Anti-inflammatory activity of small-molecule antagonists of Toll-like receptor 2 (TLR2) in mice.

Toll-like receptor 2 (TLR2) is currently investigated as a potential therapeutic target in diseases with underlying inflammation like sepsis and arthritis. We reported the discovery, by virtual screening and biological testing, of eight TLR2 antagonists (AT1-AT8) which showed TLR2-inhibitory activity in human cells (Murgueitio et al., 2014). In this study, we have deepened in the mechanism of action and selectivity (TLR2/1 or TLR2/6) of those compounds in mouse primary cells and in vivo. The antagonists reduced, in a dose-dependent way the TNFα production (e.g. AT5 IC50 7.4 µM) and also reduced the nitric oxide (NO) formation in mouse bone marrow-derived macrophages (BMDM). Treatment of BMDM with the antagonists showed that downstream of TLR2, MAPKs phosphorylation and IkBα degradation was reduced. Notably, in a mouse model of tri-acylated lipopeptide (Pam3CSK4)-induced inflammation, AT5 attenuated the TNFα and IL-6 inflammatory response. Further, the effect of AT5 in the stimulation of BMDM by the endogenous alarmin HMGB1 was investigated. Our results indicate that AT4-AT7 and, particularly AT5 appear as good starting points for the development of inhibitors targeting TLR2 in inflammatory disorders.

Decoy peptides derived from the extracellular domain of toll-like receptor 2 (TLR2) show anti-inflammatory properties.

Toll-like receptor 2 (TLR2) recognizes bacterial derived- and synthetic-lipopeptides after dimerization with TLR1 or TLR6. Hyper-activation of TLR2 has been described in several inflammatory diseases and the discovery of inhibitors of its pro-inflammatory activity represent potential starting points to develop therapeutics in such pathologies. We designed peptides derived from the TLR2 sequence comprising amino acid residues involved in ligand binding (Pam3CSK4) or heterodimerization (TLR2/TLR1) as pointed out by structural data.2 We identified several peptides (P13, P13(LL), P16, P16(LL)) which inhibited TLR2/1 signaling in HEK293-TLR2 cells (MAPK activation and NF-kB activity). Moreover, P13L and P16L decreased TNFα release in human primary PBMCs and mouse macrophages. The peptides were selective for TLR2/1 as they did not inhibit the activity of other TLRs tested. P13L and P16L inhibited the internalization of Pam3CSK4 fluorescently labeled in macrophages and the heterodimerization of TLR2 with TLR1 as demonstrated by immunoprecipitation studies. Our data demonstrate that peptides derived from the region comprising the leucine-rich repeats (LRR) 11 and 13 in the extracellular domain of TLR2 are good starting points to develop more potent anti-inflammatory peptides with TLR2 inhibitory activity.

Enhanced immunostimulatory activity of in silico discovered agonists of Toll-like receptor 2 (TLR2).

BACKGROUND: Emergent therapies in anticancer vaccination use Toll-like receptors (TLRs) agonists as dendritic cell (DC) vaccine adjuvants. DCs from the patient are isolated, stimulated with TLR agonists and tumor antigens ex vivo and then infused back into the patient. Although some TLR ligands have been tested in clinical trials, novel TLR agonists with improved immunomodulatory properties are essential to optimize treatment success. We report on the discovery of small-molecule TLR2 agonists, with favorable properties as synthetic adjuvants. METHODS: We performed a shape- and featured-based similarity virtual screening against a commercially available compound library. The selected virtual hits were experimentally tested in TLR2-reporter cells and their activity in phagocytes and DCs was characterized. A binding model of the compounds to TLR2 (docking studies) was proposed. RESULTS: Through a virtual screening approach against a library of three million compounds four virtual hits (AG1, AG2, AG3, AG4) were found to synergistically augment the NF-kB activation induced by the lipopeptide ligand Pam3CSK4 in luciferase reporter assays using HEK293-TLR2 cells. Biacore experiments indicated that AG1-AG4 are ago-allosteric modulators of TLR2 and AG2 bound TLR2 with high affinity (KD 0.8µM). The compounds induced TNF-α production in human peripheral blood mononuclear cells (PBMCs) and they activated DCs as indicated by IL-12 production and upregulation of CD83/CD86. CONCLUSIONS: Following a combined in silico/in vitro approach we have discovered TLR2-agonists (AG1-AG4) that activate human and mouse immune cells. GENERAL SIGNIFICANCE: We introduce four novel TLR2 ago-allosteric modulators that stimulate myeloid cell activity and constitute promising candidates as synthetic adjuvants.

Functional interactions between chpB and parD, two homologous conditional killer systems found in the Escherichia coli chromosome and in plasmid R1.

parD and chpB are homologous conditional killer systems of plasmid and chromosomal origin, respectively. They are bicistronic operons encoding a killer protein (Kid and ChpBK) and an antidote (Kis and ChpBI). It is shown that the antidote of the chpB system can neutralize the toxin of the parD system. This activity is improved by particular amino acid changes at the amino end of the ChpBI antidote. It is further shown that the chpB system is weakly autoregulated and that the activity of a second promoter, previously identified upstream of the regulated promoter, can modulate the functional interactions between the chpB and parD systems.

Functional interactions between homologous conditional killer systems of plasmid and chromosomal origin.

parD and chpA are homologous conditional killer systems of plasmid and chromosomal origin, respectively, encoding a killer protein (Kid and ChpAK) and an antidote (Kis and ChpAI). Here it is shown that these systems can functionally interact. A multicopy chpA recombinant partially complements two mutations in the antidote of the parD system. These mutations affect either autoregulation or neutralization of the killer component. Following in vitro mutagenesis with hydroxylamine, chpA mutants that improve this complementation were isolated. Sequence analysis shows that these mutants are clustered in the 5' end of the chpAI gene and structure predictions suggest that they affect a putative loop in the secondary structure of the ChpAI antidote. It is proposed that this region is part of a protein-protein interface required for the functional interaction between the antidote and the killer components in the two homologous systems.