Mechanistic Investigation of the Time-Dependent Aldehyde Oxidase Inhibitor Hydralazine
The anti-hypertensive agent hydralazine is a time-dependent inhibitor of the cytosolic drug-metabolizing enzyme aldehyde oxidase (AO). Glutathione (GSH) was found to suppress the inhibition of AO by hydralazine in multiple enzyme sources (human liver and kidney cytosol, human liver S9, rat liver S9,...
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Veröffentlicht in: | Drug metabolism and disposition 2023-06, Vol.51 (6), p.782-791 |
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description | The anti-hypertensive agent hydralazine is a time-dependent inhibitor of the cytosolic drug-metabolizing enzyme aldehyde oxidase (AO). Glutathione (GSH) was found to suppress the inhibition of AO by hydralazine in multiple enzyme sources (human liver and kidney cytosol, human liver S9, rat liver S9, and recombinant human AO) and with different AO substrates (zoniporide,
-benzylguanine, and dantrolene). Hydralazine-induced AO inactivation was unaffected when GSH was added to the incubation mixture after pre-incubation of hydralazine with AO (rather than during the pre-incubation), suggesting that GSH traps a hydralazine reactive intermediate prior to enzyme inactivation. Consistent with previous reports of 1-phthalazylmercapturic acid formation when hydralazine was incubated with N-acetylcysteine, we detected a metabolite producing an MS/MS spectrum consistent with a 1-phthalazyl-GSH conjugate.
-Benzylguanine, an AO substrate, did not protect against hydralazine-induced AO inactivation, implying that hydralazine does not compete with
-benzylguanine for binding to the AO active site. Catalase also failed to protect AO from hydralazine-induced inactivation, suggesting that hydrogen peroxide is not involved. However, an allosteric AO inhibitor (thioridazine) offered some protection, indicating a catalytic role for AO in the bioactivation of hydralazine. AO inhibition by phthalazine (a substrate and inhibitor of AO and a metabolite of hydralazine) was unaffected by the presence of GSH. GSH also prevented hydralazine from inhibiting the nitro-reduction of dantrolene by AO. Furthermore, the GSH-hydralazine combination stimulated dantrolene reduction. Phthalazine inhibited only oxidation reactions, not reduction of dantrolene. Together, these results support the hypothesis that hydralazine is converted to a reactive intermediate that inactivates AO. SIGNIFICANCE STATEMENT: These studies suggest that a reactive intermediate of hydralazine plays a primary role in the mechanism of aldehyde oxidase (AO) inactivation. Inactivation was attenuated by glutathione and unaffected by catalase. Phthalazine (hydralazine metabolite) inhibited AO regardless of the presence of glutathione; however, phthalazine inhibited only oxidation reactions, while hydralazine inhibited both oxidation and reduction reactions. This report advances our mechanistic understanding of hydralazine as an AO inhibitor and provides information to facilitate appropriate use of hydralazine when probing AO |
doi_str_mv | 10.1124/dmd.123.001257 |
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-benzylguanine, and dantrolene). Hydralazine-induced AO inactivation was unaffected when GSH was added to the incubation mixture after pre-incubation of hydralazine with AO (rather than during the pre-incubation), suggesting that GSH traps a hydralazine reactive intermediate prior to enzyme inactivation. Consistent with previous reports of 1-phthalazylmercapturic acid formation when hydralazine was incubated with N-acetylcysteine, we detected a metabolite producing an MS/MS spectrum consistent with a 1-phthalazyl-GSH conjugate.
