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 <i>Candida</i> 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...

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Main Authors: Tatsiana V. Tsybruk, Leonid A. Kaluzhskiy, Yuri V. Mezentsev, Tatyana N. Makarieva, Kseniya M. Tabakmaher, Natalia V. Ivanchina, Pavel S. Dmitrenok, Alexander V. Baranovsky, Andrei A. Gilep, Alexis S. Ivanov
Format: Article
Language:English
Published: MDPI AG 2023-10-01
Series:Biomedicines
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Online Access:https://www.mdpi.com/2227-9059/11/11/2873
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author Tatsiana V. Tsybruk
Leonid A. Kaluzhskiy
Yuri V. Mezentsev
Tatyana N. Makarieva
Kseniya M. Tabakmaher
Natalia V. Ivanchina
Pavel S. Dmitrenok
Alexander V. Baranovsky
Andrei A. Gilep
Alexis S. Ivanov
author_facet Tatsiana V. Tsybruk
Leonid A. Kaluzhskiy
Yuri V. Mezentsev
Tatyana N. Makarieva
Kseniya M. Tabakmaher
Natalia V. Ivanchina
Pavel S. Dmitrenok
Alexander V. Baranovsky
Andrei A. Gilep
Alexis S. Ivanov
author_sort Tatsiana V. Tsybruk
collection DOAJ
description Due to the increasing prevalence of fungal diseases caused by fungi of the genus <i>Candida</i> 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 <i>C. krusei</i> 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 <i>C. krusei</i> CYP51 in <i>E. coli</i> cells and obtained a highly purified protein. Application of the method of spectrophotometric titration allowed us to study the interaction of <i>C. krusei</i> CYP51 with various ligands. In the present work, the interaction of <i>C. krusei</i> CYP51 with azole inhibitors, and natural and synthesized steroid derivatives was evaluated. The obtained data indicate that the resistance of <i>C. krusei</i> 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 <i>C. krusei</i> 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 <i>C. krusei</i> CYP51 can be taken into account in the further development of new selective modulators of the activity of this enzyme.
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spelling doaj.art-2405999c00fc4866a1202feaf5e17b592023-11-24T14:30:32ZengMDPI AGBiomedicines2227-90592023-10-011111287310.3390/biomedicines11112873Molecular Cloning, Heterologous Expression, Purification, and Evaluation of Protein–Ligand Interactions of CYP51 of <i>Candida krusei</i> Azole-Resistant Fungal StrainTatsiana V. Tsybruk0Leonid A. Kaluzhskiy1Yuri V. Mezentsev2Tatyana N. Makarieva3Kseniya M. Tabakmaher4Natalia V. Ivanchina5Pavel S. Dmitrenok6Alexander V. Baranovsky7Andrei A. Gilep8Alexis S. Ivanov9Institute of Bioorganic Chemistry NASB, 5 Building 2, V.F. Kuprevich Street, 220084 Minsk, BelarusInstitute of Biomedical Chemistry, Pogodinskaya Str. 10 Building 8, 119121 Moscow, RussiaInstitute of Biomedical Chemistry, Pogodinskaya Str. 10 Building 8, 119121 Moscow, RussiaG.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Pr. 100-let Vladivostoku 159, 690022 Vladivostok, RussiaG.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Pr. 100-let Vladivostoku 159, 690022 Vladivostok, RussiaG.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Pr. 100-let Vladivostoku 159, 690022 Vladivostok, RussiaG.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Pr. 100-let Vladivostoku 159, 690022 Vladivostok, RussiaInstitute of Bioorganic Chemistry NASB, 5 Building 2, V.F. Kuprevich Street, 220084 Minsk, BelarusInstitute of Bioorganic Chemistry NASB, 5 Building 2, V.F. Kuprevich Street, 220084 Minsk, BelarusInstitute of Biomedical Chemistry, Pogodinskaya Str. 10 Building 8, 119121 Moscow, RussiaDue to the increasing prevalence of fungal diseases caused by fungi of the genus <i>Candida</i> 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 <i>C. krusei</i> 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 <i>C. krusei</i> CYP51 in <i>E. coli</i> cells and obtained a highly purified protein. Application of the method of spectrophotometric titration allowed us to study the interaction of <i>C. krusei</i> CYP51 with various ligands. In the present work, the interaction of <i>C. krusei</i> CYP51 with azole inhibitors, and natural and synthesized steroid derivatives was evaluated. The obtained data indicate that the resistance of <i>C. krusei</i> 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 <i>C. krusei</i> 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 <i>C. krusei</i> CYP51 can be taken into account in the further development of new selective modulators of the activity of this enzyme.https://www.mdpi.com/2227-9059/11/11/2873lanosterol 14-alpha demethylaseCYP51cytochrome P450azole inhibitorsheterocyclic analogues of steroidsmarine steroids
spellingShingle Tatsiana V. Tsybruk
Leonid A. Kaluzhskiy
Yuri V. Mezentsev
Tatyana N. Makarieva
Kseniya M. Tabakmaher
Natalia V. Ivanchina
Pavel S. Dmitrenok
Alexander V. Baranovsky
Andrei A. Gilep
Alexis S. Ivanov
Molecular Cloning, Heterologous Expression, Purification, and Evaluation of Protein–Ligand Interactions of CYP51 of <i>Candida krusei</i> Azole-Resistant Fungal Strain
Biomedicines
lanosterol 14-alpha demethylase
CYP51
cytochrome P450
azole inhibitors
heterocyclic analogues of steroids
marine steroids
title Molecular Cloning, Heterologous Expression, Purification, and Evaluation of Protein–Ligand Interactions of CYP51 of <i>Candida krusei</i> Azole-Resistant Fungal Strain
title_full Molecular Cloning, Heterologous Expression, Purification, and Evaluation of Protein–Ligand Interactions of CYP51 of <i>Candida krusei</i> Azole-Resistant Fungal Strain
title_fullStr Molecular Cloning, Heterologous Expression, Purification, and Evaluation of Protein–Ligand Interactions of CYP51 of <i>Candida krusei</i> Azole-Resistant Fungal Strain
title_full_unstemmed Molecular Cloning, Heterologous Expression, Purification, and Evaluation of Protein–Ligand Interactions of CYP51 of <i>Candida krusei</i> Azole-Resistant Fungal Strain
title_short Molecular Cloning, Heterologous Expression, Purification, and Evaluation of Protein–Ligand Interactions of CYP51 of <i>Candida krusei</i> Azole-Resistant Fungal Strain
title_sort molecular cloning heterologous expression purification and evaluation of protein ligand interactions of cyp51 of i candida krusei i azole resistant fungal strain
topic lanosterol 14-alpha demethylase
CYP51
cytochrome P450
azole inhibitors
heterocyclic analogues of steroids
marine steroids
url https://www.mdpi.com/2227-9059/11/11/2873
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