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1.
Biochimie ; 222: 169-194, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38494106

RESUMEN

We discuss the diverse functions of proteases in the context of their biotechnological and medical significance, as well as analytical approaches used to determine the functional activity of these enzymes. An insight into modern approaches to studying the kinetics and specificity of proteases, based on spectral (absorption, fluorescence), mass spectrometric, immunological, calorimetric, and electrochemical methods of analysis is given. We also examine in detail electrochemical systems for determining the activity and specificity of proteases. Particular attention is given to exploring innovative electrochemical systems based on the detection of the electrochemical oxidation signal of amino acid residues, thereby eliminating the need for extra redox labels in the process of peptide synthesis. In the review, we highlight the main prospects for the further development of electrochemical systems for the study of biotechnologically and medically significant proteases, which will enable the miniaturization of the analytical process for determining the catalytic activity of these enzymes.


Asunto(s)
Péptido Hidrolasas , Péptido Hidrolasas/metabolismo , Péptido Hidrolasas/química , Humanos , Técnicas Electroquímicas/métodos , Animales , Biocatálisis , Oxidación-Reducción , Catálisis , Cinética
2.
Biomedicines ; 12(1)2024 Jan 11.
Artículo en Inglés | MEDLINE | ID: mdl-38255257

RESUMEN

We describe a bielectrode system for evaluation of the electrocatalytic activity of cytochrome P450 2E1 (CYP2E1) towards chlorzoxazone. One electrode of the system was employed to immobilize Bactosomes with human CYP2E1, cytochrome P450 reductase (CPR), and cytochrome b5 (cyt b5). The second electrode was used to quantify CYP2E1-produced 6-hydroxychlorzoxazone by its direct electrochemical oxidation, registered using square-wave voltammetry. Using this system, we determined the steady-state kinetic parameters of chlorzoxazone hydroxylation by CYP2E1 of Bactosomes immobilized on the electrode: the maximal reaction rate (Vmax) was 1.64 ± 0.08 min-1, and the Michaelis constant (KM) was 78 ± 9 µM. We studied the electrochemical characteristics of immobilized Bactosomes and have revealed that electron transfer from the electrode occurs both to the flavin prosthetic groups of CPR and the heme iron ions of CYP2E1 and cyt b5. Additionally, it has been demonstrated that CPR has the capacity to activate CYP2E1 electrocatalytic activity towards chlorzoxazone, likely through intermolecular electron transfer from the electrochemically reduced form of CPR to the CYP2E1 heme iron ion.

3.
Talanta ; 257: 124341, 2023 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-36821964

RESUMEN

In this work, we proposed a biosensor for trypsin proteolytic activity assay using immobilization of model peptides on screen-printed electrodes (SPE) modified with gold nanoparticles (AuNPs) prepared by electrosynthetic method. Sensing of proteolytic activity was based on electrochemical oxidation of tyrosine residues of peptides. We designed peptides containing N-terminal cysteine residue for immobilization on an SPE, modified with gold nanoparticles, trypsin-specific cleavage site and tyrosine residue as a redox label. The peptides were immobilized on SPE by formation of chemical bonds between mercapto groups of the N-terminal cysteine residues and AuNPs. After the incubation with trypsin, time-dependent cleavage of the immobilized peptides was observed by decline in tyrosine electrochemical oxidation signal. The kinetic parameters of trypsin, such as the catalytic constant (kcat), the Michaelis constant (KM) and the catalytic efficiency (kcat/KM), toward the CGGGRYR peptide were determined as 0.33 ± 0.01 min-1, 198 ± 24 nM and 0.0016 min-1 nM-1, respectively. Using the developed biosensor, we demonstrated the possibility of analysis of trypsin specificity toward the peptides with amino acid residues disrupting proteolysis. Further, we designed the peptides with proline or glutamic acid residues after the cleavage site (CGGRPYR and CGGREYR), and trypsin had reduced activity toward both of them according to the existing knowledge of the enzyme specificity. The developed biosensor system allows one to perform a comparative analysis of the protease steady-state kinetic parameters and specificity toward model peptides with different amino acid sequences.


