Confirming therapeutic target of protopine using immobilized β2‐adrenoceptor coupled with site‐directed molecular docking and the target‐drug interaction by frontal analysis and injection amount–dependent method

Drug‐protein interaction analysis is pregnant in designing new leads during drug discovery. We prepared the stationary phase containing immobilized β₂‐adrenoceptor (β ₂‐ AR) by linkage of the receptor on macroporous silica gel surface through N ,N ′‐carbonyldiimidazole method. The stationary phase was applied in identifying antiasthmatic target of protopine guided by the prediction of site‐directed molecular docking. Subsequent application of immobilized β ₂‐ AR in exploring the binding of protopine to the receptor was realized by frontal analysis and injection amount–dependent method. The association constants of protopine to β ₂‐ AR by the 2 methods were (1.00 ± 0.06) × 10⁵M⁻¹ and (1.52 ± 0.14) × 10⁴M⁻¹. The numbers of binding sites were (1.23 ± 0.07) × 10⁻⁷M and (9.09 ± 0.06) × 10⁻⁷M, respectively. These results indicated that β ₂‐ AR is the specific target for therapeutic action of protopine in vivo. The target‐drug binding occurred on Ser¹⁶⁹ in crystal structure of the receptor. Compared with frontal analysis, injection amount–dependent method is advantageous to drug saving, improvement of sampling efficiency, and performing speed. It has grave potential in high‐throughput drug‐receptor interaction analysis.     

Target‐directed screening of the bioactive compounds specifically binding to β2‐adrenoceptor in Semen brassicae by high‐performance affinity chromatography

The bioactive ingredients in Semen sinapis were rapidly screened by immobilized β₂‐adrenoceptor (β₂‐AR) and target‐directed molecular docking. The methods involved the attachment of β₂‐AR using any amino group in the receptor, the simultaneous separation and identification of the retention compounds by high‐performance affinity chromatography; the binding mechanism of the interesting compound to the receptor was investigated by zonal elution and molecular docking. Sinapine in Semen sinapis was proved to be the bioactive compound that specifically binds to the immobilized receptor. The association constant of sinapine to β₂‐AR was determined to be 1.36 × 10⁵ M^?1 with a value of 1.27 × 10^?6 M for the number of binding sites. Ionic bond was believed to be the driving force during the interaction between sinapine and β₂‐AR. It is possible to become a powerful alternative for rapid screening of bioactive compounds from a complex matrix such as traditional Chinese medicine and further investigation on the drug–receptor interaction. Copyright © 2015 John Wiley & Sons, Ltd.     

Preparation of extracellular domain 3 of human VEGF receptor‐2 and the monitoring of its real‐time binding to VEGF by biosensors

Vascular endothelial growth factor receptor‐2 (VEGFR‐2) plays an important role in stimulating the proliferation of endothelial cells and improving the permeability of blood vessels, which is involved in tumor angiogenesis, a process that is essential for tumor growth and metastasis. In this study, we describe a method for high yield of recombinant extracellular domain 3 (KDR3) of human VEGFR‐2 in an Escherichia coli system with further purification by cation exchange chromatography and immobilized metal affinity chromatography (IMAC). The biological activity of recombinant KDR3 was performed by sequestering VEGF in HUVEC proliferation assay. The real‐time binding of human VEGF to immobilized KDR3 was monitored by a label‐free biosensor, Optical waveguide lightmode spectroscopy (OWLS). Under the given experimental conditions, the association rate constant k_a was 4.2 × 10³ M^?1 s^?1 and the dissociation rate k_d was 5.1 × 10^?3 s^?1. The dissociation constant K_D was then calculated to be 1.2 × 10^?6 M. The obtained values will serve as baseline parameters for the design of improved versions of recombinant soluble VEGF receptors and the evaluation of developed anti‐KDR antibodies. In addition, such a scenario established by the use of OWLS will potentiate the kinetic study of ligand/receptor and antigen/antibody. The receptor discussed here, which block VEGF binding to cell membrane KDR, have potential clinical application in the treatment of cancer and other diseases where pathological angiogenesis is involved. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Identifying the antiasthmatic target of doxofylline using immobilized β2‐adrenoceptor based high‐performance affinity chromatography and site‐directed molecular docking

