Fig. 1 A phenotypic HTS platform based on hierarchically acting TLR signaling proteins. (A) Model of the MyD88 signaling pathway activated by different TLR and IL-1R family members. Established protein interactions are highlighted (yellow circles). Proteins activated by coumermycin (CM)–induced Gyrase B (GyrB) dimerization are indicated with green bolts. TIR domains are shown in purple, and DDs in dark blue. KD, kinase domain; AP1, activator protein 1. (B) NF-κB luciferase (Luc.) reporter activity in human embryonic kidney (HEK) 293T cells expressing the indicated GyrB fusion proteins and treated as indicated. Fold change data normalized to vehicle-treated controls (Ctrl) are means ± SD from three independent experiments. (C) Western blot analysis of MyD88 expression in TIRAP-GyrB–expressing HEK293T cells that were infected with lentivirus expressing Cas9 and control sgRNA (ctrl) or sgRNA against MyD88 (MyD). Blots are representative of three independent experiments. (D) NF-κB luciferase reporter activity of TIRAP-GyrB–expressing HEK293T cells transduced with Cas9/indicated sgRNA and stimulated as indicated. RLU, relative light units; Co, control. Data are means ± SD from three independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001 by Mann-Whitney U test (B) or two-way analysis of variance (ANOVA) with Sidak’s multiple comparison test (D).
Fig. 2 Identification of MPP as TLR inhibitory compound using HTS based on inducible TLR signaling proteins. (A) Schematic of HTS workflow. (B) NF-κB luciferase reporter activity (left) and Alamar blue–determined cell viability (right) for the indicated reporter cell lines after treatment with MPP. Data are means ± SD from three independent experiments. (C) Enzyme-linked immunosorbent assay (ELISA) analysis of TNFα production by mouse BMM stimulated with TLR agonists for 6 hours in the presence of the indicated concentration of MPP. Data are means ± SD of three independent experiments. Pam, Pam3Cys. (D) Western blot analysis of the indicated proteins in lysates from CpG-DNA–, R848-, and Curdlan (Curd)–treated BMM in the presence or absence of MPP. Antibodies against total p38 were used as loading control. Blots are representative of three independent experiments. (E) ELISA analysis of TNFα production by wild-type (WT) and ERα-deficient BMM that were treated for 6 hours with CpG-DNA in the presence of the indicated concentration of MPP. Data are means ± SD of three independent experiments. (F) Western blot analysis of IκBα and MAPK phosphorylation in lysates from ERα-deficient BMM that were treated for 20 min with CpG-DNA in the presence or absence of MPP. Antibodies against total p38 were used as loading control. Blots are representative of three independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with Dunett’s multiple comparisons test (C) or a paired t test (two-tailed; E).
Fig. 3 TSI block formation of the MyD88 signaling complex by interference with TIR-mediated dimerization. (A) Quantitative MS analysis of proteins copurifying with MyD88 in CpG-DNA–stimulated RAW264.7 cells treated with MPP, as indicated. Data are the fold change in peptide abundance from CpG-DNA–treated versus control cells (open bars) and CpG-DNA plus MPP-treated versus control cells (closed bars). Numbers in parenthesis indicate the number of unique peptides identified. Data are from one experiment. (B and C) Western blot analysis of the indicated proteins coprecipitating with MyD88 in lysates (Ly) from MyD88-GyrB-F (Flag)–expressing RAW264.7 cells treated with CpG-DNA (45 min) (B) or CM (20 min) (C) and MPP, as indicated. Endo, endogenous. Blots (left) are representative of four independent experiments. Normalized band intensities (right) are pooled from all experiments. (D) Western blot analysis of MyD88 domains coprecipitating with MyD88 in lysates from HEK293T cells transfected with full-length (MyD88-F) or TIR domain [TIR–hemagglutinin (HA)] of MyD88 and treated with MPP, as indicated. Blots (left) are representative of four independent experiments. Normalized band intensities (right) are pooled from all experiments. (E) Western blot analysis of MyD88 domains coprecipitating with MyD88 in lysates from HEK293T cells transfected with full-length (MyD88-F) or death domain of MyD88 (MyD88-DD-HA) and treated with MPP, as indicated. Blots are representative of four independent experiments. (F to I) CETSA analysis of the stability of endogenous MyD88 in HEK293T cells exposed to 50 μM TSI-13-57 and the indicated temperatures in degree Celsius (F and G) or the indicated concentrations of TSI-13-57 and 43.8°C (H and I). Western blots (F and H) are representative of three independent experiments. MyD88 band intensities (G and I) normalized to β-ACTIN relative to controls [37°C (G) and 12.5 μM (I)] are means ± SD pooled from three independent experiments. TCL, total cell lysate. *P < 0.05, **P < 0.01, ****P < 0.0001 by two-way ANOVA with Sidak’s multiple comparison test (B and D), one-way ANOVA with Sidak’s multiple comparison test (C), paired t test (two-tailed) (G), and one-way ANOVA with Dunett’s multiple comparisons test (I).
