Further hit optimization of 6-(trifluoromethyl)pyrimidin-2-amine based TLR8 modulators: Synthesis, biological evaluation and structure-activity relationships.

Toll-like receptor 8 (TLR8) is an endosomal TLR that has an important role in the innate human immune system, which is involved in numerous pathological conditions. Excessive activation of TLR8 can lead to inflammatory and autoimmune diseases, which highlights the need for development of TLR8 modulators. However, only a few small-molecule modulators that selectively target TLR8 have been developed. Here, we report the synthesis and systematic investigation of the structure-activity relationships of a series of novel TLR8 negative modulators based on previously reported 6-(trifluoromethyl)pyrimidin-2-amine derivatives. Four compounds showed low-micromolar concentration-dependent inhibition of TLR8-mediated signaling in HEK293 cells. These data confirm that the 6-trifluoromethyl group and two other substituents on positions 2 and 4 are important structural elements of pyrimidine-based TLR8 modulators. Substitution of the main scaffold at position 2 with a methylsulfonyl group or para hydroxy/hydroxymethyl substituted benzylamine is essential for potent negative modulation of TLR8. Our best-in-class TLR8-selective modulator 53 with IC50 value of 6.2 µM represents a promising small-molecule chemical probe for further optimization to a lead compound with potent immunomodulatory properties.

Biological Characterization, Mechanistic Investigation and Structure-Activity Relationships of Chemically Stable TLR2 Antagonists.

Toll-like receptors (TLRs) build the first barrier in the innate immune response and therefore represent promising targets for the modulation of inflammatory processes. Recently, the pyrogallol-containing TLR2 antagonists CU-CPT22 and MMG-11 were reported; however, their 1,2,3-triphenol motif renders them highly susceptible to oxidation and excludes them from use in extended experiments under aerobic conditions. Therefore, we have developed a set of novel TLR2 antagonists (1-9) based on the systematic variation of substructures, linker elements, and the hydrogen-bonding pattern of the pyrogallol precursors by using chemically robust building blocks. The novel series of chemically stable and synthetically accessible TLR2 antagonists (1-9) was pharmacologically characterized, and the potential binding modes of the active compounds were evaluated structurally. Our results provide new insights into structure-activity relationships and allow rationalization of structural binding characteristics. Moreover, they support the hypothesis that this class of TLR ligands bind solely to TLR2 and do not directly interact with TLR1 or TLR6 of the functional heterodimer. The most active compound from this series (6), is chemically stable, nontoxic, TLR2-selective, and shows a similar activity with regard to the pyrogallol starting points, thus indicating the variability of the hydrogen bonding pattern.

Identification and validation of a novel dual small-molecule TLR2/8 antagonist.

Toll-like receptor 2 (TLR2) and TLR8 are involved in the recognition of bacterial and viral components and are linked not only to protective antimicrobial immunity but also to inflammatory diseases. Recently, increasing attention has been paid to the receptor crosstalk between TLR2 and TLR8 to fine-tune innate immune responses. In this study, we report a novel dual TLR2/TLR8 antagonist, compound 24 that was developed by a modeling-guided synthesis approach. The modulator was optimized from the previously reported 1,3-benzothiazole derivative, compound 8. Compound 24 was pharmacologically characterized for the ability to inhibit TLR2- and TLR8-mediated responses in TLR-overexpressing reporter cells and THP-1 macrophages. The modulator showed high efficacy with IC50 values in the low micromolar range for both TLRs, selectivity towards other TLRs and low cytotoxicity. At TLR2, a slight predominance for the TLR2/1 heterodimer was found in reporter cells selectively expressing TLR2/1 or TLR2/6 heterodimers. Concentration ratio analysis in the presence of Pam3CSK4 or Pam2CSK4 indicated non-competitive antagonist behavior at hTLR2. In computational docking studies, a plausible alternative binding mode of compound 24 was predicted for both TLR2 and TLR8. Our results provide evidence that it is feasible to simultaneously and selectively target endosomal- and surface-located TLRs. We identified a small-molecule dual TLR2/8 antagonist that may serve as a valuable pharmacological tool to decipher the role of TLR2/8 co-signaling in inflammation.

The novel small-molecule antagonist MMG-11 preferentially inhibits TLR2/1 signaling.

Toll-like receptor 2 (TLR2) forms heterodimers with either TLR1 or TLR6 to induce protective early inflammatory responses to pathogen- and damage-associated molecular patterns. However, excessive activation is associated with inflammatory and metabolic diseases. Several TLR2 antagonists have been described but pharmacological characterization is still at an early stage. Previously, we identified the potent and selective TLR2 antagonist MMG-11 by computational modelling and experimental validation. Here, we characterized the TLR2 antagonists MMG-11 and CU-CPT22 as well as the TIR-domain binding TLR2 antagonist C29 in TLR-overexpressing promoter cells as well as human and mouse macrophages. In line with our recent studies, MMG-11 abrogated pro-inflammatory cytokine secretion and NF-κB activation induced by different bacterial TLR2 agonists. MMG-11 preferentially inhibited TLR2/1 signaling in promoter cells stably expressing TLR2 heterodimers and mouse macrophages. Furthermore, the TLR2 antagonist blocked ligand-induced interaction of TLR2 with MyD88 and reduced MAP kinase and NF-κB activation. MMG-11 and CU-CPT22 but not C29 displaced Pam3CSK4 in an indirect binding assay confirming the competitive mode of action of MMG-11 and CU-CPT22. Isobologram analysis revealed additive and synergistic effects when the non-competitive antagonist C29 was combined with the competitive antagonist MMG-11 or CU-CPT22, respectively. In conclusion, we provide evidence that MMG-11 acts as a competitive antagonist with a predominance for the TLR2/1 heterodimer in human and mouse cells. Our results also indicate that MMG-11 is a model compound for studying TLR2 signaling.

