Rhomboids are relatively recently discovered intramembrane serine proteases that are conserved throughout evolution. They have a wide range of biological functions, and there is also much speculation about their potential medical relevance. Although rhomboids are weakly inhibited by some broad-spectrum serine protease inhibitors, no potent and specific inhibitors have been identified for these enzymes, which are mechanistically distinct from and evolutionarily unrelated to the classical soluble serine proteases. Here we report a new biochemical assay for rhomboid function based on the use of quenched fluorescent substrate peptides. We have developed this assay into a high-throughput format and have undertaken an inhibitor and activator screen of approximately 58,000 small molecules. This has led to the identification of a new class of rhomboid inhibitors, a series of monocyclic β-lactams, which are more potent than any previous inhibitor. They show selectivity, both for rhomboids over the soluble serine protease chymotrypsin and also, importantly, between different rhomboids; they can inhibit mammalian as well as bacterial rhomboids; and they are effective both in vitro and in vivo. These compounds represent important templates for further inhibitor development, which could have an impact both on biological understanding of rhomboid function and potential future drug development.

Deregulation of the transcriptional repressor BCL6 enables tumorigenesis of germinal center B-cells, and hence BCL6 has been proposed as a therapeutic target for the treatment of diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of benzimidazolone inhibitors of the protein–protein interaction between BCL6 and its co-repressors. A subset of these inhibitors were found to cause rapid degradation of BCL6, and optimization of pharmacokinetic properties led to the discovery of 5-((5-chloro-2-((3R,5S)-4,4-difluoro-3,5-dimethylpiperidin-1-yl)­pyrimidin-4-yl)­amino)-3-(3-hydroxy-3-methylbutyl)-1-methyl-1,3-dihydro-2H-benzo­[d]­imidazol-2-one (CCT369260), which reduces BCL6 levels in a lymphoma xenograft mouse model following oral dosing.

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A plane gold-supported bilayer was prepared on an electrode by fusion of phospholipid (dimyristoylphosphatidylcholine (DMPC)) vesicles onto an alkanethiol (octadecylmercaptan (OM)) self-assembled monolayer (SAM). Escherichia coli pyruvate oxidase (Pox), a peripheral membrane enzyme, was incorporated into the supported bilayer. This supramolecular assembly was characterized by contact angle goniometry, electrochemical blocking studies, double-layer capacitance, and BIAlite (surface plasmon resonance) measurements. Electrochemistry of ferrocenemethanol at the gold surface was blocked by the well-ordered alkane chains of the OM monolayer. In order to prevent this blocking effect, dibenzyl disulfide (DBDS) was used to produce defect sites in the OM monolayer and to allow the reversibility of ferrocene electrochemistry. BIAlite measurements showed that fusion of DMPC on the OM + DBDS monolayer was not significantly different from the fusion of DMPC on the OM monolayer. Pox incorporation into the (OM + DBDS)/DMPC gold-supported bilayer was detected by BIAlite measurements. The activity of incorporated Pox was detected by the electrocatalytic current produced when substrate and the electron acceptor, ferricinium methanol, were present in solution.