JQ1 is a BET-bromodomain inhibitor that has immunomodulatory effects. However, the precise molecular mechanism that JQ1 targets to elicit changes in antibody production is not understood. Our results show that JQ1 induces apoptosis, reduces cell proliferation, and as a consequence, inhibits antibody-secreting cell differentiation. ChIP-sequencing reveals a selective displacement of Brd4 in response to acute JQ1 treatment (<2 h), resulting in specific transcriptional repression. After 8 h, subsequent alterations in gene expression arise as a result of the global loss of Brd4 occupancy. We demonstrate that apoptosis induced by JQ1 is solely attributed to the pro-apoptotic protein Bim (Bcl2l11). Conversely, cell-cycle regulation by JQ1 is associated with multiple Myc-associated gene targets. Our results demonstrate that JQ1 drives temporal changes in Brd4 displacement that results in a specific transcriptional profile that directly affects B cell survival and proliferation to modulate the humoral immune response.

Abstract The role of VEGFR3 in lymphangiogenesis has been well established. Targeting VEGFR3 has been shown to curtail tumor progression mediated via lymphatic dissemination. More recently VEGFR3 was shown to play an important role in the mediation of tumor-induced immune cell tolerance. We have identified BL-011256, a novel inhibitor of VEGFR3 that exhibits 17-fold selectivity over VEGFR2 and a narrow tyrosine kinase inhibition spectrum. A 7-day b.i.d repeat oral dose study showed that BL-011256 is well tolerated in mice. Mouse plasma exposure experiments demonstrated that BL-011256 attains free drug plasma concentration levels that exceed the concentration required for IC50 activity on VEGFR3 in vitro but are considerably lower than the in vitro IC50 required for activity on VEGFR2. In the B16F10 mouse melanoma model, animals bearing melanoma tumors displayed considerably attenuated signs of tumor progression when treated with BL-011256. BL-011256 caused a 70% reduction in primary lesion growth and a 50% reduction in metastasis to the draining lymph node. Furthermore, BL-011256 was active in reducing the number of satellite in-transit metastases. Immunohistochemical whole mount analyses on ears with primary tumor lesions derived from BL-011256 treated and vehicle-treated mice using Lyve-1 for the identification of lymphatic vessels and CD31 for the identification of blood vessels was conducted. Tumors in vehicle-treated mice displayed a peri-tumoral area densely populated by lymphatic vessels. In contrast, tumors derived from BL-011256 treated mice were devoid of peri-tumoral lymphatic vessels. Notably, both vehicle-treated and BL-011256-treated animals displayed similar staining for peritumoral blood vessels, suggesting no effect on blood vessels (this is consistent with no activity on VEGFR2). Furthermore, PK sampling during the last day of dosing in a 14-day dosing schedule demonstrated that there is no compound accumulation during the repeat dosing schedule utilised in the B16F10 tumour efficacy experiment. In conclusion BL-011256 has been identified as a selective inhibitor of VEGFR3 that supresses both primary tumor growth and lymph node metastasis. Citation Format: Annabell Leske, Richard Foitzik, Donna Beaumont, John Bentley, Ylva Bergman, Chloe Brown, Michelle Camerino, Susan Charman, Neil Choi, Melanie De Silva, Matthew Chung, Hendrik Falk, Danny Ganame, Alison Gregg, Julian Grusovin, Andrew Harvey, Catherine Hemley, Ian Holmes, Belinda Huff, Daniel Inglis, Wilhelmus Kersten, Tina Lavranos, Romina Lessene, Gillian Lunniss, Brendon Monahan, Benjamin Morrow, Marica Nikac, George Nikolakopoulos, Dharam Paul, Tom Peat, Justin Ripper, Michaela Scherer, Paul Stupple, Karen White, Ian Street, Gabriel Kremmidiotis. BL-011256 is a novel VEGFR3 selective inhibitor, which suppresses tumor lymphatics and lymph node metastasis in an animal model of melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4029. doi:10.1158/1538-7445.AM2014-4029

A library of 14-helical hexa β(3)-peptides was synthesized in order to determine the influence of sequence variation as well as staple size and location on conformational stability. From this study we show that appropriately stapled hexa-β(3)-peptides can allow for a number of variations without significant perturbation of the 14-helix.

