mRNA display reveals a class of high-affinity bromodomain-binding motifs that are not found in the human proteome.

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.

Cyclic peptides can engage a single binding pocket through highly divergent modes.

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 ß-sheet structures as well as bivalent binding modes. The peptides can also exhibit a high degree of structural preorganization. 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.

The characterization of protein interactions - what, how and how much?

Protein interactions underlie most molecular events in biology. Many methods have been developed to identify protein partners, to measure the affinity with which these biomolecules interact and to characterise the structures of the complexes. Each approach has its own advantages and limitations, and it can be difficult for the newcomer to determine which methodology would best suit their system. This review provides an overview of many of the techniques most widely used to identify protein partners, assess stoichiometry and binding affinity, and determine low-resolution models for complexes. Key methods covered include: yeast two-hybrid analysis, affinity purification mass spectrometry and proximity labelling to identify partners; size-exclusion chromatography, scattering methods, native mass spectrometry and analytical ultracentrifugation to estimate stoichiometry; isothermal titration calorimetry, biosensors and fluorometric methods (including microscale thermophoresis, anisotropy/polarisation, resonance energy transfer, AlphaScreen, and differential scanning fluorimetry) to measure binding affinity; and crosslinking and hydrogen-deuterium exchange mass spectrometry to probe the structure of complexes.

Adventures in Defining Roles of Oxygenases in the Regulation of Protein Biosynthesis.

The 2-oxoglutarate (2OG) dependent oxygenases were first identified as having roles in the post-translational modification of procollagen in animals. Subsequently in plants and microbes, they were shown to have roles in the biosynthesis of many secondary metabolites, including signalling molecules and the penicillin/cephalosporin antibiotics. Crystallographic studies of microbial 2OG oxygenases and related enzymes, coupled to DNA sequence analyses, led to the prediction that 2OG oxygenases are widely distributed in aerobic biology. This personal account begins with examples of the roles of 2OG oxygenases in antibiotic biosynthesis, and then describes efforts to assign functions to other predicted 2OG oxygenases. In humans, 2OG oxygenases have been found to have roles in small molecule metabolism, as well as in the epigenetic regulation of protein and nucleic acid biosynthesis and function. The roles and functions of human 2OG oxygenases are compared, focussing on discussion of their substrate and product selectivities. The account aims to emphasize how scoping the substrate selectivity of, sometimes promiscuous, enzymes can provide insights into their functions and so enable therapeutic work.

Human UTY(KDM6C) is a male-specific Nϵ-methyl lysyl demethylase.

The Jumonji C lysine demethylases (KDMs) are 2-oxoglutarate- and Fe(II)-dependent oxygenases. KDM6A (UTX) and KDM6B (JMJD3) are KDM6 subfamily members that catalyze demethylation of N(ϵ)-methylated histone 3 lysine 27 (H3K27), a mark important for transcriptional repression. Despite reports stating that UTY(KDM6C) is inactive as a KDM, we demonstrate by biochemical studies, employing MS and NMR, that UTY(KDM6C) is an active KDM. Crystallographic analyses reveal that the UTY(KDM6C) active site is highly conserved with those of KDM6B and KDM6A. UTY(KDM6C) catalyzes demethylation of H3K27 peptides in vitro, analogously to KDM6B and KDM6A, but with reduced activity, due to point substitutions involved in substrate binding. The results expand the set of human KDMs and will be of use in developing selective KDM inhibitors.

Total synthesis of the antitumor antibiotic (±)-streptonigrin: first- and second-generation routes for de novo pyridine formation using ring-closing metathesis.

The total synthesis of (±)-streptonigrin, a potent tetracyclic aminoquinoline-5,8-dione antitumor antibiotic that reached phase II clinical trials in the 1970s, is described. Two routes to construct a key pentasubstituted pyridine fragment are depicted, both relying on ring-closing metathesis but differing in the substitution and complexity of the precursor to cyclization. Both routes are short and high yielding, with the second-generation approach ultimately furnishing (±)-streptonigrin in 14 linear steps and 11% overall yield from inexpensive ethyl glyoxalate. This synthesis will allow for the design and creation of druglike late-stage natural product analogues to address pharmacological limitations. Furthermore, assessment of a number of chiral ligands in a challenging asymmetric Suzuki-Miyaura cross-coupling reaction has enabled enantioenriched (up to 42% ee) synthetic streptonigrin intermediates to be prepared for the first time.

