Publications
37 results found
Low JKK, Patel K, Jones N, et al., 2023, mRNA display reveals a class of high-affinity bromodomain-binding motifs that are not found in the human proteome., J Biol Chem, Vol: 299
Bromodomains (BDs) regulate gene expression by recognizing protein motifs containing acetyllysine. Although originally characterized as histone-binding proteins, it has since become clear that these domains interact with other acetylated proteins, perhaps most prominently transcription factors. The likely transient nature and low stoichiometry of such modifications, however, has made it challenging to fully define the interactome of any given BD. To begin to address this knowledge gap in an unbiased manner, we carried out mRNA display screens against a BD-the N-terminal BD of BRD3-using peptide libraries that contained either one or two acetyllysine residues. We discovered peptides with very strong consensus sequences and with affinities that are significantly higher than typical BD-peptide interactions. X-ray crystal structures also revealed modes of binding that have not been seen with natural ligands. Intriguingly, however, our sequences are not found in the human proteome, perhaps suggesting that strong binders to BDs might have been selected against during evolution.
Williams J, Walport L, 2023, PADI6: What we know about the elusive fifth member of the peptidyl arginine deiminase family, Philosophical Transactions of the Royal Society B: Biological Sciences, Vol: 378, ISSN: 0962-8436
Peptidyl arginine deiminase 6 (PADI6) is a maternal factor that is vital for early embryonic development. Deletion and mutations of its encoding gene in female mice or women lead to early embryonic developmental arrest, female infertility, maternal imprinting defects and hyperproliferation of the trophoblast. PADI6 is the fifth and least well characterised member of the peptidyl arginine deiminases (PADIs) that catalyse the post-translational conversion of arginine to citrulline. It is less conserved than the other PADIs, and currently has no reported catalytic activity. Whilst there are many suggested functions of PADI6 in the early mouse embryo including in embryonic genome activation, cytoplasmic lattice formation, maternal mRNA and ribosome regulation, and organelle distribution, the molecular mechanisms of its function remain unknown. In this review we discuss what is known about the function of PADI6 and highlight key outstanding questions that must be answered if we are to understand the crucial role it plays in early embryo development and female fertility.
Franck C, Patel K, Walport L, et al., 2023, Discovery and characterization of cyclic peptides selective for the C-terminal bromodomains of BET family proteins, Structure, Vol: 31, Pages: 912-923.e4, ISSN: 0969-2126
DNA-encoded cyclic peptide libraries can yield high-potency, high-specificity ligands against target proteins. We used such a library to seek ligands that could distinguish between paralogous bromodomains from the closely related Bromodomain and ExtraTerminal domain(BET) family of epigenetic regulators. Several peptides isolated from a screen against the C-terminal bromodomain of BRD2, together with new peptides discovered in previous screens against the corresponding domain from BRD3 and BRD4, bound their targets with nanomolar and sub-nanomolar affinities. X-ray crystal structures of several of these bromodomain-peptidecomplexes reveal diverse structures and binding modes, which nevertheless display severalconserved features. Some peptides demonstrate significant paralogue-level specificity, thoughthe physicochemical explanations for this specificity are often not clear. Our data demonstratethe power of cyclic peptides to discriminate between very similar proteins with high potencyand hint that differences in conformational dynamics might modulate the affinity of thesedomains for particular ligands.
Walport L, Wu Y, Bertran MT, et al., 2023, Identification of photocrosslinking peptide ligands by mRNA display, Communications Chemistry, Vol: 6, Pages: 1-9, ISSN: 2399-3669
Photoaffinity labelling is a promising method for studying protein-ligand interactions. However, obtaining a specific, efficient crosslinker can require significant optimisation. We report a modified mRNA display strategy, photocrosslinking-RaPID (XL-RaPID), and exploit its ability to accelerate the discovery of cyclic peptides that photocrosslink to a target of interest. As a proof of concept, we generated a benzophenone-containing library and applied XL-RaPID screening against a model target, the second bromodomain of BRD3. This crosslinking screening gave two optimal candidates that selectively labelled the target protein in cell lysate. Overall, this work introduces direct photocrosslinking screening as a versatile technique for identifying covalent peptide ligands from mRNA display libraries incorporating reactive warheads.