Asunto(s)
Técnicas Biosensibles , Nanopartículas del Metal , Tripsina/metabolismo , Oro/química , Tirosina , Cisteína , Nanopartículas del Metal/química , Péptidos/química , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Electrodos
4.
Biophys Chem ; 291: 106894, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36174335

RESUMEN

The possibility of the detection of atypical kinetic profiles of drug biotransformation using electrochemical systems based on immobilized cytochromes P450 with phenytoin hydroxylation by cytochrome P450 2C19 (CYP2C19) as an example was evaluated for the first time. For this purpose, we developed an electrochemical system, where one of the electrodes was modified by didodecyldimethylammonium bromide (DDAB) and was used as an electron donor for reduction of heme iron ion of the immobilized CYP2C19 and initiation of the catalytic reaction, while the second electrode was not modified and served for an electrochemical quantitation of 4-hydroxyphenytoin, which is a metabolite of antiepileptic drug phenytoin, by its oxidation peak. It was revealed that the dependence of the rate of 4-hydroxyphenytoin formation on phenytoin concentration is described by the equation for two enzymes or two binding sites indicating the existing of high- and low-affinity forms of the enzyme. The atypical kinetics and the kinetic parameters of CYP2C19-mediated phenytoin hydroxylation in the electrochemical system correlate to the same characteristics obtained by other authors in an alternative enzymatic system. Our results demonstrate the possibility of electrochemical systems based on cytochromes P450 to be applied for the detection of atypical kinetic profiles of drug metabolism.


Asunto(s)
Sistema Enzimático del Citocromo P-450 , Fenitoína , Citocromo P-450 CYP2C19/metabolismo , Sistema Enzimático del Citocromo P-450/química , Hidroxilación , Biotransformación
5.
Biochemistry (Mosc) ; 86(Suppl 1): S140-S151, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33827405

RESUMEN

Methods of electrochemical analysis of biological objects based on the reaction of electro-oxidation/electro-reduction of molecules are presented. Polymer nanocomposite materials that modify electrodes to increase sensitivity of electrochemical events on the surface of electrodes are described. Examples of applications electrochemical biosensors constructed with nanocomposite material for detection of biological molecules are presented, advantages and drawbacks of different applications are discussed.


Asunto(s)
Técnicas Biosensibles , Técnicas Electroquímicas/métodos , ADN/análisis , Nanocompuestos , Nanotubos
6.
Bioelectrochemistry ; 140: 107736, 2021 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-33494014

RESUMEN

The interactions of dsDNA with rifampicin (RF) or with rifampicin after encapsulation in phospholipid micelles (nanosome/rifampicin) (NRF) were studied electrochemically. Screen-printed electrodes (SPEs) modified by stable dispersions of multi-wolled carbon nanotubes (MWCNTs) in aqueous solution of poly(1,2-butadiene)-block-poly(2-(dimethylamino)ethyl methacrylate) (PB290-b-PDMAEMA240) diblock copolymer were used for quantitative electrochemical investigation of direct electrochemical oxidation of guanine at E = 0.591 V (vs. Ag/AgCl) and adenine at E = 0.874 V (vs. Ag/AgCl) of dsDNA and its change in the presence of RF or NRF. Due to RF or NRF interaction with dsDNA, the differential pulse voltammetry (DPV) peak currents of guanine and adenine decreased and the peak potentials shifted to more positive values with increasing drug concentration (RF or NRF). Binding constants (Kb) of complexes RF-dsDNA and NRF-dsDNA were calculated based on adenine and guanine oxidation signals. The Kb values for RF-dsDNA were 1.48 × 104 M-1/8.56 × 104 M-1, while for NRF-dsDNA were 2.51 × 104 M-1/1.78 × 103 M-1 (based on adenine or guanine oxidation signals, respectively). The values of Kb revealed intercalation mode of interaction with dsDNA for RF and mixed type of interaction (intercalation and electrostatic mode) for NRF. The estimated values of ΔG (Gibbs free energy) of the complex formation confirmed that drug-dsDNA interactions are spontaneous and favourable reactions.


