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Project titleImproving the Efficiency of Catalytic Processes in Electrochemical Cytochrome P450 Systems

Research area 05 - FUNDAMENTAL RESEARCH IN MEDICINE, 05-109 - Clinical laboratory diagnostics and nanotechnologies in medicine

KeywordsCytochrome P450, riboflavin, reductase, microsomes, electrodes, electrocatalysis, carbon nanomaterials, metabolites, bioreactor, anodiscs

Annotation
Cytochromes P450 are actively studied due to their high functional and medical significance. The wide substrate specificity of cytochromes P450 allows them to be used to study the metabolic transformations of drugs, to obtain pharmacologically significant chemical compounds [1, 2]. A limitation in the implementation of biocatalytic synthesis is the need to use additional redox partner proteins and NADPH as an electron source. In electrochemical systems, the electron donor is the electrode. Electrochemical methods are a modern highly sensitive analytical tool for studying various functional aspects of cytochromes P450: screening of substrates, inhibitors, effectors, activators, metabolites. Electrochemical cytochrome P450 systems are actively used both in the biosensor mode to detect the substrate-inhibitory potential of this class of hemoproteins and to study the complex and multistage mechanism of cytochrome P450 [3, 4]. So far, a little-studied area is the operation of P450 cytochromes in the mode of electrochemical bioreactors. This aspect of the functioning of P450 cytochromes is important for the production of synthetic drug precursors, for the synthesis of optically active compounds and isomers, and for the production of prodrugs with greater pharmacological activity. In addition, cytochromes P450 are important for the modification and biodegradation of chemical compounds that pollute the environment. The aim of this research project is to develop approaches to improve the efficiency of cytochrome P450 electrocatalysis. The relevance of the project lies in the creation of new approaches to improve the efficiency of the functioning of cytochrome P450 electrodes as model non invasive systems for metabolic transformations and synthesis of drugs.. The scientific novelty of the project lies in the study of electrocatalysis of cytochromes P450 3A4 and P450 2C9 as the most functionally significant isoforms in the transition mode from 2D to 3D sensor due to the inclusion of the enzyme in nanopore membranes, obtaining bioconjugates with flavin nucleotides and riboflavin, as well as combining the most effective electrode modification and the most effective bioconjugate. To create the most favorable microenvironment, we will study the inclusion of the enzyme or its complex with flavin nucleotides or riboflavin in the three-dimensional structure of a nanopore membrane with different nanopore diameters based on anodized aluminum membrane placed on the working electrode. 1. Singh, R.S., Singh, T., Singh, A.K. (2019) Enzymes as Diagnostic Tools, Advances in Enzyme Technology, Elsevier B.V., doi: 10.1016/B978-0-444-64114-4.00009-1 2. R. Bernhardt, V.B. Urlacher, Cytochromes P450 as promising catalysts for biotechnological application: chances and limitations, Appl Microbiol Biotechnol. 98 (2014)6185–6203. https://doi.org/10.1007/s00253-014-5767-7 3. E. Schneider, D.S. Clark Cytochrome P450 (CYP) enzymes and the development of CYP biosensors, Biosensors and Bioelectronics. 39 (2013) 1-13. https://doi.org/10.1016/j.bios.2012.05.043. 4. Shumyantseva, V.V.; Kuzikov, A.V.; Masamrekh, R.A.; Bulko, T.V.; Archakov, A.I. From electrochemistry to enzyme kinetics of cytochrome P450. Biosens. Bioelectron. 2018, 121, 192–204. https://doi.org/10.1016/j.bios.2018.08.040

Expected results
As a result of the project, effective cytochrome P450 electrochemical systems with increased efficiency of biocatalytic processes will be developed to obtain functionally significant metabolites such as hydroxycortisol, 6β-hydroxytestosterone, O-desmethylnaproxen.

