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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2023-10-01
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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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