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.

Presynaptic Ca2+-activated K+ channels in glutamatergic hippocampal terminals and their role in spike repolarization and regulation of transmitter release.

Large-conductance Ca(2+)-activated K(+) channels (BK, also called Maxi-K or Slo channels) are widespread in the vertebrate nervous system, but their functional roles in synaptic transmission in the mammalian brain are largely unknown. By combining electrophysiology and immunogold cytochemistry, we demonstrate the existence of functional BK channels in presynaptic terminals in the hippocampus and compare their functional roles in somata and terminals of CA3 pyramidal cells. Double-labeling immunogold analysis with BK channel and glutamate receptor antibodies indicated that BK channels are targeted to the presynaptic membrane facing the synaptic cleft in terminals of Schaffer collaterals in stratum radiatum. Whole-cell, intracellular, and field-potential recordings from CA1 pyramidal cells showed that the presynaptic BK channels are activated by calcium influx and can contribute to repolarization of the presynaptic action potential (AP) and negative feedback control of Ca(2+) influx and transmitter release. This was observed in the presence of 4-aminopyridine (4-AP, 40-100 microm), which broadened the presynaptic compound action potential. In contrast, the presynaptic BK channels did not contribute significantly to regulation of action potentials or transmitter release under basal experimental conditions, i.e., without 4-AP, even at high stimulation frequencies. This is unlike the situation in the parent cell bodies (CA3 pyramidal cells), where BK channels contribute strongly to action potential repolarization. These results indicate that the functional role of BK channels depends on their subcellular localization.

High-conductance calcium-activated potassium channels in rat brain: pharmacology, distribution, and subunit composition.

In rat brain, high-conductance Ca2+-activated K+ (BK) channels are targeted to axons and nerve terminals [Knaus, H. G., et al. (1996) J. Neurosci. 16, 955-963], but absolute levels of their regional expression and subunit composition have not yet been fully established. To investigate these issues, an IbTX analogue ([125I]IbTX-D19Y/Y36F) was employed that selectively binds to neuronal BK channels with high affinity (Kd = 21 pM). Cross-linking experiments with [125I]IbTX-D19Y/Y36F in the presence of a bifunctional reagent led to covalent incorporation of radioactivity into a protein with an apparent molecular mass of 25 kDa. Deglycosylation and immunoprecipitation studies with antibodies raised against alpha- and smooth muscle beta-subunits of the BK channel suggest that the beta-subunit that is associated with the neuronal BK channel is a novel protein. Quantitative receptor autoradiography reveals the highest levels of BK channel expression in the outer layers of the neocortex, hippocampal perforant path projections, and the interpeduncular nucleus. This distribution pattern has also been confirmed in immunocytochemical experiments with a BK channel-selective antibody. Taken together, these findings imply that neuronal BK channels exhibit a restricted distribution in brain and have a subunit composition different from those of their smooth muscle congeners.