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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.

Graphical abstract, ERAP1 is a zinc-dependent M1-aminopeptidase that trims lipophilic amino acids from the N-terminus of peptides. Owing to its importance in the processing of antigens and regulation of the adaptive immune response, dysregulation of the highly polymorphic ERAP1 has been implicated in autoimmune disease and cancer. To test this hypothesis and establish the role of ERAP1 in these disease areas, high affinity, cell permeable and selective chemical probes are essential. DG013A 1, is a phosphinic acid tripeptide mimetic inhibitor with reported low nanomolar affinity for ERAP1. However, this chemotype is a privileged structure for binding to various metal-dependent peptidases and contains a highly charged phosphinic acid moiety, so it was unclear whether it would display the high selectivity and passive permeability required for a chemical probe. Therefore, we designed a new stereoselective route to synthesize a library of DG013A 1 analogues to determine the suitability of this compound as a cellular chemical probe to validate ERAP1 as a drug discovery target.

The exon junction complex (EJC) is the main mechanism by which cells select specific mRNAs\ud for translation into protein. We hypothesized that the EJC is involved in the regulation\ud of gene expression during the stress response in cardiac myocytes, with implications for\ud the failing heart. In cultured rat neonatal myocytes, we examined the cellular distribution\ud of two EJC components eukaryotic translation initiation factor 4A isoform 3 (eIF4A3) and\ud mago nashi homologue (Mago) in response to metabolic stress. There was significant relocalization\ud of eIF4A3 and Mago from the nucleus to cytoplasm following 18 h of hypoxia.\ud Treating myocytes with 50 mM NaN3 for 4 h to mimic the metabolic stress induced by hypoxia\ud also resulted in significant relocalization of eIF4A3 and Mago to the cytoplasm. To\ud examine whether the effects of metabolic stress on the EJC proteins were dependent on\ud the metabolic sensor AMP kinase (AMPK), we treated myocytes with 1 μM dorsomorphin\ud (DM) in combination with NaN3. DM augmented the translocation of Mago and eIF4A3 from\ud the nucleus to the cytoplasm. Knockdown of eIF4A3 resulted in cessation of cell contractility\ud 96 h post-treatment and a significant reduction in the number of intact sarcomeres.\ud Cell area was significantly reduced by both hypoxia and eIF4A3 knockdown, whilst eIF4A3\ud knockdown also significantly reduced nuclear size. The reduction in nuclear size is unlikely\ud to be related to apoptosis as it was reversed in combination with hypoxia. These data suggest\ud for the first time that eIF4A3 and potentially other EJC members play an important role\ud in the myocyte stress response, cell contractility and morphology.

We have examined the effects of the bacterial toxin microcin B17 (MccB17) on the reactions of Escherichia coli DNA gyrase. MccB17 slows down but does not completely inhibit the DNA supercoiling and relaxation reactions of gyrase. A kinetic analysis of the cleavage-religation equilibrium of gyrase was performed to determine the effect of the toxin on the forward (cleavage) and reverse (religation) reactions. A simple mechanism of two consecutive reversible reactions with a nicked DNA intermediate was used to simulate the kinetics of cleavage and religation. The action of MccB17 on the kinetics of cleavage and religation was compared with that of the quinolones ciprofloxacin and oxolinic acid. With relaxed DNA as substrate, only a small amount of gyrase cleavage complex is observed with MccB17 in the absence of ATP, whereas the presence of the nucleotide significantly enhances the effect of the toxin on both the cleavage and religation reactions. In contrast, ciprofloxacin, oxolinic acid, and Ca2+ show lesser dependence on ATP to stabilize the cleavage complex. MccB17 enhances the overall rate of DNA cleavage by increasing the forward rate constant (k 2) of the second equilibrium. In contrast, ciprofloxacin increases the amount of cleaved DNA by a combined effect on the forward and reverse rate constants of both equilibria. Based on these results and on the observations that MccB17 only slowly inhibits the supercoiling and relaxation reactions, we suggest a model of the interaction of MccB17 with gyrase.

Microcin B17 (MccB17) is a peptide-based bacterial toxin that targets DNA gyrase, the bacterial enzyme that introduces supercoils into DNA. The site and mode of action of MccB17 on gyrase are unclear. We review what is currently known about MccB17-gyrase interactions and summarise approaches to understanding its mode of action that involve modification of the toxin. We describe experiments in which treatment of the toxin at high pH leads to the deamidation of two asparagine residues to aspartates. The modified toxin was found to be inactive in vivo and in vitro, suggesting that the Asn residues are essential for activity. Following on from these studies we have used molecular modelling to suggest a 3D structure for microcin B17. We discuss the implications of this model for MccB17 action and investigate the possibility that it binds metal ions.