As a xanthine derivative, doxofylline is believed to be dominant for fighting against asthma in practice. Unlike other xanthines, the antiasthmatic effects of doxofylline lack any definite proof of target and mediating mechanism according to previous reports. In this work, the interaction between doxofylline and β₂‐AR was investigated by high performance affinity chromatography using frontal analysis and nonlinear model. The methodology involved the immobilization of β₂‐AR on the silica gel by a random linking method, the determination of the binding parameters by frontal analysis and nonlinear chromatography and the exploration of the binding mechanism by site‐directed molecular docking. The association constant for doxofylline binding to immobilized β₂‐AR was determined to be 7.70 × 10⁴ M^?1 by nonlinear chromatography and 5.91 × 10⁴ M^?1 by frontal analysis. Ser¹⁶⁹ and Ser¹⁷³ were the binding sites for the receptor–drug interaction on which hydrogen bond was believed to be the main driven force during the interaction. These results indicated that the antiasthmatic effects of doxofylline may be behind the mediating mechanism of β₂‐AR. High performance affinity chromatography based on immobilized receptor has potential to become an alternative for drug target confirmation and drug–receptor interaction analysis. Copyright © 2016 John Wiley & Sons, Ltd.     

2‐Hydroxynaphthalene‐1‐carbaldehyde‐ and 2‐(Aminomethyl)pyridine‐Based Schiff Base CuII Complexes for DNA Binding and Cleavage

Three mononuclear Cu^II complexes, [CuCl(naph‐pa)] ( 1 ), [Cu(bipy)(naph‐pa)]Cl ( 2 ), and [Cu(naph‐pa)(phen)]Cl ( 3 ) ((naph‐pa)=Schiff base derived from the condensation of 2‐hydroxynaphthalene‐1‐carbaldehyde and 2‐picolylamine (=2‐(aminomethyl)pyridine), bipy=2,2′‐bypiridine, and phen=1,10‐phenanthroline) were synthesized and characterized. Complex 1 exhibits square‐planar geometry, and 2 and 3 exhibit square pyramidal geometry, where Schiff base and bipy/phen act as NNO and as NN donor ligands, respectively. CT (Calf thymus)‐DNA‐binding studies revealed that the complexes bind through intercalative mode and show good binding propensity (intrinsic binding constant K_b: 0.98×10⁵, 2.22×10⁵, and 2.67×10⁵ M ^?1 for 1 – 3 , resp.). The oxidative and hydrolytic DNA‐cleavage activity of these complexes has been studied by gel electrophoresis: all the complexes displayed chemical nuclease activity in the presence and absence of H₂O₂. From the kinetic experiments, hydrolytic DNA cleavage rate constants were determined as 2.48, 3.32, and 4.10 h^?1 for 1 – 3 , respectively. It amounts to (0.68–1.14)×10⁸‐fold rate enhancement compared to non‐catalyzed DNA cleavage, which is impressive. The complexes display binding and cleavage propensity to DNA in the order of 3 > 2 > 1 .     

Investigation of the binding characteristics between ligands and epidermal growth factor receptor by cell membrane chromatography