Walport LJ, Low JKK, Matthews JM, et al., 2021, The characterization of protein interactions - what, how and how much?, CHEMICAL SOCIETY REVIEWS, Vol: 50, Pages: 12292-12307, ISSN: 0306-0012
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- Citations: 13
Wu Y, Bertran MT, Rowley J, et al., 2021, Fluorescent amino acid initiated de novo cyclic peptides for the label‐free assessment of cell permeability, ChemMedChem, Vol: 16, Pages: 3185-3188, ISSN: 1860-7179
The major obstacle in applying peptides to intracellular targets is their low inherent cell permeability. Standard approaches to attach a fluorophore (e.g. FITC, TAMRA) can change the physicochemical properties of the parent peptide and influence their ability to penetrate and localize in cells. We report a label-free strategy for evaluating the cell permeability of cyclic peptide leads. Fluorescent tryptophan analogues 4-cyanotryptophan (4CNW) and beta-(1-azulenyl)-L-alanine (AzAla) were incorporated into in vitro translated macrocyclic peptides by initiator reprogramming. We then demonstrate these efficient blue fluorescent emitters are good tools for monitoring peptide penetration into cells.
Wu Y, Bertran Domingo T, Rowley J, et al., 2021, Fluorescent Amino Acid Initiated De Novo Cyclic Peptides for the Label-Free Assessment of Cell Permeability
<jats:p>The major obstacle in applying peptides to intracellular targets is their low inherent cell permeability. Standard approaches to attach a fluorophore (e.g. FITC, TAMRA) can change the physicochemical properties of the parent peptide and influence their ability to penetrate and localize in cells. We report a label-free strategy for evaluating the cell permeability of cyclic peptide leads. Fluorescent tryptophan analogues 4-cyanotryptophan (4CNW) and beta-(1-azulenyl)-L-alanine (AzAla) were incorporated into <i>in vitro</i> translated macrocyclic peptides by initiator reprogramming. We then demonstrate these efficient blue fluorescent emitters are good tools for monitoring peptide penetration into cells.</jats:p>
Wu Y, Bertran Domingo T, Rowley J, et al., 2021, Fluorescent Amino Acid Initiated De Novo Cyclic Peptides for the Label-Free Assessment of Cell Permeability, Publisher: Elsevier BV
<jats:p>The major obstacle in applying peptides to intracellular targets is their low inherent cell permeability. Standard approaches to attach a fluorophore (e.g. FITC, TAMRA) can change the physicochemical properties of the parent peptide and influence their ability to penetrate and localize in cells. We report a label-free strategy for evaluating the cell permeability of cyclic peptide leads. Fluorescent tryptophan analogues 4-cyanotryptophan (4CNW) and beta-(1-azulenyl)-L-alanine (AzAla) were incorporated into <i>in vitro</i> translated macrocyclic peptides by initiator reprogramming. We then demonstrate these efficient blue fluorescent emitters are good tools for monitoring peptide penetration into cells.</jats:p>
Wu Y, Williams J, Calder EDD, et al., 2021, Strategies to expand peptide functionality through hybridisation with a small molecule component, RSC Chemical Biology, Vol: 2, Pages: 151-165, ISSN: 2633-0679
Combining different compound classes gives molecular hybrids that can offer access to novel chemical space and unique properties. Peptides provide ideal starting points for such molecular hybrids, which can be easily modified with a variety of molecular entities. The addition of small molecules can improve the potency, stability and cell permeability of therapeutically relevant peptides. Furthermore, they are often applied to create peptide-based tools in chemical biology. In this review, we discuss general methods that allow the discovery of this compound class and highlight key examples of peptide–small molecule hybrids categorised by the application and function of the small molecule entity.
Hsu K-F, Wilkins S, Hopkinson R, et al., 2021, Hypoxia and hypoxia mimetics differentially modulate histone post-translational modifications, Epigenetics, Vol: 16, Pages: 14-27, ISSN: 1559-2294
Post-translational modifications (PTMs) to the tails of the core histone proteins are critically involved in epigenetic regulation. Hypoxia affects histone modifications by altering the activities of histone-modifying enzymes and the levels of hypoxia-inducible factor (HIF) isoforms. Synthetic hypoxia mimetics promote a similar response, but how accurately the hypoxia mimetics replicate the effects of limited oxygen availability on the levels of histone PTMs is uncertain. Here we report studies on the profiling of the global changes to PTMs on intact histones in response to hypoxia/hypoxia-related stresses using liquid chromatography-mass spectrometry (LC-MS). We demonstrate that intact protein LC-MS profiling is a relatively simple and robust method for investigating potential effects of drugs on histone modifications. The results provide insights into the profiles of PTMs associated with hypoxia and inform on the extent to which hypoxia and hypoxia mimetics cause similar changes to histones. These findings imply chemically-induced hypoxia does not completely replicate the substantial effects of physiological hypoxia on histone PTMs, highlighting that caution should be used in interpreting data from their use.