Asunto(s)
Antibióticos Antituberculosos/farmacología , ADN/metabolismo , Nanocápsulas/química , Rifampin/farmacología , Antibióticos Antituberculosos/administración & dosificación , Técnicas Electroquímicas , Micelas , Modelos Moleculares , Fosfolípidos/química , Rifampin/administración & dosificación
7.
Fundam Clin Pharmacol ; 35(2): 423-431, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33012006

RESUMEN

We have investigated interactions of galeterone and its pharmacologically active metabolite - 3-keto-Δ4-galeterone (D4G) - with one of the key enzymes of corticosteroid biosynthesis - steroid 21-monooxygenase (CYP21A2). It was shown by absorption spectroscopy that both compounds induce type I spectral changes of CYP21A2. Spectral dissociation constants (KS ) of complexes of CYP21A2 with galeterone or D4G were calculated as 3.1 ± 0.7 µm and 4.6 ± 0.4 µm, respectively. It was predicted by molecular docking that both ligands similarly bind to the active site of CYP21A2. We have revealed using reconstituted monooxygenase system that galeterone is a competitive inhibitor of CYP21A2 with the inhibition constant (Ki ) value of 12 ± 3 µm, while D4G at the concentrations of 10 and 25 µm does not inhibit the enzyme. Summarizing, based on the in vitro analyses we detected inhibition of CYP21A2 by galeterone and lack of the influence of D4G on this enzyme.


Asunto(s)
Androstadienos/química , Bencimidazoles/química , Inhibidores Enzimáticos/química , Esteroide 21-Hidroxilasa/química , Interacciones Farmacológicas , Humanos , Masculino , Simulación del Acoplamiento Molecular , Neoplasias de la Próstata/tratamiento farmacológico
8.
Polymers (Basel) ; 12(7)2020 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-32650434

RESUMEN

We demonstrate the application of amphiphilic ionic poly(n-butylmethacrylate)-block- poly(2-(dimethylamino)ethyl methacrylate) diblock copolymers (PnBMA40-b-PDMAEMA40, PnBMA40-b-PDMAEMA120, PnBMA70-b-PDMAEMA120) for dispersing multiwalled carbon nanotubes (MWCNTs) in aqueous media, a subsequent efficient surface modification of screen-printed electrodes (SPEs), and the application of the modified SPEs for DNA electrochemistry. Stable and fine aqueous dispersions of MWCNTs were obtained with PnBMAx-b-PDMAEMAy diblock copolymers, regardless of the structure of the copolymer and the amount of MWCNTs in the dispersions. The effect of the diblock copolymer structure was important when the dispersions of MWCNTs were deposited as modifying layers on surfaces of SPEs, resulting in considerable increases of the electroactive surface areas and great acceleration of the electron transfer rate. The SPE/(PnBMAx-b-PDMAEMAy + MWCNT) constructs were further exploited for direct electrochemical oxidation of the guanine (G) and adenine (A) residues in a model salmon sperm double-stranded DNA (dsDNA). Two well-defined irreversible oxidation peaks were observed at about +600 and +900 mV, corresponding to the electrochemical oxidation of G and A residues, respectively. A multi-parametric optimization of dsDNA electrochemistry enables one to get the limits of detection (LOD) as low as 5 µg/mL (0.25 µM) and 1 µg/mL (0.05 µM) for G and A residues, respectively. The achieved sensitivity of DNA assay enables quantification of the A and G residues of dsDNA in the presence of human serum and DNA in isolated human leukocytes.

