{"title":"胰岛素调节的氨基肽酶的新变构抑制剂的发现为酶的机制提供了新的见解。","authors":"Galateia Georgaki, Nikoletta-Maria Koutroumpa, Panagiotis Lagarias, Antreas Afantitis, Athanasios Papakyriakou, Efstratios Stratikos","doi":"10.1021/acsomega.5c01169","DOIUrl":null,"url":null,"abstract":"<p><p>Insulin-regulated aminopeptidase (IRAP) is a transmembrane zinc metalloprotease with various important biological roles, including fibrosis, septic thrombosis, cognitive functions, and immune system regulation. As a result, IRAP is an emerging pharmacological target for several diseases. However, the development of selective inhibitors that specifically regulate its activity remains challenging due to its high sequence and functional homology with many other enzymes that have highly conserved active sites. To circumvent this limitation, we targeted the malate allosteric site, a site that has yielded highly selective inhibitors of the homologous enzyme ERAP1. We performed virtual screening to discover drug-like compounds that bind with high affinity to this allosteric site in IRAP. A database of 38 million diverse, drug-like compounds from ENAMINE was employed for screening at three conformations of the targeted site. A subset of the top-ranked compounds was subsequently evaluated using molecular dynamics simulations and comparative MM/GBSA free energy calculation, from which 17 were selected for further <i>in vitro</i> evaluation of their inhibitory activity for IRAP by two orthogonal assays. Three hits, one for each enzyme conformation and substrate class, were selected for further mechanistic evaluation revealing substrate-dependent uncompetitive or noncompetitive mechanisms of action, consistent with the conformationally sensitive nature of the allosteric site. Our results support the tractability of the malate site for the discovery of novel selective IRAP inhibitors, establish novel hits for further development, and suggest that it may be possible to target specific biological functions of IRAP by targeting distinct conformations of the enzyme by allosteric inhibitors.</p>","PeriodicalId":22,"journal":{"name":"ACS Omega","volume":"10 17","pages":"17960-17972"},"PeriodicalIF":4.3000,"publicationDate":"2025-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12059914/pdf/","citationCount":"0","resultStr":"{\"title\":\"Discovery of Novel Allosteric Inhibitor Hits for Insulin-Regulated Aminopeptidase Provides Insights on Enzymatic Mechanism.\",\"authors\":\"Galateia Georgaki, Nikoletta-Maria Koutroumpa, Panagiotis Lagarias, Antreas Afantitis, Athanasios Papakyriakou, Efstratios Stratikos\",\"doi\":\"10.1021/acsomega.5c01169\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Insulin-regulated aminopeptidase (IRAP) is a transmembrane zinc metalloprotease with various important biological roles, including fibrosis, septic thrombosis, cognitive functions, and immune system regulation. As a result, IRAP is an emerging pharmacological target for several diseases. However, the development of selective inhibitors that specifically regulate its activity remains challenging due to its high sequence and functional homology with many other enzymes that have highly conserved active sites. To circumvent this limitation, we targeted the malate allosteric site, a site that has yielded highly selective inhibitors of the homologous enzyme ERAP1. We performed virtual screening to discover drug-like compounds that bind with high affinity to this allosteric site in IRAP. A database of 38 million diverse, drug-like compounds from ENAMINE was employed for screening at three conformations of the targeted site. A subset of the top-ranked compounds was subsequently evaluated using molecular dynamics simulations and comparative MM/GBSA free energy calculation, from which 17 were selected for further <i>in vitro</i> evaluation of their inhibitory activity for IRAP by two orthogonal assays. Three hits, one for each enzyme conformation and substrate class, were selected for further mechanistic evaluation revealing substrate-dependent uncompetitive or noncompetitive mechanisms of action, consistent with the conformationally sensitive nature of the allosteric site. Our results support the tractability of the malate site for the discovery of novel selective IRAP inhibitors, establish novel hits for further development, and suggest that it may be possible to target specific biological functions of IRAP by targeting distinct conformations of the enzyme by allosteric inhibitors.</p>\",\"PeriodicalId\":22,\"journal\":{\"name\":\"ACS Omega\",\"volume\":\"10 17\",\"pages\":\"17960-17972\"},\"PeriodicalIF\":4.3000,\"publicationDate\":\"2025-04-23\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12059914/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"ACS Omega\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://doi.org/10.1021/acsomega.5c01169\",\"RegionNum\":3,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2025/5/6 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"Q2\",\"JCRName\":\"CHEMISTRY, MULTIDISCIPLINARY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Omega","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1021/acsomega.5c01169","RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/5/6 0:00:00","PubModel":"eCollection","JCR":"Q2","JCRName":"CHEMISTRY, MULTIDISCIPLINARY","Score":null,"Total":0}
Discovery of Novel Allosteric Inhibitor Hits for Insulin-Regulated Aminopeptidase Provides Insights on Enzymatic Mechanism.
Insulin-regulated aminopeptidase (IRAP) is a transmembrane zinc metalloprotease with various important biological roles, including fibrosis, septic thrombosis, cognitive functions, and immune system regulation. As a result, IRAP is an emerging pharmacological target for several diseases. However, the development of selective inhibitors that specifically regulate its activity remains challenging due to its high sequence and functional homology with many other enzymes that have highly conserved active sites. To circumvent this limitation, we targeted the malate allosteric site, a site that has yielded highly selective inhibitors of the homologous enzyme ERAP1. We performed virtual screening to discover drug-like compounds that bind with high affinity to this allosteric site in IRAP. A database of 38 million diverse, drug-like compounds from ENAMINE was employed for screening at three conformations of the targeted site. A subset of the top-ranked compounds was subsequently evaluated using molecular dynamics simulations and comparative MM/GBSA free energy calculation, from which 17 were selected for further in vitro evaluation of their inhibitory activity for IRAP by two orthogonal assays. Three hits, one for each enzyme conformation and substrate class, were selected for further mechanistic evaluation revealing substrate-dependent uncompetitive or noncompetitive mechanisms of action, consistent with the conformationally sensitive nature of the allosteric site. Our results support the tractability of the malate site for the discovery of novel selective IRAP inhibitors, establish novel hits for further development, and suggest that it may be possible to target specific biological functions of IRAP by targeting distinct conformations of the enzyme by allosteric inhibitors.
ACS OmegaChemical Engineering-General Chemical Engineering
CiteScore
6.60
自引率
4.90%
发文量
3945
审稿时长
2.4 months
期刊介绍:
ACS Omega is an open-access global publication for scientific articles that describe new findings in chemistry and interfacing areas of science, without any perceived evaluation of immediate impact.