Research ArticlePHARMACOLOGY

Specific activation of the TLR1-TLR2 heterodimer by small-molecule agonists

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Science Advances  10 Apr 2015:
Vol. 1, no. 3, e1400139
DOI: 10.1126/sciadv.1400139
  • Fig. 1 Selection of CU-T12-9 as a TLR2 signaling agonist.

    (A) Role of TLRs in the innate and adaptive immunity responses. Recognition of PAMPs by TLRs expressed on APCs, such as dendritic cells, up-regulates cell surface expression of costimulatory molecules (CD80 and CD86), major histocompatibility complex class II (MHC II), and T cell receptor (TCR). Induction of CD80/86 on APCs by TLRs leads to the activation of T cells specific to pathogens. TLRs also induce expression of cytokines, such as IL-12, IL-6, and TNF-α, as well as chemokines and their receptors, triggering many other events associated with dendritic cell maturation. The above cytokines will contribute to the differentiation of activated T cells into T helper effector cells, building long-term protective immunity (51). (B) Chemical structure of CU-T12-9. (C) CU-T12-9 activates SEAP signaling in a dose-dependent manner. HEK-Blue hTLR2 cells were incubated with CU-T12-9 or GA for 24 hours, and activation was evaluated by SEAP secretion in the culture supernatants by the luminescence assay. (D) Human TLR2, TLR3, TLR4, TLR5, TLR7, and TLR8 HEK-Blue cells were incubated with CU-T12-9 (0 to 20 μM) or TLR-specific agonist for 24 hours, and activation was evaluated by the luminescence assay. (E to I) As positive control, agonists that selectively activate a specific TLR were used: TLR1/TLR2, Pam3CSK4 (0 to 66 nM or 0 to 100 ng/ml); (E) TLR3, polyinosinic-polycytidylic acid [poly(I:C)] (0 to 10.9 μg/ml); (F) TLR4, lipopolysaccharide (LPS) (0 to 36.5 ng/ml); (G) TLR5, FLA-BS (0 to 10 μg/ml); (H) TLR7 and (I) TLR8, R848 (0 to 6 μg/ml). Data are means ± SD of triplicate and representative of three independent experiments.

  • Fig. 2 Characterizations of CU-T12-9 as a TLR1/2 agonist, not TLR2/6.

    (A) HEK-Blue hTLR2 cells were treated with CU-T12-9 and anti-hTLR1, anti-hTLR2, or anti-hTLR6 antibodies for 24 hours. CU-T12-9 strongly activates QUANTI-Blue SEAP signaling at 60 nM, and anti-hTLR1–IgG (immunoglobulin G) and anti-hTLR2–IgA antibodies can dose-dependently inhibit CU-T12-9–triggered SEAP signaling, whereas anti-hTLR6–IgG has no influence. This demonstrates that CU-T12-9 can activate TLR1/2 signaling, with no activation of TLR2/6. (B) The positive control of Pam3CSK4, a TLR1/2 agonist, showed similar activation to CU-T12-9. (C) The positive control of Pam2CSK4, a TLR2/6 agonist, showed that anti-hTLR2–IgA and anti-hTLR6–IgG dose-dependently inhibited the Pam2CSK4-induced TLR2 and TLR6 QUANTI-Blue SEAP signaling, whereas anti-hTLR1–IgG had no influence. (D) The human monocyte cell line U937 was stably transfected with a GFP-labeled NF-κB reporter gene. The cells sensitive to TLR1/2 activation were sorted using a MoFlo Cytomation fluorescence-activated cell sorter, and 10% of activated cells were collected. The flow cytometric analysis of 0, 1, and 5 μM CU-T12-9 and 66 nM (100 ng/ml) Pam3CSK4 triggered NF-κB expression in U937 cells. (E) Flow cytometric analysis for 20 μM CU-T12-9 and GA, along with the blank and positive control in U937 cells. The result demonstrated that CU-T12-9 had excellent NF-κB activation compared with GA. (F) The NF-κB activation of CU-T12-9 can be inhibited by an NF-κB inhibitor, triptolide, in HEK-Blue hTLR2 cells.

  • Fig. 3 The downstream cytokines activated by CU-T12-9.