The binding property between a ligand and its receptor is very important for numerous biological processes. In this study, we developed a high epidermal growth factor receptor (EGFR)‐expression cell membrane chromatography (CMC) method to investigate the binding characteristics between EGFR and the ligands gefitinib, erlotinib, canertinib, afatinib, and vandetanib. Competitive binding analysis using gefitinib as the marker was used to investigate the interactions that occurred at specific binding sites on EGFR. The ability of displacement was measured from the HEK293‐EGFR/CMC column on the binding sites occupied by gefitinib for these ligands, which revealed the following order: gefitinib (K_D, 8.49 ± 0.11 × 10^?7 M) > erlotinib (K_D, 1.07 ± 0.02 × 10^?6 M) > canertinib (K_D, 1.41 ± 0.07 × 10^?6 M) > afatinib (K_D, 1.80 ± 0.12 × 10^?6 M) > vandetanib (K_D, 1.99 ± 0.03 × 10^?6 M). This order corresponded with the values estimated by frontal displacement analysis and the scores obtained with molecular docking. Furthermore, thermodynamic analysis indicated that the hydrogen bond or Van der Waals force was the main interaction force in the process of EGFR binding to all 5 ligands. Overall, these results demonstrate that a CMC method could be an effective tool to investigate the binding characteristics between ligands and receptors.     

The optical biosensor study of the redox partner interaction with the cytochrome P450 2B4-containing monooxygenase system under hydroxylation conditions

The interactions between cytochrome P450 2B4 (d-2B4), NADPH:cytochrome P450 reductase and cytochrome b5 have been investigated in the monomeric reconstituted P450 2B4-containing monooxygenase system in the presence of a substrate (7-pentoxyresorufin) and an electron donor, NADPH. Each partner was immobilized via its amino groups on the carboxymethyldextran biochip surface of the optical biosensor IAsys+. Such mode immobilization was not accompanied by any loss of activities of the immobilized proteins. The formation of binary d-Fp/d-2B4 complexes was registered. The association/dissociation rate constants (k_on/k_off) were (0.013 ± 0.005) × 10⁶ M^?1 s^?1/0.05 ± 0.02 s^?1, and dissociation constant (K_D) was (0.26 ± 0.13) × 10^?6 M. Comparison of k_on, k_off and K_D values for d-Fp/d-2B4 complexes formed under hydroxylation (O-dealkylation) with corresponding constants obtained for the oxidized proteins of (0.10 ± 0.03) × 10⁶ M^?1 s^?1/(0.14 ± 0.06) s^?1, and (0.71 ± 0.37) × 10^?6 M, respectively shows that the decrease in k_on and an insignificant decrease in K_D are associated with the increase of complex lifetime during transition from the oxidized to hydroxylation conditions. Complex formation between d-Fp and d-b5 was not registered in both hydroxylation conditions and in the case of oxidized forms of these proteins. In both cases formation of the ternary d-Fp/d-2B4/d-b5 complexes occurred.     

Overpotentials and Faraday Efficiencies in CO2 Electrocatalysis–the Impact of 1‐Ethyl‐3‐Methylimidazolium Trifluoromethanesulfonate

The mixtures of room temperature ionic liquid 1‐ethyl‐3‐methylimidazolium trifluoromethanesulfonate ([EMIM]TFO) and water as electrolytes for reduction of CO₂ to CO are reported. Linear sweep voltammetry shows overpotentials for CO₂ reduction and the competing hydrogen evolution reaction (HER), both of which vary as a function of [EMIM]TFO concentration in the range from 4 × 10^?3m (0.006 mol%) to 4869 × 10^?3m (50 mol%). A steady lowering of overpotentials up to an optimum for 334 × 10?³m is identified. At 20 mol% and more of [EMIM]TFO, a significant CO₂ reduction plateau and inhibition of HER, which is limited by H₂O diffusion, is noted. Such a plateau in CO₂ reduction correlates to high CO Faraday efficiencies. In case of 50 mol% [EMIM]TFO, a broad plateau spanning over a potential range of 0.58 V evolves. At the same time, the overpotential for HER is increased by 1.20 V when compared to 334 × 10^?3m and, in turn, HER is largely inhibited. The Faraday efficiencies for CO and H₂ formation feature 95.6% ± 6.8% and 0.5% ± 0.3%, respectively, over a period of 3 h in a separator divided cell. Cathodic as well as anodic electrochemical stability of the electrolyte throughout this time period is corroborated in ¹H NMR spectroscopic measurements.     

