Light-driven post-translational installation of reactive protein side chains.

Post-translational modifications (PTMs) greatly expand the structures and functions of proteins in nature1,2. Although synthetic protein functionalization strategies allow mimicry of PTMs3,4, as well as formation of unnatural protein variants with diverse potential functions, including drug carrying5, tracking, imaging6 and partner crosslinking7, the range of functional groups that can be introduced remains limited. Here we describe the visible-light-driven installation of side chains at dehydroalanine residues in proteins through the formation of carbon-centred radicals that allow C-C bond formation in water. Control of the reaction redox allows site-selective modification with good conversions and reduced protein damage. In situ generation of boronic acid catechol ester derivatives generates RH2C• radicals that form the native (ß-CH2-γ-CH2) linkage of natural residues and PTMs, whereas in situ potentiation of pyridylsulfonyl derivatives by Fe(II) generates RF2C• radicals that form equivalent ß-CH2-γ-CF2 linkages bearing difluoromethylene labels. These reactions are chemically tolerant and incorporate a wide range of functionalities (more than 50 unique residues/side chains) into diverse protein scaffolds and sites. Initiation can be applied chemoselectively in the presence of sensitive groups in the radical precursors, enabling installation of previously incompatible side chains. The resulting protein function and reactivity are used to install radical precursors for homolytic on-protein radical generation; to study enzyme function with natural, unnatural and CF2-labelled post-translationally modified protein substrates via simultaneous sensing of both chemo- and stereoselectivity; and to create generalized 'alkylator proteins' with a spectrum of heterolytic covalent-bond-forming activity (that is, reacting diversely with small molecules at one extreme or selectively with protein targets through good mimicry at the other). Post-translational access to such reactions and chemical groups on proteins could be useful in both revealing and creating protein function.

Selective targeting of human TET1 by cyclic peptide inhibitors: Insights from biochemical profiling.

Ten-Eleven Translocation (TET) enzymes are Fe(II)/2OG-dependent oxygenases that play important roles in epigenetic regulation, but selective inhibition of the TETs is an unmet challenge. We describe the profiling of previously identified TET1-binding macrocyclic peptides. TiP1 is established as a potent TET1 inhibitor (IC50 = 0.26 µM) with excellent selectivity over other TETs and 2OG oxygenases. TiP1 alanine scanning reveals the critical roles of Trp10 and Glu11 residues for inhibition of TET isoenzymes. The results highlight the utility of the RaPID method to identify potent enzyme inhibitors with selectivity over closely related paralogues. The structure-activity relationship data generated herein may find utility in the development of chemical probes for the TETs.

Engineered anti-inflammatory peptides inspired by mapping an evasin-chemokine interaction.

Chemokines mediate leukocyte migration and homeostasis and are key targets in inflammatory diseases including atherosclerosis, cytokine storm, and chronic autoimmune disease. Chemokine redundancy and ensuing network robustness has frustrated therapeutic development. Salivary evasins from ticks bind multiple chemokines to overcome redundancy and are effective in several preclinical disease models. Their clinical development has not progressed because of concerns regarding potential immunogenicity, parenteral delivery, and cost. Peptides mimicking protein activity can overcome the perceived limitations of therapeutic proteins. Here we show that peptides possessing multiple chemokine-binding and anti-inflammatory activities can be developed from the chemokine-binding site of an evasin. We used hydrogen-deuterium exchange MS to map the binding interface of the evasin P672 that physically interacts with C-C motif chemokine ligand (CCL) 8 and synthesized a 16-mer peptide (BK1.1) based on this interface region in evasin P672. Fluorescent polarization and native MS approaches showed that BK1.1 binds CCL8, CCL7, and CCL18 and disrupts CCL8 homodimerization. We show that a BK1.1 derivative, BK1.3, has substantially improved ability to disrupt P672 binding to CCL8, CCL2, and CCL3 in an AlphaScreen assay. Using isothermal titration calorimetry, we show that BK1.3 directly binds CCL8. BK1.3 also has substantially improved ability to inhibit CCL8, CCL7, CCL2, and CCL3 chemotactic function in vitro We show that local as well as systemic administration of BK1.3 potently blocks inflammation in vivo Identification and characterization of the chemokine-binding interface of evasins could thus inspire the development of novel anti-inflammatory peptides that therapeutically target the chemokine network in inflammatory diseases.