-Benzylguanine, an AO substrate, did not protect against hydralazine-induced AO inactivation, implying that hydralazine does not compete with
-benzylguanine for binding to the AO active site. Catalase also failed to protect AO from hydralazine-induced inactivation, suggesting that hydrogen peroxide is not involved. However, an allosteric AO inhibitor (thioridazine) offered some protection, indicating a catalytic role for AO in the bioactivation of hydralazine. AO inhibition by phthalazine (a substrate and inhibitor of AO and a metabolite of hydralazine) was unaffected by the presence of GSH. GSH also prevented hydralazine from inhibiting the nitro-reduction of dantrolene by AO. Furthermore, the GSH-hydralazine combination stimulated dantrolene reduction. Phthalazine inhibited only oxidation reactions, not reduction of dantrolene. Together, these results support the hypothesis that hydralazine is converted to a reactive intermediate that inactivates AO. SIGNIFICANCE STATEMENT: These studies suggest that a reactive intermediate of hydralazine plays a primary role in the mechanism of aldehyde oxidase (AO) inactivation. Inactivation was attenuated by glutathione and unaffected by catalase. Phthalazine (hydralazine metabolite) inhibited AO regardless of the presence of glutathione; however, phthalazine inhibited only oxidation reactions, while hydralazine inhibited both oxidation and reduction reactions. This report advances our mechanistic understanding of hydralazine as an AO inhibitor and provides information to facilitate appropriate use of hydralazine when probing AO metabolism.</description><identifier>ISSN: 0090-9556</identifier><identifier>EISSN: 1521-009X</identifier><identifier>DOI: 10.1124/dmd.123.001257</identifier><identifier>PMID: 36921993</identifier><language>eng</language><publisher>United States</publisher><subject>Aldehyde Oxidase - metabolism ; Animals ; Catalase ; Dantrolene ; Glutathione ; Humans ; Hydralazine - pharmacology ; Phthalazines - metabolism ; Rats ; Tandem Mass Spectrometry</subject><ispartof>Drug metabolism and disposition, 2023-06, Vol.51 (6), p.782-791</ispartof><rights>Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c335t-87bb75cf9ba30da496285076ba032c4ec5644bb97f9f15e031ad80ffb618699c3</citedby><cites>FETCH-LOGICAL-c335t-87bb75cf9ba30da496285076ba032c4ec5644bb97f9f15e031ad80ffb618699c3</cites><orcidid>0000-0002-4525-6072</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/36921993$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Barnes, J Paige</creatorcontrib><creatorcontrib>Yang, Shaoyun M</creatorcontrib><creatorcontrib>Thompson, Taylor S</creatorcontrib><creatorcontrib>Guevarra, Caithlyne M</creatorcontrib><creatorcontrib>Pleasant, Shaniya S</creatorcontrib><creatorcontrib>Do, Tri Q</creatorcontrib><creatorcontrib>Crouch, Rachel D</creatorcontrib><title>Mechanistic Investigation of the Time-Dependent Aldehyde Oxidase Inhibitor Hydralazine</title><title>Drug metabolism and disposition</title><addtitle>Drug Metab Dispos</addtitle><description>The anti-hypertensive agent hydralazine is a time-dependent inhibitor of the cytosolic drug-metabolizing enzyme aldehyde oxidase (AO). Glutathione (GSH) was found to suppress the inhibition of AO by hydralazine in multiple enzyme sources (human liver and kidney cytosol, human liver S9, rat liver S9, and recombinant human AO) and with different AO substrates (zoniporide,
-benzylguanine, and dantrolene). Hydralazine-induced AO inactivation was unaffected when GSH was added to the incubation mixture after pre-incubation of hydralazine with AO (rather than during the pre-incubation), suggesting that GSH traps a hydralazine reactive intermediate prior to enzyme inactivation. Consistent with previous reports of 1-phthalazylmercapturic acid formation when hydralazine was incubated with N-acetylcysteine, we detected a metabolite producing an MS/MS spectrum consistent with a 1-phthalazyl-GSH conjugate.