Annotation of the results obtained in 2023
- An analysis of the problems of using alternative electron sources to create bioreactors based on cytochrome P450 was carried out. The following main sources of electrons, different from the natural electron transport system, have been identified: creation of a “biogalvanic couple” using metals whose redox potential is more negative than the reduction potential of heme, use as a reducing agent, irradiation of photosensitizing substances with subsequent transfer of electrons to the active center of the enzyme, use electrochemical techniques for studying the properties of cytochromes P450 and creating bioreactors, with electrons coming directly from the electrode. A comparative analysis of the approaches was carried out, the pros and cons of each method were highlighted. - The prospects and difficulties in creating bioreactors based on cytochrome P450 are studied, the main methods for creating reconstructed (as cytochrome P450 and reductase) and electrochemical catalytic systems based on cytochrome P450 are considered, as well as modern trends and approaches to the practical use of cytochrome P450. A major trend for both reconstituted and electrochemical systems is the incorporation of cytochrome P450 into a confined volume with three-dimensional structures based on nanoporous materials. In this case, various materials and their combinations can be used, for example, mesoporous materials or porous membranes. Electrochemical methods for analyzing the functional activity of enzymes have received widespread development due to the information content of the methods and their multi-parameter nature. In addition, an important aspect is the technological development of the instrumentation base and the availability of software. This makes it possible to standardize research and conduct a comparative analysis of an ensemble of various biological objects. - A system has been developed with a transition from a 2D electrode surface to a 3D structure by modifying a printed graphite electrode with a membrane-like substance didodecyldimethylammonium bromide and the membrane protein streptolysin O, and a study of the electrochemical properties of cytochrome P450 3A4 has been carried out. It has been shown that streptolysin O forms a highly developed surface with cavities on the film of the membrane-like substance DDAB, which contributes to the immobilization of cytochrome P450 3A4 for the manifestation of electrochemical and electrocatalytic properties. The formation of a developed structure was demonstrated using atomic force microscopy. The electroanalytical parameters of immobilized cytochrome P450 3A4 under anaerobic conditions were obtained; the formal potential value was -0.325 ± 0.024. The efficiency of cytochrome P450 3A4 dependent N-demethylation of erythromycin in an electrocatalytic system was assessed as 297 ± 7% for modification with streptolysin O and DDAB, and 100 ± 22% for modification with DDAB.

Publications

1. Polina I. Koroleva, Andrei A. Gilep, Sergey V. Kraevskiy, Tatiana V. Tsybruk, Victoria V. Shumyantseva 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 Biosensors, 13, 457 (https:// creativecommons.org/licenses/by/ 4.0/) (year - 2023) https://doi.org/10.3390/bios13040457

2. Victoria V. Shumyantseva, Polina I. Koroleva, Tatiana V. Bulko, Lyubov E. Agafonova Alternative electron sources for cytochrome P450s catalytic cycle: biosensing and biosynthetic application Processes, https://www.mdpi.com/journal/processes, 00, 11, 1801 (https:// creativecommons.org/licenses/by/4.0/) (year - 2023) https://doi.org/10.3390/pr11061801

3. Polina I. Koroleva, Tatiana V. Bulko, Lyubov E. Agafonova, Victoria V. Shumyantseva Catalytic and Electrocatalytic Mechanisms of Cytochromes P450 in the Development of Biosensors and Bioreactors Biochemistry (Moscow), 10,. 88,. 1645-1657 (year - 2023) https://doi.org/10.1134/S0006297923100176

4. Polina I. Koroleva, Tatiana V. Bulko, Lyubov’ E. Agafonova, Victoria V. Shumyantseva ЦИТОХРОМЫ Р450: ОСОБЕННОСТИ МЕХАНИЗМОВ КАТАЛИЗА И ЭЛЕКТРОКАТАЛИЗА ДЛЯ СОЗДАНИЯ БИОСЕНСОРОВ И БИОРЕАКТОРОВ Биохимия Pleiades Publishing, том 88, вып. 10, с. 1985 – 2001. EDN: OVNWSI (year - 2023) https://doi.org/10.31857/S0320972523100172