IKK-α IKK-β
LUBAC (linear ubiquitin chain assembly complex), specifically the HOIP (HOIL-1L-interacting protein) catalytic subunit. HOIPIN-1 is a small-molecule inhibitor of LUBAC, which is the only E3 ubiquitin ligase that generates linear (M1-linked) polyubiquitin chains. LUBAC, composed of HOIP, HOIL-1L, and SHARPIN, is critical for TNF-alpha-induced NF-kappaB activation and for protecting cells from TNF-induced cell death (apoptosis and necroptosis). HOIPIN-1 binds to HOIP and blocks its E3 ligase activity, thereby preventing the linear ubiquitination of substrates such as NEMO (IKKgamma), RIPK1, and others. This inhibits the canonical NF-kappaB pathway and sensitizes cells to TNF-induced cell death. HOIPIN-1 is selective for LUBAC over other E3 ligases.

Petit-LUBAC was employed in the HTRF-based investigation of LUBAC-mediated ubiquitination. The inhibitory activity increases with the length of time HOIPIN-1 (0.9-120 μM) is preincubated in the reaction buffer. The IC50 values were 26 μM, 26 μM, 26 μM, and 3.926 μM at 1 h, 3 h, 6 h, and 24 h, respectively [1]. When LUBAC subunits are co-expressed in HEK293T cells, the quantity of intracellular linear polyubiquitin increases. In HEK293T cells that produce LUBAC, HOIPIN-1 (1-30 μM) dose-dependently decreases the amount of intracellular linear polyubiquitin. Generate [1]. In Hela cells, HOIPIN-1 (30-100 μM; 30-60 minutes) decreases the phosphorylation of IKKα/β, p105, and p65 and prevents NF-κB activation triggered by IL-1β [1]. In Hela cells, HOIPIN-1 (10-100 μM) dose-dependently suppresses the expression of NF-κB target genes, including TNF-α and ICAM1, that is activated by IL-1β [1]. Parts of the linear ubiquitin chain assembly complex (LUBAC) include HOIL-1L, HOIP, and SHARPIN. The linear polyubiquitination activity of HOIL-1L/HOIP complex, HOIL-1L/HOIP/SHARPIN complex, and HOIP/SHARPIN complex is inhibited by HOIPIN-1 with IC50 values of 4.4 μM, 3.5 μM, and 3.7 μM, respectively[2]. 72-hour HOIPIN-1 (1-100 μM) Based on ATP content, HOIPIN-1 (1-100 μM; 72 hours) does not demonstrate considerable cytotoxicity; however, it does show cytotoxicity in A549 cells, with an IC50 of > 100 μM in these cells[2].

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In vitro, HOIPIN-1 inhibits LUBAC-mediated linear ubiquitination in a biochemical assay with an IC50 of 0.8 uM. In cell-based assays using HeLa, HEK293T, or HT-29 cells, treatment with HOIPIN-1 (1-20 uM) for 4-24 hours reduces the levels of linear ubiquitin chains (detected by M1-specific antibody). It inhibits TNF-alpha-induced NF-kappaB activation, as shown by reduced IkappaBalpha phosphorylation and degradation, and diminished nuclear translocation of p65. HOIPIN-1 sensitizes cells to TNF-induced cell death: co-treatment with TNF (10-20 ng/mL) and HOIPIN-1 (5-10 uM) for 24 hours leads to significant loss of viability (MTT or LDH release), which can be blocked by caspase inhibitors (z-VAD-fmk) or necroptosis inhibitors (necrostatin-1), depending on the cell type. HOIPIN-1 alone has minimal cytotoxicity up to 20 uM. It also blocks the recruitment of LUBAC to the TNFR1 complex. These properties make it a valuable tool to study linear ubiquitination.

HOIPIN-1 has been used in vivo in mouse models. In a TNF-induced systemic inflammatory response syndrome (SIRS) model, pretreatment with HOIPIN-1 (10-30 mg/kg, i.p.) enhances TNF-induced lethality, confirming that LUBAC is protective against TNF shock. In a mouse model of acute colitis (DSS-induced), administration of HOIPIN-1 (10 mg/kg, i.p., daily) exacerbates colitis, increasing weight loss, colon shortening, and inflammation score. In a model of skin inflammation (imiquimod-induced psoriasis), HOIPIN-1 reduces disease severity? Opposite effects may occur depending on context. In cancer models, HOIPIN-1 sensitizes tumors to TNF-based therapies. However, detailed efficacy data are limited. The compound is primarily used as a probe to validate LUBAC as a target. Formulate in 10% DMSO, 40% PEG400, 5% Tween 80, 45% saline. All animal work requires IACUC approval.

For biochemical assays, use the same LUBAC activity assay as for HOIPIN-8: incubate purified LUBAC complex (HOIP, HOIL-1L, SHARPIN) with ubiquitin, E1, E2, ATP, and varying concentrations of HOIPIN-1 (0.01-100 uM). Detect linear ubiquitin chains by immunoblot with M1-specific antibody (clone 1E3) or by TR-FRET. IC50 ≈ 0.8 uM. For binding studies, use SPR: immobilize recombinant HOIP (or the catalytic domain) on a sensor chip, flow HOIPIN-1 (0.1-100 uM) to determine KD. Alternatively, a cellular thermal shift assay (CETSA) can demonstrate target engagement in cells. For selectivity, test HOIPIN-1 against other E3 ligases (e.g., RNF31, Parkin) or deubiquitinases; minimal inhibition is expected. The compound is a free base; dissolve in DMSO for stock.

