Stereochemical Dependence of the Self-Assembly of the Immunoadjuvants Pam<sub>3</sub>Cys and Pam<sub>3</sub>Cys-Ser

Reichel, Frank; Roelofsen, A.M.; Geurts, Hubertus P. M.; Hämäläinen, Taina I.; Feiters, Martinus C.; Boons, Geert‐Jan

doi:10.1021/ja991319q

Cited by 30 publications

(22 citation statements)

References 33 publications

(31 reference statements)

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“…Considering all the results, which are summarized in Table 1, our findings indicate that not only the number of acyl‐residues and the peptide sequence but also the whole molecular structure of the LP are responsible for TLR6‐dependent signaling. Like LPS, LP are amphiphilic molecules and are able to form supramolecular structures in aqueous media, air‐water interfaces, and lipid bilayer membranes 22–25. For LPS it has been shown that the molecular shape and the supramolecular conformation are important for its biological activity and the recognition by LPS‐binding molecules, e.g.…”

Section: Resultsmentioning

confidence: 99%

Toll‐like receptor 6‐independent signaling by diacylated lipopeptides

et al. 2004

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Bacterial lipopeptides are strong immune modulators that activate early host responses after infection as well as initiating adjuvant effects on the adaptive immune system. These lipopeptides induce signaling in cells of the immune system through Toll-like receptor 2 (TLR2)-TLR1 or TLR2-TLR6 heteromers. So far it has been thought that triacylated lipopeptides, such as the synthetic N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam3)-CSK4, signal through TLR2-TLR1 heteromers, whereas diacylated lipopeptides, like the macrophage-activating lipopeptide from Mycoplasma fermentans (MALP2) or S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam2)-CGNNDESNISFKEK, induce signaling through TLR2-TLR6 heteromers. Using new synthetic lipopeptide derivatives we addressed the contribution of the lipid and, in particular, the peptide moieties with respect to TLR2 heteromer usage. In contrast to the current model of receptor usage, not only triacylated lipopeptides, but also diacylated lipopeptides like Pam2CSK4 and the elongated MALP2 analog Pam2CGNNDESNISFKEK-SK4 (MALP2-SK4) induced B lymphocyte proliferation and TNF-a secretion in macrophages in a TLR6-independent manner as determined with cells from TLR6-deficient mice. Our results indicate that both the lipid and the Nterminal peptides of lipoproteins contribute to the specificity of recognition by TLR2 heteromers and are responsible for the ligand-receptor interaction on host cells. IntroductionToll-like receptors (TLR) are prominent pattern-recognition receptors (PRR) of the innate immune system, recognizing various invading microorganisms through conserved molecular structures, so-called pathogenassociated molecular patterns (PAMP) [1]. The receptors differ from each other not only in ligand specificity but also in their expression pattern on different cells of the innate immune system. In addition, distinct TLR are able to address different sets of inflammatory target genes [2]. [3] to attain specificity for a given stimulus [4][5][6][7].LP have been described as strong immunostimulatory compounds initiating early innate host defense reactions in cells such as macrophages and epithelial cells [8,9]. In addition, they are potent polyclonal activators of murine B lymphocytes [10]. Signaling through TLR2-TLRx heteromers by LP leads to the formation of an active signaling complex that includes MyD88 and the IL-1R-associated kinase (IRAK) proteins, finally resulting in the nuclear translocation of NF-jB and subsequent cytokine production [11]. As a consequence TLR2-dependent signaling initiates a variety of host defense responses including the activation of the adaptive immune system [12][13][14].Among the TLR2-dependent PAMP, LP are primary candidates for analyzing the structural requirements for heteromer usage, for several reasons. Firstly, these molecules are available from various different bacterial species expressing different molecular structures. Secondly, the chemical synthesis of LP analogues provides a vast variety of chemically defined m...

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Section: Resultsmentioning

confidence: 99%

Toll‐like receptor 6‐independent signaling by diacylated lipopeptides

et al. 2004

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“…For compound 3 , 8 - 13 , an Fmoc-protected version of PEGO (Novabiochem, San Diego, CA) was used and an Fmoc protected derivative of Cys, Fmoc-Cys( S -[2,3-bisacyloxy-(R)-propyl])-OH (Fmoc-Dhc(Pam 2 )-OH) was synthesized as described in Supporting information. 27 All compounds were fully deprotected and cleaved from the resin by treatment with 91% TFA (3% water, 3% EDT, and 3% TA) or 90% TFA (5% water, 5% TIS). After ether extraction of scavengers, compounds were purified by HLPC and/or size-exclusion chromatography (Sephadex G-25, 0.1 M acetic acid) to > 95% purity.…”

Section: Methodsmentioning

confidence: 99%

Novel Toll-like Receptor 2 Ligands for Targeted Pancreatic Cancer Imaging and Immunotherapy

et al. 2012

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Toll-like receptor 2 (TLR2) is a target for immune system stimulation during cancer immunotherapy and a cell-surface marker for pancreatic cancer. To develop targeted agents for cancer imaging and therapy, we designed, synthesized and characterized thirteen novel, fully synthetic high affinity TLR2 agonists. Analog 10 had the highest agonist activity (NF-κB functional assay, EC50=20 nM) and binding affinity (competitive binding assay, Ki=25 nM). As an immune adjuvant, compound 10 stimulated the immune system in vivo by generation and persistence of antigen-specific CD8+ T cells indicating its potential use in cancer immunotherapy. After conjugation of near-infrared dye to 10, agonist activity (EC50=34 nM) and binding affinity (Ki =11 nM) were retained in 13. Fluorescence signal was present in TLR2 expressing pancreatic tumor xenografts 24 h post-injection of 13; while an excess of unlabeled ligand blocked 13 from binding to the tumor resulting in significantly decreased signal (p<0.001) demonstrating in vivo selectivity.

