ЛИГАНД-СВЯЗЫВАЮЩИЕ И КАТАЛИТИЧЕСКИЕ СВОЙСТВА РЕКОМБИНАНТНОЙ ТРОМБОКСАНСИНТАЗЫ ЧЕЛОВЕКА

To study the spectrum of modulators of the human thromboxane synthase activity, the interaction of recombinant protein with various low-molecular weight ligands was analyzed. It was shown that thromboxane synthase interacts with a number of fatty acids and their derivatives (potential substrates or concurrent inhibitors), being a target for nonselective inhibition by imidazole and triazole derivatives used in medical practice and agriculture. Thus, another mechanism of action of endocrine-disrupting chemicals (EDC) was established. For the first time, the reduction of heme iron of thromboxane synthase by cytochrome P450 reductase was shown. This interaction accompanied by a partial inhibitory effect on the enzyme suppresses the formation of reaction by-products 12-hydroxyheptadecatenoic acid (12-HHT) and malonic dialdehyde (MDA). It is likely that this mechanism can participate in the regulation of the enzyme activity in vivo. С целью выявления модуляторов активности тромбоксансинтазы человека осуществлен анализ взаимодействия рекомбинантного гемопротеида с различными низкомолекулярными лигандами. Показано, что тромбоксансинтаза взаимодействует с рядом жирных кислот и их производных (потенциальные субстраты или конкурентные ингибиторы), а также является мишенью для ингибирования производными имидазола и триазола, применяющимися в медицинской практике и сельском хозяйстве, что позволило установить механизм, по которому могут реализовывать свое токсическое действие соединения, нарушающие работу эндокринной системы человека (endocrine-disrupting chemicals, EDC). Впервые показано восстановление атома железа гема тромбоксансинтазы NАDPH-цитохром P450 редуктазой, что оказывает частичное ингибирующее действие на фермент и подавляет образование побочных продуктов реакции – 12-гидроксигептадекатриеноевой кислоты (12-HHT) и малонового диальдегида (МДА). Предполагается, что данный механизм может участвовать в регуляции активности фермента in vivo.

Improving the Efficiency of Electrocatalysis of Cytochrome P450 3A4 by Modifying the Electrode with Membrane Protein Streptolysin O for Studying the Metabolic Transformations of Drugs

In the present work, screen-printed electrodes (SPE) modified with a synthetic surfactant, didodecyldimethylammonium bromide (DDAB) and streptolysin O (SLO) were prepared for cytochrome P450 3A4 (CYP3A4) immobilization, direct non-catalytic and catalytic electrochemistry. The immobilized CYP3A4 demonstrated a pair of redox peaks with a formal potential of −0.325 ± 0.024 V (vs. the Ag/AgCl reference electrode). The electron transfer process showed a surface-controlled mechanism (“protein film voltammetry”) with an electron transfer rate constant (ks) of 0.203 ± 0.038 s−1. Electrochemical CYP3A4-mediated reaction of N-demethylation of erythromycin was explored with the following parameters: an applied potential of −0.5 V and a duration time of 20 min. The system with DDAB/SLO as the electrode modifier showed conversion of erythromycin with an efficiency higher than the electrode modified with DDAB only. Confining CYP3A4 inside the protein frame of SLO accelerated the enzymatic reaction. The increases in product formation in the reaction of the electrochemical N-demethylation of erythromycin for SPE/DDAB/CYP3A4 and SPE/DDAB/SLO/CYP3A4 were equal to 100 ± 22% and 297 ± 7%, respectively. As revealed by AFM images, the SPE/DDAB/SLO possessed a more developed surface with protein cavities in comparison with SPE/DDAB for the effective immobilization of the CYP3A4 enzyme

Molecular Cloning, Heterologous Expression, Purification, and Evaluation of Protein–Ligand Interactions of CYP51 of <i>Candida krusei</i> Azole-Resistant Fungal Strain

Due to the increasing prevalence of fungal diseases caused by fungi of the genus Candida and the development of pathogen resistance to available drugs, the need to find new effective antifungal agents has increased. Azole antifungals, which are inhibitors of sterol-14α-demethylase or CYP51, have been widely used in the treatment of fungal infections over the past two decades. Of special interest is the study of C. krusei CYP51, since this fungus exhibit resistance not only to azoles, but also to other antifungal drugs and there is no available information about the ligand-binding properties of CYP51 of this pathogen. We expressed recombinant C. krusei CYP51 in E. coli cells and obtained a highly purified protein. Application of the method of spectrophotometric titration allowed us to study the interaction of C. krusei CYP51 with various ligands. In the present work, the interaction of C. krusei CYP51 with azole inhibitors, and natural and synthesized steroid derivatives was evaluated. The obtained data indicate that the resistance of C. krusei to azoles is not due to the structural features of CYP51 of this microorganism, but rather to another mechanism. Promising ligands that demonstrated sufficiently strong binding in the micromolar range to C. krusei CYP51 were identified, including compounds 99 (Kd = 1.02 ± 0.14 µM) and Ch-4 (Kd = 6.95 ± 0.80 µM). The revealed structural features of the interaction of ligands with the active site of C. krusei CYP51 can be taken into account in the further development of new selective modulators of the activity of this enzyme

Affinity Isolation and Mass Spectrometry Identification of Prostacyclin Synthase (PTGIS) Subinteractome

Prostacyclin synthase (PTGIS; EC 5.3.99.4) catalyzes isomerization of prostaglandin H2 to prostacyclin, a potent vasodilator and inhibitor of platelet aggregation. At present, limited data exist on functional coupling and possible ways of regulating PTGIS due to insufficient information about protein&ndash;protein interactions in which this crucial enzyme is involved. The aim of this study is to isolate protein partners for PTGIS from rat tissue lysates. Using CNBr-activated Sepharose 4B with covalently immobilized PTGIS as an affinity sorbent, we confidently identified 58 unique proteins by mass spectrometry (LC-MS/MS). The participation of these proteins in lysate complex formation was characterized by SEC lysate profiling. Several potential members of the PTGIS subinteractome have been validated by surface plasmon resonance (SPR) analysis. SPR revealed that PTGIS interacted with full-length cytochrome P450 2J2 and glutathione S-transferase (GST). In addition, PTGIS was shown to bind synthetic peptides corresponding to sequences of for GSTA1, GSTM1, aldo-keto reductase (AKR1A1), glutaredoxin 3 (GLRX3) and histidine triad nucleotide binding protein 2 (HINT2). Prostacyclin synthase could potentially be involved in functional interactions with identified novel protein partners participating in iron and heme metabolism, oxidative stress, xenobiotic and drugs metabolism, glutathione and prostaglandin metabolism. The possible biological role of the recognized interaction is discussed in the context of PTGIS functioning

PharmVar GeneFocus: CYP3A5

The Pharmacogene Variation Consortium (PharmVar) catalogs star (*) allele nomenclature for the polymorphic human CYP3A5 gene. Genetic variation within the CYP3A5 gene locus impacts the metabolism of several clinically important drugs, including the immunosuppressants tacrolimus, sirolimus, cyclosporine, and the benzodiazepine midazolam. Variable CYP3A5 activity is of clinical importance regarding tacrolimus metabolism. This GeneFocus provides a CYP3A5 gene summary with a focus on aspects regarding standardized nomenclature. In addition, this review also summarizes recent changes and updates, including the retirement of several allelic variants and provides an overview of how PharmVar CYP3A5 star allele nomenclature is utilized by the Pharmacogenomics Knowledgebase (PharmGKB) and the Clinical Pharmacogenetics Implementation Consortium (CPIC)