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To identify new chemical series with enhanced binding affinity to the BTB domain of B-cell lymphoma 6 protein, we targeted a subpocket adjacent to Val18. With no opportunities for strong polar interactions, we focused on attaining close shape complementarity by ring fusion onto our quinolinone lead series. Following exploration of different sized rings, we identified a conformationally restricted core which optimally filled the available space, leading to potent BCL6 inhibitors. Through X-ray structure-guided design, combined with efficient synthetic chemistry to make the resulting novel core structures, a300-fold improvement in activity was obtained by the addition of seven heavy atoms.

The transcriptional repressor BCL6 is an oncogenic driver found to be deregulated in lymphoid malignancies. Herein, we report the optimization of our previously reported benzimidazolone molecular glue-type degrader

We describe the optimization of modestly active starting points to potent inhibitors of BCL6 by growing into a subpocket, which was occupied by a network of five stably bound water molecules. Identifying potent inhibitors required not only forming new interactions in the subpocket but also perturbing the water network in a productive, potency-increasing fashion while controlling the physicochemical properties. We achieved this goal in a sequential manner by systematically probing the pocket and the water network, ultimately achieving a 100-fold improvement of activity. The most potent compounds displaced three of the five initial water molecules and formed hydrogen bonds with the remaining two. Compound 25 showed a promising profile for a lead compound with submicromolar inhibition of BCL6 in cells and satisfactory pharmacokinetic (PK) properties. Our work highlights the importance of finding productive ways to perturb existing water networks when growing into solvent-filled protein pockets.

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.

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.

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.

Summary Rhomboids are evolutionarily conserved serine proteases that cleave transmembrane proteins within the membrane. The increasing number of known rhomboid functions in prokaryotes and eukaryotes makes them attractive drug targets. Here, we describe structures of the Escherichia coli rhomboid GlpG in complex with β-lactam inhibitors. The inhibitors form a single bond to the catalytic serine and the carbonyl oxygen of the inhibitor faces away from the oxyanion hole. The hydrophobic N-substituent of β-lactam inhibitors points into a cavity within the enzyme, providing a structural explanation for the specificity of β-lactams on rhomboid proteases. This same cavity probably represents the S2′ substrate binding site of GlpG. We suggest that the structural changes in β-lactam inhibitor binding reflect the state of the enzyme at an initial stage of substrate binding to the active site. The structural insights from these enzyme-inhibitor complexes provide a starting point for structure-based design for rhomboid inhibitors., Graphical Abstract, Highlights • Structures of E. coli GlpG, in complex with three different β-lactam inhibitors • The acyl enzyme structures define the S2′ substrate binding site in GlpG • Biochemical analysis reveals a preference for large hydrophobic groups at S2′ cavity The GlpG-β-lactam structures reveal the changes essential for formation of S2′ cavity, The structures of the E. coli rhomboid GlpG, an intramembrane serine protease in complex with β-lactam inhibitors, reported by Vinothkumar et al., reveal the nature and formation of the S2′ cavity. The differences in this cavity may form the basis of substrate/inhibitor specificity and structure-based drug design.

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.

Microcin B17 (MccB17) is a DNA gyrase poison; in previous work, this bacterial toxin was found to slowly and incompletely inhibit the reactions of supercoiling and relaxation of DNA by gyrase and to stabilize the cleavage complex, depending on the presence of ATP and the DNA topology. We now show that the action of MccB17 on the gyrase ATPase reaction and cleavage complex formation requires a linear DNA fragment of more than 150 base pairs. MccB17 is unable to stimulate the ATPase reaction by stabilizing the weak interactions between short linear DNA fragments (70 base pairs or less) and gyrase, in contrast with the quinolone ciprofloxacin. However, MccB17 can affect the ATP-dependent relaxation of DNA by gyrase lacking its DNA-wrapping or ATPase domains. From these findings, we propose a mode of action of MccB17 requiring a DNA molecule long enough to allow the transport of a segment through the DNA gate of the enzyme. Furthermore, we suggest that MccB17 may trap a transient intermediate state of the gyrase reaction present only during DNA strand passage and enzyme turnover. The proteolytic signature of MccB17 from trypsin treatment of the full enzyme requires DNA and ATP and shows a protection of the C-terminal 47-kDa domain of gyrase, indicating the involvement of this domain in the toxin mode of action and consistent with its proposed role in the mechanism of DNA strand passage. We suggest that the binding site of MccB17 is in the C-terminal domain of GyrB.

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.

Ornithine is an allosteric activator of carbamoyl phosphate synthetase (CPS) from Escherichia coli. Nine amino acids in the vicinity of the binding sites for ornithine and potassium were mutated to alanine, glutamine, or lysine. The residues E783, T1042, and T1043 were found to be primarily responsible for the binding of ornithine to CPS, while E783 and E892, located within the carbamate domain of the large subunit, were necessary for the transmission of the allosteric signals to the active site. In the K loop for the binding of the monovalent cation potassium, only E761 was crucial for the exhibition of the allosteric effects of ornithine, UMP, and IMP. The mutations H781K and S792K altered significantly the allosteric properties of ornithine, UMP, and IMP, possibly by modifying the conformation of the K-loop structure. Overall, these mutations affected the allosteric properties of ornithine and IMP more than those of UMP. The mutants S792K and D1041A altered the allosteric regulation by ornithine and IMP in a similar way, suggesting common features in the activation mechanism exhibited by these two effectors.

The catalytic activity of carbamoyl phosphate synthetase (CPS) from Escherichia coli is allosterically regulated by UMP, IMP, and ornithine. Thirteen amino acids within the domain that harbors the overlapping binding sites for IMP and UMP were mutated to alanine and characterized. The four residues that interact directly with the phosphate moiety of IMP in the X-ray crystal structure (K954, T974, T977, and K993) were shown to have the greatest impact on the dissociation constants for the binding of IMP and UMP and the associated allosteric effects on the kinetic constants of CPS. Of the four residues that interact with the ribose moiety of IMP (S948, N1015, T1017, and S1026), S1026 was shown to be more important for the binding of IMP than UMP. Five residues (V994, I1001, D1025, V1028, and I1029) were mutated in the region of the allosteric domain that surrounds the hypoxanthine ring of IMP. With the exception of V994A, these mutations had a modest influence on the binding and subsequent allosteric effects by UMP and IMP.