The mammalian 26S proteasome is a 2500 kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome used on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin–proteasome system in cells. We show that these compounds are entirely selective for the β5 (chymotrypsin-like) site over the β1 (caspase-like) and β2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC50 values for the human 20S β5 site in vitro, and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin–luciferase reporter, activation of NFκB (nuclear factor κB) in response to TNF-α (tumour necrosis factor-α) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the β5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the β5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.

This article describes the discovery of a series of potent inhibitors of Polo-like kinase 1 (PLK1). Optimization of this benzolactam-derived chemical series produced an orally bioavailable inhibitor of PLK1 (12c, MLN0905). In vivo pharmacokinetic−pharmacodynamic experiments demonstrated prolonged mitotic arrest after oral administration of 12c to tumor bearing nude mice. A subsequent efficacy study in nude mice achieved tumor growth inhibition or regression in a human colon tumor (HT29) xenograft model.

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A screen for HDAC6 inhibitors identified acyl derivatives of 4-(aminomethyl)-N-hydroxybenzamide as potent leads with unexpected selectivity over the other subtypes. We designed and synthesized constrained heterocyclic analogues such as tetrahydroisoquinolines that show further enhanced HDAC6 selectivity and inhibitory activity in cellular assays. Selectivity may be attributed to the benzylic spacer more effectively accessing the wider channel of HDAC6 compared to other HDAC subtypes as well as hydrophobic capping groups interacting with the protein surface near the rim of the active site.

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Inhibition of the proteasome by covalent inhibitors is a clinically proven anti-cancer therapy. We report here that dipeptides with a P3 neopentyl Asn residue are potent, reversible, non-covalent inhibitors selective for the chymotryptic activity of the 20S proteasome in vitro and in cells. The X-ray structure of compound 20 in complex with yeast 20S reveals the importance of hydrophobic bonding interactions of the neopentyl group within the S3 binding pocket of the 20S b5 sub-unit. Four compounds show comparable potencies to boronic acid inhibitors in a panel of assays.

Adenine phosphoribosyltransferase plays a role in purine salvage by catalyzing the direct conversion of adenine to adenosine monophosphate. The involvement of the purine salvage pathway in tumor proliferation and angiogenesis makes adenine phosphoribosyltransferase a potential target for oncology drug discovery. We have expressed and characterized recombinant, N-terminally His-tagged human adenine phosphoribosyltransferase. Two assay formats were assessed for use in a high throughput screen: a spectrophotometric-based enzyme-coupled assay system and a radiometric ionic capture scintillation proximity bead assay format. Ultimately, the scintillation proximity assay format was chosen because of automated screening compatibility limitations of the coupled assay. We describe here the biochemical characterization of adenine phosphoribosyltransferase and the development of a robust, homogeneous, 384-well assay suitable for high throughput screening.