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Conservation and cloning of CYP51: a sterol 14α-demethylase from Mycobacterium smegmatis

Colin J Jackson, David C Lamb, Timothy H Marczylo, Josie Parker, Nigel L Manning, Diane Kelly, Steven L Kelly

Biochemical and Biophysical Research Communications, Volume: 301, Issue: 2, Start page: 558

Swansea University Authors: Josie Parker, Diane Kelly

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DOI (Published version): 10.1016/S0006-291X(02)03078-4

Abstract

<p><span>Inhibition of sterol-14&alpha;-demethylase, a cytochrome P450 (CYP51, Erg11p), is the mode of action of azole antifungal drugs, and with high frequencies of fungal infections new agents are required. New drugs that target fungal CYP51 should not inhibit human CYP51, although...

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Published in: Biochemical and Biophysical Research Communications
ISSN: 0006-291X
Published: 2003
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URI: https://cronfa.swan.ac.uk/Record/cronfa6859
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fullrecord <?xml version="1.0"?><rfc1807><datestamp>2011-10-01T00:00:00.0000000</datestamp><bib-version>v2</bib-version><id>6859</id><entry>2012-01-26</entry><title>Conservation and cloning of CYP51: a sterol 14&#x3B1;-demethylase from Mycobacterium smegmatis</title><swanseaauthors><author><sid>e563ed4e1c7db8d1e131fb78a5f8d0d5</sid><firstname>Josie</firstname><surname>Parker</surname><name>Josie Parker</name><active>true</active><ethesisStudent>false</ethesisStudent></author><author><sid>5ccf81e5d5beedf32ef8d7c3d7ac6c8c</sid><firstname>Diane</firstname><surname>Kelly</surname><name>Diane Kelly</name><active>true</active><ethesisStudent>false</ethesisStudent></author></swanseaauthors><date>2012-01-26</date><deptcode>FGMHL</deptcode><abstract>&lt;p&gt;&lt;span&gt;Inhibition of sterol-14&amp;alpha;-demethylase, a cytochrome P450 (CYP51, Erg11p), is the mode of action of azole antifungal drugs, and with high frequencies of fungal infections new agents are required. New drugs that target fungal CYP51 should not inhibit human CYP51, although selective inhibitors of the human target are also of interest as anticholesterol agents. A strain of&amp;nbsp;&lt;/span&gt;&lt;em&gt;Saccharomyces cerevisiae&lt;/em&gt;&lt;span&gt;&amp;nbsp;that was humanized with respect to the amino acids encoded at the&amp;nbsp;&lt;/span&gt;&lt;em&gt;CYP51&lt;/em&gt;&lt;span&gt;&amp;nbsp;(&lt;/span&gt;&lt;em&gt;ERG11&lt;/em&gt;&lt;span&gt;) yeast locus (BY4741:huCYP51) was produced. The strain was validated with respect to gene expression, protein localization, growth characteristics, and sterol content. The MIC was determined and compared to that for the wild-type parental strain (BY4741), using clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, miconazole, and voriconazole. The humanized strain showed up to &amp;gt;1,000-fold-reduced susceptibility to the orally active azole drugs, while the topical agents showed no difference. Data from growth kinetic measurements substantiated this finding but also revealed reduced effectiveness against the humanized strain for the topical drugs. Cellular sterol profiles reflected the decreased susceptibility of BY4741:huCYP51 and showed a smaller depletion of ergosterol and accumulation of 14&amp;alpha;-methyl-ergosta-8, 24(28)-dien-3&amp;beta;-6&amp;alpha;-diol than the parental strain under the same treatment conditions. This strain provides a useful tool for initial specificity testing for new drugs targeting CYP51 and clearly differentiates azole antifungals in a side-by-side comparison.&lt;/span&gt;&lt;/p&gt;</abstract><type>Journal Article</type><journal>Biochemical and Biophysical Research Communications</journal><volume>301</volume><journalNumber>2</journalNumber><paginationStart>558</paginationStart><paginationEnd/><publisher/><placeOfPublication/><issnPrint>0006-291X</issnPrint><issnElectronic/><keywords/><publishedDay>28</publishedDay><publishedMonth>2</publishedMonth><publishedYear>2003</publishedYear><publishedDate>2003-02-28</publishedDate><doi>10.1016/S0006-291X(02)03078-4</doi><url/><notes/><college>COLLEGE NANME</college><department>Medicine, Health and Life Science - Faculty</department><CollegeCode>COLLEGE CODE</CollegeCode><DepartmentCode>FGMHL</DepartmentCode><institution>Swansea University</institution><apcterm/><lastEdited>2011-10-01T00:00:00.0000000</lastEdited><Created>2012-01-26T10:22:38.2770000</Created><path><level id="1">Faculty of Medicine, Health and Life Sciences</level><level id="2">Swansea University Medical School - Medicine</level></path><authors><author><firstname>Colin J</firstname><surname>Jackson</surname><order>1</order></author><author><firstname>David C</firstname><surname>Lamb</surname><order>2</order></author><author><firstname>Timothy H</firstname><surname>Marczylo</surname><order>3</order></author><author><firstname>Josie</firstname><surname>Parker</surname><order>4</order></author><author><firstname>Nigel L</firstname><surname>Manning</surname><order>5</order></author><author><firstname>Diane</firstname><surname>Kelly</surname><order>6</order></author><author><firstname>Steven L</firstname><surname>Kelly</surname><order>7</order></author></authors><documents/><OutputDurs/></rfc1807>
