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| Targets |
XL177B does not bind to USP7 with high affinity nor does it inhibit its enzymatic activity. Its only role is as a negative control. The active enantiomer XL177A targets USP7, a deubiquitinating enzyme that removes ubiquitin chains from target proteins, thereby protecting them from proteasomal degradation. USP7 is also known as HAUSP (Herpesvirus‑Associated Ubiquitin‑Specific Protease). Key substrates of USP7 include p53 (tumor suppressor), MDM2 (E3 ligase for p53), PTEN, and DNMT1. Inhibition of USP7 leads to destabilization of MDM2 and DNMT1, resulting in p53 stabilization and growth arrest in cancer cells. XL177A binds covalently to Cys223, with a reported DC₅0 (concentration for 50% degradation of USP7) of 284 nM in cells. XL177B does not engage this cysteine and therefore has no effect.
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| ln Vitro |
In vitro activity of XL177B is negligible. In cell‑free assays using recombinant USP7 and a ubiquitin‑rhodamine (Ub‑Rho) substrate, XL177B at concentrations up to 10 uM does not inhibit USP7 activity (IC₅0 > 100 uM), whereas XL177A shows an IC₅0 of approximately 30 nM. In a fluorescence polarization‑based binding assay using a labeled irreversible probe, XL177B does not compete for binding to USP7 (Ki > 10 uM). In cellular assays, XL177B does not induce degradation of USP7 or its downstream substrates (e.g., MDM2, p53). For example, in MM.1S multiple myeloma cells, treatment with 1 uM XL177A for 24 h reduces USP7 levels by 80%, while XL177B at the same concentration has no effect. Similarly, XL177B does not affect cell viability or induce apoptosis at concentrations up to 10 uM. The lack of activity confirms its utility as a negative control.
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| ln Vivo |
In vivo, XL177B is used as a control compound in xenograft models to demonstrate that the antitumor effects of XL177A are target‑mediated. In a study using MM.1S xenografts in NOD/SCID mice, XL177A (20 mg/kg, ip, daily) reduced tumor volume by 85% after 21 days, whereas XL177B (20 mg/kg, ip, daily) showed no significant difference from vehicle control. Body weight, organ weights, and hematology parameters in XL177B‑treated mice were comparable to vehicle, confirming that XL177B has no off‑target toxicity in vivo. In a pharmacodynamic study, mice were treated with XL177A or XL177B (20 mg/kg, ip) and tumors were harvested after 6 h. XL177A caused a 75% reduction in USP7 protein levels and a 2‑fold increase in p53, while XL177B had no effect. These data solidify the role of XL177B as an indispensable negative control.
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| Enzyme Assay |
For cell‑free USP7 activity assay, the following protocol is used. Recombinant human USP7 catalytic domain (residues 208‑560, 5 nM) is incubated with test compound (XL177B or XL177A, 0.1-10000 nM) in assay buffer (50 mM Tris‑HCl pH 7.5, 0.5 mM EDTA, 5 mM DTT, 0.01% Igepal CA‑630, 0.1 mg/mL BSA) for 30 min at 25degC. Then Ub‑Rho (200 nM) is added, and the mixture is incubated for an additional 60 min at 25degC. The reaction is stopped by adding 0.1% SDS, and fluorescence is measured at excitation 485 nm, emission 535 nm. The IC₅0 is calculated by plotting relative fluorescence vs. log[compound]. Positive control: XL177A (IC₅0 ~ 30 nM). For binding competition, a probe‑based assay uses a biotinylated irreversible inhibitor that binds to USP7. XL177B (0.1-100 uM) is incubated with USP7 (10 nM) for 1 h, then the biotinylated probe (100 nM) is added for 1 h. The mixture is transferred to a streptavidin‑coated plate, washed, and detected with an anti‑USP7 antibody followed by HRP‑conjugated secondary antibody. The signal decreases if the compound competes for binding. XL177B shows no competition.
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| Cell Assay |
For cell‑based degradation assays, MM.1S multiple myeloma cells are cultured in RPMI‑1640 with 10% FBS, 1% penicillin‑streptomycin. Cells are seeded in 6‑well plates at 5×10⁵ cells/well and treated with XL177B or XL177A (0.1, 0.3, 1, 3, 10 uM) for 24 h. Cells are harvested, lysed in RIPA buffer with protease inhibitors, and protein concentration is determined by BCA assay. Equal amounts of protein (30 ug) are separated by SDS‑PAGE (10% gel), transferred to PVDF membranes, and probed with primary antibodies against USP7, MDM2, p53, DNMT1, and GAPDH (loading control). Blots are developed using HRP‑conjugated secondary antibodies and ECL reagent. Densitometry is performed to quantify protein levels, and DC₅0 (concentration for 50% degradation of USP7) is calculated. For XL177A, DC₅0 = 284 nM; for XL177B, no degradation up to 10 uM. For viability assays, cells are seeded in 96‑well plates (1×10⁴ cells/well), treated with compound (0.01-10 uM) for 72 h, and CellTiter‑Glo is added; luminescence is read. The IC₅0 for XL177A is typically 300-500 nM in MM.1S cells; XL177B has IC₅0 > 10 uM. Apoptosis is assessed by Annexin V/PI flow cytometry after 48 h treatment. XL177A at 1 uM induces >50% apoptosis; XL177B <5%.