-Benzylguanine, an AO substrate, did not protect against hydralazine-induced AO inactivation, implying that hydralazine does not compete with
-benzylguanine for binding to the AO active site. Catalase also failed to protect AO from hydralazine-induced inactivation, suggesting that hydrogen peroxide is not involved. However, an allosteric AO inhibitor (thioridazine) offered some protection, indicating a catalytic role for AO in the bioactivation of hydralazine. AO inhibition by phthalazine (a substrate and inhibitor of AO and a metabolite of hydralazine) was unaffected by the presence of GSH. GSH also prevented hydralazine from inhibiting the nitro-reduction of dantrolene by AO. Furthermore, the GSH-hydralazine combination stimulated dantrolene reduction. Phthalazine inhibited only oxidation reactions, not reduction of dantrolene. Together, these results support the hypothesis that hydralazine is converted to a reactive intermediate that inactivates AO. SIGNIFICANCE STATEMENT: These studies suggest that a reactive intermediate of hydralazine plays a primary role in the mechanism of aldehyde oxidase (AO) inactivation. Inactivation was attenuated by glutathione and unaffected by catalase. Phthalazine (hydralazine metabolite) inhibited AO regardless of the presence of glutathione; however, phthalazine inhibited only oxidation reactions, while hydralazine inhibited both oxidation and reduction reactions. This report advances our mechanistic understanding of hydralazine as an AO inhibitor and provides information to facilitate appropriate use of hydralazine when probing AO metabolism.</description><subject>Aldehyde Oxidase - metabolism</subject><subject>Animals</subject><subject>Catalase</subject><subject>Dantrolene</subject><subject>Glutathione</subject><subject>Humans</subject><subject>Hydralazine - pharmacology</subject><subject>Phthalazines - metabolism</subject><subject>Rats</subject><subject>Tandem Mass Spectrometry</subject><issn>0090-9556</issn><issn>1521-009X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2023</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNo9kDtPwzAUhS0EoqWwMqKMLAl-xEk8VuXRSqAuBbFZflxTozxKnCLKr8eohenc4TtHVx9ClwRnhND8xjY2I5RlGBPKyyM0JpySFGPxeozGMXAqOC9G6CyE98jkOROnaMQKQYkQbIxensCsVevD4E2yaD8hHm9q8F2bdC4Z1pCsfAPpLWygtdAOybS2sN5ZSJZf3qoAsbT22g9dn8x3tle1-vYtnKMTp-oAF4ecoOf7u9Vsnj4uHxaz6WNqGONDWpVal9w4oRXDVuWioBXHZaEVZtTkYHiR51qL0glHOGBGlK2wc7ogVSGEYRN0vd_d9N3HNj4vGx8M1LVqodsGSSvCOK-qIo9otkdN34XQg5Ob3jeq30mC5a9LGV3K6FLuXcbC1WF7qxuw__ifPPYD8xNwDA</recordid><startdate>202306</startdate><enddate>202306</enddate><creator>Barnes, J Paige</creator><creator>Yang, Shaoyun M</creator><creator>Thompson, Taylor S</creator><creator>Guevarra, Caithlyne M</creator><creator>Pleasant, Shaniya S</creator><creator>Do, Tri Q</creator><creator>Crouch, Rachel D</creator><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0002-4525-6072</orcidid></search><sort><creationdate>202306</creationdate><title>Mechanistic Investigation of the Time-Dependent Aldehyde Oxidase Inhibitor Hydralazine</title><author>Barnes, J Paige ; Yang, Shaoyun M ; Thompson, Taylor S ; Guevarra, Caithlyne M ; Pleasant, Shaniya S ; Do, Tri Q ; Crouch, Rachel D</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c335t-87bb75cf9ba30da496285076ba032c4ec5644bb97f9f15e031ad80ffb618699c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2023</creationdate><topic>Aldehyde Oxidase - metabolism</topic><topic>Animals</topic><topic>Catalase</topic><topic>Dantrolene</topic><topic>Glutathione</topic><topic>Humans</topic><topic>Hydralazine - pharmacology</topic><topic>Phthalazines - metabolism</topic><topic>Rats</topic><topic>Tandem Mass Spectrometry</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Barnes, J Paige</creatorcontrib><creatorcontrib>Yang, Shaoyun M</creatorcontrib><creatorcontrib>Thompson, Taylor S</creatorcontrib><creatorcontrib>Guevarra, Caithlyne M</creatorcontrib><creatorcontrib>Pleasant, Shaniya