Identification and characterization of a novel chemotype for human TLR8 inhibitors.

The endosomal Toll-like receptor 8 (TLR8) recognizes single-stranded RNA and initiates early inflammatory responses. Despite the importance of endosomal TLRs for human host defense against microbial pathogens, extensive activation may contribute to autoimmune and inflammatory diseases. In contrast to the recent progress made in the development of modulators of plasma membrane-bound TLRs, little is known about endosomal TLR modulation and very few TLR8 inhibitors have been reported. In this study, we discovered and validated novel small-molecule TLR8 inhibitors. Fourteen potential TLR8 modulators were experimentally validated in HEK293T cells stably overexpressing human TLR8 and THP-1 macrophages. Five compounds inhibited TLR8-mediated signaling, representing a hit rate of 36%. The three most potent compounds neither cause cellular toxicity nor inhibition of TLR signaling induced by other receptor subtypes. Conclusively, we experimentally confirm novel and selective, pyrimidine-based TLR8 inhibitors with low cytotoxicity that are relevant candidates for lead optimization and further mechanistic studies.

Identification of a pyrogallol derivative as a potent and selective human TLR2 antagonist by structure-based virtual screening.

Toll-like receptor 2 (TLR2) induces early inflammatory responses to pathogen and damage-associated molecular patterns trough heterodimerization with either TLR1 or TLR6. Since overstimulation of TLR2 signaling is linked to several inflammatory and metabolic diseases, TLR2 antagonists may provide therapeutic benefits for the control of inflammatory conditions. We present virtual screening for the identification of novel TLR2 modulators, which combines analyses of known ligand sets with structure-based approaches. The 13 identified compounds were pharmacologically characterized in HEK293-hTLR2 cells, THP-1 macrophages and peripheral blood mononuclear cells for their ability to inhibit TLR2-mediated responses. Four out of 13 selected compounds show concentration-dependent activity, representing a hit rate of 31%. The most active compound is the pyrogallol derivative MMG-11 that inhibits both TLR2/1 and TLR2/6 signaling and shows a higher potency than the previously discovered CU-CPT22. Concentration ratio analysis identified both compounds as competitive antagonists of Pam3CSK4- and Pam2CSK4-induced responses. Schild plot analysis yielded apparent pA2 values of 5.73 and 6.15 (TLR2/1), and 5.80 and 6.65 (TLR2/6) for CU-CPT22 and MMG-11, respectively. MMG-11 neither shows cellular toxicity nor interference with signaling induced by other TLR agonists, IL-1ß or TNF. Taken together, we demonstrate that MMG-11 is a potent and selective TLR2 antagonist with low cytotoxicity rendering it a promising pharmacological tool for the investigation of TLR signaling and a suitable lead structure for further chemical optimization.

Development of an Instrument to Examine Nursing Attitudes Toward Fertility Preservation in Oncology.

PURPOSE/OBJECTIVES: To develop an instrument to measure staff nurse perceptions of the barriers to and benefits of addressing fertility preservation (FP) with patients newly diagnosed with cancer. 
. DESIGN: A prospective, nonrandomized instrument development approach. 
. SETTING: Harold C. Simmons Comprehensive Cancer Center at the University of Texas Southwestern Medical Center in Dallas. 
. SAMPLE: 224 RNs who care for patients with cancer. 
. METHODS: The instrument was developed with content experts and field-tested with oncology staff nurses. Responses to a web-based survey were used in exploratory factor analysis. After refining the instrument, the authors conducted a confirmatory factor analysis with 230 web-based survey responses. 
. MAIN RESEARCH VARIABLES: Self-perceived barriers to providing FP options to patients newly diagnosed with cancer.
. FINDINGS: The results supported a 15-item instrument with five domains. CONCLUSIONS: This instrument can be used to explore oncology nurses' attitudes toward FP in newly diagnosed people with cancer in their reproductive years. 
. IMPLICATIONS FOR NURSING: A more comprehensive understanding of attitudes and barriers related to FP will guide the building of optimal systems that support effective FP options, resources, and programs for individuals with cancer.

Recognition of Propionibacterium acnes by human TLR2 heterodimers.

Propionibacterium acnes has been considered as a crucial contributor to the pathogenesis of acne vulgaris. The interaction between P. acnes and the host is mainly mediated by Toll like receptor (TLR) 2 recognition. TLR2 homodimers recognize P. acnes in mice, but here we describe the prerequisite of TLR2/1 and TLR2/6 heterodimers in human cells for P. acnes recognition. P. acnes-induced NF-κB and AP-1activation observed in HEK hTLR2-transfected but not control cells confirmed the specificity of TLR2 recognition. The activation was blocked by neutralizing antibodies against TLR2, TLR1 and TLR6, as well as the TLR2 antagonist CU-CPT22, which showed no selectivity towards human TLR2 heterodimers. The combination of anti-TLR1 and anti-TLR6 antibodies completely abrogated activation by P. acnes. In primary human keratinocytes, P. acnes-increased NF-κB phosphorylation was inhibited by anti-TLR6 and anti-TLR2 antibodies. Furthermore, P. acnes-induced inflammatory responses were impaired by anti-TLR2 neutralizing antibodies and fully blocked by CU-CPT22. Our study suggests species-specific recognition of P. acnes by TLR2 heterodimers which can be exploited therapeutically by small molecules targeting TLR2 for the control of inflammatory responses.