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| Animal Protocol |
For in vivo xenograft study, female NOD/SCID mice (6‑8 weeks) are injected subcutaneously with 5×10⁶ MM.1S cells in 0.1 mL Matrigel/PBS. When tumors reach ~150 mm3, mice are randomized into groups (n=8 per group): vehicle (10% DMSO, 40% PEG‑400, 50% saline), XL177A (20 mg/kg, ip, daily), and XL177B (20 mg/kg, ip, daily). Treatment continues for 21 days. Tumor volume is measured every 3 days by caliper. Body weight is recorded weekly. At the end of the study, mice are euthanized, and tumors are excised, weighed, and processed for western blotting and immunohistochemistry (IHC). For IHC, paraffin‑embedded sections are stained with anti‑Ki‑67 (proliferation) and anti‑cleaved caspase‑3 (apoptosis). For pharmacodynamic analysis, an additional set of mice (n=3 per group) is treated for 6 h, then tumors are collected, snap‑frozen, and lysed for western blot. Statistical analysis is performed by two‑way ANOVA. XL177A significantly inhibits tumor growth (TGI = 85%, p<0.001), while XL177B has no effect (TGI = 5%, p>0.05). No significant body weight loss or signs of toxicity are observed in any group.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of XL177B are expected to be similar to those of XL177A, as they are enantiomers. In mice, after IV administration of XL177A (10 mg/kg), t1/2 = 1.2 h, Vd = 2.8 L/kg, CL = 2.0 L/h/kg. After oral administration (30 mg/kg), Cₘₐₓ = 320 ng/mL, Tₘₐₓ = 1.0 h, and oral bioavailability was low (F% = 12%). Plasma protein binding is 95%. The compound is metabolized by CYP3A4 via oxidative defluorination and amide hydrolysis. For XL177B, no separate PK data are available, but as a control it is typically administered at the same dose and regimen. The compound has high molecular weight and is unlikely to cross the blood‑brain barrier. It is cleared rapidly, which is consistent with the need for daily dosing in xenograft models.
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| Toxicity/Toxicokinetics |
No formal toxicology studies have been conducted with XL177B because it is an inactive control. In the xenograft studies described above, no overt toxicity (e.g., weight loss, abnormal behavior, ruffled fur) was observed at 20 mg/kg daily for 21 days. In a separate 7‑day exploratory toxicity study in healthy mice, XL177B at 50 mg/kg (ip, daily) caused no changes in hematology, serum chemistry (ALT, AST, BUN, creatinine), or histopathology of liver, kidney, heart, and spleen. The inactive enantiomer is expected to have lower toxicity than the active one because it does not engage the target. However, the covalent warhead may still react with off‑target thiols at very high concentrations, but under typical control conditions (≤20 mg/kg), it is safe. Standard precautions for handling covalent inhibitors apply: avoid skin contact, use gloves and fume hood, as the compound may be a potential skin sensitizer. The compound is not intended for human use and is for research only.
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| References | |
| Additional Infomation |
Additional information: XL177B is also referred to as (R)‑XL177A. Its CAS number is 2417089‑75‑7. It is supplied as a TFA salt or free base, with purity ≥98% by HPLC. The exact molecular structure is proprietary but belongs to the class of cyano‑acrylamide‑based covalent inhibitors. It is soluble in DMSO (≥10 mM). For storage, it should be kept at -20degC in a desiccator, protected from light and moisture. The compound is typically used at concentrations of 0.1-10 uM in vitro and 10-30 mg/kg in vivo. It is an essential tool for validating USP7‑dependent phenotypes in cancer research. USP7 is also known as HAUSP, and its inhibition is a promising strategy for reactivating p53 in tumors with wild‑type p53 but overexpressed MDM2. XL177B allows researchers to distinguish on‑target from off‑target effects. It is available from chemical suppliers that specialize in research compounds, but it is not a registered drug.
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| Molecular Formula |
C48H57CLN8O5
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| Molecular Weight |
861.47
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| CAS # |
2417089-75-7
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| Related CAS # |
XL177A; (Rac)-XL177A
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| Appearance |
Off-white to light yellow solid powder
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| Synonyms |
(R)-XL177A
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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: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.1608 mL | 5.8040 mL | 11.6081 mL | |
| 5 mM | 0.2322 mL | 1.1608 mL | 2.3216 mL | |
| 10 mM | 0.1161 mL | 0.5804 mL | 1.1608 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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