    (A) TLR1/2 signaling pathway after NF-κB activation. Ligands from Gram-positive bacteria, yeast, or fungi can induce the TLR1/2 dimerization, leading to the NF-κB activation through the adaptor proteins MyD88/TRAF/IKK. This triggers the SEAP promoter to secrete alkaline phosphatase or induces production of proinflammatory cytokines, such as TNF-α, IL, IFN, and NO (23, 29). (B and C) Dose-dependent activation of NO production in Raw 264.7 macrophage cells (B) and primary macrophage cells (C). Data are means ± SD. *P < 0.05 for CU-T12-9 relative to positive control. (D) ELISA assay results showed that CU-T12-9 activates the TNF-α production in Raw 264.7 macrophage cells with an EC50 of 60.46 ± 16.99 nM. Pam3CSK4 was used as a positive control in the experiment. Data are means ± SD. **P < 0.01 for CU-T12-9 (1.2 μM) relative to positive control.

  • Fig. 4 mRNA induction by CU-T12-9 and Pam3CSK4 in Raw 264.7 macrophage cells.

    (A) CU-T12-9 (1 μM) and 33 nM (50 ng/ml) Pam3CSK4 increased the TLR1 mRNA expression over time. Shown are the changes in relative gene expression levels (fold change) relative to the expression levels in dimethyl sulfoxide (DMSO)–treated controls, represented as means ± SD. *P < 0.05 for Pam3CSK4 or CU-T12-9 relative to DMSO-treated control. The statistical analyses were based on two independent biological replicates, and each biological replicate was divided into three samples for independent measurements. Changes in the relative expression of hTLR1 were standardized to the expression of the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase). (B) Dose-dependent assay of CU-T12-9 triggered TLR1 mRNA after the cells were treated for 24 hours. (C) CU-T12-9 and Pam3CSK4 increased TLR2 mRNA at 2 hours with a gradual decline in TLR2 mRNA expression by 24 hours. *P < 0.01 for Pam3CSK4 or CU-T12-9 relative to DMSO-treated control. (D) Dose-dependent activation with CU-T12-9–induced TLR2 mRNA in 2 hours. (E) TNF mRNA was activated by CU-T12-9 and Pam3CSK4 through the NF-κB pathway, and the highest signaling was seen at 8 hours. *P < 0.01 for Pam3CSK4 or CU-T12-9 relative to DMSO-treated control. (F) CU-T12-9 showed dose-dependent activation of TNF mRNA at 8 hours. (G) Gradual increase in iNOS mRNA expression over time with CU-T12-9 and Pam3CSK4 through the NF-κB pathway compared with vehicle control. **P < 0.001 for Pam3CSK4 or CU-T12-9 relative to DMSO-treated control. (H) CU-T12-9 showed dose-dependent activation of iNOS mRNA at 24 hours. (I) IL-10 mRNA up-regulated by CU-T12-9 and Pam3CSK4 through the NF-κB pathway in 2 hours, and gradual decrease in IL-10 mRNA in 8 and 24 hours. *P < 0.01 for CU-T12-9 relative to DMSO-treated control. (J) CU-T12-9 showed dose-dependent activation of IL-10 mRNA at 24 hours.

  • Fig. 5 Biophysical characterizations of CU-T12-9.