β‐Cell Function and Insulin Sensitivity in Adolescents From an OGTT

Given the increase in the incidence of insulin resistance, obesity, and type 2 diabetes in children and adolescents, it would be of paramount importance to assess quantitative indices of insulin secretion and action during a physiological perturbation, such as a meal or an oral glucose‐tolerance test (OGTT). A minimal model method is proposed to measure quantitative indices of insulin secretion and action in adolescents from an oral test. A 7 h, 21‐sample OGTT was performed in 11 adolescents. The C‐peptide minimal model was identified on C‐peptide and glucose data to quantify indices of β‐cell function: static φ_s and dynamic φ_d responsivity to glucose from which total responsivity φ was also measured. The glucose minimal model was identified on glucose and insulin data to estimate insulin sensitivity, S_I, which was compared to a reference measure, S_I^ref, provided by a tracer method. Disposition indices, which adjust insulin secretion for insulin action, were then calculated. Indices of β‐cell function were φ_s = 51.35 ± 8.89 × 10^?9min^?1, φ_d = 1,392 ± 258 × 10^?9, and φ = 82.09 ± 17.70 × 10^?9min^?1. Insulin sensitivity was S_I = 14.19 ± 2.73 × 10^?4, not significantly different from S_I^ref = 14.96 ± 3.04 × 10^?4 dl/kg·min per µU/ml, and well correlated: r = 0.98, P < 0.0001, thus indicating that S_I can be accurately measured from an oral test. Disposition indices were DI_s = 1,040 ± 201 × 10^?14 dl/kg/min² per pmol/l, DI_d = 33,178 ± 10,720 × 10^?14 dl/kg/min per pmol/l, DI = 1,844 ± 522 × 10^?14 dl/kg/min² per pmol/l. Virtually the same minimal model assessment was obtained with a reduced 3 h, 9‐sample protocol. OGTT interpreted with C‐peptide and glucose minimal model has the potential to provide novel insight regarding the regulation of glucose metabolism in adolescents, and to evaluate the effect of obesity and interventions such as diet and exercise.     

Stepwise Frontal Analysis Coupled with Affinity Chromatography: A Fast and Reliable Method for Potential Ligand Isolation and Evaluation from Mahuang-Fuzi-Xixin Decoction

Mahuang-Fuzi-Xixin Decoction (MFXD) is widely used in the treatment of asthma, however, the functional components in the decoction targeting beta2-adrenoceptor (β₂-AR) remain unclear. Herein, we immobilized the haloalkane dehalogenase (Halo)-tagged β₂-AR on the 6-chlorocaproic acid-modified microspheres. Using the affinity stationary phase, the interactions of four ligands with the receptor were analyzed by stepwise frontal analysis. The association constants were (4.75±0.28)×10⁴ M⁻¹ for salbutamol, (2.93±0.15)×10⁴ M⁻¹ for terbutaline, (1.23±0.03)×10⁴ M⁻¹ for methoxyphenamine, (5.67±0.38)×10⁴ M⁻¹ for clorprenaline at high-affinity binding site, and (2.73±0.05)×10³ M⁻¹ at low-affinity binding site. These association constants showed the same rank order as the radioligand binding assay, demonstrating that immobilized β₂-AR had capacity to screen bioactive compounds binding to the receptor while stepwise frontal analysis could predict their binding affinities. Application of the immobilized receptor in analysis of MFXD by chromatographic method revealed that ephedrine, aconifine, karakoline, and chasmanine were the bioactive compounds targeting β₂-AR. Among them, ephedrine and chasmanine exhibited association constants of (2.94±0.02)×10⁴ M^-1and (4.60±0.15)×10⁴ M⁻¹ to the receptor by stepwise frontal analysis. Molecular docking analysis demonstrated that ephedrine, chasmanine, and the other two compounds interact with β₂-AR through the same pocket involving the key amino acids such as Asn312, Asp113, Phe289, Trp286, Tyr316, and Val114. As such, we reasoned that the four compounds dominate the therapeutic effect of MFXD against asthma through β₂-AR mediating pathway. This work shed light on the potential of immobilized β₂-AR for drug discovery and provided a valuable methodology for rapid screening.     