Walport L, Patel K, Walshe JL, et al., 2020, Cyclic peptides can engage a single binding pocket through highly divergent modes, Proceedings of the National Academy of Sciences of USA, Vol: 117, Pages: 26728-46738, ISSN: 0027-8424
Cyclic peptide library screening technologies show immense promise for identifying drug leads and chemical probes for challenging targets. However, the structural and functional diversity encoded within such libraries is largely undefined. We have systematically profiled the affinity, selectivity and structural features of library-derived cyclic peptides selected to recognize three closely related targets: the acetyllysine-binding bromodomain proteins BRD2, -3 and -4. We report affinities as low as 100 pM and specificities of up to 106-fold. Crystal structures of 13 peptide-bromodomain complexes reveal remarkable diversity in both structure and binding mode, including both α-helical and β-sheet structures as well as bivalent binding modes. The peptides can also exhibit a high degree of structural pre-organization. Our data demonstrate the enormous potential within these libraries to provide diverse binding modes against a single target, which underpins their capacity to yield highly potent and selective ligands.
Bello C, Hartrampf N, Walport LJ, et al., 2019, Protein Chemistry Looking Ahead: 8th Chemical Protein Synthesis Meeting 16-19 June 2019, Berlin, Germany, CELL CHEMICAL BIOLOGY, Vol: 26, Pages: 1349-1354, ISSN: 2451-9448
Walport LJ, Schofield CJ, 2018, Adventures in Defining Roles of Oxygenases in the Regulation of Protein Biosynthesis, CHEMICAL RECORD, Vol: 18, Pages: 1760-1781, ISSN: 1527-8999
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- Citations: 2
Walport LJ, Hopkinson RJ, Chowdhury R, et al., 2018, Mechanistic and structural studies of KDM-catalysed demethylation of histone 1 isotype 4 at lysine 26, FEBS Letters, Vol: 592, Pages: 3264-3273, ISSN: 0014-5793
N‐Methylation of lysyl residues is widely observed on histone proteins. Using isolated enzymes, we report mechanistic and structural studies on histone lysine demethylase (KDM)‐catalysed demethylation of Nε‐methylated lysine 26 on histone 1 isotype 4 (H1.4). The results reveal that methylated H1.4K26 is a substrate for all members of the KDM4 subfamily and that KDM4A‐catalysed demethylation of H1.4K26me3 peptide is similarly efficient to that of H3K9me3. Crystallographic studies of an H1.4K26me3:KDM4A complex reveal a conserved binding geometry to that of H3K9me3. In the light of the high activity of the KDM4s on this mark, our results suggest JmjC KDM‐catalysed demethylation of H1.4K26 may be as prevalent as demethylation on the H3 tail and warrants further investigation in cells.
Hopkinson RJ, Langley GW, Belle R, et al., 2018, Human histone demethylase KDM6B can catalyse sequential oxidations, Chemical Communications, Vol: 54, Pages: 7975-7978, ISSN: 1359-7345
Jumonji domain-containing demethylases (JmjC-KDMs) catalyse demethylation of Nε-methylated lysines on histones and play important roles in gene regulation. We report selectivity studies on KDM6B (JMJD3), a disease-relevant JmjC-KDM, using synthetic lysine analogues. The results unexpectedly reveal that KDM6B accepts multiple Nε-alkylated lysine analogues, forming alcohol, aldehyde and carboxylic acid products.
Walport LJ, Obexer R, Suga H, 2017, Strategies for transitioning macrocyclic peptides to cell permeable drug leads, CURRENT OPINION IN BIOTECHNOLOGY, Vol: 48, Pages: 242-250, ISSN: 0958-1669
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- Citations: 50
Obexer R, Walport LJ, Suga H, 2017, Exploring sequence space: harnessing chemical and biological diversity towards new peptide leads, CURRENT OPINION IN CHEMICAL BIOLOGY, Vol: 38, Pages: 52-61, ISSN: 1367-5931
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- Citations: 56
Kawamura A, Muenzel M, Kojima T, et al., 2017, Highly selective inhibition of histone demethylases by de novo macrocyclic peptides, Nature Communications, Vol: 8, ISSN: 2041-1723
The JmjC histone demethylases (KDMs) are linked to tumour cell proliferation and are current cancer targets; however, very few highly selective inhibitors for these are available. Here we report cyclic peptide inhibitors of the KDM4A-C with selectivity over other KDMs/2OG oxygenases, including closely related KDM4D/E isoforms. Crystal structures and biochemical analyses of one of the inhibitors (CP2) with KDM4A reveals that CP2 binds differently to, but competes with, histone substrates in the active site. Substitution of the active site binding arginine of CP2 to N-ɛ-trimethyl-lysine or methylated arginine results in cyclic peptide substrates, indicating that KDM4s may act on non-histone substrates. Targeted modifications to CP2 based on crystallographic and mass spectrometry analyses results in variants with greater proteolytic robustness. Peptide dosing in cells manifests KDM4A target stabilization. Although further development is required to optimize cellular activity, the results reveal the feasibility of highly selective non-metal chelating, substrate-competitive inhibitors of the JmjC KDMs.
