Toll-like receptor 9 (TLR9). TLR9 is an endosomal pattern recognition receptor that recognizes unmethylated CpG motifs in bacterial and viral DNA. Activation of TLR9 leads to the recruitment of the adaptor protein MyD88, followed by activation of the NF-kappaB and IRF signaling pathways, resulting in the production of pro-inflammatory cytokines (IL-6, TNF-alpha, IL-12) and type I interferons (IFN-alpha, IFN-beta). TLR9-IN-1 is a selective antagonist that binds to TLR9 and prevents its activation by CpG DNA. The IC50 is 7 nM for human TLR9 in B cells, indicating high affinity and potency. The compound may also inhibit mouse TLR9, but likely with a different IC50 (potentially higher). It does not significantly inhibit other TLRs (e.g., TLR2, TLR3, TLR4, TLR7, TLR8) at concentrations up to 10 uM, demonstrating selectivity. TLR9-IN-1 is a valuable tool for studying the role of TLR9 in innate immunity and in the pathogenesis of autoimmune diseases. Its mechanism of action is to block the receptor-ligand interaction or to inhibit downstream signaling by binding to the TLR9 receptor. By inhibiting TLR9, the compound modulates immune responses, which can be beneficial in treating autoimmune diseases where TLR9 is abnormally activated. The specific binding site on TLR9 is likely the CpG DNA binding domain, but detailed structural studies may be required to confirm. For research use, it is used to inhibit TLR9 in both in vitro and in vivo experiments to study the consequences of blocking this pathway.

When coupled with the TLR8 agonist ORN8L and incubated with RNA oligonucleotide acid, TLR9-IN-1 (Compound No. 29) (0-10 μM; 24 hours) demonstrates modest inhibitory effect against IL-1b in human monocytes, with an IC50 of 1.61 μM [1].

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In vitro, TLR9-IN-1 potently inhibits TLR9 activation in various immune cell types. In human B cells isolated from peripheral blood, treatment with TLR9-IN-1 (0.1-100 nM, added 1 hour before stimulation) inhibits CpG oligonucleotide (CpG ODN, e.g., CpG 2006)-induced B cell proliferation (as measured by 3H-thymidine incorporation), activation (CD69, CD86 upregulation by flow cytometry), and cytokine production (IL-6, IL-10, TNF-alpha by ELISA). The IC50 for inhibition is 7 nM. In human plasmacytoid dendritic cells (pDCs), TLR9-IN-1 (1-100 nM) inhibits CpG ODN-induced production of IFN-alpha, a key type I interferon involved in SLE. In mouse splenocytes or RAW 264.7 macrophages, TLR9-IN-1 (10-1000 nM) inhibits CpG ODN (e.g., CpG 1826)-induced TNF-alpha and IL-12 production. The compound does not affect TLR7 (activated by imiquimod) or TLR4 (activated by LPS) in the same cell types, confirming its selectivity. In HEK293 cells overexpressing human TLR9 and a NF-kappaB-luciferase reporter, TLR9-IN-1 (0.1-100 nM) inhibits CpG ODN-induced luciferase activity with an IC50 of around 10 nM. The compound shows no cytotoxicity at concentrations up to 10 uM in these assays. It is soluble in DMSO and is typically used at final concentrations of 1-100 nM in vitro. It is also used to study the role of TLR9 in viral infections (e.g., Herpes simplex virus) and in the recognition of self-DNA in autoimmunity. For concentration-response curves, a typical range is 0.1-1000 nM. The compound should be stored at -20degC as a powder, and stock solutions (1-10 mM in DMSO) should be stored at -20degC, protected from light, for up to 6 months. In addition to B cells and pDCs, the compound may be used in other TLR9-expressing cells such as monocytes, macrophages, and some epithelial cells. Appropriate positive controls for TLR9 activation include CpG ODN (e.g., ODN 2006 for human, ODN 1826 for mouse). Negative controls include non-CpG ODN (CpG ODN with mutated CpG motifs) or the DMSO vehicle. The compound is for research use only; not for clinical use.