Inhibition of AChE by malathion and some structurally similar compounds

Inhibition of bovine erythrocyte acetylcholinesterase (free and immobilized on controlled pore glass) by separate and simultaneous exposure to malathion and malathion transformation products which are generally formed during storage or through natural or photochemical degradation was investigated. Increasing concentrations of malathion, its oxidation product malaoxon, and its isomerisation product isomalathion inhibited free and immobilized AChE in a concentration-dependent manner. K_I, the dissociation constant for the initial reversible enzyme inhibitor-complex, and k₃, the first order rate constant for the conversion of the reversible complex into the irreversibly inhibited enzyme, were determined from the progressive development of inhibition produced by reaction of native AChE with malathion, malaoxon and isomalathion. K_I values of 1.3 × 10^{? 4} M^{? 1}, 5.6 × 10^{? 6} M^{? 1} and 7.2 × 10^{? 6} M^{? 1} were obtained for malathion, malaoxon and isomalathion, respectively. The IC₅₀ values for free/immobilized AChE, (3.7 ± 0.2) × 10^{? 4} M/(1.6 ± 0.1) × 10^{? 4}, (2.4 ± 0.3) × 10^{? 6}/(3.4 ± 0.1) × 10^{? 6} M and (3.2 ± 0.3) × 10^{? 6} M/(2.7 ± 0.2) × 10^{? 6} M, were obtained from the inhibition curves induced by malathion, malaoxon and isomalathion, respectively. However, the products formed due to photoinduced degradation, phosphorodithioic O,O,S-trimethyl ester and O,O-dimethyl thiophosphate, did not noticeably affect enzymatic activity, while diethyl maleate inhibited AChE activity at concentrations > 10 mM. Inhibition of acetylcholinesterase increased with the time of exposure to malathion and its inhibiting by-products within the interval from 0 to 5 minutes. Through simultaneous exposure of the enzyme to malaoxon and isomalathion, an additive effect was achieved for lower concentrations of the inhibitors (in the presence of malaoxon/isomalathion at concentrations 2 × 10^{? 7} M/2 × 10^{? 7} M, 2 × 10^{? 7} M/3 × 10^{? 7} M and 2 × 10^{? 7} M/4.5 × 10^{? 7} M), while an antagonistic effect was obtained for all higher concentrations of inhibitors. The presence of a non-inhibitory degradation product (phosphorodithioic O,O,S-trimethyl ester) did not affect the inhibition efficiencies of the malathion by-products, malaoxon and isomalathion.     

Synthetic peptide TPLVTLFK (octarphin) reduces the corticosterone production by rat adrenal cortex through nonopioid β‐endorphin receptor

The synthetic peptide octarphin (TPLVTLFK) corresponding to the sequence 12–19 of β‐endorphin, a selective agonist of nonopioid β‐endorphin receptor, was labeled with tritium to a specific activity of 29 Ci/mmol. [³H]Octarphin was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from rat adrenal cortex (K_d = 35.7 ± 2.3 nM, B_max = 41.0 ± 3.6 pmol/mg protein). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but to α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin as well. At the same time, the [³H]octarphin‐specific binding with adrenal cortex membranes was inhibited by unlabeled β‐endorphin (K_i = 32.9 ± 3.8 nM). Octarphin at concentrations of 10^?9–10^?6 M was found to inhibit the adenylate cyclase activity in adrenocortical membranes, whereas intranasal injection of octarphin at doses of 5 and 20 µg/rat was found to reduce the secretion of corticosterone from the adrenals to the bloodstream. Thus, octarphin decreases the adrenal cortex functional activity through the high affinity binding to nonopioid receptor of β‐endorphin. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