Fig. 4 MPP analogs with selectivity against specific TLR signaling pathways and TIR domains. (A) ELISA analysis of TNFα production and Alamar blue (AB) staining for cell viability in RAW264.7 cells treated as indicated. Data are mean IC50 values ± SD from three independent experiments. (B) Interaction-dependent luciferase activity and cell toxicity in HEK293T cells transfected with the indicated M2H protein pairs and a Gal4 luciferase reporter treated with TSI-13-48 and TSI-13-57 at indicated concentrations. Data relative to maximal inhibition by staurosporine are means ± SD from three independent experiments. **P <0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA with Sidak’s multiple comparison test (A) or paired t test (two-tailed) (B).
Fig. 5 TSI-13-57–mediated inhibition of LPS-induced TNFα release in vivo. (A) ELISA analysis of TNFα concentration in the plasma of mice that were pretreated as indicated and challenged with intraperitoneal LPS. Data relative to vehicle control treatment are means ± SD of five mice per group from two independent experiments. (B) Liquid chromatography (LC)–MS/MS analysis of TSI-13-57 concentration in the plasma of mice described in (A). Data are means ± SD of five mice per group from two independent experiments. ****P < 0.0001 by one-way ANOVA with Dunett’s multiple comparisons test (A).
- Table 1 Initial SAR to differentiate TLR inhibition from ER binding affinity.
IC50 and EC50values for TLR9 were obtained using RAW264.7 cells. Values are presented as means ± SD of three independent experiments.
Entry Compound R1 R3 R4 R5 TLR9 TLR9 RBA* IC50 (μM) Toxicity
EC50 (μM)ERα ERβ 1 MPP OH CH3 BSC 2.11 ± 0.64 13.2 ± 2.4 12 ± 2 0.05 ± 0.01 2 TSI-13-03 OH Et 32.1 ± 1.43 43 ± 6.4 8.92 ± 0.53 0.324 ± 0.01 3 TSI-13-30 OCH3 CH3 BSC 1.97 ± 1.72 33.2 ± 2.7 0.013 ± 0.001 <0.001 4 TSI-13-27 OH H BSC 1.03 ± 0.8 12.3 ± 3.7 5.92 ± 0.13 0.021 ± 0.007 5 TSI-13-04 OH Et BSC 2.84 ± 0.12 11.6 ± 0.9 11.5 ± 1.5 0.49 ± 0.27 6 TSI-13-19 OH BSC 28.2 ± 0.5 35.7 ± 4.7 17.8 ± 5.1 0.044 ± 0.02 7 TSI-13-06 OH BSC Et 3.12 ± 0.103 11.2 ± 1.1 7.34 ± 0.036 0.467 ± 0.13 8 TSI-13-07 OH BSC n-Pr 2.96 ± 0.2 12.9 ± 0.3 8.54 ± 2.1 1.45 ± 0.05 9 TSI-13-05 OH BSC Et 3.33 ± 0.2 7.4 ± 0.4 0.132† 0.010† *RBA indicates relative binding affinity values, where estradiol = 100.