spelling 2011-10-01T00:00:00.0000000 v2 6859 2012-01-26 Conservation and cloning of CYP51: a sterol 14α-demethylase from Mycobacterium smegmatis e563ed4e1c7db8d1e131fb78a5f8d0d5 Josie Parker Josie Parker true false 5ccf81e5d5beedf32ef8d7c3d7ac6c8c Diane Kelly Diane Kelly true false 2012-01-26 FGMHL <p><span>Inhibition of sterol-14&alpha;-demethylase, a cytochrome P450 (CYP51, Erg11p), is the mode of action of azole antifungal drugs, and with high frequencies of fungal infections new agents are required. New drugs that target fungal CYP51 should not inhibit human CYP51, although selective inhibitors of the human target are also of interest as anticholesterol agents. A strain of&nbsp;</span><em>Saccharomyces cerevisiae</em><span>&nbsp;that was humanized with respect to the amino acids encoded at the&nbsp;</span><em>CYP51</em><span>&nbsp;(</span><em>ERG11</em><span>) yeast locus (BY4741:huCYP51) was produced. The strain was validated with respect to gene expression, protein localization, growth characteristics, and sterol content. The MIC was determined and compared to that for the wild-type parental strain (BY4741), using clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, miconazole, and voriconazole. The humanized strain showed up to &gt;1,000-fold-reduced susceptibility to the orally active azole drugs, while the topical agents showed no difference. Data from growth kinetic measurements substantiated this finding but also revealed reduced effectiveness against the humanized strain for the topical drugs. Cellular sterol profiles reflected the decreased susceptibility of BY4741:huCYP51 and showed a smaller depletion of ergosterol and accumulation of 14&alpha;-methyl-ergosta-8, 24(28)-dien-3&beta;-6&alpha;-diol than the parental strain under the same treatment conditions. This strain provides a useful tool for initial specificity testing for new drugs targeting CYP51 and clearly differentiates azole antifungals in a side-by-side comparison.</span></p> Journal Article Biochemical and Biophysical Research Communications 301 2 558 0006-291X 28 2 2003 2003-02-28 10.1016/S0006-291X(02)03078-4 COLLEGE NANME Medicine, Health and Life Science - Faculty COLLEGE CODE FGMHL Swansea University 2011-10-01T00:00:00.0000000 2012-01-26T10:22:38.2770000 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Medicine Colin J Jackson 1 David C Lamb 2 Timothy H Marczylo 3 Josie Parker 4 Nigel L Manning 5 Diane Kelly 6 Steven L Kelly 7
title Conservation and cloning of CYP51: a sterol 14α-demethylase from Mycobacterium smegmatis
spellingShingle Conservation and cloning of CYP51: a sterol 14α-demethylase from Mycobacterium smegmatis
Josie Parker
Diane Kelly
title_short Conservation and cloning of CYP51: a sterol 14α-demethylase from Mycobacterium smegmatis
title_full Conservation and cloning of CYP51: a sterol 14α-demethylase from Mycobacterium smegmatis
title_fullStr Conservation and cloning of CYP51: a sterol 14α-demethylase from Mycobacterium smegmatis
title_full_unstemmed Conservation and cloning of CYP51: a sterol 14α-demethylase from Mycobacterium smegmatis
title_sort Conservation and cloning of CYP51: a sterol 14α-demethylase from Mycobacterium smegmatis
author_id_str_mv e563ed4e1c7db8d1e131fb78a5f8d0d5
5ccf81e5d5beedf32ef8d7c3d7ac6c8c
author_id_fullname_str_mv e563ed4e1c7db8d1e131fb78a5f8d0d5_***_Josie Parker
5ccf81e5d5beedf32ef8d7c3d7ac6c8c_***_Diane Kelly
author Josie Parker
Diane Kelly
author2 Colin J Jackson
David C Lamb
Timothy H Marczylo
Josie Parker
Nigel L Manning
Diane Kelly
Steven L Kelly
format Journal article
container_title Biochemical and Biophysical Research Communications
container_volume 301
container_issue 2
container_start_page 558
publishDate 2003
institution Swansea University
issn 0006-291X
doi_str_mv 10.1016/S0006-291X(02)03078-4
college_str Faculty of Medicine, Health and Life Sciences
hierarchytype
hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Medicine{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Medicine
document_store_str 0
active_str 0
description <p><span>Inhibition of sterol-14&alpha;-demethylase, a cytochrome P450 (CYP51, Erg11p), is the mode of action of azole antifungal drugs, and with high frequencies of fungal infections new agents are required. New drugs that target fungal CYP51 should not inhibit human CYP51, although selective inhibitors of the human target are also of interest as anticholesterol agents. A strain of&nbsp;</span><em>Saccharomyces cerevisiae</em><span>&nbsp;that was humanized with respect to the amino acids encoded at the&nbsp;</span><em>CYP51</em><span>&nbsp;(</span><em>ERG11</em><span>) yeast locus (BY4741:huCYP51) was produced. The strain was validated with respect to gene expression, protein localization, growth characteristics, and sterol content. The MIC was determined and compared to that for the wild-type parental strain (BY4741), using clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, miconazole, and voriconazole. The humanized strain showed up to &gt;1,000-fold-reduced susceptibility to the orally active azole drugs, while the topical agents showed no difference. Data from growth kinetic measurements substantiated this finding but also revealed reduced effectiveness against the humanized strain for the topical drugs. Cellular sterol profiles reflected the decreased susceptibility of BY4741:huCYP51 and showed a smaller depletion of ergosterol and accumulation of 14&alpha;-methyl-ergosta-8, 24(28)-dien-3&beta;-6&alpha;-diol than the parental strain under the same treatment conditions. This strain provides a useful tool for initial specificity testing for new drugs targeting CYP51 and clearly differentiates azole antifungals in a side-by-side comparison.</span></p>
published_date 2003-02-28T03:08:27Z
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score 11.037581

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