Cell experiment:

EOL-1 cells are seeded into 384-well plates 24 h before compound addition. Cells are then incubated with compound (e.g., GNE-6776; 0.003, 0.009, 0.027, 0.082, 0.25, 0.74, 2.22, 6.67, and 20 μM) for 72 h or 120 h before assaying viability. Assays are performed in biological triplicate. Cells are incubated (37℃, 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 are as follows: IC50 is the dose at which the estimated inhibition is 50% relative to untreated wells (that is, absolute IC50). The mean viability is calculated[1].

Animal experiment:

Mice[1]GNE-6776 is administered at 200 mg/kg (body weight) by oral gavage to female C.B-17 SCID mice, aged 12-16 weeks (n=3 per time point). No randomization is used for DMPK studies. At 0.5, 1, 2, 4, 8 and 24 h post-dose, blood samples are collected by terminal cardiac puncture into anticoagulant tubes (EDTA). Clarified plasma is then transferred to a fresh tube and snap frozen. GNE-6776 plasma concentrations are determined by LC-MS/MS[1].

GNE-6776 is a selective USP7 inhibitor.

GNE-6776 non-covalently targets USP7 12 ? distant from the catalytic cysteine. GNE-6776 attenuates ubiquitin binding and thus inhibits USP7 deubiquitinase activity. GNE-6776 interacts with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain. GNE-6776 targets cellular USP7, MDM2, and p53 signalling pathways.GNE-6776 selectively inhibits recombinant USP7 relative to 36 other deubiquitinases. GNE-6776 remains selective even at 100 μM, a more than sixfold higher concentration than used in cellular assays. GNE-6776 significantly inhibits USP7 while remaining selective against 44-47 other detected deubiquitinases[1].

Although efficacious exposure is only transiently achieved, GNE-6776 causes modest, although significant, EOL-1 xenograft growth delay[1].

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