title: Structure-activity relationship studies on the immune stimulatory effects of base-modified CpG toll-like receptor 9 agonists.
abstract: Synthetic oligodeoxynucleotides containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are able to stimulate potent immune responses through a signaling pathway involving Toll-like receptor 9 (TLR9). We have investigated the structure-activity relationship (SAR) of base-modified CpG oligonucleotides with TLR9 by measuring TLR9 activation by 20-mer oligonucleotides having just a single human recognition motif (5'-GTCGTT-3') in functional cell-based TLR9 assays. Substitution of guanine by hypoxanthine and 6-thioguanine resulted in activity similar to the unmodified parent molecule, whereas purine, 2-aminopurine, 2,6-diaminopurine, and 8-oxo-7,8-dihydroguanine substitution resulted in approximately 40-60 % reduction in activity, and 7-deazaguanine substitution led to the strongest (80 %) reduction in TLR9 stimulation. Furthermore, none of the investigated modifications at C5 and N4 of cytosine were well tolerated with respect to human TLR9 stimulation. Our results are compatible with a SAR model in which guanine is recognized by the Hoogsteen site, and C5 is most critical for recognition of cytosine. In addition, we found significant species-specific differences between human and murine TLR9 recognition, which demonstrates the importance of choosing appropriate assay systems for SAR studies.
authors.affiliation: Coley Pharmaceutical GmbH, Elisabeth-Selbert-Strasse 9, 40764 Langenfeld, Germany.
authors.forename: Marion
authors.initials: M
authors.lastname: Jurk
authors.forename: Andrea
authors.initials: A
authors.lastname: Kritzler
authors.forename: Harald
authors.initials: H
authors.lastname: Debelak
authors.forename: Jörg
authors.initials: J
authors.lastname: Vollmer
authors.forename: Arthur M
authors.initials: AM
authors.lastname: Krieg
authors.forename: Eugen
authors.initials: E
authors.lastname: Uhlmann
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D018899:CpG Islands / Q000187:drug effects*; D017930:Genes, Reporter; D006147:Guanine / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D016328:NF-kappa B / Q000378:metabolism; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D013045:Species Specificity; D013329:Structure-Activity Relationship; D051217:Toll-Like Receptor 9 / Q000819:agonists*
chemical_list: D000276:Adjuvants, Immunologic; D016328:NF-kappa B; D009841:Oligonucleotides; D051217:Toll-Like Receptor 9; D006147:Guanine; D008156:Luciferases