A knottin scaffold directs the CXC-chemokine-binding specificity of tick evasins.

Tick evasins (EVAs) bind either CC- or CXC-chemokines by a poorly understood promiscuous or "one-to-many" mechanism to neutralize inflammation. Because EVAs potently inhibit inflammation in many preclinical models, highlighting their potential as biological therapeutics for inflammatory diseases, we sought to further unravel the CXC-chemokine-EVA interactions. Using yeast surface display, we identified and characterized 27 novel CXC-chemokine-binding evasins homologous to EVA3 and defined two functional classes. The first, which included EVA3, exclusively bound ELR+ CXC-chemokines, whereas the second class bound both ELR+ and ELR- CXC-chemokines, in several cases including CXC-motif chemokine ligand 10 (CXCL10) but, surprisingly, not CXCL8. The X-ray crystal structure of EVA3 at a resolution of 1.79 Å revealed a single antiparallel ß-sheet with six conserved cysteine residues forming a disulfide-bonded knottin scaffold that creates a contiguous solvent-accessible surface. Swapping analyses identified distinct knottin scaffold segments necessary for different CXC-chemokine-binding activities, implying that differential ligand positioning, at least in part, plays a role in promiscuous binding. Swapping segments also transferred chemokine-binding activity, resulting in a hybrid EVA with dual CXCL10- and CXCL8-binding activities. The solvent-accessible surfaces of the knottin scaffold segments have distinctive shape and charge, which we suggest drives chemokine-binding specificity. These studies provide structural and mechanistic insight into how CXC-chemokine-binding tick EVAs achieve class specificity but also engage in promiscuous binding.

The N-terminal domain of a tick evasin is critical for chemokine binding and neutralization and confers specific binding activity to other evasins.

Tick chemokine-binding proteins (evasins) are an emerging class of biologicals that target multiple chemokines and show anti-inflammatory activities in preclinical disease models. Using yeast surface display, we identified a CCL8-binding evasin, P672, from the tick Rhipicephalus pulchellus We found that P672 binds CCL8 and eight other CC-class chemokines with a Kd < 10 nm and four other CC chemokines with a Kd between 10 and 100 nm and neutralizes CCL3, CCL3L1, and CCL8 with an IC50 < 10 nm The CC chemokine-binding profile was distinct from that of evasin 1 (EVA1), which does not bind CCL8. We also show that P672's binding activity can be markedly modulated by the location of a StrepII-His purification tag. Combining native MS and bottom-up proteomics, we further demonstrated that P672 is glycosylated and forms a 1:1 complex with CCL8, disrupting CCL8 homodimerization. Homology modeling of P672 using the crystal structure of the EVA1 and CCL3 complex as template suggested that 44 N-terminal residues of P672 form most of the contacts with CCL8. Replacing the 29 N-terminal residues of EVA1 with the 44 N-terminal residues of P672 enabled this hybrid evasin to bind and neutralize CCL8, indicating that the CCL8-binding properties of P672 reside, in part, in its N-terminal residues. This study shows that the function of certain tick evasins can be manipulated simply by adding a tag. We conclude that homology modeling helps identify regions with transportable chemokine-binding functions within evasins, which can be used to construct hybrid evasins with altered properties.

Design, Synthesis and Characterization of Covalent KDM5 Inhibitors.

Histone lysine demethylases (KDMs) are involved in the dynamic regulation of gene expression and they play a critical role in several biological processes. Achieving selectivity over the different KDMs has been a major challenge for KDM inhibitor development. Here we report potent and selective KDM5 covalent inhibitors designed to target cysteine residues only present in the KDM5 sub-family. The covalent binding to the targeted proteins was confirmed by MS and time-dependent inhibition. Additional competition assays show that compounds were non 2-OG competitive. Target engagement and ChIP-seq analysis showed that the compounds inhibited the KDM5 members in cells at nano- to micromolar levels and induce a global increase of the H3K4me3 mark at transcriptional start sites.

Lysine-241 Has a Role in Coupling 2OG Turnover with Substrate Oxidation During KDM4-Catalysed Histone Demethylation.

