrimidines/composition chimiqueRÉSUMÉ
In the present work, we study different physicochemical properties related to LADME processes of volasertib, a Polo-like kinase 1 inhibitor in advanced clinical trials. Firstly, the protonation equilibria, the extent of ionization at the physiological pH and pKa values of this drug are studied combining spectroscopic techniques and computational calculations. Secondly, the binding process of volasertib to the human serum albumin (HSA) protein is analyzed by fluorescence spectroscopy. We report a high binding constant to HSA (Ka = 4.10 × 106 M-1) and their pharmacokinetic implications are discussed accordingly. The negative enthalpy and entropy (ΔH0 = -54.49 kJ/mol; ΔS0 = -58.90 J K-1 mol-1) determined for the binding process suggests the implication of hydrogen bonds and van der Waals interactions in the formation of the HSA-volasertib complex. Additionally, volasertib is encapsulated in an alginate/montmorillonite bionanocomposite as a proof of concept for an oral delivery nanocarrier. The physical properties of that nanocomposite as well as volasertib delivery kinetics are analyzed.
Sujet(s)
Alginates , Bentonite , Nanocomposites , Sérum-albumine humaine , Spectrométrie de fluorescence , Humains , Alginates/composition chimique , Sérum-albumine humaine/composition chimique , Sérum-albumine humaine/métabolisme , Nanocomposites/composition chimique , Bentonite/composition chimique , Ptéridines/composition chimique , Thermodynamique , Liaison aux protéines , Liaison hydrogène , Concentration en ions d'hydrogèneRÉSUMÉ
Modification of the R1 and R2 side chain structures has been used as the main strategy to expand the spectrum of cephalosporins and impart resistance to hydrolysis by ß-lactamases. These structural modifications also result in a wide range of plasma protein binding, especially with human serum albumin (HSA). Here, we determined the crystal structures of the HSA complexes with two clinically important cephalosporins, ceftriaxone and cefazolin, and evaluated the binding of cephalosporin to HSA by susceptibility testing and competitive binding assay. Ceftriaxone and cefazolin bind to subdomain IB of HSA, and their cephem core structures are recognized by Arg117 of HSA. Tyr161 of HSA changes its rotamer depending on the cephalosporin, resulting in alterations of the cavity shape occupied by the R2 side chain of cephalosporins. These findings provide structural insight into the mechanisms underlying the HSA binding of cephalosporins.
Sujet(s)
Céphalosporines , Liaison aux protéines , Sérum-albumine humaine , Humains , Sérum-albumine humaine/composition chimique , Sérum-albumine humaine/métabolisme , Céphalosporines/composition chimique , Céphalosporines/métabolisme , Cristallographie aux rayons X , Antibactériens/composition chimique , Antibactériens/métabolisme , Antibactériens/pharmacologie , Ceftriaxone/composition chimique , Ceftriaxone/métabolisme , Modèles moléculaires , Céfazoline/composition chimique , Céfazoline/métabolisme , Sites de fixation , Sérumalbumine/composition chimique , Sérumalbumine/métabolismeRÉSUMÉ
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)â2.5H2O 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.
Sujet(s)
ADN , Simulation de docking moléculaire , Liaison aux protéines , Sérumalbumine bovine , Ligands , ADN/composition chimique , ADN/métabolisme , Humains , Sérumalbumine bovine/composition chimique , Sérumalbumine bovine/métabolisme , Cristallographie aux rayons X , Thiosemicarbazones/composition chimique , Thiosemicarbazones/métabolisme , Bovins , Complexes de coordination/composition chimique , Complexes de coordination/métabolisme , Animaux , Protons , Composés du fer III/composition chimique , Sérum-albumine humaine/composition chimique , Sérum-albumine humaine/métabolisme , Sites de fixation , Fer/composition chimique , Fer/métabolismeRÉSUMÉ
Here, we report a photodynamic therapy (PDT) photosensitizer of Nâ§Câ§N-coordinated Pt(II) complexes: [Pt(L)(solv)]+ (HL = 1,3-(2-dipyridyl)benzene) and [Pt(L)]+@HSA, which is the Pt(II) complex encapsulated in human serum albumin (HSA). The quantum yield of singlet oxygen production for [Pt(L)(solv)]+ is more than 50%, while that for [Pt(L)]+@HSA is much lower. Photoimages of human umbilical vein endothelial cells (HUVECs) treated with the Pt(II) complexes suggest that [Pt(L)(solv)]+ is delocalized in the entire cell after the fast uptake by diffusion and [Pt(L)]+@HSA is taken up by endocytosis and localized on organelles and the cell membrane. [Pt(L)(solv)]+ shows high photocytotoxicity for HUVECs, while [Pt(L)]+@HSA does not show photocytotoxicity.
