Research ArticlePharmacology

Noncompetitive inhibitors of TNFR1 probe conformational activation states

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Science Signaling  30 Jul 2019:
Vol. 12, Issue 592, eaav5637
DOI: 10.1126/scisignal.aav5637
  • Fig. 1 Discovery of small molecules that perturb conformational states of preassembled TNFR1 dimer.

    (A) Schematic of ligand-induced oligomerization of TNFR1 trimers held together by the preligand assembly domain (PLAD). (B) Schematic of the TNFR1ΔCD-FRET biosensor engineered by fusing the green or red fluorescent proteins (GFPs or RFPs) to the C terminus of TNFR1 with truncated cytosolic domain. Ligand-independent association of the fluorophore-tagged receptors through PLAD-PLAD interactions results in fluorescence resonance energy transfer (FRET). The FRET biosensor can detect changes in the cytosolic spacing between receptor monomers. (C) High-throughput screening of ChemBridge DIVERSet 50,000-compound library using the TNFR1 FRET biosensor expressed in HEK293 cells. Compounds that reduced the FRET efficiency below 3SD (red line) were selected for further characterization. Data are representative of one experiment. (D) Secondary FRET analysis of the dose response of the seven hit compounds and zafirlukast (known TNFR1 inhibitor). Data are means ± SD from three independent experiments.

  • Fig. 2 Hit compounds inhibit TNFR1-stimulated IκBα degradation and NF-κB activation.

    (A to C) Western blot analysis of IκBα abundance in lysates of HEK293 cells treated with LTα and the hit compounds at the indicated doses. Western blots (A) are representative of three independent experiments. Quantified band intensity values (B and C) are means ± SD from all experiments. ****P < 0.0001 compared to control by two-tailed unpaired t test. (D and E) Luciferase assay of NF-κB activation in HEK293 cells transfected with reporter plasmids and treated with LTα and DMSO control (D) or LTα and increasing concentrations of hit compounds (E). Data are means ± SD of three independent experiments. ****P < 0.0001 compared to control by two-tailed unpaired t test.

  • Fig. 3 Hit compounds bind TNFR1 and require the receptor for their effects.

    (A) Direct binding of the hit compounds to the TNFR1 extracellular domain (ECD) was characterized by surface plasmon resonance (SPR). Data are means ± SD from three independent experiments. (B) Dose-dependent ligand-induced NF-κB activation in both wild-type (WT) and TNFR1 knockout (KO) HAP1 cells. Data are means ± SD of three independent experiments. ****P < 0.0001 compared to control by two-tailed unpaired t test, and n.s. indicates not significant. (C) NF-κB activation in WT and TNFR1 KO HAP1 cells with the optimized LTα concentration of 0.1 μg/ml. Data are means ± SD of three independent experiments. ****P < 0.0001 compared to control by two-tailed unpaired t test, and n.s. indicates not significant. (D and E) NF-κB activation in WT HAP1 cells (D) and TNFR1 KO HAP1 cells (E) treated with LTα and increasing concentration of compounds (DS41, DS42, and zafirlukast) to test the specificity of the compounds to TNFR1. Data are means ± SD of three independent experiments. N/A, not applicable.

  • Fig. 4 Small-molecule inhibitors do not block ligand-receptor interactions.

    (A) Coimmunoprecipitation between TNFR1 and ligand LTα with treatment of hit compounds (Cpd) at saturation dose of 200 μM. Equal amount of LTα is shown as pull-down controls. Western blots are representative of three independent experiments. (B) Noncompetitive binding assay of LTα (50 nM) and compounds [DS42 or zafirlukast (Zaf) at 200 μM] to TNFR1 ECD was performed by SPR. Data are means ± SD of three independent experiments. (C) Dose-dependent binding of LTα to TNFR1 ECD with increasing concentration of ligand. Data are means ± SD of three independent experiments. (D and E) Dose-dependent binding of LTα in the presence of compounds, DS42 (D) or zafirlukast (E), at saturated compound concentration of 200 μM. Data are means ± SD of three independent experiments. (F) Comparison of the binding affinity of LTα to TNFR1 in the absence and presence of compounds (DS42 or zafirlukast). Data are means ± SD of three independent experiments, and n.s. indicates not significant by two-tailed unpaired t test.

  • Fig. 5 Small-molecule inhibitors do not disrupt receptor-receptor interactions.

    (A) Native gel characterization of soluble PLAD of TNFR1 with treatment of DMSO control, zafirlukast, and hit compounds (200 μM). Gels are representative of three independent experiments. (B) Native gel characterization of soluble ECD of TNFR1 with treatment of DMSO control, zafirlukast, and hit compounds (200 μM). Gels are representative of three independent experiments. (C) Schematics illustrating the mechanism of competitive inhibition by zafirlukast in disrupting receptor-receptor interactions. (D) Schematics illustrating the mechanism of noncompetitive inhibition by the new hit compounds in stabilizing the nonfunctional conformational states of TNFR1 without disrupting receptor-receptor interactions.

  • Fig. 6 Noncompetitive inhibitors are more efficient than competitive inhibitor.

    (A) NF-κB activation in HEK293 cells treated with LTα and increasing concentration of compounds (DS42 and zafirlukast) to compare the inhibition efficiency between noncompetitive and competitive inhibitors. Data are means ± SD of three independent experiments. (B) Comparison of the binding affinity, the absolute (Abs) IC50, and the percent inhibition of NF-κB activation between DS42 and zafirlukast. Data are means ± SD of three independent experiments. ****P < 0.0001 for DS42 compared to zafirlukast by two-tailed unpaired t test, and n.s. indicates not significant.

