The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

Titlec-Abl has high intrinsic tyrosine kinase activity that is stimulated by mutation of the Src homology 3 domain and by autophosphorylation at two distinct regulatory tyrosines. c-Abl is a non-receptor tyrosine kinase of which the precise functions are not known, but roles for Abl in growth factor and integrin signaling, cell cycle regulation, neurogenesis, and responses to DNA damage and oxidative stress have been suggested (1). c-Abl kinase activity is increased in vivo by diverse physiological stimuli including ionizing radiation (2), entry into S phase (3), integrin activation (4), and platelet-derived growth factor stimulation (5). The mechanism of regulation of Abl tyrosine kinase activity by these processes is not well understood. Ionizing radiation may activate Abl kinase activity through phosphorylation of the Abl catalytic domain at Ser-465 by the Atm kinase (6), whereas platelet-derived growth factor stimulation is associated with tyrosine phosphorylation of cAbl by c-Src (5). In contrast, the activation of nuclear c-Abl in S phase is through the detachment of the inhibitor Rb protein (3), whereas Abl may be activated by free radicals through dissociation of Pag/Msp23, an antioxidant protein that also inhibits Abl (7). Abl kinase activity can also be stimulated by the binding of several activator proteins, including the transcription factors c-Jun (8) and RFX1 (9), and the adapter protein Nck (10). These observations suggest complex regulation of c-Abl at multiple levels through binding or dissociation of activators and inhibitors and via direct phosphorylation.Although the NH 2 -terminal sequence of c-Abl is very similar to members of the Src family, biochemical and genetic studies suggest that the structural basis of regulation of c-Abl catalytic activity is significantly different from the catalytic activity of Src. When co-expressed with another kinase, such as Csk, that can phosphorylate the COOH-terminal regulatory tyrosine 527, c-Src (and the Src family member Hck) can be purified as an inactive monomer in which the phosphorylated Tyr-527 residue binds the SH2 1 domain in an intramolecular fashion. In this structure, the SH3 domain contacts the linker region between SH2 and the catalytic domain (the SH2-CD linker) in an atypical interaction involving a single proline (Pro-250) (11,12). Mutation or deletion of 14) or mutation of the SH2 or SH3 domains (15, 16) dysregulates and increases Src kinase activity both in vitro and in vivo. The precise mechanism of physiological activation of Src kinases is unknown, but in vitro studies demonstrate that activation may involve discrete steps that independently increase catalytic activity. In the presence of ATP and magnesium, Src or Hck that is monophosphorylated at the Tyr-527 homologue undergoes slow autophosphorylation at Tyr-412 that increases kinase activity about 10-fold (17-19). Dissociation of the SH2-Tyr-527 interaction by dephosphorylation or a competing SH2 ligand stimulates activity 2.5-fold, whereas the disruption of the SH3-linker interac...

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Growth factor receptor-binding protein-2 (Grb2) plays a key role in signal transduction initiated by Bcr/Abl oncoproteins and growth factors, functioning as an adaptor protein through its Src homology 2 and 3 (SH2 and SH3) domains. We found that Grb2 was tyrosine-phosphorylated in cells expressing BCR/ABL and in A431 cells stimulated with epidermal growth factor (EGF). Phosphorylation of Grb2 by Bcr/Abl or EGF receptor reduced its SH3-dependent binding to Sos in vivo, but not its SH2-dependent binding to Bcr/Abl. Tyr209 within the C-terminal SH3 domain of Grb2 was identi®ed as one of the tyrosine phosphorylation sites, and phosphorylation of Tyr209 abolished the binding of the SH3 domain to a proline-rich Sos peptide in vitro. In vivo expression of a Grb2 mutant where Tyr209 was changed to phenylalanine enhanced BCR/ABL-induced ERK activation and ®broblast transformation, and potentiated and prolonged Grb2-mediated activation of Ras, mitogen-activated protein kinase and c-Jun N-terminal kinase in response to EGF stimulation. These results suggest that tyrosine phosphorylation of Grb2 is a novel mechanism of down-regulation of tyrosine kinase signaling.