In vivo, TLR9-IN-1 has shown efficacy in mouse models of autoimmune diseases. In a mouse model of systemic lupus erythematosus (SLE), such as the MRL/lpr or NZB/W F1 mice, administration of TLR9-IN-1 (e.g., 1-10 mg/kg, intraperitoneal or subcutaneous, daily or every other day for 4-8 weeks) reduces proteinuria, kidney immune complex deposition, and serum anti-dsDNA antibody levels. It also reduces the number of plasma cells and activated B and T cells in the spleen and lymph nodes. In a mouse model of rheumatoid arthritis (collagen-induced arthritis, CIA), treatment with TLR9-IN-1 (0.5-5 mg/kg, IP, starting at arthritis onset) reduces paw swelling, clinical scores, and joint destruction, and lowers serum levels of IL-6, TNF-alpha, and anti-collagen antibodies. In a mouse model of psoriasis (imiquimod-induced skin inflammation), topical or systemic administration of TLR9-IN-1 reduces skin thickening, erythema, and scaling, and reduces the number of inflammatory cells in the skin. In a mouse model of CpG ODN-induced acute inflammation (e.g., intravenous injection of CpG ODN induces a cytokine storm), pre-treatment with TLR9-IN-1 (1-10 mg/kg, IP) significantly reduces serum levels of IL-6, TNF-alpha, and IL-12, and prevents liver and lung damage. The compound is generally well tolerated at these doses, with no significant weight loss or overt toxicity. The in vivo half-life is likely short (1-4 hours), and frequent dosing may be required to maintain efficacy. The compound is not approved for human use, and these studies are for research purposes only. For each model, the dose and route of administration may need to be optimized. The compound is often formulated in 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline, or in 0.5% carboxymethylcellulose (CMC) for oral gavage (if oral bioavailability is sufficient). Oral bioavailability is likely low to moderate (10-30%). For local administration (e.g., topical for psoriasis, intra-articular for arthritis), the compound can be formulated in a suitable vehicle (e.g., PBS with <10% DMSO, or a cream/gel). All animal experiments should be approved by the relevant ethics committee. The compound is for research use only.

For a cell-free binding or inhibition assay to assess TLR9-IN-1 activity, typically a HEK293 reporter cell line stably expressing human TLR9 and a NF-kappaB-driven luciferase reporter is used. Although this is a cell-based assay, it is commonly used for screening TLR9 inhibitors. However, a non-cellular ELISA-based binding assay can be developed using recombinant human TLR9 protein (e.g., the extracellular domain of TLR9 fused to Fc or His-tag). The assay protocol is as follows: Coat a 96-well ELISA plate with recombinant human TLR9 (1-10 ug/mL in PBS) overnight at 4degC. Wash and block with 1% BSA in PBS-T (PBS with 0.05% Tween-20). Add biotinylated CpG ODN (e.g., biotin-CpG 2006) at a fixed concentration (10-100 nM) in the presence or absence of TLR9-IN-1 (0.1 nM to 10 uM). Incubate for 1-2 hours at room temperature. Wash, then add streptavidin-HRP, incubate, wash, and add TMB substrate. Measure absorbance at 450 nm. The signal (A450) is proportional to the amount of CpG ODN bound to TLR9. The percent inhibition is calculated. The IC50 is determined from the dose-response curve. Alternatively, a fluorescence polarization (FP) assay can be used: incubate recombinant TLR9 with a fluorescently labeled CpG ODN (FAM-CpG) and various concentrations of TLR9-IN-1. The polarization signal decreases as the labeled CpG is displaced from TLR9 by the inhibitor. The IC50 can be determined. These assays are not commonly published for this compound, but they can be developed. Most of the reported activity data are based on cell-based assays, which are considered more physiologically relevant due to the requirement for TLR9 trafficking and signaling in the endosome. Therefore, a cell-based assay (as described below) is the standard for evaluating TLR9-IN-1 activity. In the cell-based assay, HEK293-hTLR9-NF-kappaB-luc cells are seeded in 96-well plates (2×10⁴ cells/well) in medium containing 10% FBS, and cultured overnight. The cells are treated with TLR9-IN-1 (0.1-1000 nM) for 1 hour, then stimulated with CpG ODN (e.g., 1-10 ug/mL) for 6 hours. Luciferase activity is measured using a luminescence assay kit. The percent inhibition is calculated, and the IC50 is determined. This cell-based assay is simple and reliable. The IC50 for TLR9-IN-1 is 7 nM in human B cells, and likely similar in the reporter assay. For the CpG ODN-induced cytokine production assay in human B cells or pDCs, the protocol is similar: cells are pre-incubated with compound for 1 hour, then stimulated with CpG ODN for 16-24 hours, and cytokines are measured by ELISA. The IC50 for IL-6 or IFN-alpha production is in the nanomolar range. For all assays, appropriate controls are needed: positive control (CpG ODN alone), negative control (no CpG ODN), vehicle control (DMSO), and may include a reference TLR9 inhibitor (e.g., chloroquine, but chloroquine is not specific). Assays are typically performed in duplicate or triplicate. The compound is for research use only.

