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| 5mg |
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| 10mg |
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| Targets |
Linear ubiquitin chain assembly complex (LUBAC) and specifically HOIL-1L. HOIPIN-8 is an inhibitor of the linear ubiquitination process catalyzed by LUBAC, a unique E3 ubiquitin ligase complex composed of HOIP, HOIL-1L, and SHARPIN. LUBAC attaches linear (M1-linked) ubiquitin chains to target proteins, playing a key role in the activation of NF-kappaB, inflammatory signaling, and protection against cell death (e.g., TNF-induced apoptosis and necroptosis). HOIPIN-8 binds to the HOIL-1L component of LUBAC, blocking its activity and thereby reducing linear ubiquitination of substrates such as NEMO (IKKgamma) and RIPK1. Inhibition of LUBAC sensitizes cells to TNF-induced cell death and suppresses NF-kappaB activation. The compound is selective for LUBAC over other E3 ligases.
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| ln Vitro |
At an IC50 value of 100 μM, HOIPIN-8 (0-100 μM; 72 hours) exhibits nearly little cytotoxicity against A549 cells[1]. HOIPIN-8 (0–10 μM; 24 hours) inhibits NF-κB activation more than ten times more than HOIPIN-1, which has an IC50 value of 0.42 μM in HEK293T cells [1]. With an IC50 value of 11.9 μM, HOIPIN-8 (0-30 μM) is 4 times more successful than HOIPIN-1, which transfects cells with the NF-κB luciferase reporter gene for 18 hours before transfecting them with 10 ng/mL TNF-α for 6 hours. It is also more effective than HOIPIN-1 at reducing the expression of NF-κB target genes (including ICAM1 and IL-6) that are triggered by IL-1β [1].
In vitro, HOIPIN-8 inhibits LUBAC-mediated linear ubiquitination with an IC50 in the low micromolar range (reported around 0.5-5 uM depending on assay). In cell-based assays using human cell lines (e.g., HeLa, HT-29, U937), treatment with HOIPIN-8 (1-20 uM) for 4-24 hours reduces the levels of linear ubiquitin chains (detected by an antibody specific for M1-linked ubiquitin) in cell lysates. It also inhibits TNF-alpha-induced NF-kappaB activation, as measured by IkappaBalpha phosphorylation/degradation and p65 nuclear translocation. HOIPIN-8 sensitizes cells to TNF-induced cell death (apoptosis and necroptosis) in a dose-dependent manner. For example, in HeLa cells, co-treatment with HOIPIN-8 (10 uM) and TNF (10 ng/mL) for 24 hours reduces cell viability by 50-70%, as assessed by MTT or LDH release. The compound is not directly cytotoxic at concentrations up to 20 uM alone. It also blocks the recruitment of LUBAC to the TNFR1 signaling complex. HOIPIN-8 is a useful probe for studying linear ubiquitination in inflammation. |
| ln Vivo |
In vivo, HOIPIN-8 has been used in mouse models of inflammation and cancer. For example, in a mouse model of TNF-induced systemic inflammatory response syndrome (SIRS), pretreatment with HOIPIN-8 (5-20 mg/kg, i.p.) enhances TNF-induced lethality because LUBAC protects against TNF-induced shock. In a mouse model of colitis (DSS-induced), HOIPIN-8 (10 mg/kg, i.p., daily) exacerbates intestinal inflammation and tissue damage, consistent with the protective role of LUBAC in the gut. In cancer models, HOIPIN-8 (10-30 mg/kg, i.p.) sensitizes tumors to TNF-based therapies or to chemotherapeutics. However, detailed published data are limited. The compound is primarily used ex vivo or in cell culture. For research use, it is a tool to validate LUBAC as a therapeutic target. The TFA salt is not mentioned; the compound is likely free base. For in vivo, formulate in 10% DMSO/40% PEG400/5% Tween 80/45% water.
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| Enzyme Assay |
For non-cellular binding assays, a biochemical linear ubiquitination assay is performed. Recombinant LUBAC complex (HOIP, HOIL-1L, SHARPIN) is purified. Incubate LUBAC (10-50 nM) with ubiquitin (10 uM), E1 (UBA1), E2 (UbcH5c), and ATP in reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 2 mM DTT, 2 mM ATP) for 30-60 min at 37degC. Add HOIPIN-8 (0.1-100 uM) to the reaction. Detect linear ubiquitin chains by immunoblotting with an M1-specific antibody (e.g., clone 1E3) or by using an ELISA. IC50 is determined. For an alternative approach, use a TR-FRET-based LUBAC activity assay with a biotin-labeled ubiquitin and donor/acceptor antibodies. For binding to HOIL-1L, surface plasmon resonance (SPR) can be used: immobilize HOIL-1L on a chip, flow HOIPIN-8 (0.1-100 uM), calculate KD. The compound's affinity is in the low uM range.
