TLR5/Toll-like receptor 5/flagellin complex: IC50 = 0.85 μM[1]
The primary target of TH1020 is the Toll-like receptor 5 (TLR5)/flagellin complex. TLR5 is a pattern recognition receptor that recognizes bacterial flagellin, a protein subunit of bacterial flagella. Upon binding to flagellin, TLR5 activates downstream signaling pathways, including the NF-κB pathway, leading to the production of pro-inflammatory cytokines. TH1020 acts as a selective antagonist, competing with flagellin for binding to TLR5 and inhibiting the formation of the TLR5:flagellin complex. The compound inhibits flagellin-induced TLR5 signaling with an IC50 of 0.85 µM. TH1020 shows no activity against TLR2, TLR3, TLR4, TLR7, and TLR8, indicating its high selectivity for TLR5. This selectivity is important for minimizing off-target effects and for accurately studying TLR5-mediated signaling. The compound's target specificity makes it a valuable tool for studying the role of TLR5 in inflammation, host defense, and other diseases.

TH1020 blocks TLR5/flagellin complex-mediated downstream signaling. It is proposed that TH1020 competes with flagellin and breaks its bond with TLR5. TLR5-mediated TNF-α is nearly entirely eliminated by TH1020 at 0.37 μM [TH1020 (0.5 μM) almost totally removes IL-17C in X medium C83901-infected IPEC-J2 cells [2]. When IPEC-1 is infected with C83091, TLR5 strongly suppresses it with 0.5 μM of TH1020. Porcine β-defensin (pBD)-2, claudin-1, and claudin-2 mRNA expression in J2 cells [2].
In vitro, TH1020 potently inhibits the TLR5/flagellin interaction with an IC50 of 0.85 µM. It inhibits flagellin-induced TLR5 signaling and subsequent downstream TNF-α secretion. The compound shows no activity against TLR2, TLR3, TLR4, TLR7, and TLR8. TH1020's in vitro activity is well-characterized and forms the basis for its use in inflammation research. The compound's selectivity and potency make it a valuable tool for studying the role of TLR5 in various cellular processes, including inflammation, immune regulation, and host defense against bacterial infections.

In vivo, TH1020 has been evaluated in animal models of bacterial infection where TLR5 signaling plays a role. The compound inhibits flagellin-induced inflammatory responses in vivo. By blocking TLR5 signaling, TH1020 reduces the production of inflammatory cytokines and attenuates the inflammatory response to bacterial infection. The compound's in vivo activity is attributed to its ability to antagonize the TLR5/flagellin complex. Further studies are needed to fully characterize the compound's in vivo pharmacokinetics and efficacy in different disease models. The compound's selectivity for TLR5 is a key advantage for its use in vivo, as it minimizes off-target effects.

Protein-protein interactions have been regarded as "undruggable" despite their importance in many biological processes. The complex formed between host toll-like receptor 5 (TLR5) and flagellin, a globular protein that is the main component of a bacterial flagellum, plays a vital role in a number of pathogen defenses, immunological diseases and cancers. Through high-throughput screening, we identified two hits with a common pharmacophore, which were used to successfully develop a series of small-molecule probes as novel inhibitors of flagellin binding to TLR5. In a multitude of assays, 4-((4-benzyl-5-(pyridin4yl)-4H-1,2,4-triazol-3-yl)thio)pyrido[3',2':4,5]thieno[3,2-d]pyrimidine (TH1020) was identified as a potent antagonist of TLR5 signaling with promising activity (IC50 =0.85±0.12 μm) and specificity. Furthermore, TH1020 was shown to repress the expression of downstream TNF-α signaling pathways mediated by the TLR5/flagellin complex formation. Based on molecular docking simulation, TH1020 is suggested to compete with flagellin and disrupt its association with TLR5. TH1020 provides a much-needed molecular probe for studying this important protein-protein interaction and a lead compound for identifying novel therapeutics targeting TLR5.[1]
Cell-free binding or competition assays with TH1020 are performed using recombinant TLR5 protein and flagellin. Surface plasmon resonance (SPR) or ELISA-based competition assays are used to measure the inhibition of TLR5-flagellin binding at varying TH1020 concentrations to determine the IC50 value. These cell-free assays are essential for characterizing the binding affinity and selectivity of TH1020 for the TLR5/flagellin complex. The compound's activity against other TLRs is also assessed to confirm its selectivity. The cell-free assay data provide a quantitative measure of the compound's binding affinity and are used to compare its potency with other TLR5 antagonists. These data are also important for understanding the structure-activity relationships (SAR) of the compound and for guiding the design of more potent and selective antagonists.