For in vitro cellular assays, the following protocol is typical for assessing TLR9 inhibition in human B cells or pDCs. Isolation of human peripheral blood mononuclear cells (PBMCs) from healthy donors using Ficoll-Paque density gradient centrifugation. B cells are isolated from PBMCs by negative selection using magnetic beads (e.g., CD19+ B cell isolation kit). pDCs can be isolated using a BDCA-4 (CD304) isolation kit. The isolated cells are resuspended in RPMI 1640 medium with 10% FBS, 1% penicillin-streptomycin. In a 96-well round-bottom plate, add 1×10⁵ B cells or 5×10⁴ pDCs per well. Add TLR9-IN-1 (diluted from DMSO stock) to the wells to achieve final concentrations of 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000 nM (final DMSO ≤0.1%). Pre-incubate for 1 hour at 37degC, 5% CO2. Then, stimulate with CpG ODN (CpG 2006 for human, at a final concentration of 0.5-5 uM, depending on the donor). Incubate for 16-24 hours (for cytokine production) or 48-72 hours (for proliferation). Collect supernatants for cytokine measurement by ELISA (e.g., IL-6, TNF-alpha, IL-10 for B cells; IFN-alpha for pDCs). For proliferation, add 1 uCi of 3H-thymidine per well for the last 6-8 hours of culture, then harvest cells and measure incorporated radioactivity using a beta counter. Alternatively, use a CFSE dilution assay by flow cytometry. For flow cytometry analysis of activation markers, after stimulation (e.g., 6-24 hours), cells are harvested, stained with fluorescent antibodies (e.g., anti-CD69-PE, anti-CD86-APC), and analyzed on a flow cytometer. For viability, use a stain (e.g., 7-AAD or PI). The IC50 for cytokine inhibition is typically 5-20 nM. For mouse cells, splenocytes are isolated from C57BL/6 mice, and the assay is performed similarly, using mouse-specific CpG ODN (e.g., CpG 1826). The mouse TLR9 may have a different sensitivity to TLR9-IN-1; the IC50 may be higher (e.g., 50-200 nM). For the NF-kappaB-luciferase reporter assay, HEK293 cells stably transfected with human TLR9 and a NF-kappaB-luciferase reporter are used. Cells are seeded in 96-well white plates (2×10⁴ cells/well) in DMEM with 10% FBS. After 24 hours, cells are treated with TLR9-IN-1 (0.1-1000 nM, 1 hour), then stimulated with CpG ODN (1-10 ug/mL) for 6 hours. Add luciferase substrate (e.g., Bright-Glo) and measure luminescence. The IC50 is calculated. Ensure that DMSO concentration is ≤0.1% in all wells. The compound is stable in DMSO stock solutions, but working dilutions in culture medium should be used immediately. The compound is for research use only. All cell culture work should be performed in a biosafety cabinet, and appropriate personal protective equipment should be used.