Abstract Abstract 2129 The developmental switch in human β-like globin gene subtype from fetal (γ) to adult (β) that begins at birth foreshadows the onset of the hemoglobinopathies, β-thalassemia and sickle cell disease (SCD). In the clinical setting it is established that β-thalassemia and SCD patients with hereditary persistence of fetal hemoglobin mutations enjoy a significant amelioration of disease severity due to the continued expression of γ-globin. This has prompted the search for therapeutic strategies to reverse γ-globin gene silencing. Central to the mechanism of γ-gene silencing is DNA methylation, which marks critical CpG dinucleotides flanking the γ-gene transcriptional initiation site in adult bone marrow erythroid cells. These marks are established by recruitment of DNMT3A, a DNA methyltransferase, to the γ-globin promoter by protein arginine methyltransferase 5 (PRMT5)[Zhao Q et al. Nat Struct Mol Biol. 2009;16(3):304–311]. PRMT5 catalyses the symmetric dimethylation of arginine 3 of Histone 4 (H4R3me2), which serves as a template for direct binding of DNMT3A and the subsequent DNA methylation of the γ-gene promoter. Loss of PRMT5 or its enzymatic activity is sufficient to induce demethylation of the CpG dinucleotides and reactivation of γ-globin gene expression [Rank, G., et al. Blood, 116(9), 1585–92]. Based on these observations we hypothesize that small molecule inhibitors of PRMT5 activity could provide a beneficial treatment for β-thalassemia and SCD. To identify small molecule inhibitors of PRMT5 a high throughput screen (HTS) was performed. Both radiometric and non-radiometric assay formats were developed to support the screening campaign. The radiometric assay format measures the ability of PRMT5 purified from K562 cells to catalyse the labelling of a short peptide based on the N-terminal sequence of Histone H4 by 3H-Methyl-S-Adenosyl-L-methionine (SAM). In contrast, the non-radiometric assay format employs recombinant PRMT5/MEP50 and measures the production of S-adenosyl-L-homocysteine (SAH), which is generated by PRMT5-catalysed methylation of H4 peptide. SAH is measured with Transcreener EPIGEN” and the assay is formatted in 1536-well microtitre plates in a total assay volume of 4 μL. Using these assays, a chemical library of 350,000 lead-like molecules and known pharmacologically active agents was screened to identify inhibitors of PRMT5 methyltransferase activity. A number of compounds with low micromolar or submicromolar inhibitory activity were identified by the HTS campaign, and six were selected for re-synthesis. The inhibitory activity of five of the six compounds was confirmed. To provide an initial appraisal of inhibitor selectivity the five active compounds were subsequently tested against a panel of enzymes consisting of 23 protein and DNA methyltransferases and 12 kinases. These compounds were found to be remarkably selective PRMT5 inhibitors, inhibition of MLL4 being the only significant off-target activity noted for one of the scaffolds. We have established a critical path for selection and progression of new chemical analogues which entails testing the compounds for: i) inhibition of PRMT5, other protein methyl transferases and kinases; ii) the ability to induce expression of γ-globin mRNA in the K562 erythroleukemic cell line; iii) the ability to induce expression of γ-globin mRNA in adult bone marrow erythroid cells; and iv) the induction of γ-globin in vivo in β-YAC mice, a transgenic model which carries the 250-kb human globin locus. In parallel, the physicochemical, metabolism, and pharmacokinetic properties of the most promising compounds are also determined. Medicinal chemistry efforts have now produced molecules with > 100-fold increased inhibitory potency against PRMT5 compared to the original hits, and preliminary results indicate that the more potent compounds have the ability to induce γ-globin mRNA in our cell based models. These early results illustrate the potential of PRMT5 inhibitors as a novel approach for the treatment of β-thalassemia and sickle cell disease. Disclosures: No relevant conflicts of interest to declare.

The key tricyclic intermediate 3a, for the total synthesis of the C(20)-nor analogue of salvinorin A, was prepared in seven steps from 3-furaldehyde. Key steps involved a highly regio- and diastereoselective Lewis acid assisted Diels-Alder reaction followed by base-promoted epimerization and a completely stereoselective conjugate reduction.

The first synthesis of carbon-stapled beta(3)-peptides is reported. The precursor beta(3)-peptides, with O-allyl beta-serines located in an i/i+3 relationship, were prepared on solid phase. We show that efficient ring-closing metathesis (RCM) of these new beta(3)-peptides proceeds smoothly either in solution or on an appropriate solid support. All products were generated with high selectivity for the E-isomer.

An extremely simple method for preparing imines from aryl and alkylaryl ketones and a variety of amines is presented. The process involves the diffusion of the appropriate amine vapor from one arm of an H-tube into the other arm containing the ketone. Most reactions are quantitative, and, in many cases, there is no real work-up or purification required.

To understand how folded polymers, such as proteins behave has inspired widespread interest in unnatural oligomers that are known to display similar folding patterns and are referred to as foldamers1. β3-Peptides foldamers, (oligomers of β3-amino acids) are among the most widely studied foldamers at present, and have been reported to adopt discrete and stable secondary helical structures. Of the many types of helices formed by β3-peptides, 14-helices are particularly interesting as they have almost 3 residues per helical turn (with a pitch of 5.0A). This allows the side chains to be aligned perfectly in three faces. The residues at i/i+3 positions are not only on the same face of the helix but are also are relatively close in proximity to each other. The aim of this study was to explore synthetic strategies for the synthesis of novel β3-peptides on which further functionalisation on specific faces of the helix can be performed and used to create novel foldamer assemblies. β3-Peptides were synthesised incorporating O-allyl-β3-serines and allyl-β3-glycines at i/i+3 positions and efficiently “stapled” the allyl groups on β3-serines and β3-glycines via Ring Closing Metathesis (RCM) on a solid support. The CD and 2D NMR analysis showed that β3-peptides, “stapled” as well as “unstapled”, exhibited spectra in various solvents consistent with 14 helix signature. Thus, the “stapled” side chains on the β-peptides can now be used as a template to attach functional groups to generate new molecules of biological significance.

Two concise routes for the synthesis of the title amino acid have been developed. The first route employs Seebach's general approach [Seebach, D.; Lelais, G.; Micuch, P.; Josien-Lefebvre, D.; Rossi, F. Helv. Chim. Acta 2004, 87, 3131] with Arndt Eistert homologation of Boc-alpha-allyl alpha-serine as the key process. The second route employs an approach using Boc-alpha-aspartic acid as a starting material [Salzmann, T.N.; Ratcliffe, R.W.; Christensen, B.G.; Bouffard, F.A.J Am. Chem. Soc. 1980, 102, 6163; Rodriguez, M.; Llinares, M.; Doulut, S.; Heitz, A.; Martinez, J. Tetrahedron. Lett. 1991, 32, 923] with a selective palladium-catalysed O-allylation [Haight, A.R: Stoner, EJ.; Peterson, MJ.: Grover, V.K.J. Org. Chem. 2003, 68, 8092] as key processes. These two routes are evaluated for their relative efficiency and safety. (C) 2008 Elsevier Ltd. All rights reserved.

Primary imines can be readily prepared from (2-hydroxyaryl) ketones by reaction with ammonium iodide and piperidine under solvent-free conditions.