Sujet(s)
Cellules endothéliales de la veine ombilicale humaine , Photothérapie dynamique , Photosensibilisants , Humains , Cellules endothéliales de la veine ombilicale humaine/effets des médicaments et des substances chimiques , Photosensibilisants/pharmacologie , Photosensibilisants/composition chimique , Photosensibilisants/synthèse chimique , Composés organiques du platine/pharmacologie , Composés organiques du platine/composition chimique , Composés organiques du platine/synthèse chimique , Survie cellulaire/effets des médicaments et des substances chimiques , Structure moléculaire , Oxygène singulet/métabolisme , Sérum-albumine humaine/composition chimique , Sérum-albumine humaine/métabolisme , Platine/composition chimique , Platine/pharmacologie , Tensioactifs/composition chimique , Tensioactifs/pharmacologie , Tensioactifs/synthèse chimique , Complexes de coordination/pharmacologie , Complexes de coordination/composition chimique , Complexes de coordination/synthèse chimiqueRÉSUMÉ
Serine proteases (SPs) are distributed among all living cells accounting for almost one-third of all proteases. Dysregulation of SPs during inflammation and/or infection can result in devastating consequences, such as skin and lung inflammation, neuroinflammation, arthritis, as well as metastasis of cancerous cells. Such activities are tightly regulated by various inhibitors known as serine protease inhibitors (SERPIN). The thermodynamic investigations previously revealed that L-ascorbic acid binds to trypsin more firmly than pepsin and the binding force of L-ascorbic acid is driven by hydrogen bonds and van der Waals forces. However, the physiochemical effects of such interaction on trypsin and/or pepsin have not yet been reported. Ascorbic acid, also known as vitamin C, is one of the essential nutrients and most common food supplements, fortificants, and preservatives. The aim of this study was to explore the inhibitory effects of ascorbic acid on serine proteases at various concentrations on the in-vitro digestion and/or hydrolysis of intercellular matrix of cell monolayer and human serum albumin (HSA). The inhibitory effects of ascorbic on trypsin are investigated by qualitative and quantitative analysis using SDS-PAGE imaging and NIH densitometric software. Upon the addition of ascorbic acid in both indicator systems, the detachment and/or dissociation of cell monolayer and the digestion of HSA were inhibited in the presence of EDTA-Trypsin. The inhibitory effect of ascorbic acid on the digestion of intercellular matrix and/or hydrolysis of HSA showed a dose-dependent trend until it reached the maximum extent of inhibition. At an equal concentration (2.5mg/mL) ascorbic acid and EDTA-Trypsin exhibited the most potent inhibitory effect on the in vitro digestion of protein either in the form of intercellular matrix in cell monolayer and/or HSA respectively. Overall, our results based on two indicator systems strongly indicate that ascorbic acid may function as a serine protease inhibitor (SERPIN) beyond other important functions.
Sujet(s)
Acide ascorbique , Inhibiteurs de la sérine protéinase , Humains , Acide ascorbique/pharmacologie , Acide ascorbique/composition chimique , Inhibiteurs de la sérine protéinase/pharmacologie , Inhibiteurs de la sérine protéinase/composition chimique , Inhibiteurs de la sérine protéinase/métabolisme , Tumeurs du poumon/métabolisme , Tumeurs du poumon/anatomopathologie , Sérum-albumine humaine/métabolisme , Sérum-albumine humaine/composition chimique , Trypsine/métabolisme , Trypsine/composition chimique , Lignée cellulaire tumorale , Cellules A549RÉSUMÉ
Biotherapeutics is the fastest growing class of drugs administered by subcutaneous injection. In vitro release testing mimicking physiological conditions at the injection site may guide formulation development and improve biopredictive capabilities. Here, anin vitrorelease cartridge (IVR cartridge) comprising a porous agarose matrix emulating subcutaneous tissue was explored. The objective was to assess effects of medium composition and incorporation of human serum albumin into the matrix. Drug disappearance was assessed for solution, suspension and in situ precipitating insulin products (Actrapid, Levemir, Tresiba, Mixtard 30, Insulatard, Lantus) using the flow-based cartridge. UV-Vis imaging and light microscopy visualized dissolution, precipitation and albumin binding phenomena at the injection site. Divalent cations present in the release medium resulted in slower insulin disappearance for suspension-based and in situ precipitating insulins. Albumin-binding acylated insulin analogs exhibited rapid disappearance from the cartridge; however, sustained retention was achieved by coupling albumin to the matrix. An in vitro-in vivorelation was established for the non-albumin-binding insulins.The IVR cartridge is flexible with potential in formulation development as shown by the ability to accommodate solutions, suspensions, and in situ forming formulations while tailoring of the system to probe in vivo relevant medium effects and tissue constituent interactions.
Sujet(s)
Libération de médicament , Injections sous-cutanées , Humains , Insuline/administration et posologie , Insuline/composition chimique , Hypoglycémiants/administration et posologie , Hypoglycémiants/composition chimique , Hypoglycémiants/pharmacocinétique , Sérum-albumine humaine/composition chimique , Sérum-albumine humaine/métabolisme , Agarose/composition chimique , Liaison aux protéines , Chimie pharmaceutique/méthodes , MâleRÉSUMÉ
"Purple Drank", a soft drink containing promethazine (PMZ) and codeine (COD), has gained global popularity for its hallucinogenic effects. Consuming large amounts of this combination can lead to potentially fatal events. The binding of these drugs to plasma proteins can exacerbate the issue by increasing the risk of drug interactions, side effects, and/or toxicity. Herein, the binding affinity to human serum albumin (HSA) of PMZ and its primary metabolites [N-desmethyl promethazine (DMPMZ) and promethazine sulphoxide (PMZSO)], along with COD, was investigated by high-performance affinity chromatography (HPAC) though zonal approach. PMZ and its metabolites exhibited a notable binding affinity for HSA (%b values higher than 80%), while COD exhibited a %b value of 65%. To discern the specific sites of HSA to which these compounds were bound, displacement experiments were performed using warfarin and (S)-ibuprofen as probes for sites I and II, respectively, which revealed that all analytes were bound to both sites. Molecular docking studies corroborated the experimental results, reinforcing the insights gained from the empirical data. The in silico data also suggested that competition between PMZ and its metabolites with COD can occur in both sites of HSA, but mainly in site II. As the target compounds are chiral, the enantioselectivity for HSA binding was also explored, showing that the binding for these compounds was not enantioselective.