In vitro effects of 2‐methoxyestradiol on cell numbers,morphology, cell cycle progression,and apoptosis induction in oesophageal carcinoma cells

The influence of 2‐methoxyestradiol (2‐ME) was investigated on cell numbers, morphology, cell cycle progression, and apoptosis induction in an oesophageal carcinoma cell line (WHCO3). Dose‐dependent studies (1 × 10^?9M–1 × 10^?6M) revealed that 2‐ME significantly reduced cell numbers to 60% in WHCO3 after 72 h of exposure at a concentration of 1 × 10^?6M compared to vehicle‐treated cells. Morphological studies entailing light‐, fluorescent‐, as well as transmission electron microscopy (TEM) confirmed 2‐ME's antimitotic effects. These results indicated hallmarks of apoptosis including cell shrinkage, hypercondensation of chromatin, cell membrane blebbing, and apoptotic bodies in treated cells. Flow cytometric analyses demonstrated an increase in the G₂/M‐phase after 2‐ME exposure; thus preventing cells from proceeding through the cell cycle. β‐tubulin immunofluorescence revealed that 2‐ME caused spindle disruption. In addition, increased expression of death receptor 5 protein was observed further supporting the proposed mechanism of apoptosis induction via the extrinsic pathway in 2‐ME‐exposed oesophageal carcinoma cells. Copyright © 2009 John Wiley & Sons, Ltd.     

Estimation of interaction between oriented immobilized green fluorescent protein and its antibody by high performance affinity chromatography and molecular docking

Although green fluorescence protein (GFP) and its antibody are widely used to track a protein or a cell in life sciences, the binding behavior between them remains unclear. In this work, diazo coupling method that synthesized a new stationary GFP was oriented immobilized on the surface of macro‐porous silica gel by a phase. The stationary phase was utilized to confirm the validation of injection amount‐dependent analysis in exploring protein–protein interaction that use GFP antibody as a probe. GFP antibody was proved to have one type of binding site on immobilized GFP. The number of binding site and association constant were calculated to be (6.41 ± 0.76) × 10^‐10 M and (1.39 ± 0.12) × 10⁹ M^‐1. Further analysis by molecular docking showed that the binding of GFP to its antibody is mainly driven by hydrogen bonds and salt bridges. These results indicated that injection amount‐dependent analysis is capable of exploring the protein–protein interactions with the advantages of ligand and time saving. It is a valuable methodology for the ligands, which are expensive or difficult to obtain. Copyright © 2015 John Wiley & Sons, Ltd.     

Binding of synthetic fragments of β‐endorphin to nonopioid β‐endorphin receptor

Selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin (SLTCLVKGFY) was labeled with tritium (the specific activity of 24 Ci/mmol). [³H]Immunorphin was found to bind to nonopioid β‐endorphin receptor of mouse peritoneal macrophages (K_d = 2.0 ± 0.1 nM ). The [³H]immunorphin specific binding with macrophages was inhibited by unlabeled β‐endorphin (K_i = 2.9 ± 0.2 nM ) and was not inhibited by unlabeled naloxone, α‐endorphin, γ‐endorphin and [Met⁵]enkephalin (K_i > 10 µM ). Thirty fragments of β‐endorphin have been synthesized and their ability to inhibit the [³H]immunorphin specific binding to macrophages was studied. Unlabeled fragment 12–19 (TPLVTLFK, the author's name of the peptide octarphin) was found to be the shortest peptide possessing practically the same inhibitory activity as β‐endorphin (K_i = 3.1 ± 0.3 nM ). The peptide octarphin was labeled with tritium (the specific activity of 28 Ci/mmol). [³H]Octarphin was found to bind to macrophages with high affinity (K_d = 2.3 ± 0.2 nM ). The specific binding of [³H]octarphin was inhibited by unlabeled immunorphin and β‐endorphin (K_i = 2.4 ± 0.2 and 2.7 ± 0.2 nM , respectively). Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Vitronectin binds to the head region of Moraxella catarrhalis ubiquitous surface protein A2 and confers complement‐inhibitory activity