  • Fig. 7 Long-range perturbation of TNFR1 conformational dynamics by noncompetitive inhibitors is mediated by residues in the ligand-binding loop.

    (A) Crystal structure of the TNFR1 ECD (PDB: 1NCF). The distance between the membrane distal domain, including PLAD, and the membrane proximal domain is estimated to be 73.6 Å, which suggests a potential long-range signal propagation between noncompetitive inhibitors binding at the PLAD or the ECD and the perturbation of the membrane proximal domain as shown by FRET change. Four different cysteine-rich domains (CRD1 to CRD4) are colored in blue, gray, red, and orange, respectively. (B) Surface representation showing the coupling motions between residues in the ligand-binding loop and the membrane proximal domain (PDB: 1NCF). The key ligand binding residues Trp107, Ser108, and Met80 form four hydrogen bonds with Leu111, Gln113, and Cys114, which stabilize the conformation of the region to behave like a hinge in aiding the opening of the receptor. Abolishing the hydrogen bonds may decouple the domains and prevent conformational change acting through the hinge. (C) The amount of FRET decrease in HEK293 cells expressing WT and mutant TNFR1 FRET biosensors (W107A, S108A, WS107/108AA, M80A, and V90A) treated with noncompetitive inhibitors (200 μM) in the absence of ligand. Values were normalized to the DMSO-only control, and data are means ± SD of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 for WT compared to mutant biosensors by two-tailed unpaired t test, and n.s. indicates not significant. (D) Native gel characterization of soluble PLAD in cells cotreated with DMSO control or hit compounds (1000 μM) and zafirlukast (200 μM) to test the competition between the hit compounds and zafirlukast in interacting with PLAD. Gels are representative of three independent experiments.

  • Fig. 8 Lead compounds are optimizable for binding affinity, potency and specificity.

    (A) SPR characterization of the binding affinity of the hit compound DS41 or its analog DSA114 to TNFR1 ECD. Data are means ± SD of three independent experiments. (B and C) NF-κB activation in WT HAP1 cells (B) and TNFR1 KO HAP1 cells (C) treated with LTα and increasing concentration of DS41 or DSA114 to test the improvements in the potency and specificity of the analog. Data are means ± SD of three independent experiments. N/A, not applicable. (D) Noncompetitive binding test of LTα (50 nM) and DSA114 (200 μM) to TNFR1 ECD was performed by SPR. Data are means ± SD of three independent experiments. (E) Native gel characterization of soluble PLAD with treatment of DMSO control, zafirlukast, DS41, or DS114 (200 μM) to test the disruption of PLAD dimerization by the compounds. Gels are representative of three independent experiments. (F) FRET measurements in HEK293 cells expressing WT TNFR1 FRET biosensor treated with DMSO control, DS41 (50 μM), and DSA114 (50 μM) in the absence of ligand to compare the extent of receptor perturbation by the hit compound and its analog. Data are means ± SD of three independent experiments. *P < 0.05 and ****P < 0.0001 compared to control by two-tailed unpaired t test.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/592/eaav5637/DC1

    Fig. S1. Chemical structures of the novel small-molecule inhibitors of TNFR1 and a negative control compound.

    Fig. S2. The seven hit compounds and zafirlukast bind the TNFR1 ECD as characterized by SPR measurements.

    Fig. S3. Some hit compounds nonspecifically inhibit TNFR1-stimulated NF-κB activation.

    Fig. S4. DS41, but not DS42 and zafirlukast, inhibits TRADD-induced NF-κB activation in HEK293 cells.

    Fig. S5. Small-molecule inhibitors do not disrupt ligand-receptor interactions as characterized by SPR measurements.

    Fig. S6. Hit compounds do not disrupt either the TNFR1 PLAD dimer or the LTα trimer.

    Fig. S7. Hit compounds reduce FRET mediated by the TNFR1 mutant biosensors.

    Fig. S8. Hit compounds compete with zafirlukast in binding to the TNFR1 PLAD and in inhibiting NF-κB activation.

    Fig. S9. Hit compounds do not compete with the H398 antibody in modulating TNFR1 signaling.

    Fig. S10. DSA114, an analog of DS41, shows improved potency and specificity by acting through the same noncompetitive inhibition mechanism.

    Table S1. Functional characterization of DS41 and its analogs.

  • This PDF file includes:

    • Fig. S1. Chemical structures of the novel small-molecule inhibitors of TNFR1 and a negative control compound.
    • Fig. S2. The seven hit compounds and zafirlukast bind the TNFR1 ECD as characterized by SPR measurements.
    • Fig. S3. Some hit compounds nonspecifically inhibit TNFR1-stimulated NF-κB activation.
    • Fig. S4. DS41, but not DS42 and zafirlukast, inhibits TRADD-induced NF-κB activation in HEK293 cells.
    • Fig. S5. Small-molecule inhibitors do not disrupt ligand-receptor interactions as characterized by SPR measurements.
    • Fig. S6. Hit compounds do not disrupt either the TNFR1 PLAD dimer or the LTα trimer.
    • Fig. S7. Hit compounds reduce FRET mediated by the TNFR1 mutant biosensors.
    • Fig. S8. Hit compounds compete with zafirlukast in binding to the TNFR1 PLAD and in inhibiting NF-κB activation.
    • Fig. S9. Hit compounds do not compete with the H398 antibody in modulating TNFR1 signaling.
    • Fig. S10. DSA114, an analog of DS41, shows improved potency and specificity by acting through the same noncompetitive inhibition mechanism.
    • Table S1. Functional characterization of DS41 and its analogs.

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