USP7 (IC50 = 0.75 μM); USP7 (IC50 = 0.43 μM); USP43 (IC50 = 20.3 μM); Ub-MDM2 (IC50 = 0.23 μM)
GNE-6640 encourages the natural ubiquitination of MDM2 through Lys48 (K48)-linked polyubiquitin chains, thereby guiding the process of proteasomal degradation13. GNE-6640 focuses on the p53, MDM2, and USP7 signaling pathways in cells. With an IC50 ≤ 10 μM, GNE-6640 reduces the viability of 108 cell lines. Combining GNE-6640 with DNA-damaging drugs like doxorubicin or cisplatin may trigger the p53 response and improve the effectiveness of USP7 inhibitors. GNE-6640 may cause the death of tumor cells. PIM kinase inhibitors and other targeted substances, such as chemotherapeutic agents, increase the cytotoxicity of GNE-6640[1].

GNE-6640 encourages the natural ubiquitination of MDM2 through Lys48 (K48)-linked polyubiquitin chains, thereby guiding the process of proteasomal degradation13. GNE-6640 focuses on the p53, MDM2, and USP7 signaling pathways in cells. With an IC50 ≤ 10 μM, GNE-6640 reduces the viability of 108 cell lines. Combining GNE-6640 with DNA-damaging drugs like doxorubicin or cisplatin may trigger the p53 response and improve the effectiveness of USP7 inhibitors. GNE-6640 may cause the death of tumor cells. PIM kinase inhibitors and other targeted substances, such as chemotherapeutic agents, increase the cytotoxicity of GNE-6640[1].

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GNE‑6640 selectively inhibits USP7 over USP47 (IC₅₀ >200 µM) and USP5 (IC₅₀ >200 µM).
In cellular assays, it enhances endogenous MDM2 ubiquitination (EC₅₀ ≈ 0.23 µM in Ub‑MDM2 MSD assay) and promotes K48‑linked polyubiquitination of MDM2.
It stabilizes p53 and upregulates p21 in wild‑type cells but not in p53‑null or USP7‑null cells.
The compound decreases viability of 108 out of 441 cell lines (3‑day assay) with IC₅₀ ≤10 µM; in a 5‑day assay, 54 cell lines showed IC₅₀ ≤10 µM.
Acute myeloid leukemia (AML) cell lines exhibited increased sensitivity to GNE‑6640.
Combination with DNA‑damaging agents (e.g., doxorubicin, cisplatin) enhances its efficacy.
GNE‑6640 also shows synergy with PIM kinase inhibitors (e.g., GDC‑0339, GDC‑0570) in Bliss analysis.[1]

GNE-6640, another novel selective and non-covalent inhibitor of USP7, has IC50 values of 0.75 μM (full length USP7), 0.43 μM (USP7 catalytic domain), and 20.3 μM (full length USP43). Ubiquitin-specific protease-7 (USP7) controls the stability of the p53 tumor suppressor and various other proteins critical for tumor cell survival. GNE-6640 facilitates endogenous MDM2 ubiquitination and targets cellular USP7 and p53 signaling pathways, inducing tumor cell death, enhancing cytotoxicity, and decreasing the viability of around 108 cell lines with IC50 ≤ 10 μM with chemotherapeutic agents such as PIM kinase inhibitors[2].

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USP7 enzymatic analysis[1]
Michaelis–Menten kinetic measurements with full-length USP7 were performed using 1 nM USP7 with a series of ubiquitin–AMC substrate titrations. The initial rate of substrate hydrolysis was determined using the Magellan software on a Tecan Safire2 plate reader, and kinetic parameters modelled using nonlinear regression analysis with GraphPad Prism software. Standard error was calculated from three technical replicates. For studies using the USP7 D305/E308 mutant, samples were reacted in a buffer consisting of 50 mM HEPES (pH 7.5), 100 mM NaCl, 2.5 mM dithiothreitol, and 0.1% (w/v) bovine gamma globulin. The starting substrate concentration of ubiquitin-Rho110 used for the Michaelis–Menten analysis was 100 μM serial diluted to 781 nM. Reactions were performed for 1 h at room temperature with a final enzyme concentration of 100 nM (three independent experiments, see symbols in plots), in black 100-μl volume 96-well half area plates. The enzymatic activity was calculated by fitting the data using the initial velocity with the linear V0 values measured by analysing the fluorescence signal of cleaved Rho-110 using excitation at 485 nm and emission at 535 nm.