The serum resistance of the common respiratory pathogen Moraxella catarrhalis is mainly dependent on ubiquitous surface proteins (Usp) A1 and A2 that interact with complement factor 3 (C3) and complement inhibitor C4b binding protein (C4BP) preventing the alternative and classical pathways of the complement system respectively. UspA2 also has the capacity to attract vitronectin that in turn binds C9 and hereby inhibits membrane attack complex (MAC) formation. We found UspA2 as a major vitronectin binding protein and hence the UspA2/vitronectin interaction was studied in detail. The affinity constant (K_D) for vitronectin binding to UspA2 was 2.3 × 10^?8 M, and the N‐terminal region encompassing residues UspA2 30–170 bound vitronectin with a K_D of 7.9 × 10^?8 M. Electron microscopy verified that the active binding domain (UspA2^30–177) was located at the head region of UspA2. Experiments with recombinantly expressed vitronectin also revealed that UspA2^30–177 bound to the C‐terminal region of vitronectin residues 312–396. Finally, when human serum was pre‐incubated with UspA2, bacteria showed significantly less serum resistance. Our study directly reveals the binding mode between the N‐terminal domain of UspA2 and the C‐terminal part of vitronectin and thus sheds light upon the mechanism of M. catarrhalis‐dependent serum resistance.     

A biomimetic Protein G affinity adsorbent: an Ugi ligand for immunoglobulins and Fab fragments based on the third IgG‐binding domain of Protein G

This work reports the development of a synthetic affinity adsorbent for immunoglobulins based on the Fab‐binding domain of Streptococcal Protein G (SpG‐domain III). The ligand (A2C7I1) was synthesized by the four‐component Ugi reaction to generate a substituted peptoidal scaffold mimicking key amino acid residues of SpG. Computer‐aided analysis suggests a putative binding site on the C_H1 domain of the Fab molecule. In silico studies, supported by affinity chromatography in comparison with immobilized SpG, as well as analytical characterization by liquid chromatography/electrospray ionization–mass spectrometry and ¹H nuclear magnetic resonance of the ligand synthesized in solution, indicated the authenticity and suitability of the designed ligand for the purification of immunoglobulins. The immobilized ligand displayed an apparent static binding capacity of ~17 mg IgG ml^?1 and a dissociation constant of 5.34 × 10^?5 M. Preparative chromatography demonstrated the ability of the immobilized ligand to purify IgG and Fab fragments from crude mammalian and yeast cell cultures, under near physiological ionic strength and pH, to yield proteins of 99% and 93% purity, respectively. Copyright © 2013 John Wiley & Sons, Ltd.     

Cytochalasin B binding by human platelets

Intact human platelets bind cytochalasin B (CB) with a capacity of 100– 120 p mols CB/mg protein or approximately 7 × 10⁴ molecules/cell and dissociation constants (K_D) ranging from 2 × 10^?8 to 10^?6 M. Up to 85% of this saturable binding is displaced by 10^?5 M cytochalasin E (CE). This CE-sensitive binding also appears heterogeneous with K_D similar to those of the overall binding. The CE-insensitive binding, however, appears as a single component with K_D ≌ 4 × 10^?7 M. The sedimentable constituents from frozen, thawed, and washed cells also bind CB with K_D ranging from 2.4 × 10^?8 to 1.5 × 10^?6 M and a total capacity of approximately 39 p mols/mg protein which accounts for only 4% of the ligand binding to the intact cell. The major portion (60–80%) of this CB binding is displaceable by 500 mM D-glucose and has a K_D of 1.5 × 10^?6M, while only 10–15% is CE-sensitive with a K_D of 2.4 ± 10^?8 M. It is concluded that 95% of the saturable CB binding in platelets is associated with the cytosol of which 80–85% is sensitive to CE and that only 3% of the cellular binding is glucose sensitive, membrane-associated binding. If the CE-sensitive binding associated with the cytosol is entirely to actin, the stoichiometry of this binding is approximately one CB to 30 actin monomers, which is greater by an order of magnitude than that for CB binding to muscle actin.     

