TLRs activate immune responses by sensing microbial structures such as bacterial LPS, viral RNA, and endogenous "danger" molecules released by damaged host cells. MyD88 is an adapter protein that mediates signal transduction for most TLRs and leads to activation of NF-kappaB and MAPKs and production of proinflammatory cytokines. TLR4-mediated signaling also leads to rapid activation of PI3K, one of a family of kinases involved in regulation of cell growth, apoptosis, and motility. LPS stimulates phosphorylation of Akt, a downstream target of PI3K, in wild-type (WT) mouse macrophages. LPS-induced phosphorylation of Akt serine 473 was blunted in MyD88(-/-) macrophages and was completely TLR4-dependent. MyD88 and p85 were shown previously to co-immunoprecipitate, and a YXXM motif within the Toll-IL-1 resistance (TIR) domain of MyD88 was suggested to be important for this interaction. To test this hypothesis, we compared expressed MyD88 variants with mutations within the YXXM motif or lacking the TIR domain or death domain and measured their capacities to bind PI3K p85, MyD88, and TLR4 by co-immunoprecipitation analyses. The YXXM --> YXXA mutant MyD88 bound more strongly to p85, TLR4, and WT MyD88 than the other variants, yet was significantly less active than WT MyD88, suggesting that sustained interaction of MyD88/PI3K with the TLR4 intracellular "signaling platform" negatively regulates signaling. We propose a hypothetical model in which sustained PI3K activity at the membrane limits the availability of the PI3K substrate, thereby negatively regulating signaling.

Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs except TLR3, recruitment of the adapter, myeloid differentiation primary response gene 88 (MyD88), to TLR TIR domains results in downstream signaling culminating in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein-protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a pocket adjacent to the highly conserved P681 and G682 BB loop residues. Using computer-aided drug design (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this pocket and potentially disrupt TLR2 signaling. In silico screening identified 149 compounds and 20 US Food and Drug Administration-approved drugs based on their predicted ability to bind in the BB loop pocket. These compounds were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. C 16 H 15 NO 4 (C29) was identified as a potential TLR2 inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an effective approach for identification of TLR2 signaling inhibitors.small molecule inhibitor | BB loop | TLR2 pocket | CADD T oll-like receptors (TLRs) are type I transmembrane receptors that detect conserved "pathogen-associated molecular patterns" from microbes, as well as host-derived "danger-associated molecular patterns" (1). TLR2 heterodimerizes with TLR6 or TLR1 to recognize diacyl lipopeptides or triacyl lipopeptides, respectively (2, 3), present in gram-positive and gram-negative bacteria (4-9).Ligand engagement of TLR2/1 or TLR2/6 activates the myeloid differentiation primary response gene 88 (MyD88)-dependent pathway (i.e., nuclear translocation of NF-κB, activation of MAPKs), resulting in production of proinflammatory cytokines (10). Dysregulated TLR2 signaling has been implicated in numerous diseases (e.g., sepsis, atherosclerosis, tumor metastasis, ischemia/reperfusion injury) (11)(12)(13)(14). Several inhibitors of TLR2 signaling have been developed (15-18), yet none is licensed for human use. A better understanding of the Toll/IL-1 receptor resistance (TIR) domain interactions involved in TLR2 signaling could lead to novel therapeutic agents.Both TLRs and adapter proteins contain a cytoplasmic TIR domain that mediates homotypic and heterotypic interactions ...

TLR2 plays a central role in the activation of innate immunity in response to Ft, the causative agent of tularemia. We reported previously that Ft LVS elicited strong, dose-dependent NF-kappaB reporter activity in TLR2-expressing human embryo kidney 293 T cells and that Ft LVS-induced murine macrophage proinflammatory cytokine gene and protein expression is TLR2-dependent. We demonstrated further that Ft can signal through TLR2 from within the phagosome and that phagosomal retention of Ft leads to greatly increased expression of a subset of proinflammatory genes. The two adaptor proteins associated with TLR2-mediated signaling are MyD88 and TIRAP. Although MyD88 is absolutely required for the Ft-induced macrophage cytokine response, the requirement for TIRAP can be overcome through retention of Ft within the phagosome. TIRAP-independent signaling was observed whether Ft was retained in the phagosome as a result of bacterial mutation (LVSDeltaiglC) or BFA-mediated inhibition of phagosome acidification. The requirement for TIRAP in TLR2 signaling could also be overcome by increasing the concentrations of synthetic bacterial TLR2 agonists. Taken together, these data suggest that prolonging or enhancing the interaction between TLR2 and its agonist overcomes the "bridging" function ascribed previously to TIRAP.

Toll-like receptors (TLRs) are a family of transmembrane innate immune molecules that play a key role in the detection of microbial and self ligands released from damaged cells. At their C-termini, Toll/Interleukin-1 receptor (TIR) domains dimerize upon ligand binding and initiate a signaling cascade that leads to upregulation of inflammatory cytokines. Overexuberant signaling leads to a “cytokine storm,” causing extensive tissue damage that can be fatal. The ability to block TLR signaling may ameliorate such a scenario. Using CADD, a panel of ~150 low molecular-weight compounds was identified based on their predicted ability to bind a pocket on the TLR2 TIR surface adjacent to BB loop. We performed preliminary screening of these compounds in TLR2-HEK293T stable transfectants and secondary screening of promising candidates in thioglycollate-elicited peritoneal murine macrophages. The impact of compounds on signaling was determined using quantitative RT-PCR of cytokines in response to TLR2-specific agonists (P2C and P3C) and TNF-α, which does not utilize TIR domains for signaling. Compound cytotoxicity was assessed by lactate dehydrogenase (LDH) release. To date, we have identified 10 putative TIR-specific inhibitors, 1 TNF-α-specific inhibitor, and 4 inhibitors of both TIR and TNF-α signaling. This project may lead to the identification of potent small molecule inhibitors of TIR-specific TLR signaling.

Overexuberant TLR2 signaling has been implicated in numerous diseases. Visual analysis of the TLR2 TIR domain crystal structure suggested the presence of a “pocket” adjacent to the highly conserved and functionally important proline and glycine residues of the BB loop. Given the importance of the BB loop in mediating TLR signal transduction, we hypothesized that Computer-Aided Drug Design (CADD) could be used to identify a small molecule inhibitor(s) that would fit within this “pocket” and blunt TLR2 signaling. Using CADD, ~150 small compounds were identified based on their predicted ability to bind in this pocket. Based on inhibition of IL-8 mRNA induced by TLR2 agonists, compound “C29” was found to inhibit TLR2/1 and TLR2/6 signaling in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 blocks heat-killed and live bacterial TLR2 agonist-induced proinflammatory cytokine mRNA. C29 prevents the early activation of TLR2-mediated signaling, including NF-κB and MAPKs, as well as MyD88 recruitment to TLR2 in THP-1 cells. C29 is cleavable and the byproduct, o-vanillin, reproduces comparable TLR2 inhibitory activity. O-vanillin covalently interacts with recombinant human TLR2 TIR domain dose-dependently. Mutagenesis of “pocket” residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Collectively, these results provide proof-of-principle for using CADD to identify inhibitors of TLR2 signaling.