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Deubiquitinase selectivity analysis[1]
Recombinant deubiquitinase di-ubiquitin mass spectrometry cleavage assay. The MALDI–TOF DUB assay was performed using the indicated concentrations of recombinant deubiquitinases, di-ubiquitin substrates, and USP7 inhibitor compounds as described previously. The inhibition efficiency for GNE-6640 and GNE-6776 against the UCHl family members was monitored on Ub-Ube2W (Ub-E2), an alternative substrate to di-ubiquitin.

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USP7 activity was measured using a TR‑FRET‑based assay with GST‑UbA10‑His as substrate. The assay buffer contained HEPES (pH 7.5), detergent, DTT, and carrier protein. Compounds were pre‑incubated with recombinant full‑length USP7, then substrate was added. After reaction, detection antibodies (anti‑His‑europium and anti‑GST‑d2) were used to measure TR‑FRET signal. Inhibition was calculated relative to controls, and IC₅₀ values were determined by curve‑fitting.
A di‑ubiquitin FRET assay with internally quenched K63‑linked di‑ubiquitin substrate was used as an orthogonal activity assay. Fluorescence increase upon cleavage was monitored.
A ubiquitin‑rhodamine‑110 assay was also employed, where cleavage releases rhodamine‑110 and increases fluorescence.
Di‑ubiquitin cleavage was further validated by mass spectrometry, monitoring conversion to ubiquitin using MRM.[1]

Tumour cell-line panel viability. [1]
GNE-6640 and GNE-6641 were profiled for 3 days across 441 cell lines, and GNE-6776, GNE-6640, and GNE-6641 were profiled for 5 days across a subset of 185 cell lines as previously described26. In brief, compounds were screened in nine-point dose–response using a threefold dilution. Cells were seeded into 384-well plates 24 h before compound addition. Cells were then incubated with compound for 72 h or 120 h before assaying viability. Assays were performed in biological triplicate. Cells were incubated (37 °C, 5% CO2) in RPMI-1640, 2.5% FBS (72 h assay) or 5% FBS (120 h assay), and 2 mM glutamine throughout the assay. The reported IC50 and mean viability metrics were as follows: IC50 was the dose at which the estimated inhibition was 50% relative to untreated wells (that is, absolute IC50).

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Primary combination screen. [1]
A compound library comprising 589 compounds arrayed in nine-point dose–response was screened in the absence or presence of fixed doses of GNE-6776 (0 nm, 125 nM, 250 nM, 500 nM, 1,000 nM, and 2,000 nM) or GNE-6640 (400 nM). In brief, 5,000 EOL-1 cells were seeded into 384-well plates, and compound was added 24 h later. Cell viability was determined 120 h after compound addition (CellTiter-Glo). Curves were fitted, and both IC50 and mean viability metrics were calculated. The IC50 was the dose at which inhibition was 50% relative to untreated wells. The mean viability was the average of the fitted viabilities at each tested dose. Mean viability was equivalent to the area under the log-dose/viability curve divided by the total number of tested doses. Mean viability values were used for the analysis described in Extended Data Fig. 6g. All data were fitted using Genedata Screener software.

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Primary combination screen analysis. [1]
Normalized mean viabilities were determined in the EOL-1 cell line for 574 compounds that have known protein or mechanistic targets, in the presence of DMSO or increasing concentrations of GNE-6776 (100 nM, 250 nM, 500 nM, 1,000 nM or 2,000 nM) or 400 nM of GNE-6640. For each compound, we assessed the difference in mean viability between USP7 inhibitor treatment and the DMSO treatment. For targets targeted by three or more compounds, we calculated the enrichment of high mean viability difference for each concentration of USP7 inhibitor by using a Wilcoxon rank-sum test. For visualization purposes, we combined the results of all concentrations by taking the mean of the −log10(transformed P values) for each target.