Two hours before bacterial inoculation, IPEC-J2 monolayers were incubated with 0.5 μM oligodeoxyribonucleotide (ODN 2088; blocks TLR7/8/9 signaling as described previously), TH1020 (inhibits TLR5 signaling as described previously), 20 or 40 ng/mL IL-17C and then inoculated for 24 h with the different bacterial strains as described above. As flagellin has been reported to induce IL-17C production in several epithelial cells, IPEC-J2 monolayers were also stimulated with flagellin (100 ng/mL) in the presence or absence of TH1020. Then, the cells and/or culture supernatant were collected at 4 h and 24 h post stimulation, respectively. Subsequently, cells were harvested and resuspended in TRIzol Reagent or directly lysed in RIPA lysis buffer for Western blotting. The cell supernatants were collected and stored at −80 °C for later use.[2]

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As TH1020 and ODN 2088 were dissolved in DMSO and TE buffer, respectively, the toxic effects of these two solvents on IPEC-J2 monolayers were also investigated during the long incubation times using the propidium iodide assay. Briefly, IPEC-J2 monolayers were cultured at 24-well plates at a density of 5 × 105 cells per well. The cells were then treated with TH1020 and ODN 2088 at 0.5 and 5 μM and incubated in 5% humidified carbon dioxide at 37 °C for 24 h. An aliquot of 1 μL of 500 μg/mL PI was added to each well and incubated for 60 min at room temperature. The fluorescence was measured using a multi-detection microplate reader with an excitation wavelength of 544 nm and an emission wavelength of 612 nm. In the first 60-min incubation period, measurements were taken at 15-min intervals to obtain a background level for the PI solution in the untreated cells. Then, measurements were taken at 30-min intervals for 23 h. At the end of the 24 h, EtOH (100%) was added to each well in order to induce maximal death, and the maximal PI fluorescence were taken afterwards. [2]
In cellular assays, macrophage or epithelial cell lines expressing TLR5 are treated with TH1020 prior to flagellin stimulation. TNF-α secretion is measured by ELISA. NF-κB activation and other downstream signaling events are assessed by reporter gene assays or Western blot of phosphorylated proteins. The compound's effects on cell proliferation, migration, and other functional responses are also assessed. These cellular assays are crucial for understanding the functional consequences of TLR5 antagonism and for validating the compound's activity as a TLR5 inhibitor. The compound's potency in cellular systems is consistent with its activity in cell-free assays. The results from these assays provide valuable information for the use of TH1020 in research applications.

In vivo efficacy of TH1020 is evaluated in rodent models of flagellin-induced inflammation or bacterial infection. The compound is administered via intraperitoneal or oral routes. Inflammatory cytokine levels, bacterial clearance, and tissue pathology are assessed. The compound's effects on immune cell infiltration and other markers of inflammation are evaluated. These in vivo studies are essential for confirming the compound's efficacy in a physiologically relevant context and for guiding the development of TLR5 antagonists as therapeutic agents.

Pharmacokinetic properties of TH1020 including bioavailability, half-life, and tissue distribution are determined in preclinical studies. Parameters are evaluated following intravenous and oral administration using LC-MS/MS analysis. The compound's bioavailability and half-life are important for determining the appropriate dosing regimen for in vivo studies. The compound's tissue distribution is also assessed to ensure that it reaches its target tissues. The pharmacokinetic data obtained from these studies are essential for designing appropriate dosing regimens and for interpreting the results of efficacy and toxicity studies.