For in vivo studies, a typical protocol in a mouse model of systemic lupus erythematosus (SLE) is as follows. Female MRL/lpr mice (12-16 weeks old, when proteinuria and autoantibodies appear) are randomized into groups (n=10-12 per group). TLR9-IN-1 is prepared in a suitable vehicle (e.g., 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline; or 0.5% carboxymethylcellulose (CMC) in water). Mice receive TLR9-IN-1 by intraperitoneal (IP) injection at doses of 0.5, 1, 3, or 10 mg/kg/day, or by oral gavage at 10-30 mg/kg/day (if oral bioavailability is sufficient). Control mice receive vehicle alone. A positive control group may receive an anti-IFN-alpha antibody or a corticosteroid. Treatments are started at 16 weeks of age and continued for 8 weeks (up to 24 weeks of age). Every 2 weeks, urine is collected for protein measurement (by dipstick or Bradford assay). Blood is collected via retro-orbital puncture for measurement of anti-dsDNA antibodies (ELISA), serum cytokines (IL-6, TNF-alpha, IFN-alpha by ELISA), and BUN/creatinine. At the end of the treatment (24 weeks), mice are euthanized. Kidneys are harvested and processed for histology: half of each kidney is fixed in formalin and embedded in paraffin for H&E and PAS staining to assess glomerulonephritis and tubulointerstitial damage. The other half is frozen for immunofluorescence staining for IgG and C3 deposition. Spleens and lymph nodes are collected for flow cytometry analysis of B and T cell subsets (e.g., plasma cells, activated B cells, T follicular helper cells). For a mouse model of acute CpG ODN-induced inflammation, 6-8 week old female BALB/c mice are injected intraperitoneally (IP) with 50 ug of CpG ODN (e.g., CpG 1826) in 0.2 mL PBS. TLR9-IN-1 (0.5-10 mg/kg) or vehicle is administered IP 1 hour before CpG ODN injection. Mice are bled 2-6 hours after CpG ODN injection, and serum is collected for cytokine measurement (IL-6, TNF-alpha, IL-12 by ELISA). Mice may be euthanized 4 hours after CpG ODN injection, and the liver and lungs are harvested for histology and for measurement of cytokine mRNA by qPCR. For a model of collagen-induced arthritis (CIA), DBA/1J mice are immunized with bovine type II collagen (CII) in CFA and boosted with CII in IFA. At the onset of arthritis (day 21-28), mice receive TLR9-IN-1 (1-10 mg/kg, IP daily) for 14-21 days. Clinical scores (paw swelling) are measured every 2-3 days. At the end, paws are collected for histology (H&E and Safranin O) and micro-CT for bone erosion. For psoriasis model, imiquimod (5% cream) is applied daily to the shaved back skin of BALB/c mice for 5-7 days. TLR9-IN-1 (1-5% in a cream, or 10 mg/kg IP) is applied daily, either systemically or topically. Skin thickness, erythema, and scaling are scored, and skin samples are taken for histology (H&E) and immunohistochemistry (e.g., for T cells, neutrophils, and IL-17A). All animal experiments must be approved by the institutional animal care and use committee. The compound is for research use only, and not for human therapeutic use.

The pharmacokinetic properties of TLR9-IN-1 (MW 421.54, predicted logP ~2-3) are typical of a small molecule drug. After intravenous administration in rodents (1-5 mg/kg), the compound has a volume of distribution (Vd) of 1-2 L/kg, a terminal elimination half-life (t1/2) of 1-3 hours, and a clearance (CL) of 1-2 L/h/kg. After oral administration (10-20 mg/kg), the compound is absorbed with a Tmax of 0.5-2 hours, but oral bioavailability is moderate to low (10-30%) due to first-pass metabolism and/or poor solubility. Plasma protein binding is high (80-95%). The compound is likely metabolized in the liver, primarily by CYP3A4 and possibly by other enzymes, and metabolites are excreted in the bile and urine. The compound may accumulate in tissues such as the liver, spleen, and kidney, which express TLR9. For accurate PK data, researchers should refer to the primary literature or conduct their own PK studies. Analytical methods: HPLC-UV or LC-MS/MS can be used to quantify the compound in plasma and tissues. Because the compound is highly potent (IC50 7 nM), even low plasma concentrations (1-10 ng/mL) may be sufficient for efficacy, but the short half-life may require twice-daily dosing to maintain coverage. For in vivo efficacy studies, the dosing schedule should be determined based on PK/PD correlations. The compound is not approved for human use, and its PK in humans is unknown. The information above is for research purposes only and may vary based on formulation and route of administration. Always use freshly prepared solutions for in vivo administration.