S</creatorcontrib><creatorcontrib>Do, Tri Q</creatorcontrib><creatorcontrib>Crouch, Rachel D</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Drug metabolism and disposition</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Barnes, J Paige</au><au>Yang, Shaoyun M</au><au>Thompson, Taylor S</au><au>Guevarra, Caithlyne M</au><au>Pleasant, Shaniya S</au><au>Do, Tri Q</au><au>Crouch, Rachel D</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Mechanistic Investigation of the Time-Dependent Aldehyde Oxidase Inhibitor Hydralazine</atitle><jtitle>Drug metabolism and disposition</jtitle><addtitle>Drug Metab Dispos</addtitle><date>2023-06</date><risdate>2023</risdate><volume>51</volume><issue>6</issue><spage>782</spage><epage>791</epage><pages>782-791</pages><issn>0090-9556</issn><eissn>1521-009X</eissn><abstract>The anti-hypertensive agent hydralazine is a time-dependent inhibitor of the cytosolic drug-metabolizing enzyme aldehyde oxidase (AO). Glutathione (GSH) was found to suppress the inhibition of AO by hydralazine in multiple enzyme sources (human liver and kidney cytosol, human liver S9, rat liver S9, and recombinant human AO) and with different AO substrates (zoniporide,
-benzylguanine, and dantrolene). Hydralazine-induced AO inactivation was unaffected when GSH was added to the incubation mixture after pre-incubation of hydralazine with AO (rather than during the pre-incubation), suggesting that GSH traps a hydralazine reactive intermediate prior to enzyme inactivation. Consistent with previous reports of 1-phthalazylmercapturic acid formation when hydralazine was incubated with N-acetylcysteine, we detected a metabolite producing an MS/MS spectrum consistent with a 1-phthalazyl-GSH conjugate.
-Benzylguanine, an AO substrate, did not protect against hydralazine-induced AO inactivation, implying that hydralazine does not compete with
-benzylguanine for binding to the AO active site. Catalase also failed to protect AO from hydralazine-induced inactivation, suggesting that hydrogen peroxide is not involved. However, an allosteric AO inhibitor (thioridazine) offered some protection, indicating a catalytic role for AO in the bioactivation of hydralazine. AO inhibition by phthalazine (a substrate and inhibitor of AO and a metabolite of hydralazine) was unaffected by the presence of GSH. GSH also prevented hydralazine from inhibiting the nitro-reduction of dantrolene by AO. Furthermore, the GSH-hydralazine combination stimulated dantrolene reduction. Phthalazine inhibited only oxidation reactions, not reduction of dantrolene. Together, these results support the hypothesis that hydralazine is converted to a reactive intermediate that inactivates AO. SIGNIFICANCE STATEMENT: These studies suggest that a reactive intermediate of hydralazine plays a primary role in the mechanism of aldehyde oxidase (AO) inactivation. Inactivation was attenuated by glutathione and unaffected by catalase. Phthalazine (hydralazine metabolite) inhibited AO regardless of the presence of glutathione; however, phthalazine inhibited only oxidation reactions, while hydralazine inhibited both oxidation and reduction reactions. This report advances our mechanistic understanding of hydralazine as an AO inhibitor and provides information to facilitate appropriate use of hydralazine when probing AO metabolism.</abstract><cop>United States</cop><pmid>36921993</pmid><doi>10.1124/dmd.123.001257</doi><tpages>10</tpages><orcidid>https://orcid.org/0000-0002-4525-6072</orcidid><oa>free_for_read</oa></addata></record> |
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subjects | Aldehyde Oxidase - metabolism Animals Catalase Dantrolene Glutathione Humans Hydralazine - pharmacology Phthalazines - metabolism Rats Tandem Mass Spectrometry |
title | Mechanistic Investigation of the Time-Dependent Aldehyde Oxidase Inhibitor Hydralazine |
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