The JmjC histone lysyl demethylases (KDMs) play important roles in modulating histone methylation states and have the potential to be regulated by oxygen availability. Lys241 of the KDM4 subfamily is proposed to be important in oxygen binding by KDM4A. We report evidence that, although Lys241 is unlikely to be directly involved in oxygen binding, it has an important role in coupling 2-oxoglutarate cosubstrate oxidation with lysine demethylase activity. The results suggest that compounds promoting the uncoupling of substrate oxidation are of interest as JmjC-KDM inhibitors.

Thioether Macrocyclic Peptides Selected against TET1 Compact Catalytic Domain Inhibit TET1 Catalytic Activity.

The ten-eleven translocation (TET) protein family, consisting of three isoforms (TET1/2/3), have been found in mammalian cells and have a crucial role in 5-methylcytosine demethylation in genomic DNA through the catalysis of oxidation reactions assisted by 2-oxoglutarate (2OG). DNA methylation/demethylation contributes to the regulation of gene expression at the transcriptional level, and recent studies have revealed that TET1 is highly elevated in malignant cells of various diseases and related to malignant alteration. TET1 inhibitors based on a scaffold of thioether macrocyclic peptides, which have been discovered by the random nonstandard peptide integrated discovery (RaPID) system, are reported. The affinity-based selection was performed against the TET1 compact catalytic domain (TET1CCD) to yield thioether macrocyclic peptides. These peptides exhibited inhibitory activity of the TET1 catalytic domain (TET1CD), with an IC50 value as low as 1.1 µm. One of the peptides, TiP1, was also able to inhibit TET1CD over TET2CD with tenfold selectivity, although it was likely to target the 2OG binding site; this provides a good starting point to develop more selective inhibitors.

Structure-activity studies of a macrocyclic peptide inhibitor of histone lysine demethylase 4A.

The combination of genetic code reprogramming and mRNA display is a powerful approach for the identification of macrocyclic peptides with high affinities to a target of interest. We have previously used such an approach to identify a potent inhibitor (CP2) of the human KDM4A and KDM4C lysine demethylases; important regulators of gene expression. In the present study, we have used genetic code reprogramming to synthesise very high diversity focused libraries (>1012 compounds) based on CP2 and, through affinity screening, used these to delineate the structure activity relationship of CP2 binding to KDM4A. In the course of these experiments we identified a CP2 analogue (CP2f-7) with ∼4-fold greater activity than CP2 in in vitro inhibition assays. This work will facilitate the development of more potent, selective inhibitors of lysine demethylases.

Investigations on small molecule inhibitors targeting the histone H3K4 tri-methyllysine binding PHD-finger of JmjC histone demethylases.

Plant homeodomain (PHD) containing proteins are important epigenetic regulators and are of interest as potential drug targets. Inspired by the amiodarone derivatives reported to inhibit the PHD finger 3 of KDM5A (KDM5A(PHD3)), a set of compounds were synthesised. Amiodarone and its derivatives were observed to weakly disrupt the interactions of a histone H3K4me3 peptide with KDM5A(PHD3). Selected amiodarone derivatives inhibited catalysis of KDM5A, but in a PHD-finger independent manner. Amiodarone derivatives also bind to H3K4me3-binding PHD-fingers from the KDM7 subfamily. Further work is required to develop potent and selective PHD finger inhibitors.

Selective recognition of the di/trimethylammonium motif by an artificial carboxycalixarene receptor.

Chemical tools that recognise post-translational modifications have promising applications in biochemistry and in therapy. We report a simple carboxycalixarene that selectively binds molecules containing di/trimethylammonium moieties in isolation, in cell lysates and when incorporated in histone peptides. Our findings reveal the potential of using carboxycalixarene-based receptors to study epigenetic regulation.

Discovery of a Highly Selective Cell-Active Inhibitor of the Histone Lysine Demethylases KDM2/7.

Histone lysine demethylases (KDMs) are of critical importance in the epigenetic regulation of gene expression, yet there are few selective, cell-permeable inhibitors or suitable tool compounds for these enzymes. We describe the discovery of a new class of inhibitor that is highly potent towards the histone lysine demethylases KDM2A/7A. A modular synthetic approach was used to explore the chemical space and accelerate the investigation of key structure-activity relationships, leading to the development of a small molecule with around 75-fold selectivity towards KDM2A/7A versus other KDMs, as well as cellular activity at low micromolar concentrations.