    (A) TLR1 (5 μg/ml) and TLR2 (4 μg/ml) or BSA (5 μg/ml) (used as the control) were coated onto the plate. Different concentrations of biotin-labeled Pam3CSK4 (Biotin-Pam3) were added to the plate, and the bound Biotin-Pam3 was detected by streptavidin conjugated with HRP. Absorbance with TLR1/2 in the presence of 150 nM (300 ng/ml) Biotin-Pam3 was set as 100%. (B) TLR1 (5 μg/ml) and TLR2 (4 μg/ml) were coated onto the plate, and 50 nM (100 ng/ml) Biotin-Pam3 and different concentrations of CU-T12-9 (0 to 7 μM) were added. Absorbance with 0 μM CU-T12-9 was set as 100%. CU-T12-9 can compete with Biotin-Pam3 binding to TLR1/2. (C) Binding curve of TLR1/2 with rhodamine-labeled Pam3CSK4 (Rho-Pam3) measured by fluorescence anisotropy. Different concentrations of TLR1/2 were titrated into 10 nM (~20 ng/ml) Rho-Pam3; 549 nm was chosen as the excitation wavelength, and 566 nm was chosen as the emission wavelength (InvivoGen). A Kd of 34.97 ± 1.98 nM was obtained by fitting the binding curve to a one-site saturation model. (D) Titration of the TLR1/TLR2 protein into the Rho-Pam3 results in a significant increase of fluorescence anisotropy. Added CU-T12-9 competes with Rho-Pam3 and results in lower fluorescence anisotropy, demonstrating competitive binding between CU-T12-9 and Pam3CSK4 for TLR1/TLR2. (E) Compound T12-29 was used as a negative control in the fluorescence anisotropy experiment. The results showed no competitive binding between T12-29 and Pam3CSK4 for TLR1/TLR2 up to 10 μM. (F) The hTLR2 protein (60 μl at 0.8 mg/ml) and different concentrations of CU-T12-9 (0 to 40 μM) or Pam3CSK4 were incubated at room temperature for 2 hours. Then, hTLR1 protein (60 μl at 1 mg/ml) was added to the reaction mixture, which was incubated at 37°C for an additional 2 hours before the SEC-LS experiment. (G to I) SEC-LS results for the TLR1/2 protein heterodimer formation by different doses of CU-T12-9: 0 μM, (G) 1 μM, (H) 10 μM, and (I) 40 μM. Increasing CU-T12-9 concentrations lead to more heterodimerization. (J) Pam3CSK4 (260 nM or 400 ng/ml) (positive control) also induces TLR1/2 heterodimerization in this experiment. (K) Molar mass of the three peaks appearing in (H). The TLR1/2 heterodimer peak was present at the molecular weight of about 180 kD.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/3/e1400139/DC1

    General Methods

    Fig. S1. Dose-dependent activation of SEAP signaling by analogs in HEK-Blue hTLR2 cells after 24 hours.

    Fig. S2. The MTT cell viability of HEK-Blue hTLR2 cells after 24 hours of incubation with CU-T12-9 and antibodies.

    Fig. S3. NO activation of CU-T12-9 can be suppressed by a TLR1/2 antagonist, but not by a TLR4 antagonist.

    Fig. S4. Anisotropy assays for TLR1, TLR2, or TLR1/2 protein binding to rhodamine-labeled Pam3CSK4 (Rho-Pam3).

    Fig. S5. Binding of CU-T12-9 to TLR1 by MST.

    Fig. S6. Binding of CU-T12-9 to TLR2 by MST.

    Fig. S7. TLR1 and TLR2 oligomeric states as seen by SEC-LS.

    Fig. S8. Concentration-dependent 1H NMR experiments.

    Fig. S9. HEK-Blue hTLR2 and Raw 264.7 cell viability upon CU-T12-9 treatment.

    Fig. S10. Protein expression and characterization.

    Table S1. SAR studies of the GA analogs in activation of SEAP signaling in HEK-Blue hTLR2 cells.

    Scheme S1. General synthesis of TLR1/2 agonist GA.

    Synthesis and experimental data.

    References (5254)

  • Supplementary Materials

    This PDF file includes:

    • General Methods
    • Fig. S1. Dose-dependent activation of SEAP signaling by analogs in HEK-Blue hTLR2 cells after 24 hours.
    • Fig. S2. The MTT cell viability of HEK-Blue hTLR2 cells after 24 hours of incubation with CU-T12-9 and antibodies.
    • Fig. S3. NO activation of CU-T12-9 can be suppressed by a TLR1/2 antagonist, but not by a TLR4 antagonist.
    • Fig. S4. Anisotropy assays for TLR1, TLR2, or TLR1/2 protein binding to rhodamine-labeled Pam3CSK4 (Rho-Pam3).
    • Fig. S5. Binding of CU-T12-9 to TLR1 by MST.
    • Fig. S6. Binding of CU-T12-9 to TLR2 by MST.
    • Fig. S7. TLR1 and TLR2 oligomeric states as seen by SEC-LS.
    • Fig. S8. Concentration-dependent 1H NMR experiments.
    • Fig. S9. HEK-Blue hTLR2 and Raw 264.7 cell viability upon CU-T12-9 treatment.
    • Fig. S10. Protein expression and characterization.
    • Table S1. SAR studies of the GA analogs in activation of SEAP signaling in HEK-Blue hTLR2 cells.
    • Scheme S1. General synthesis of TLR1/2 agonist GA.
    • Synthesis and experimental data.
    • References (52–54)

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