†Single determinations.
- Table 2 Initial hit compounds that demonstrate selective TLR inhibition.
IC50 and EC50 values were obtained using RAW264.7 cells. n.d., not done. Values are presented as means ± SD of three independent experiments.
Entry Compound R1 R2 BSC IC50 (μM)
TLR9TLR7 TLR2 TLR4 EC50 (μM)
toxicity1 TSI-13-50 p-OH H 1-piperidinyl 6.82 ± 0.65 15.6 ± 1.0 8.11 ± 2.19 15.8 ± 3.1 21.4 ± 5.2 2 TSI-13-60 H p-OH 1-piperidinyl 3.04 ± 1.37 2.07 ± 0.62 5.9 ± 1.97 5.56 ± 0.64 33.8 ± 2.6 3 TSI-13-57 p-OH p-OH 1-piperidinyl 2.7 ± 0.39 6.03 ± 1.41 7.37 ± 3.88 6.38 ± 0.66 32.6 ± 3.2 4 TSI-13-58 p-OH m-OH 1-piperidinyl 30.2 ± 0.1 n.d. 50 n.d. 50 5 TSI-13-48 p-NO2 p-OCH3 1-piperidinyl 3.02 ± 0.11 30.0 ± 0.9 26.4 ± 1.6 28.6 ± 0.3 30.6 ± 3.1 6 TSI-14-44 p-OCH3 3,4,5-(OCH3)3 1-piperidinyl 3.43 ± 0.49 2.24 ± 0.63 2.28 ± 1.10 1.15 ± 0.39 14.6 ± 0.1 7 TSI-15-17 H 5-OCH3-3,4-dioxymethylene 1-piperidinyl 6.59 ± 0.29 0.66 ± 0.09 50 50 50 8 TSI-15-16 p-OCH3 p-OCH3 1-pyrazolyl 50 0.85 ± 0.95 50 50 50 - Table 3 In vivo PK of TSI-13-57.
Mice were intraperitoneally treated with TSI-13-57, and the serum concentrations of compound were determined during time to assess Cmax (peak plasma concentration), Tmax (time to reach Cmax), AUCall (total area from zero to the last sampling time point), Vd (volume of distribution), Cl (clearance), and t1/2 (half-life). i.p., intraperitoneally; N.A., not analyzed.
10 mg/kg (i.p.) 20 mg/kg (i.p.) Tmax (hours) 0.25 4 Cmax (nM) 669 1,260 AUCall (hour·nmol) 4,781.9 19,644.3 Vd (ml) 1,190.3 N.A. Cl (ml/hour) 84.2 42.5 t1/2 (hours) 9.8 9 - Table 4 In vitro early ADMET analysis of TSI-13-57.
Absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of TSI-13-57 were analyzed in vitro to assess suitability for in vivo applications. CLint, intrinsic clearance.
Cytotoxicity EC50 (μM) HEK293 >18.5 HepG2 >18.5 Raji >18.5 Solubility (μM) pH 7.4 14.4 ± 5 Permeability (×10−6 cm/s) pH 7.4 112.6 ± 35 Stability mouse liver microsomes (t1/2) (hours) >4 CLint (ml min−1 kg−1) <13.5
Supplementary Materials
www.sciencesignaling.org/cgi/content/full/11/543/eaaq1077/DC1
Fig. S1. DMSO tolerance of reporter cell lines during stimulation.
Fig. S2. Z′ values of the HEK293T cell–based screening system.
Table S1. Complementary data for Table 1.
Table S2. Complementary data for Table 2.
Methods
References (80–82)
Additional Files
This PDF file includes:
- Fig. S1. DMSO tolerance of reporter cell lines during stimulation.
- Fig. S2. Z′ values of the HEK293T cell–based screening system.
- Table S1. Complementary data for Table 1.
- Table S2. Complementary data for Table 2.
- Methods
- References (80–82)