9.
Steroids ; 162: 108693, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32645328

RESUMEN

The interactions of pharmacologically active 3-keto-Δ4-metabolite of anticancer drug abiraterone (D4A) with steroid-metabolizing cytochromes P450 (CYP51A1, CYP11A1, CYP19A1) was studied by absorption spectroscopy and molecular docking. Both abiraterone and D4A induce type I spectral changes of CYP51A1, one of the enzymes of cholesterol biosynthesis. We have revealed that D4A did not induce spectral changes of CYP11A1, the key enzyme of pregnenolone biosynthesis, unlike abiraterone (type II ligand of CYP11A1). On the contrary, D4A interacts with the active site of CYP19A1, the key enzyme of estrogen biosynthesis, inducing type II spectral changes, while abiraterone does not. Spectral analysis allowed us to calculate spectral dissociation constant (KS) for each complex of cytochrome P450 with respective ligands. The data were supported by molecular docking. The obtained results broaden understanding of interactions of D4A with some of the key steroid-metabolizing cytochromes P450 and allow one to predict possible disproportions of steroid metabolism.


Asunto(s)
Androstenos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Simulación del Acoplamiento Molecular , Sistema Enzimático del Citocromo P-450/química , Unión Proteica , Conformación Proteica , Análisis Espectral
10.
Fundam Clin Pharmacol ; 34(1): 120-130, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31286572

RESUMEN

Potential drug-drug interactions of the antitumor drug abiraterone and the macrolide antibiotic erythromycin were studied at the stage of cytochrome P450 3A4 (CYP3A4) biotransformation. Using differential spectroscopy, we have shown that abiraterone is a type II ligand of CYP3A4. The dependence of CYP3A4 spectral changes on the concentration of abiraterone is sigmoidal, which indicates cooperative interactions of CYP3A4 with abiraterone; these interactions were confirmed by molecular docking. The dissociation constant (Kd ) and Hill coefficient (h) values for the CYP3A4-abiraterone complex were calculated as 3.8 ± 0.1 µM and 2.3 ± 0.2, respectively. An electrochemical enzymatic system based on CYP3A4 immobilized on a screen-printed electrode was used to show that abiraterone acts as a competitive inhibitor toward erythromycin N-demethylase activity of CYP3A4 (apparent Ki  = 8.1 ± 1.2 µM), while erythromycin and its products of enzymatic metabolism do not affect abiraterone N-oxidation by CYP3A4. In conclusion, the inhibition properties of abiraterone toward CYP3A4-dependent N-demethylation of erythromycin and the biologically inert behavior of erythromycin toward abiraterone hydroxylation were demonstrated.


Asunto(s)
Androstenos/farmacología , Antibacterianos/farmacocinética , Citocromo P-450 CYP3A/efectos de los fármacos , Eritromicina/farmacocinética , Antineoplásicos/farmacología , Citocromo P-450 CYP3A/metabolismo , Inhibidores del Citocromo P-450 CYP3A/farmacología , Interacciones Farmacológicas , Humanos , Hidroxilación , Simulación del Acoplamiento Molecular
11.
Biosens Bioelectron ; 121: 192-204, 2018 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-30218927

RESUMEN

This review is an attempt to describe advancements in the electrochemistry of cytochrome P450 enzymes (EC 1.14.14.1) and to study molecular aspects and catalytic behavior of enzymatic electrocatalysis. Electroanalysis of cytochrome P450 demonstrates how to translate theoretical laws and equations of classical electrochemistry for the calculation of the kinetic parameters of enzymatic reactions and then translation of kinetic parameters to interpretation of drug-drug interactions. The functional significance of cytochrome P450s (CYPs) includes the metabolism of drugs, foreign chemicals, and endogenic compounds. The pharmaceutical industry needs sensitive and cost-effective systems for screening new drugs and investigation of drug-drug interactions. The development of different types of CYP-based biosensors is now in great demand. This review also highlights the characteristics of electrode processes and electrode properties for optimization of the cytochrome P450 electroanalysis. Electrochemical cytochrome P450-biosensors are the most studied. In this review, we analyzed electrode/cytochrome P450 systems in terms of the mechanisms underlying P450-catalyzed reactions. Screening of potential substrates or inhibitors of cytochromes P450 by means of electrodes were described.