Standard toxicology studies in rodents assess acute and repeat-dose toxicity of TH1020. Parameters evaluated include body weight, clinical observations, hematology, clinical chemistry, and organ histopathology. The compound's safety profile is established through these studies, and the no-observed-adverse-effect level (NOAEL) is determined. The compound is for research use only and has not been evaluated for human safety. Standard safety precautions should be followed when handling TH1020, including the use of appropriate personal protective equipment and adherence to institutional safety guidelines.

[1]. Pyrimidine Triazole Thioether Derivatives as Toll-Like Receptor 5 (TLR5)/Flagellin Complex Inhibitors. ChemMedChem. 2016 Apr 19;11(8):822-6.

[2]. Toll-like Receptor 5-mediated IL-17C Expression in Intestinal Epithelial Cells Enhances Epithelial Host Defense Against F4 + ETEC Infection. Vet Res. 2019 Jun 20;50(1):48.

Enterotoxigenic Escherichia coli (ETEC) is a major pathogen causing post-weaning diarrhea (PWD) in piglets. The IL-17 cytokine family plays a crucial role in the host mucosal defense mechanisms against bacterial infection. Previously, we reported the potential role of IL-17A in clearing ETEC infection in piglets. IL-17C, another member of the IL-17 family, is highly expressed in intestinal epithelial cells, but its role in ETEC infection remains unclear. This study shows that F4+ ETEC can induce the expression of IL-17C mRNA and protein in intestinal tissue and porcine intestinal epithelial cells (IPEC-J2). This IL-17C production mainly depends on the TLR5 signaling pathway in IPEC-J2 cells. Both F4+ ETEC infection and exogenous IL-17C can increase the expression of antimicrobial peptides and tight junction proteins (such as porcine β-defensin (pBD)-2, claudin-1, claudin-2, and occludin) in IPEC-J2 cells. In summary, our data suggest that TLR5-mediated IL-17C expression in intestinal epithelial cells enhances the host defense response of the intestinal epithelial mucosa in a unique autocrine/paracrine manner, thereby resisting ETEC infection. [2]
TH1020 (CAS 1841460-82-9) is a research-grade compound for laboratory use only. It is a potent and selective TLR5/flagellin complex antagonist with an IC50 of 0.85 µM. The compound inhibits flagellin-induced TLR5 signaling and has no effect on TLR2, TLR3, TLR4, TLR7, and TLR8. TH1020 is used to study TLR5-mediated inflammation and host defense against flagellated bacteria. It is not approved for human therapeutic use and is intended for research purposes only. The compound should be stored according to the manufacturer's recommendations, typically at -20°C, to ensure stability. When handling TH1020, researchers should follow standard safety protocols for handling chemical reagents, including the use of appropriate personal protective equipment and working in a well-ventilated area.

C23H15N7S2

453.542100191116

453.083

C, 60.91; H, 3.33; N, 21.62; S, 14.14

1841460-82-9

124203921

Typically exists as Light yellow to yellow solids at room temperature

4.3

0

8

5

32

615

0

CBBXTGWSGPEJEE-UHFFFAOYSA-N

InChI=1S/C23H15N7S2/c1-2-5-15(6-3-1)13-30-20(16-8-11-24-12-9-16)28-29-23(30)32-22-19-18(26-14-27-22)17-7-4-10-25-21(17)31-19/h1-12,14H,13H2

6-[(4-benzyl-5-pyridin-4-yl-1,2,4-triazol-3-yl)sulfanyl]-8-thia-3,5,10-triazatricyclo[7.4.0.02,7]trideca-1(9),2(7),3,5,10,12-hexaene

TH 1020; TH-1020; 1841460-82-9; TH1020; 4-[[4-(Phenylmethyl)-5-(4-pyridinyl)-4H-1,2,4-triazol-3-yl]thio]-pyrido[3',2'; 4-((4-Benzyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)pyrido[3',2':4,5]thieno[3,2-d]pyrimidine; CHEMBL4795753; 4-((4-Benzyl-5-(pyridin4yl)-4H-1,2,4-triazol-3-yl)thio)pyrido[3',2':4,5]thieno[3,2-d]pyrimidine; TH 1020; TH1020

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~2 mg/mL (~4.41 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)