Novel renin inhibitors containing derivatives of N‐alkylleucyl‐β‐hydroxy‐γ‐amino acids

In search for new drugs lowering arterial blood pressure, which could be applied in anti‐hypertensive therapy, research concerning agents blocking of renin‐angiotensin‐aldosteron system has been conducted. Despite many years of research conducted at many research centers around the world, aliskiren is the only one renin inhibitor, which is used up to now. Four novel potential renin inhibitors, having structure based on the peptide fragment 8–13 of human angiotensinogen, a natural substrate for renin, were designed and synthesized. All these inhibitors contain unnatural moieties that are derivatives of N‐methylleucyl‐β‐hydroxy‐γ‐amino acids at the P₂‐P₁' position: 4‐[N‐(N‐methylleucyl)‐amino]‐3‐hydroxy‐7‐(3‐nitroguanidino)‐heptanoic acid (AHGHA), 4‐[N‐(N‐methylleucyl)‐amino]‐3‐hydroxy‐5‐phenyl‐pentanoic acid (AHPPA) or 4‐[N‐(N‐methylleucyl)‐amino]‐8‐benzyloxycarbonylamino‐3‐hydroxyoctanoic acid (AAHOA). The previously listed synthetic β‐hydroxy‐γ‐amino acids constitute pseudodipeptidic units that correspond to the P₁‐P₁' position of the inhibitor molecule. An unnatural amino acid, 4‐methoxyphenylalanin (Phe(4‐OMe)), was introduced at the P₃ position of the obtained compounds. Three of these compounds contain isoamylamide of 6‐aminohexanoic acid (ε‐Ahx‐Iaa) at the P₂'‐P₃' position. The proposed modifications of the selected human angiotensinogen fragment are intended to increase bioactivity, bioavailability, and stability of the inhibitor molecule in body fluids and tissues. The inhibitor Boc‐Phe(4‐OMe)‐MeLeu‐AHGHA‐OEt was obtained in the form of an ethyl ester. The hydrophobicity coefficient, expressed as log P varied between 3.95 and 8.17. In vitro renin inhibitory activity of all obtained compounds was contained within the range 10^?6‐10^?9 M. The compound Boc‐Phe(4‐OMe)‐MeLeu‐AHPPA‐Ahx‐Iaa proved to be the most active (IC₅₀ = 1.05 × 10^?9 M). The compounds Boc‐Phe(4‐OMe)‐MeLeu‐AHGHA‐Ahx‐Iaa and Boc‐Phe(4‐OMe)‐MeLeu‐AHPPA‐Ahx‐Iaa are resistant to chymotrypsin. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Temperature‐controlled down‐conversion luminescence behavior of Eu3+‐doped transparent MF2 (M = Ba,Ca, Sr) glass ceramics

Eu³⁺‐doped transparent glass ceramics containing MF₂ (M = Ba, Ca, Sr) nanocrystals were fabricated using a melt–quenching method, and the resulting structures were studied using X‐ray diffraction. Levels ⁵D₁ and ⁵D₀ of Eu³⁺ ions were verified as thermally coupled levels using the fluorescence intensity ratio method. The fluorescence intensity ratios, optical temperature sensitivity and thermal quenching ratios of the transparent glass ceramics were studied as a function of temperature. With an increase in temperature, the relative sensitivity (S _R) decreased sharply at first, then slowly increased, before finally decreasing. The minimum S _R values of GCBaF₂ (GCB), GCCaF₂ (GCC) and GCSrF₂ (GCS) were 2.8 × 10^?4, 0.8 × 10^?4 and 1.9 × 10^?4 K^?1 at 360, 269 and 319 K, respectively. Glass ceramics with an intense emission intensity can be used to convert the measured spectrum into temperature and may have an important role in temperature detectors.