Given the importance of ubiquitin-specific protease 7 (USP7) in oncogenic pathways, identification of USP7 inhibitors has attracted considerable interest. Despite substantial efforts, however, the development of validated deubiquitinase (DUB) inhibitors that exhibit drug-like properties and a well-defined mechanism of action has proven particularly challenging. In this article, we describe the identification, optimization and detailed characterization of highly potent (IC < 10 nM), selective USP7 inhibitors together with their less active, enantiomeric counterparts. We also disclose, for the first time, co-crystal structures of a human DUB enzyme complexed with small-molecule inhibitors, which reveal a previously undisclosed allosteric binding site. Finally, we report the identification of cancer cell lines hypersensitive to USP7 inhibition (EC < 30 nM) and demonstrate equal or superior activity in these cell models compared to clinically relevant MDM2 antagonists. Overall, these findings demonstrate the tractability and druggability of DUBs, and provide important tools for additional target validation studies.
An X-ray crystal structure of Kelch-like ECH-associated protein (Keap1) co-crystallised with (1S,2R)-2-[(1S)-1-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,2,3,4-tetrahydroisoquinolin-2-carbonyl]cyclohexane-1-carboxylic acid (compound (S,R,S)-1 a) was obtained. This X-ray crystal structure provides breakthrough experimental evidence for the true binding mode of the hit compound (S,R,S)-1 a, as the ligand orientation was found to differ from that of the initial docking model, which was available at the start of the project. Crystallographic elucidation of this binding mode helped to focus and drive the drug design process more effectively and efficiently.
The class A G-protein-coupled receptors
(GPCRs) Orexin-1 (OX1)
and Orexin-2 (OX2) are located predominantly in the brain and are
linked to a range of different physiological functions, including
the control of feeding, energy metabolism, modulation of neuro-endocrine
function, and regulation of the sleep–wake cycle. The natural
agonists for OX1 and OX2 are two neuropeptides, Orexin-A and Orexin-B,
which have activity at both receptors. Site-directed mutagenesis (SDM)
has been reported on both the receptors and the peptides and has provided
important insight into key features responsible for agonist activity.
However, the structural interpretation of how these data are linked
together is still lacking. In this work, we produced and used SDM
data, homology modeling followed by MD simulation, and ensemble-flexible
docking to generate binding poses of the Orexin peptides in the OX
receptors to rationalize the SDM data. We also developed a protein
pairwise similarity comparing method (ProS) and a GPCR-likeness assessment
score (GLAS) to explore the structural data generated within a molecular
dynamics simulation and to help distinguish between different GPCR
substates. The results demonstrate how these newly developed methods
of structural assessment for GPCRs can be used to provide a working
model of neuropeptide–Orexin receptor interaction.
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