| Description |
FT671 is a potent and selective USP7 inhibitor with an IC50 of 52 nM and binds to the USP7 catalytic domain with a Kd of 65 nM.
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| Related Catalog |
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| Target |
IC50: 52 nM (USP7)[1] Kd: 65 nM (USP7)[1]
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| In Vitro |
FT671 binds to the USP7 catalytic domain (USP7CD; residues 208-560) with apparent dissociation constant (Kd) value of 65nM (s.e.m. range: 45–92). FT671 inhibits USP7 with half-maximal inhibitory concentration (IC50) value of 52 (29-94) nM (USP7CD). Cell lines derived from colorectal carcinoma (HCT116) or bone osteosarcoma (U2OS) respond to USP7 knockdown with p53 stabilization and p21 induction, leading to growth arrest and apoptosis. Similarly, FT671 increases p53 protein levels in these cell lines, leading to induction of p53 target genes including BBC3 (which encodes PUMA), CDKN1A (p21), RPS27L (S27L) and MDM2. The increase in p53 correlates with increased MDM2 degradation, which is initially balanced by p53-induced MDM2 expression, but has an effect on MDM2 protein levels after prolonged compound treatment. FT671 leads to the degradation of N-Myc and upregulation of p53 in the neuroblastoma cell line IMR-32. FT671 also stabilizes p53 in the MM.1S multiple myeloma cell line, which correlates with increased MDM2 ubiquitination and leads to expression of p53 target genes. FT671 blocks the proliferation of MM.1S cells, with an IC50 value of 33 nM[1].
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| In Vivo |
Treatment of mice with FT671 leads to a significant dose-dependent inhibition of tumour growth. FT671 is well-tolerated even at high doses, and no significant weight loss or cachexia is observed during the study[1].
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| Kinase Assay |
To determine compound IC50 values, compounds (including FT671) are diluted in 100% DMSO in threefold 12-point dilution series from 100 μM. 100 nL of 100-fold concentrated solutions are dispensed into black 384-well Proxiplates using an Echo. 25nM ubiquitin-rhodamine 110, along with recombinant USP7CD (3nM), or USP7C-term (30-125pM, depending on batch activity) are added and the plates incubated at room temperature for 1h. The reaction is terminated by adding 2.5 μL citric acid to a final concentration of 10 mM before measuring fluorescence intensity on a Pherastar with a 485nm excitation/520nm emission optic module[1].
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| Cell Assay |
MM.1S are plated at a starting density of 3,500 cells per well in 40 μL of RPMI-1640 supplemented with 2mM l-glutamine, 10% fetal bovine serum, 1% penicillin/streptomycin and 10mM HEPES, pH 7.4. Cells are plated in 384-well black plates and incubated at 37°C, 5% CO2 in a humidified atmosphere for 12h before DMSO-solubilised FT671 is added using an ECHO 525 acoustic dispenser. The final DMSO concentration is 0.5% (v/v). After compound addition, cells are incubated for 120 h at 37°C, 5% CO2 in humidified atmosphere. Experiments are performed in triplicate, simultaneously on different plates. The viability assay is performed using CellTiter-Glo 2.0. Luminescence is measured using an Envision plate reader and normalized to the samples treated with 0.5% (v/v) DMSO. The IC50 values are calculated using nonlinear regression algorithms in ActivityBase[1].
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| Animal Admin |
Mice[1] For the pharmacokinetic/pharmacodynamic (PK/PD) study, a single administration of 200, 75 or 25 mg/kg FT671 is given per os by oral gavage when tumours reach an average volume of 318mm3. 10% DMA, 90% PEG 400 serve as a vehicle control (n= 5 mice per group per time point). Tumour samples are collected at 0.5h, 2h, 6h and 24h time points and flash-frozen for subsequent analysis. Vehicle control samples are obtained at the 24h time point[1].
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| References |
[1]. Turnbull AP, et al. Molecular basis of USP7 inhibition by selective small-molecule inhibitors. Nature. 2017 Oct 26;550(7677):481-486.
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