Physical validation

To explore the activity of the synthesized and selected compounds 1 to 6 and 9 to 11 on the TLR2 activity, reporter cells overexpressing hTLR2 were used. 7, 10a, 11 Except for 1, all selected compounds decreased TLR2-mediated NF-?B activation in a primary screen (Figure step threeA). Additionally, cuatro, 6, 9 and 10 reduced Pam₃CSK₄-induced TLR2/1 responses to less than 40 % at fifty ?M and 2–6, 9 and 10 diminished Pam₂CSK₄-induced TLR2/6 responses to less than 50 % at 50 ?Mpound 6 and 11 abrogated the TLR2 response of both heterodimers almost completely and therefore showed a similar TLR2 inhibition as CU-CPT22 (Figure 3A). None of the tested compounds appeared to have any agonistic TLR2 activity (Figure S1A in the Supporting Information).

Inhibition of TLR2-dependent NF-?B activation and IL-8 secretion. A) HEK-Blue hTLR2 cells were pre-incubated with CU-CPT22 or compounds 1–6 and 9–11 for 1 h and then stimulated additionally with TLR2/1 agonist Pam₃CSK₄ (10 ng/mL) or TLR2/6 agonist Pam₂CSK₄ (1 ng/mL) for 24 h. B) HEK-Blue hTLR2 cells were pre-incubated with increasing concentrations of 4, 6, 9, 10 and then stimulated additionally with Pam₃CSK₄ or Pam₂CSK₄ for 24 h. Supernatants were tested for TLR2-mediated NF-?B/AP-1 activity by quantification of SEAP release (OD₆₄₀). C) THP-1 macrophages were incubated with CU-CPT22 (25 ?M) or compound 6 for 1 h and afterwards stimulated with Pam₃CSK₄ (10 ng/mL), Pam₂CSK₄ (1ng/mL), LPS (10 ng/mL), flagellin (1 ?g/mL), CL075 (8 ?M) or ODN2006 (5 ?M) for 24 h. Supernatants were analyzed for IL-8 secretion by ELISA. Mean+SD or ±SD (n=3).

No cytotoxic effects were observed within the activity range except for 11 which reduced cell viability starting at 50 ?M (Figure S1B). Hence, the apparent inhibition of TLR2-mediated responses by 11 might be due its cytotoxicity. Consequently, compound 11 was excluded from further investigation. The remaining most active hits, 4, 6, 9 and 10, were analyzed regarding their potency. For this purpose, concentration-response curves were assessed and IC₅₀ values were calculated (Figure 2B). All four compounds showed IC₅₀ values in the ?M range, thus confirming their antagonistic effect as MMG-11 analogues (Table 1)pound 6 turned out to have the lowest IC₅₀ value of the identified TLR2 inhibitors with similar results for both heterodimers (TLR2/1: 15.4 ?M; TLR2/6: 13.6 ?M). This suggests binding of compound 6 to the TLR2 cavity but no interaction towards TLR1 or TLR6. While compound 6 effectively inhibits TLR2 signaling, we could show that it has no influence on other TLR subtypes (Figure 2C). While keeping the activity of compound 6 in a comparable range as for CU-CPT22 and MMG-11, we could replace the highly sensitive 1,2,3-trihydroxyphenyl structure of pyrogallol in the starting compounds by the chemically stable 1,3-dihydroxyphenyl substructure of resorcinol. This is essential for further optimization of TLR2 antagonists because 6 is robust, synthetically accessible and no longer susceptibility toward oxidation.

• [a] 95 % depend on interval revealed for the mounts.

SAR off novel antagonists

In order to rationalize the bioactivity and elucidate the potential binding modes of the tested compounds docking studies were performed. As all characterized compounds both inhibited TLR2/1 and TLR2/6 signaling, without showing effects at other TLRs, we hypothesized that they would bind ourteennetwork alternatif to the TLR2 monomer and share the same ligand binding pocket like its pyrogallol-containing precursor, MMG-11. 10a This compound has been previously shown to be a competitive antagonist for the binding of Pam₃CSK₄ and Pam₂CSK₄, which further supports our proposed binding mode. However, an experimental confirmation of the binding mode is beyond the scope of this study. Extensive docking analyses were performed and plausible binding poses selected based on their similarity to the binding mode of MMG-11 and the amount of performed interactions between the ligand and the TLR2 binding site. The resulting proposed binding mode for the most active compound 6 is shown in Figure 3B. Selected docking poses for the remaining compounds can be found in the Figures S2 to S5. 6 is embedded in the front part of the binding pocket with the dihydroxy-benzamide moiety forming several H-Bonds to the backbone atoms of TLR2. The benzene ring forms hydrophobic contacts to the Phe325 side chain.