Is JmjC oxygenase catalysis limited to demethylation?

Jobs on the side: Substrate selectivity studies indicate that members of the biomedically important JmjC demethylase family of histone N(ε)-methyllysine demethylases are capable of catalyzing the de-N-alkylation of groups other than N-methyl and can catalyze reactions that form stable hydroxylated products. The differences in binding preferences in this set of enzymes may be helpful in the design of selective inhibitors.

PADI6: What we know about the elusive fifth member of the peptidyl arginine deiminase family.

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-characterized member of the peptidyl arginine deiminases (PADIs), which catalyse the post-translational conversion of arginine to citrulline. It is less conserved than the other PADIs, and currently has no reported catalytic activity. While 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. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Identification of photocrosslinking peptide ligands by mRNA display.

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.

Discovery and characterization of cyclic peptides selective for the C-terminal bromodomains of BET family proteins.

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 extra-terminal domain 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-peptide complexes reveal diverse structures and binding modes, which nevertheless display several conserved features. Some peptides demonstrate significant paralog-level specificity, although the physicochemical explanations for this specificity are often not clear. Our data demonstrate the power of cyclic peptides to discriminate between very similar proteins with high potency and hint that differences in conformational dynamics might modulate the affinity of these domains for particular ligands.

Strategies to expand peptide functionality through hybridisation with a small molecule component.

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.

Fluorescent Amino Acid Initiated de novo Cyclic Peptides for the Label-Free Assessment of Cell Permeability*.

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 ß-(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.

Hypoxia and hypoxia mimetics differentially modulate histone post-translational modifications.

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.

Protein Chemistry Looking Ahead: 8th Chemical Protein Synthesis Meeting 16-19 June 2019, Berlin, Germany.

The 8th Chemical Protein Synthesis meeting took place in Berlin in June 2019, covering broad topics in protein chemistry, ranging from synthetic methodology to applications in medicine and biomaterials. The meeting was also the culmination of the Priority Program SPP1623 on "Chemoselective Reactions for the Synthesis and Application of Functional Proteins" funded by the German Science Foundation (DFG) from 2012 to 2018. We present highlights from presentations at the forefront of the field, grouped into broad themes that illustrate how the field of protein chemistry is looking ahead to new discoveries and applications.

Mechanistic and structural studies of KDM-catalysed demethylation of histone 1 isotype 4 at lysine 26.

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.

Human histone demethylase KDM6B can catalyse sequential oxidations.

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.

Strategies for transitioning macrocyclic peptides to cell-permeable drug leads.

The ready availability of potent peptide binders for any desired target highlights their potential impact as therapeutic agents. Despite their versatility, however, peptides tend to display unfavourable pharmacological properties, such as low bioavailability, high renal clearance and proteolytic degradation rates, and low cell permeability. Fortunately, an increasing number of promising strategies to produce novel peptides and furnish pre-existing scaffolds with more drug-like properties are now becoming available. These strategies include incorporation of non-proteinogenic amino acids, tag appendage to existing peptides and grafting onto scaffolds already possessing desirable pharmacokinetic properties. As a consequence, a variety of promising bioactive macrocyclic peptides have recently been discovered highlighting the promise of this class of molecules as future medicines.

Exploring sequence space: harnessing chemical and biological diversity towards new peptide leads.

From their early roots in natural products, peptides now represent an expanding class of novel drugs. Their modular structures make them ideal candidates for pooled library screening approaches. Key technologies for library generation and screening, such as SICLOPPS, phage display and mRNA display, give unparalleled access to tight binding peptides. Through combination with genetic code reprogramming and chemical modifications, access to more natural product-like libraries, spanning non-canonical peptide space, is readily achievable. Recent advances in these fields enable introduction of diverse non-standard motifs, such as cyclisation and backbone methylations. Peptide discovery platforms now allow robust access to potent, highly functionalised peptides against virtually any protein of interest, with typical binding constants in the nanomolar range. Application of these optimised platforms in a drug discovery setting has the potential to significantly accelerate identification of new leads.

Highly selective inhibition of histone demethylases by de novo macrocyclic peptides.

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.