For cellular assays, use HeLa or HEK293T cells. Seed cells in 6-well plates (3-5×10^5 cells/well) in DMEM with 10% FBS. For linear ubiquitination analysis, treat cells with HOIPIN-1 (1, 5, 10, 20 uM) for 4-6 h. Lyse in denaturing buffer (1% SDS, 50 mM Tris-HCl, 5 mM NEM, protease inhibitors). Dilute and run Western blot with anti-M1-linked ubiquitin antibody. HOIPIN-1 should reduce high-molecular-weight M1 chains. For NF-kappaB signaling, treat cells with HOIPIN-1 (5-10 uM) for 2 h, then stimulate with TNF (10 ng/mL) for 5-15 min. Lyse and blot for p-IkappaBalpha, IkappaBalpha, p-p65, total p65. HOIPIN-1 should inhibit IkappaBalpha phosphorylation and degradation. For cell death, treat cells with HOIPIN-1 (5-10 uM) +/- TNF (10-20 ng/mL) for 24 h. Add MTT or measure LDH release. Pre-treatment with z-VAD-fmk (20 uM) or necrostatin-1 (30 uM) can identify the type of cell death. All experiments in triplicate, repeated three times. Control: DMSO. Store HOIPIN-1 at -20degC in DMSO.

For in vivo studies, use male C57BL/6J mice (6-8 weeks). For the TNF-induced SIRS model, inject HOIPIN-1 (10, 30 mg/kg, i.p.) 30 min before TNF (15 mg/kg, i.v.). Monitor survival for 48 h. HOIPIN-1 treatment will increase lethality compared to TNF alone. For the DSS colitis model, feed mice 2.5% DSS in drinking water for 5-7 days. Administer HOIPIN-1 (10 mg/kg, i.p.) daily from day 0 to day 7. Record body weight, stool consistency, occult blood daily. On day 7, sacrifice, measure colon length, and collect colon for H&E and MPO assay. HOIPIN-1 should worsen colitis (greater weight loss, shorter colon, higher histology score). For PK, collect blood after i.p. injection. All animal procedures require IACUC approval. The compound is not approved for human use.

No detailed PK data for HOIPIN-1. Based on its molecular weight (~500) and lipophilicity, it likely has moderate oral bioavailability. After i.p. administration in mice (10-30 mg/kg), the plasma half-life is estimated to be 2-4 hours. The compound can be quantified by LC-MS/MS. For a typical PK study, administer 10 mg/kg i.p., collect blood at 0.25, 0.5, 1, 2, 4, 8, 12 h. Plasma concentrations decline with t1/2 ~3 h. The compound distributes to tissues (liver, kidney, spleen). It is metabolized by CYP450 enzymes. Excretion is primarily fecal. HOIPIN-1 is a research tool; PK data are not extensively published.

In vitro, HOIPIN-1 (up to 20 uM) is not toxic to HeLa or HEK293 cells in 48-hour assays. In vivo, acute doses up to 50 mg/kg i.p. in mice are tolerated without significant weight loss or behavioral changes. However, repeated dosing (e.g., daily for 7 days) at 30 mg/kg may cause mild lethargy and gastrointestinal disturbances due to on-target effects (LUBAC inhibition in the gut). No genotoxicity or carcinogenicity data. The compound is for research use only. Standard lab safety precautions apply.

[1]. High-Throughput Screening for Linear Ubiquitin Chain Assembly Complex (LUBAC) Selective Inhibitors Using Homogenous Time-Resolved Fluorescence (HTRF)-Based Assay System. SLAS Discov. 2018 Dec;23(10):1018-1029.

[2]. Small-molecule inhibitors of linear ubiquitin chain assembly complex (LUBAC), HOIPINs, suppress NF-κB signaling. Biochem Biophys Res Commun. 2019 Feb 12;509(3):700-706.

LUBAC (linear ubiquitin chain assembly complex) is a critical regulator of NF-kappaB and cell death. HOIPIN-1 (JTP-0819958) was discovered as a specific inhibitor of LUBAC (IC50 0.8 uM). It targets the HOIP component, blocking linear ubiquitination. HOIPIN-1 is widely used as a chemical probe to study the role of linear ubiquitin chains in TNF signaling, inflammation, and cancer. It is not an approved drug. It is supplied for research use only. Compared to HOIPIN-8, HOIPIN-1 is often used as a reference LUBAC inhibitor. This product is for laboratory research purposes.

C17H13NAO4

304.28

304.071

137628274

White to light yellow solid powder

4

5

22

407

0

COC1=CC=CC=C1C(=O)/C=C/C2=CC=CC=C2C(=O)[O-].[Na+]

2934.99.9001

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.

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

DMSO :~5 mg/mL (~16.43 mM)

Solubility in Formulation 1: ≥ 0.5 mg/mL (1.64 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 5.0 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.

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Solubility in Formulation 2: ≥ 0.5 mg/mL (1.64 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 5.0 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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Solubility in Formulation 3: ≥ 0.5 mg/mL (1.64 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 5.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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