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“…N-palmitoyl-S-[2,3-bis (palmitoyloxy)-(2R)-propyl]-(R)-cysteine (PAM 3 Cys) was synthesized as described earlier. 34 N-palmitoyl-S-[2,3-bis (palmitoyloxy)-(2R)-propyl]-(R)-cysteinyl-(S)-alanyl-glycine (PAM 3 CAG) was assembled manually by conventional solid-phase peptide methodology 35 using Fmoc chemistry on a polystyrene resin functionalized with p-benzyloxybenzyl alcohol (Wang Resin). The resin preloaded with N-Fmoc-L-glycine (173 mg, loading 0.690 mmol/g, 0.12 mmol) was placed in a reaction vessel and pre-swollen in DMF (4 ml) for 1 hour.…”

Section: Synthesis Of Pam 3 Cys and Pam 3 Cagmentioning

confidence: 99%

An Angiogenic Switch in Macrophages Involving Synergy between Toll-Like Receptors 2, 4, 7, and 9 and Adenosine A2A Receptors

Pinhal‐Enfield

Ramanathan

Haskó

et al. 2003

The American Journal of Pathology

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Adenosine A 2A receptor (A 2A R) agonists synergize with Escherichia coli (E. coli) LPS [toll-like receptor (TLR)4 agonist] to up-regulate vascular endothelial growth factor (VEGF) expression in murine macrophages. Here, we demonstrate that TLR2, TLR7, and TLR9, but not TLR3 and TLR5 agonists, also synergize with A 2A R agonists and adenosine to up-regulate VEGF, while simultaneously strongly down-regulating TNF␣ expression. In the absence of adenosine or A 2A R agonists, Porphyromonas gingivalis (P. gingivalis) LPS and PAM 3 CAG (TLR2 agonists), resiquimod (R848) (TLR7 agonist), and non-methylated CpG DNA (TLR9 agonist) strongly up-regulate TNF␣ expression, with no effect on VEGF. In the presence of adenosine or A 2A R agonists, but not A 1 R agonists, TLR2, 4, 7, and 9 agonists strongly up-regulate VEGF expression, while simultaneously down-regulating TNF␣. C57BL/ 10ScN (TLR4 deletion mutant) macrophages produce TNF␣ in response to TLR2, 3, 7, and 9 agonists, but not the TLR4 agonist E. coli LPS. With adenosine or A 2A R agonists, TLR2, 7, and 9, but not TLR4 agonists, also synergistically up-regulate VEGF, while downregulating TNF␣ expression. Polyinosinic-polycytidilic acid (poly(I:C)) (TLR3 agonist) stimulates TNF␣ expression in macrophages from both C57BL/10ScSn and C57BL/10ScN mice, but has little effect on VEGF expression in the presence of adenosine or A 2A R agonists. R-flagellins from Serratia marcescens (S. marcescens) and Salmonella muenchen (S. muenchen) do not stimulate TNF␣ expression in either C57BL/ 10ScSn or C57BL10/ScN mice, and have no effect on VEGF production in the presence of adenosine or A We have shown previously that VEGF expression by murine macrophages is synergistically up-regulated by Escherichia coli (E. coli) lipopolysaccharide (LPS) acting through TLR4 receptors, and adenosine A 2A agonists acting through A 2A Rs. 3 Treatment of macrophages with 2-[p-(2-carboxylethyl)-phenylethyl amino]-5Ј-N-ethyl-carboxamido-adenosine (CGS21680) (a specific adenosine A 2A R agonist), or 5Ј-N-ethyl-carboxamido-adenosine (NECA) (a non-specific adenosine A 2 R agonist), together with E. coli LPS, strongly up-regulates VEGF expression above the level induced by CGS21680 or NECA alone, while LPS alone does not induce VEGF expression. This synergistic up-regulation is independent of hypoxia, NO, and protein kinase-A and is stronger than that induced by hypoxia alone. 3 Mammalian toll-like receptors (TLRs) are members of a family of proteins that resemble the Drosophila toll protein. 4,5 Toll plays a role in dorsal-ventral patterning in the developing fly embryo, and also plays a key role in regulating the innate immune response of adult flies to fungi. 4,5 In mammals, TLR receptors also play a key role in the innate immune response. TLR receptors respond to bacteria and bacterial products by transmitting a ligandinduced trans-membrane signal that induces the expression of cytokines such as TNF␣, IL-1,

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Stereochemical Dependence of the Self-Assembly of the Immunoadjuvants Pam₃Cys and Pam₃Cys-Ser

Cited by 30 publications

References 33 publications

Toll‐like receptor 6‐independent signaling by diacylated lipopeptides

Toll‐like receptor 6‐independent signaling by diacylated lipopeptides

Novel Toll-like Receptor 2 Ligands for Targeted Pancreatic Cancer Imaging and Immunotherapy

An Angiogenic Switch in Macrophages Involving Synergy between Toll-Like Receptors 2, 4, 7, and 9 and Adenosine A2A Receptors

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Stereochemical Dependence of the Self-Assembly of the Immunoadjuvants Pam3Cys and Pam3Cys-Ser

Cited by 30 publications

References 33 publications

Toll‐like receptor 6‐independent signaling by diacylated lipopeptides

Toll‐like receptor 6‐independent signaling by diacylated lipopeptides

Novel Toll-like Receptor 2 Ligands for Targeted Pancreatic Cancer Imaging and Immunotherapy

An Angiogenic Switch in Macrophages Involving Synergy between Toll-Like Receptors 2, 4, 7, and 9 and Adenosine A2A Receptors

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Stereochemical Dependence of the Self-Assembly of the Immunoadjuvants Pam₃Cys and Pam₃Cys-Ser