Asunto(s)
Técnicas Biosensibles/métodos , Sistema Enzimático del Citocromo P-450/metabolismo , Electroquímica , Electrodos , Inhibidores Enzimáticos/análisis , Cinética , Oxidación-Reducción , Especificidad por Sustrato
13.
Steroids ; 129: 24-34, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29183745

RESUMEN

Four new 4,5-dihydro-1,3-oxazole, and four new benzo-[d]-oxazole derivatives of [17(20)E]-21-norpregnene, differing in the structure of steroid moiety, were synthesized and evaluated for their potency to inhibit 17α-hydroxylase/17,20-lyase (CYP17A1) activity. Among new compounds, the only oxazolinyl derivative comprising 5-oxo-4,5-seco-3-yn- moiety potently inhibited CYP17A1. Binding modes of the oxazolinyl derivatives of [17(20)E]-21-norpregnene were analyzed by molecular dynamics simulations, and model of alternate, water-bridged type II interaction was proposed for these compounds. Eight new compounds, together with two CYP17A1-inhibiting oxazolinyl derivatives synthesized earlier, abiraterone and galeterone were evaluated for their potency to inhibit prostate carcinoma PC-3 and LNCaP cells growth. Oxazolinyl and benzoxazolyl derivatives comprising 3ß-hydroxy-5-ene moieties potently inhibited prostate carcinoma cell growth; inhibitory potencies of 3-oxo-4-en- and 5-oxo-4,5-seco-3-yn- derivatives were significantly lower.


Asunto(s)
Benzoxazoles/química , Benzoxazoles/farmacología , Norpregnenos/química , Oxazoles/química , Oxazoles/farmacología , Neoplasias de la Próstata/patología , Esteroide 17-alfa-Hidroxilasa/antagonistas & inhibidores , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Benzoxazoles/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos del Citocromo P-450/química , Inhibidores Enzimáticos del Citocromo P-450/metabolismo , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Electroquímica , Humanos , Masculino , Simulación del Acoplamiento Molecular , Oxazoles/metabolismo , Conformación Proteica , Esteroide 17-alfa-Hidroxilasa/química , Esteroide 17-alfa-Hidroxilasa/metabolismo
14.
Biosens Bioelectron ; 99: 216-222, 2018 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-28763782

RESUMEN

Electroanalysis of myoglobin (Mb) in 10 plasma samples of healthy donors (HDs) and 14 plasma samples of patients with acute myocardial infarction (AMI) was carried out with screen-printed electrodes modified first with multi-walled carbon nanotubes (MWCNT) and then with a molecularly imprinted polymer film (MIP), viz., myoglobin-imprinted electropolymerized poly(o-phenylenediamine). The differential pulse voltammetry (DPV) parameters, such as a maximum amplitude of reduction peak current (A, nA), a reduction peak area (S, nA × V), and a peak potential (P, V), were measured for the MWCNT/MIP-sensors after their incubation with non-diluted plasma. The relevance of the multi-parameter electrochemical data for accurate discrimination between HDs and patients with AMI was assessed on the basis of electrochemical threshold values (this requires the reference standard method (RAMP® immunoassay)) or alternatively on the basis of the computational cluster assay (this does not require any reference standard method). The multi-parameter electrochemical analysis of biosamples combined with computational cluster assay was found to provide better accuracy in classification of plasma samples to the groups of HDs or AMI patients.


Asunto(s)
Técnicas Biosensibles , Infarto del Miocardio/sangre , Mioglobina/sangre , Nanotubos de Carbono/química , Técnicas Electroquímicas , Humanos , Impresión Molecular , Mioglobina/aislamiento & purificación , Fenilendiaminas/química
15.
Mater Sci Eng C Mater Biol Appl ; 68: 52-58, 2016 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-27523995

