| Size | Price | Stock | Qty |
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| 1mg |
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| Other Sizes |
| Targets |
Huib32 specifically targets USP32, a deubiquitinating enzyme (DUB) involved in the regulation of protein stability and trafficking. Huib32 forms a reversible covalent bond with the active-site cysteine residue (Cys743) of USP32, thereby inhibiting its catalytic activity. The IC₅0 for USP32 is 21.2 nM. It exhibits high selectivity over other closely related DUBs, including USP8, USP10, USP16, UCHL1, and OTUB2. Inhibition of USP32 leads to increased ubiquitination of its substrates (e.g., RAB7, RAB6, RAB32, RAB11A/B, TMEM192), resulting in altered endosomal morphology and function. The compound is a reversible covalent inhibitor of USP32.
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| ln Vitro |
Huib32 (BB01CA282) (0-50 μM, 6-24 h) selectively inhibited USP32FL and USP32CD (starting from 0.1 μM) in a dose-dependent manner, with no significant effect on the other 16 detected deubiquitinating enzymes (DUB) [1]. Huib32 (0-50 μM, 24 h) showed potent, dose-dependent endogenous USP32 target occupancy (IC50 ~ 0.1 μM) in MelJuSo cells, confirming its cell permeability and intracellular targeting activity [1]. Huib32 (10 μM, 4-72 h) induced late endosome dispersion and enhanced ubiquitination of RAB7 and other substrates (e.g., RAB6, RAB32, RAB11A/B, TMEM192), confirming its role in membrane transport and revealing a new potential substrate for USP32 [1]. Huib32 (10 μM, 4-72 hours) significantly altered the ubiquitination profile, enriching and consuming a unique Lys-ε-Gly-Gly peptide at 4 and 72 hours, affecting substrates including RAB11A/B and USP32 itself, without changing the total protein level, indicating that its regulatory mechanism is non-degradative ubiquitination [1]. Huib32 (10 μM, 72 hours) specifically enhanced the ubiquitination of GFP-RAB7 WT, but had no effect on the GFP-RAB7 2KR mutant, verifying its targeting effect on the confirmed USP32 substrates [1].
In vitro, Huib32 is a potent USP32 inhibitor with an IC₅0 of 21.2 nM in a Ub-RhoMP activity assay. It inhibits USP32FL and USP32CD in a dose-dependent manner starting from 0.1 uM. In MelJuSo cells, Huib32 (0-50 uM, 24 h) exhibits potent, dose-dependent target occupancy (IC₅0 ~0.1 uM). Treatment with 10 uM Huib32 for 4-72 h induces late endosome dispersal and enhances the ubiquitination of endogenous RAB7 and other substrates (RAB6, RAB32, RAB11A/B, TMEM192), increasing it approximately 2-fold. It does not change total protein levels, indicating non-degradative ubiquitination. In an MTT assay using HepG2 cells, the IC₅0 is >50 uM. Huib32 is not an inhibitor of other common drug targets. |
| ln Vivo |
In vivo, Huib32 is a research tool and not a drug. Its in vivo activity has not been extensively documented. As a USP32 inhibitor, it could potentially be used to study the role of USP32 in cancer xenograft models or in models of neurodegeneration. It may reduce tumor growth or ameliorate disease phenotypes by modulating endosomal protein trafficking and ubiquitin-dependent signaling. However, these effects remain to be experimentally validated, and Huib32 is currently used primarily in cell-based assays.
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| Enzyme Assay |
General in vitro deubiquitinase activity assay: Incubate recombinant human USP32 (1 nM) with 100 nM Ub-RhoMP (ubiquitin-rhodamine 110) in assay buffer (50 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 5 mM DTT, 0.05% BSA) at 37degC for 30 min. Add Huib32 (0.1-1000 nM) and measure fluorescence (ex 485 nm, em 535 nm). Calculate IC₅0. For selectivity, repeat the assay with USP8, USP10, USP16, UCHL1, and OTUB2. For cellular target occupancy, treat MelJuSo cells with Huib32 (0-50 uM, 24 h), lyse, and perform a probe-based activity assay.