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For ubiquitin‑MDM2 assays, HCT116 or SJSA‑1 cells were seeded in 96‑well plates, treated with compound for 24 h, then exposed to proteasome inhibitor MG132 for 1 h. Cells were lysed, and ubiquitinated‑MDM2 and total MDM2 were quantified using a multiplexed mesoscale immunoassay.
Total MDM2 immunofluorescence: HCT‑116 cells were seeded in 384‑well plates, treated with compound for 24 h, fixed, permeabilized, stained with anti‑MDM2 primary and AlexaFluor‑555‑conjugated secondary antibodies, and imaged using a high‑content imager. Nuclear MDM2 intensity was quantified.
Cell viability (IncuCyte): Cells were treated with compound in low‑serum medium containing caspase‑3/7 reagent, imaged every 2 h for 68–72 h, and confluence/caspase activity were analyzed.
CellTiter‑Glo assays were performed after 72 h or 120 h of compound treatment to assess viability.
Combination screens with 589 chemotherapeutic/targeted agents were conducted in EOL‑1 cells, and synergy was evaluated using Bliss analysis.[1]

In vitro pharmacokinetic parameters of GNE-6640: molecular weight 330.38 Da, logD₇.₄ 4.40, total polar surface area 87.0 Ų.
Hepatic microsomal stability (CLₕₑₚ): Human 14 mL/min/kg, Rat 46 mL/min/kg, Mouse 75 mL/min/kg, Dog 24 mL/min/kg, Cynomolgus monkey 33 mL/min/kg.
Hepatocyte stability (CLₕₑₚ): Human 16 mL/min/kg, Rat 45 mL/min/kg, Mouse 68 mL/min/kg, Dog 24 mL/min/kg, Cynomolgus monkey 39 mL/min/kg.
Plasma protein binding: Human 99.6%, Rat 98.7%, Mouse 99.0%.
MDCK cell permeability: B to A 12.17 × 10⁻⁶ cm/s, A to B 14.7 × 10⁻⁶ cm/s, ratio 0.83. [1]

[1]. USP7 small-molecule inhibitors interfere with ubiquitin binding. Nature. 2017 Oct 26;550(7677):534-538.

[2]. The role of deubiquitinating enzymes in cancer drug resistance. Cancer Chemother Pharmacol. 2020 Apr;85(4):627-639

The ubiquitin system regulates important cellular processes in eukaryotes. Ubiquitins are linked to substrate proteins in monomeric or chain form, and the topological structure modified by ubiquitin regulates the interaction between substrates and specific proteins. Therefore, ubiquitination determines the fate of a variety of substrates, including proteasome degradation. Deubiquitinating enzymes cleave ubiquitin from substrates and are associated with a variety of diseases; for example, ubiquitin-specific protease-7 (USP7) regulates the stability of p53 tumor suppressor protein and other proteins essential for tumor cell survival. However, developing selective deubiquitinating enzyme inhibitors has been extremely challenging, and the co-crystal structures of small molecule inhibitors and deubiquitinating enzymes have not yet been resolved. In this paper, we describe the development of the selective USP7 inhibitors GNE-6640 and GNE-6776 using NMR-based screening and structure-based drug design. These compounds induce tumor cell death and enhance the cytotoxicity of chemotherapeutic drugs and targeted compounds, including PIM kinase inhibitors. Structural studies revealed that GNE-6640 and GNE-6776 non-covalently target USP7, with their interaction sites located 12 Å from the catalytic cysteine residue. These compounds weaken ubiquitin binding, thereby inhibiting the deubiquitinating activity of USP7. GNE-6640 and GNE-6776 interact with acidic residues mediating hydrogen-bonded interactions with the Lys48 side chain of ubiquitin, suggesting that USP7 preferentially interacts with and cleaves ubiquitin molecules with free Lys48 side chains. We validated this hypothesis by constructing biubiquitin chains with different proximal and distal isotopic labels and measuring USP7 binding using NMR. This preferential binding elongates the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked ubiquitin chains. In summary, the design of compounds that inhibit USP7 activity by weakening ubiquitin binding provides an opportunity to develop other deubiquitinating enzyme inhibitors and may be a more broadly applicable strategy for inhibiting proteins that require ubiquitin binding to exert their full functional activity. [1]