RESUMEN

We report the construction and characterization of a novel, level free impedimetric immunosensor for rapid, sensitive and selective detection of myoglobin (Mb). Monoclonal anti-myoglobin (anti-Mb-IgG) antibody was immobilized on screen-printed multiwalled carbon nanotubes electrode for signal amplification without the need of natural enzymes. The fabrication of resulting immunosensor was extensively characterized by using scanning electron microscopy (SEM), fourier transform infrared (FT-IR) spectroscopy, cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). Electrochemical impedance spectroscopy (EIS) technique offered a linear detection range (0.1-90ngmL(-1)) of myoglobin with sensitivity of 0.74kΩngmL(-1) (correlation coefficient, R(2)=0.97) and detection limit of 0.08ngmL(-1) (S/N=3). The mean percentage recovery of Mb in serum samples using this working biosensor is 97.33%. Furthermore, the proposed strategy can be a promising alternative for detection of Mb related cardiovascular disorders.


Asunto(s)
Técnicas Biosensibles/métodos , Enfermedades Cardiovasculares/sangre , Espectroscopía Dieléctrica/métodos , Inmunoglobulina G/química , Mioglobina/sangre , Animales , Biomarcadores/sangre , Caballos
16.
Anal Biochem ; 513: 28-35, 2016 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-27567992

RESUMEN

Direct electrochemistry and bioelectrocatalysis of a newly discovered C-19 steroid 1α-hydroxylase (CYP260A1) from the myxobacterium Sorangium cellulosum So ce56 were investigated. CYP260A1 was immobilized on screen-printed graphite electrodes (SPE) modified with gold nanoparticles, stabilized by didodecyldimethylammonium bromide (SPE/DDAB/Au). Cyclic voltammograms in argon-saturated substrate free 0.1 M potassium phosphate buffer, pH 7.4, and in enzyme-substrate complex with androstenedione demonstrated a redox processes with a single redox couple of E(0') of -299 ± 16 mV and -297.5 ± 21 mV (vs. Ag/AgCl), respectively. CYP260A1 exhibited an electrocatalytic activity detected by an increase of the reduction current in the presence of dissolved oxygen and upon addition of the substrate (androstenedione) in the air-saturated buffer. The catalytic current of the enzyme correlated with substrate concentration in the electrochemical system and this dependence can be described by electrochemical Michaelis-Menten model. The products of CYP260A1-depended electrolysis at controlled working electrode potential of androstenedione were analyzed by mass-spectrometry. MS analysis revealed a mono-hydroxylated product of CYP260A1-dependent electrocatalytic reaction towards androstenedione.


Asunto(s)
Androsterona/análisis , Proteínas Bacterianas/química , Sistema Enzimático del Citocromo P-450/química , Técnicas Electroquímicas , Enzimas Inmovilizadas/química , Myxococcales/enzimología , Catálisis , Oro/química , Grafito/química , Nanopartículas del Metal/química
17.
Steroids ; 115: 114-122, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27505042

RESUMEN

Five 4,5-dihydro-1,3-oxazole derivatives of [17(20)E]-21-norpregnene, comprising 3ß-hydroxy-5-ene (1), 3,6-dioxo-4-ene (2), 3-oxo-4-ene (3), 3α,5α-cyclo-6-oxo (4), 3ß-hydroxy-6-oxo (5) fragments were synthesized. Synthesis was conducted with improved procedure, based on reaction of suitably protected [17(20)E]-pregnen-21-oic acids with ethanolamine in presence of triphenyl phosphine, carbon tetrachloride, and triethyl amine. Potency of the compounds 1-5 to inhibit 17α-hydroxylase/17,20-lyase (CYP17A1) activity was studied by highly sensitive electrochemical method, using the enzyme immobilization technique. Compounds 1 and 3 were found to be potent CYP17A1 inhibitors, compounds 2 and 5 were not active, compound 4 strongly and irreversibly suppressed the enzyme activity. Molecular docking of compounds 1-5 in the active site of CYP17A1 showed that positions of all compounds in the enzyme active site were similar.


Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Oxazoles/química , Esteroide 17-alfa-Hidroxilasa/antagonistas & inhibidores , Esteroide 17-alfa-Hidroxilasa/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Relación Estructura-Actividad
18.
Biosens Bioelectron ; 86: 330-336, 2016 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-27392234

RESUMEN

Electrosynthesis of molecularly imprinted polymer (MIP) templated with myoglobin (Mb) and the reference non-imprinted polymer (NIP) was examined with o-phenylenediamine (o-PD) as a monomer. Mass-sensitive quartz crystal microbalance with dissipation monitoring supplied by an electrochemical module (EQCM-D) was applied to characterize and optimize MIP/NIP electrosynthesis. Mb rebinding was detected by direct electrocatalytic reduction of Mb by square wave voltammetry (SWV) or differential pulse voltammetry (DPV). The results obtained showed high specificity of polymeric antibodies to template Mb, with an imprinting factor determined as a ratio Imax(MIP)/Imax(NIP) of 2-4. The prepared MIP sensor is characterized by an apparent dissociation constant of (3.3±0.5)×10(-9)M and has a broad range of working concentrations of 1nM-1µÐœ, with the detection limit of 0.5nM (9ng/ml). Mb rebinding was examined in Mb-free diluted human serum spiked with Mb as well as in plasma samples of patients with acute myocardial infarction (AMI) and in control plasma of healthy donors in order to demonstrate the potential medical application of developed MIP sensors.


Asunto(s)
Conductometría/instrumentación , Galvanoplastia/métodos , Impresión Molecular/métodos , Infarto del Miocardio/sangre , Mioglobina/sangre , Fenilendiaminas/química , Absorción Fisicoquímica , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Infarto del Miocardio/diagnóstico , Mioglobina/química , Unión Proteica , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
19.
Steroids ; 88: 66-71, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24971814

RESUMEN

New oxazolinyl derivatives of [17(20)E]-pregna-5,17(20)-diene: 2'-{[(E)-3ß-hydroxyandrost-5-en-17-ylidene]methyl}-4',5'-dihydro-1',3'-oxazole 1 and 2'-{[(E)-3ß-hydroxyandrost-5-en-17-ylidene]methyl}-4',4'-dimethyl-4',5'-dihydro-1',3'-oxazole 2 were evaluated as potential CYP17A1 inhibitors in comparison with 17-(pyridin-3-yl)androsta-5,16-dien-3ß-ol 3 (abiraterone). Differential absorption spectra of human recombinant CYP17A1 in the presence of compound 1 (λmax=422 nm, λmin=386 nm) and compound 2 (λmax=416 nm) indicated significant differences in enzyme/inhibitors complexes. CYP17A1 activity was measured using electrochemical methods. Inhibitory activity of compound 1 was comparable with abiraterone 3 (IC50=0.9±0.1 µM, and IC50=1.3±0.1 µM, for compounds 1 and 3, respectively), while compound 2 was found to be weaker inhibitor (IC50=13±1 µM). Docking of aforementioned compounds to CYP17A1 revealed that steroid fragments of compound 1 and abiraterone 3 occupied close positions; oxazoline cycle of compound 1 was coordinated with heme iron similarly to pyridine cycle of abiraterone 3. Configuration of substituents at 17(20) double bond in preferred docked position corresponded to Z-isomers of compounds 1 and 2. Presence of 4'-substituents in oxazoline ring of compound 2 prevents coordination of oxazoline nitrogen with heme iron and worsens its docking score in comparison with compound 1. These data indicate that oxazolinyl derivative of [17(20)E]-pregna-5,17(20)-diene 1 (rather than 4',4'-dimethyl derivative 2) may be considered as potential CYP17A1 inhibitor and template for development of new compounds affecting growth and proliferation of prostate cancer cells.


Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450/química , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Diseño de Fármacos , Oxazoles/química , Pregnanos/química , Pregnanos/farmacología , Esteroide 17-alfa-Hidroxilasa/antagonistas & inhibidores , Sitios de Unión , Inhibidores Enzimáticos del Citocromo P-450/metabolismo , Electroquímica , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Pregnanos/metabolismo , Conformación Proteica , Esteroide 17-alfa-Hidroxilasa/química , Esteroide 17-alfa-Hidroxilasa/metabolismo , Relación Estructura-Actividad
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