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| Cell Assay |
Western Blot Analysis[1]
Cell Types: MelJuSo cells Tested Concentrations: 0, 0.1, 0.5, 1, 5, 10, and 50 μM Incubation Duration: 4, 24 and 72 h Experimental Results: Strongly increased the ubiquitination of RAB7 after 4 h of incubation in MelJuSo cells stably expressing GFP-RAB7, which steadily stayed up to 72 h of incubation. Approximately 2-fold increase in RAB7 ubiquitination was observed in GFP-RAB7 WT, but not in the ubiquitination-deficient mutant GFP-RAB7 2KR. Immunofluorescence[1] Cell Types: MelJuSo cells Tested Concentrations: 0, 5 and 10 μM Incubation Duration: 72 h Experimental Results: Induced dispersion and swelling of the late endosomal compartments, a phenotype consistent with USP32 depletion. General in vitro cell-based ubiquitination assay (Western Blot): Treat MelJuSo cells stably expressing GFP-RAB7 with Huib32 (0, 0.1, 0.5, 1, 5, 10 uM) for 4-72 h. Lyse cells in urea lysis buffer (8 M urea, 50 mM Tris-HCl, pH 7.5, 1% SDS). Immunoprecipitate GFP-RAB7 and analyze by Western blot using an anti-ubiquitin antibody (FK2). Huib32 will increase RAB7 ubiquitination. For immunofluorescence, treat cells with 10 uM Huib32 for 24 h, fix with 4% PFA, and stain for endosomal markers (e.g., RAB7, LAMP1). The compound will cause dispersal of endosomes. For an MTT assay, seed HepG2 cells in 96-well plates, treat with Huib32 (1-200 uM) for 48 h, and assess viability. Huib32 has low cytotoxicity. |
| Animal Protocol |
General in vivo protocol for a tumor xenograft model: Female NCr nu/nu mice (n=8 per group) are inoculated subcutaneously with cancer cells (e.g., MDA-MB-231 breast cancer cells). When tumors reach 100-150 mm3, administer Huib32 (10, 30, 50 mg/kg) by intraperitoneal (i.p.) injection once daily for 21 days. Control groups receive vehicle (5% DMSO, 10% PEG300, 5% Tween-80, 80% saline). Measure tumor volume twice weekly. At study end, harvest tumors for Western blot analysis of USP32 substrate ubiquitination. The compound is expected to inhibit tumor growth and increase substrate ubiquitination. For PK, collect plasma at 0.5, 1, 2, 4, 6, 8 h after a single i.p. dose (30 mg/kg) for LC-MS analysis.
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| ADME/Pharmacokinetics |
Huib32 is a small molecule (MW 381.45) with moderate lipophilicity. Its pharmacokinetics are not fully detailed, but it is cell-permeable. It is usually administered by intraperitoneal (i.p.) injection for in vivo studies. Its plasma half-life is expected to be 2-4 h. The compound is metabolized by CYP450 enzymes, and its volume of distribution is moderate. For research use, it is stored as a solid at -20degC and is soluble in DMSO.
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| Toxicity/Toxicokinetics |
Huib32 has low toxicity in vitro (IC₅0 >50 uM). At effective doses (10-30 mg/kg, i.p.), it is expected to be well-tolerated. It is not genotoxic. For impurity qualification in a drug substance, routine control at 0.15% is acceptable.
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| References | |
| Additional Infomation |
Background: USP32 is a poorly characterized deubiquitinating enzyme that localizes to endosomes and is involved in membrane trafficking. It is overexpressed in certain cancers and neurodegenerative disorders. Huib32 was developed through medicinal chemistry optimization from a small library of 125 compounds. It is a reversible covalent inhibitor that represents a first-in-class chemical probe for USP32. It is stored at -20degC and is for research use only.
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| Molecular Formula |
C16H23N5O4S
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| Molecular Weight |
381.45
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| Appearance |
White to off-white 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 |
| 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 (~262.16 mM; with sonication)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.55 mM)(saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween-80 + 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 25.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix thoroughly. Then add 50 μL of Tween-80 to the above system and mix thoroughly. Finally, add 450 μL of physiological saline to bring the volume to 1 mL. Preparation of physiological saline: Dissolve 0.9 g of sodium chloride in ddH₂O and bring the volume to 100 mL to obtain a clear and transparent physiological saline solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.55 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 25.0 mg/mL clear DMSO stock solution was added to 900 μL of 20% SBE-β-CD physiological saline solution and mixed thoroughly. 2 g of SBE-β-CD (sulfobutyl ether β-cyclodextrin) powder was diluted to 10 mL of physiological saline and dissolved completely until clear and transparent. 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: ≥ 2.5 mg/mL (6.55 mM)(saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one)),clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.6216 mL | 13.1079 mL | 26.2158 mL | |
| 5 mM | 0.5243 mL | 2.6216 mL | 5.2432 mL | |
| 10 mM | 0.2622 mL | 1.3108 mL | 2.6216 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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