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This paper describes GNE-6640 and GNE-6776, selective USP7 inhibitors with well-defined inhibitory mechanisms. Establishing a rigorous screening process is essential for selecting and optimizing targeted inhibitors. The joint study revealed previously undescribed interactions between USP7 deubiquitinating enzyme activity and PIM kinase in regulating cell viability. The co-crystal structures of GNE-6640 or GNE-6776 suggest that complementary charged interactions between USP7-D305/E308 and the ubiquitin-K48 side chain are crucial, which we confirmed through mutation analysis. Notably, D305G has been identified as a somatic loss-of-function mutant from patients with acute lymphoblastic leukemia²¹. NMR analysis of USP7 binding to natural monoubiquitin and differentially labeled diubiquitin showed that USP7 preferentially interacts with ubiquitin moieties having free K48 side chains. Some studies have suggested that certain deubiquitinating enzymes cannot effectively depolymerize long substrate-bound K48 linker chains, thus setting a threshold for proteasome-targeted polyubiquitination²²; our study confirms this view and provides a biophysical mechanism. Many proteins, including other deubiquitinating enzymes, ubiquitin ligases, DNA repair and endocytosis mechanisms, and epigenetic regulators, depend on ubiquitin binding for their function²³. Developing selective inhibitors that can weaken ubiquitin binding is an effective strategy for inhibiting USP7. Our study demonstrates the feasibility of this approach and suggests that it may have broader applications in inhibiting other types of ubiquitin-binding proteins. [1] Drug resistance is a well-known phenomenon that leads to reduced efficacy of drug treatments. Resistance to chemotherapy drugs may involve a variety of intrinsic cellular processes, including drug efflux, enhanced resistance to apoptosis, enhanced DNA damage repair capacity against platinum-based or other DNA-damaging drugs, drug inactivation, altered drug targets, epithelial-mesenchymal transition (EMT), inherent cellular heterogeneity, epigenetic effects, or any combination of these mechanisms. Deubiquitinating enzymes (DUBs) reverse ubiquitination of target proteins, maintaining the balance between protein ubiquitination and deubiquitination, thereby maintaining cellular homeostasis. Increasing evidence suggests that altered DUB activity is closely associated with the development and progression of various cancers. Therefore, DUBs are potential candidate targets for targeted drug development. This article reviews the role of DUBs, particularly ubiquitin-specific proteases, in resistance to drugs in different types of cancer. We also review potential small molecule DUB inhibitors for cancer treatment. [2]

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GNE-6640 is a selective non-covalent USP7 inhibitor with a binding site approximately 12 Å away from catalytic cysteine, which weakens ubiquitin binding. It preferentially inhibits the activity of USP7 on ubiquitin moieties with free Lys48 side chains, leading to a prolonged depolymerization process of K48-linked polyubiquitin chains. This compound was developed through NMR-based fragment screening and structure-based drug design. It exhibits synergistic effects with PIM kinase inhibitors and DNA-damaging chemotherapeutic agents. The somatic loss-of-function mutation D305G of USP7 was discovered in patients with acute lymphoblastic leukemia, highlighting the clinical significance of targeting this site. [1]

C20H18N4O

330.383123874664

330.15

C, 72.71; H, 5.49; N, 16.96; O, 4.84

2009273-67-8

122531786

White to off-white solid powder

3.8

3

4

3

25

439

0

ZHYXJQQBKROZDX-UHFFFAOYSA-N

InChI=1S/C20H18N4O/c1-2-16-17(13-5-8-18-14(9-13)10-23-24-18)11-22-20(21)19(16)12-3-6-15(25)7-4-12/h3-11,25H,2H2,1H3,(H2,21,22)(H,23,24)

4-[2-amino-4-ethyl-5-(1H-indazol-5-yl)pyridin-3-yl]phenol

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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