Langley GW, Brinko A, Munzel M, et al., 2017, Probing the Charge and Conformational Requirements of JmjC Demethylases, Experimental Biology (EB) Annual Meeting, Publisher: WILEY, ISSN: 0892-6638
Walport LJ, Hopkinson RJ, Chowdhury R, et al., 2016, Arginine demethylation is catalysed by a subset of JmjC histone lysine demethylases, Nature Communications, Vol: 7, Pages: 1-12, ISSN: 2041-1723
While the oxygen-dependent reversal of lysine Nɛ-methylation is well established, the existence of bona fide Nω-methylarginine demethylases (RDMs) is controversial. Lysine demethylation, as catalysed by two families of lysine demethylases (the flavin-dependent KDM1 enzymes and the 2-oxoglutarate- and oxygen-dependent JmjC KDMs, respectively), proceeds via oxidation of the N-methyl group, resulting in the release of formaldehyde. Here we report detailed biochemical studies clearly demonstrating that, in purified form, a subset of JmjC KDMs can also act as RDMs, both on histone and non-histone fragments, resulting in formaldehyde release. RDM catalysis is studied using peptides of wild-type sequences known to be arginine-methylated and sequences in which the KDM’s methylated target lysine is substituted for a methylated arginine. Notably, the preferred sequence requirements for KDM and RDM activity vary even with the same JmjC enzymes. The demonstration of RDM activity by isolated JmjC enzymes will stimulate efforts to detect biologically relevant RDM activity.
Langley GW, Brinko A, Munzel M, et al., 2016, Analysis of JmjC Demethylase-Catalyzed Demethylation Using Geometrically-Constrained Lysine Analogues, ACS CHEMICAL BIOLOGY, Vol: 11, Pages: 755-762, ISSN: 1554-8929
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- Citations: 11
Walport LJ, Morra R, Mancini EJ, et al., 2015, <SUP>1</SUP>H, <SUP>13</SUP>C, and <SUP>15</SUP>N resonance assignments for the tandem PHD finger motifs of human CHD4, BIOMOLECULAR NMR ASSIGNMENTS, Vol: 9, Pages: 239-242, ISSN: 1874-2718
Walport LJ, Hopkinson RJ, Williams S, et al., 2015, Expanding the substrate scope of the Jumonji C histone demethylases, 40th Congress of the Federation-of-European-Biochemical-Societies (FEBS) - The Biochemical Basis of Life, Publisher: WILEY-BLACKWELL, Pages: 69-69, ISSN: 1742-464X
Thinnes CC, Tumber A, Yapp C, et al., 2015, Betti reaction enables efficient synthesis of 8-hydroxyquinoline inhibitors of 2-oxoglutarate oxygenases, CHEMICAL COMMUNICATIONS, Vol: 51, Pages: 15458-15461, ISSN: 1359-7345
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- Citations: 33
Hancock RL, Dunne K, Walport LJ, et al., 2015, Epigenetic regulation by histone demethylases in hypoxia, EPIGENOMICS, Vol: 7, Pages: 791-811, ISSN: 1750-1911
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- Citations: 96
Aik WS, Chowdhury R, Clifton IJ, et al., 2015, Introduction to Structural Studies on 2-Oxoglutarate-Dependent Oxygenases and Related Enzymes, 2-OXOGLUTARATE-DEPENDENT OXYGENASES, Editors: Hausinger, Schofield, Publisher: ROYAL SOC CHEMISTRY, Pages: 59-94, ISBN: 978-1-84973-950-4
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- Citations: 38
Williams ST, Walport LJ, Hopkinson RJ, et al., 2014, Studies on the catalytic domains of multiple JmjC oxygenases using peptide substrates, EPIGENETICS, Vol: 9, Pages: 1596-1603, ISSN: 1559-2294
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- Citations: 60
Walport LJ, Hopkinson RJ, Vollmar M, et al., 2014, Human UTY(KDM6C) Is a Male-specific <i>N</i><SUP>ε</SUP>-Methyl Lysyl Demethylase, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 289, Pages: 18302-18313, ISSN: 0021-9258
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- Citations: 140
Donohoe TJ, Jones CR, Kornahrens AF, et al., 2013, Total Synthesis of the Antitumor Antibiotic (±)-Streptonigrin: First- and Second-Generation Routes for de Novo Pyridine Formation Using Ring-Closing Metathesis, JOURNAL OF ORGANIC CHEMISTRY, Vol: 78, Pages: 12338-12350, ISSN: 0022-3263
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- Citations: 49
Suzuki T, Ozasa H, Itoh Y, et al., 2013, Identification of the KDM2/7 Histone Lysine Demethylase Subfamily Inhibitor and its Antiproliferative Activity, JOURNAL OF MEDICINAL CHEMISTRY, Vol: 56, Pages: 7222-7231, ISSN: 0022-2623
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- Citations: 62
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