Afzaal Tufail, Matthew E Warnes, Nathalie Signoret, Martin A Fascione
{"title":"Convergent construction of N-terminally modified CCL5 chemokines for photoaffinity receptor pull-down using cross-aldol bioconjugations.","authors":"Afzaal Tufail, Matthew E Warnes, Nathalie Signoret, Martin A Fascione","doi":"10.1039/d5cb00162e","DOIUrl":null,"url":null,"abstract":"<p><p>Chemokines such as CCL5 (RANTES) mediate immune responses <i>via</i> interaction with G-protein-coupled receptors like CCR5, which also serves as a co-receptor for HIV-1 entry into host cells. Modified CCL5 analogues have shown promise as CCR5 antagonists for anti-HIV strategies, but current approaches involve hydrolytically unstable linkages or laborious synthesis. Here, we demonstrate the use of an organocatalyst-mediated protein aldol ligation (OPAL) to construct N-terminally modified CCL5 analogues bearing hydrolytically stable carbon-carbon linkages. Using a recombinant CCL5 P2G mutant and selective oxidation to introduce an α-oxo aldehyde at the N-terminus, we achieved efficient OPAL bioconjugation with various aldehyde donors, including alkyl and aryl acetaldehydes. Notably, a 4-azido aryl acetaldehyde CCL5 OPAL product was utilised as a CCR5 photoaffinity probe. This modified chemokine successfully captured CCR5 from mammalian cells <i>via</i> photo-crosslinking, enabling receptor pull-down for biochemical analysis. Our work showcases cross-aldol bioconjugations as a versatile and convergent strategy for stable chemokine functionalisation, with potential applications in therapeutic development and mechanistic studies of chemokine-receptor interactions. This method offers a promising chemical biology platform for modulating or probing the CCL5-CCR5 axis with enhanced precision and synthetic accessibility.</p>","PeriodicalId":40691,"journal":{"name":"RSC Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.1000,"publicationDate":"2025-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12455665/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"RSC Chemical Biology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1039/d5cb00162e","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Chemokines such as CCL5 (RANTES) mediate immune responses via interaction with G-protein-coupled receptors like CCR5, which also serves as a co-receptor for HIV-1 entry into host cells. Modified CCL5 analogues have shown promise as CCR5 antagonists for anti-HIV strategies, but current approaches involve hydrolytically unstable linkages or laborious synthesis. Here, we demonstrate the use of an organocatalyst-mediated protein aldol ligation (OPAL) to construct N-terminally modified CCL5 analogues bearing hydrolytically stable carbon-carbon linkages. Using a recombinant CCL5 P2G mutant and selective oxidation to introduce an α-oxo aldehyde at the N-terminus, we achieved efficient OPAL bioconjugation with various aldehyde donors, including alkyl and aryl acetaldehydes. Notably, a 4-azido aryl acetaldehyde CCL5 OPAL product was utilised as a CCR5 photoaffinity probe. This modified chemokine successfully captured CCR5 from mammalian cells via photo-crosslinking, enabling receptor pull-down for biochemical analysis. Our work showcases cross-aldol bioconjugations as a versatile and convergent strategy for stable chemokine functionalisation, with potential applications in therapeutic development and mechanistic studies of chemokine-receptor interactions. This method offers a promising chemical biology platform for modulating or probing the CCL5-CCR5 axis with enhanced precision and synthetic accessibility.