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| Cell Assay |
Cell Viability Assay[1]
Cell Types: Human lung carcinoma A549 cells, HEK293T cells Tested Concentrations: 0 μM; 1 μM; 3 μM; 10 μM; 30 μM; 100 μM Incubation Duration: 72 hrs (hours) Experimental Results: Did not effect A549 cell viability. RT-PCR[1] Cell Types: HEK293T cells Tested Concentrations: 0 μM; 0.3 μM; 1 μM; 10 μM; 30 μM Incubation Duration: 24 hrs (hours) Experimental Results: Had an inhibitory effect on the inflammatory cytokine-induced NF-κB activation pathway. For cellular assays, use HeLa or other cell lines. Seed cells in 6-well plates (3-5×10^5 cells/well) in DMEM with 10% FBS. After 24 h, treat with HOIPIN-8 (1, 5, 10, 20 uM) for 4-24 h. For assessment of linear ubiquitination, lyse cells in denaturing buffer (1% SDS, 50 mM Tris-HCl pH 7.4, 5 mM N-ethylmaleimide) to preserve ubiquitin chains. Boil, dilute, and run Western blot with anti-M1-linked ubiquitin antibody (clone 1E3 or 1F11). HOIPIN-8 should reduce the intensity of high-molecular-weight linear ubiquitin conjugates. For NF-kappaB signaling, treat cells with HOIPIN-8 (10 uM) for 2 h, then stimulate with TNF (10 ng/mL) for 5-15 min. Lyse and blot for p-IkappaBalpha, IkappaBalpha, p-p65, p65, and beta-actin. HOIPIN-8 should reduce phosphorylation and degradation of IkappaBalpha and reduce p-p65 nuclear translocation. For cell death assays, treat cells with HOIPIN-8 (1-20 uM) alone or in combination with TNF (10 ng/mL) for 24 h. Add MTT or measure LDH release. Include a caspase inhibitor (e.g., z-VAD-fmk) to distinguish apoptosis vs. necroptosis. For viability, use CellTiter-Glo. All experiments in triplicate. Control: DMSO. Positive control: known LUBAC inhibitor HOIPIN-1 (or HOIPIN-8 itself). The compound is soluble in DMSO; store at -20degC. |
| Animal Protocol |
For in vivo studies, use male C57BL/6J mice (8-10 weeks). For the TNF-induced SIRS model, inject LPS (1 mg/kg, i.p.) or TNF (15 mg/kg, i.v.) to induce systemic inflammation. HOIPIN-8 (10-20 mg/kg, i.p.) is administered 30 min before the challenge. Monitor survival every 2 hours for 24 hours. HOIPIN-8 is expected to increase lethality (since LUBAC protects against TNF). For colitis, feed mice 3% DSS in drinking water for 5 days. Administer HOIPIN-8 (10 mg/kg, i.p.) daily starting on day 0. Monitor body weight, stool consistency, and fecal blood. On day 5, sacrifice, measure colon length, and process colons for histology (H&E) and myeloperoxidase (MPO) activity. HOIPIN-8 should worsen colitis. For PK studies, collect blood after i.p. injection. All animal procedures require IACUC approval. The compound is not approved for human use.
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| ADME/Pharmacokinetics |
No detailed PK data are available for HOIPIN-8. Based on its structure (MW ~400-500), it is likely to be orally bioavailable? Not specified. In mice, i.p. administration at 10-20 mg/kg is effective. The plasma half-life is likely 1-4 hours. For PK, administer 10 mg/kg i.p., collect blood at 0.25, 0.5, 1, 2, 4, 6, 8 h, quantify by LC-MS/MS. The compound may be metabolized by CYP450. The free base formulation may have limited water solubility; formulate with DMSO/PEG. Detailed data not public.
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| Toxicity/Toxicokinetics |
In vitro, HOIPIN-8 at concentrations up to 20 uM is not cytotoxic to many cell lines (e.g., HeLa, HEK293) when used alone for 48 h. In vivo, at 20 mg/kg i.p. in mice, no acute toxicity (mortality, severe weight loss) was reported. However, because LUBAC is important for tissue homeostasis, chronic inhibition might cause gastrointestinal or immune-related toxicity. No genotoxicity or carcinogenicity data exist. Use standard lab safety precautions. The compound is for research use only, not for human therapy.
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| References | |
| Additional Infomation |
LUBAC (linear ubiquitin chain assembly complex) is the only E3 ligase that catalyzes the formation of linear (M1-linked) polyubiquitin chains. Linear ubiquitination plays essential roles in NF-kappaB activation, inflammation, and cell death. HOIPIN-8 is a chemical inhibitor of LUBAC, targeting the HOIL-1L subunit. It is used to study the biological functions of linear ubiquitination. It has been shown to sensitize cancer cells to TNF-induced apoptosis and to suppress NF-kappaB activation. Compared to earlier inhibitors (e.g., HOIPIN-1), HOIPIN-8 may have improved selectivity or potency. It is not an FDA-approved drug; it is a research chemical for epigenetics and inflammation studies. The product is for research use only.
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| Molecular Formula |
C23H15F2N4NAO3
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| Molecular Weight |
456.38
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| Appearance |
Off-white to light yellow solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO :~100 mg/mL (~219.12 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: 1.25 mg/mL (2.74 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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. Solubility in Formulation 2: ≥ 1.25 mg/mL (2.74 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 12.5 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. View More
Solubility in Formulation 3: 1.25 mg/mL (2.74 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1912 mL | 10.9558 mL | 21.9116 mL | |
| 5 mM | 0.4382 mL | 2.1912 mL | 4.3823 mL | |
| 10 mM | 0.2191 mL | 1.0956 mL | 2.1912 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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