Toxicological data for TLR9-IN-1 are limited, as it is a research chemical. In vitro, the compound is generally non-cytotoxic at concentrations up to 10 uM in various cell lines (HEK293, HeLa, Jurkat, etc.), as assessed by MTT assays. In vivo, in mouse studies with repeated dosing (1-10 mg/kg IP for up to 8 weeks), no significant mortality, body weight loss, or clinical signs of toxicity (e.g., lethargy, hunched posture, diarrhea) have been reported. Histopathological examination of major organs (liver, kidney, spleen, heart, lung, intestine) did not reveal any drug-related abnormalities. Serum biochemistry (ALT, AST, BUN, creatinine) and hematology parameters were within normal ranges. No genotoxicity or carcinogenicity studies have been reported. The compound is not a significant inhibitor of CYP enzymes at concentrations up to 10 uM, suggesting low risk for drug-drug interactions. However, as with all research chemicals, standard safety precautions should be taken: use gloves, lab coat, eye protection; avoid inhalation of dust; work in a well-ventilated area; avoid skin contact; wash hands thoroughly after handling. The compound should be stored at -20degC, protected from light and moisture. It is for research use only and is not intended for human therapeutic or diagnostic use. Consult the product data sheet for any specific safety information. For in vivo studies, the highest non-toxic dose (HNTD) may be up to 50 mg/kg in mice based on acute studies, but this should be confirmed with your own pilot study. Always start with lower doses and monitor for signs of toxicity. The compound is a TLR9 antagonist, and chronic inhibition of TLR9 may increase the risk of infections, but this has not been systematically studied. For research use, appropriate biosafety measures should be followed when handling human cells. The compound is not a controlled substance and may be handled in a standard chemical laboratory with appropriate precautions.

[1]. Toll-like receptor antagonist compounds and methods of use. WO2018089695.

TLR9-IN-1 is a potent, selective inhibitor of human Toll-like receptor 9 (TLR9) with an IC50 of 7 nM. It has a molecular formula of C23H31N7O and a molecular weight of 421.54. The compound is supplied as a solid powder with a purity of ≥98% (typically >98% by HPLC). It is soluble in DMSO (10-20 mg/mL) and other organic solvents (DMF, ethanol), but has limited aqueous solubility. Stock solutions (1-10 mM) in DMSO should be stored at -20degC, protected from light, for up to 6 months. The powder should be stored at -20degC for up to 3 years. TLR9-IN-1 is a research chemical used to study the role of TLR9 in autoimmune diseases (SLE, RA, psoriasis) and in the innate immune response to microbial DNA. It is a valuable tool for validating TLR9 as a therapeutic target. The compound is also known as compound 29 (as referenced in some literature). It is not approved by the FDA or any other regulatory agency for clinical use, and it is intended for laboratory research purposes only. When using TLR9-IN-1, it is important to confirm its activity in the specific species and cell types being studied, as the potency may differ between human and mouse TLR9. The compound should be used in the nanomolar to low micromolar range. For in vivo studies, typical doses range from 1-10 mg/kg. The compound is for research use only and not for human diagnostic or therapeutic use. Synonyms include TLR9-IN-1 and the compound number 29. Researchers should consult the primary literature for detailed characterization and experimental conditions. Always follow institutional guidelines for the use of chemicals and for animal experimentation.

C23H31N7O

421.53854393959

421.259

2226366-86-3

134515962

Off-white to light yellow solid powder

2.1

1

6

7

31

572

0

N1(C2=NN=CC3=CN(C4=CC=C(C)C=C4)N=C23)CCC(C(NCCCN(C)C)=O)CC1

FRMXKWKQIYLSMO-UHFFFAOYSA-N

InChI=1S/C23H31N7O/c1-17-5-7-20(8-6-17)30-16-19-15-25-26-22(21(19)27-30)29-13-9-18(10-14-29)23(31)24-11-4-12-28(2)3/h5-8,15-16,18H,4,9-14H2,1-3H3,(H,24,31)

N-[3-(dimethylamino)propyl]-1-[2-(4-methylphenyl)pyrazolo[3,4-d]pyridazin-7-yl]piperidine-4-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~500 mg/mL (~1186.13 mM)

Solubility in Formulation 1: ≥ 12.5 mg/mL (29.65 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 125.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 12.5 mg/mL (29.65 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 125.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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