Targeting histone lysine demethylases - progress, challenges, and the future.

N-Methylation of lysine and arginine residues has emerged as a major mechanism of transcriptional regulation in eukaryotes. In humans, N(ε)-methyllysine residue demethylation is catalysed by two distinct subfamilies of demethylases (KDMs), the flavin-dependent KDM1 subfamily and the 2-oxoglutarate- (2OG) dependent JmjC subfamily, which both employ oxidative mechanisms. Modulation of histone methylation status is proposed to be important in epigenetic regulation and has substantial medicinal potential for the treatment of diseases including cancer and genetic disorders. This article provides an introduction to the enzymology of the KDMs and the therapeutic possibilities and challenges associated with targeting them, followed by a review of reported KDM inhibitors and their mechanisms of action from kinetic and structural perspectives.

Cyclic ß2,3-amino acids improve the serum stability of macrocyclic peptide inhibitors targeting the SARS-CoV-2 main protease.

Due to their constrained conformations, cyclic ß2,3-amino acids (cßAA) are key building blocks that can fold peptides into compact and rigid structures, improving peptidase resistance and binding affinity to target proteins, due to their constrained conformations. Although the translation efficiency of cßAAs is generally low, our engineered tRNA, referred to as tRNAPro1E2, enabled efficient incorporation of cßAAs into peptide libraries using the flexible in vitro translation (FIT) system. Here we report on the design and application of a macrocyclic peptide library incorporating 3 kinds of cßAAs: (1R,2S)-2-aminocyclopentane carboxylic acid (ß1), (1S,2S)-2-aminocyclohexane carboxylic acid (ß2), and (1R,2R)-2-aminocyclopentane carboxylic acid. This library was applied to an in vitro selection against the SARS-CoV-2 main protease (Mpro). The resultant peptides, BM3 and BM7, bearing one ß2 and two ß1, exhibited potent inhibitory activities with IC50 values of 40 and 20 nM, respectively. BM3 and BM7 also showed remarkable serum stability with half-lives of 48 and >168 h, respectively. Notably, BM3A and BM7A, wherein the cßAAs were substituted with alanine, lost their inhibitory activities against Mpro and displayed substantially shorter serum half-lives. This observation underscores the significant contribution of cßAA to the activity and stability of peptides. Overall, our results highlight the potential of cßAA in generating potent and highly stable macrocyclic peptides with drug-like properties.

Focused Screening Identifies Different Sensitivities of Human TET Oxygenases to the Oncometabolite 2-Hydroxyglutarate.

Ten-eleven translocation enzymes (TETs) are Fe(II)/2-oxoglutarate (2OG) oxygenases that catalyze the sequential oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine in eukaryotic DNA. Despite their roles in epigenetic regulation, there is a lack of reported TET inhibitors. The extent to which 2OG oxygenase inhibitors, including clinically used inhibitors and oncometabolites, modulate DNA modifications via TETs has been unclear. Here, we report studies on human TET1-3 inhibition by a set of 2OG oxygenase-focused inhibitors, employing both enzyme-based and cellular assays. Most inhibitors manifested similar potencies for TET1-3 and caused increases in cellular 5hmC levels. (R)-2-Hydroxyglutarate, an oncometabolite elevated in isocitrate dehydrogenase mutant cancer cells, showed different degrees of inhibition, with TET1 being less potently inhibited than TET3 and TET2, potentially reflecting the proposed role of TET2 mutations in tumorigenesis. The results highlight the tractability of TETs as drug targets and provide starting points for selective inhibitor design.

Identification and engineering of potent cyclic peptides with selective or promiscuous binding through biochemical profiling and bioinformatic data analysis.

As our understanding of biological systems grows, so does the need to selectively target individual or multiple members of specific protein families in order to probe their function. Many targets of current biological and pharmaceutical interest are part of a large family of closely related proteins and achieving ligand selectivity often remains either an elusive or time-consuming endeavour. Cyclic peptides (CPs) occupy a key niche in ligand space, able to achieve high affinity and selectivity while retaining synthetic accessibility. De novo cyclic peptide ligands can be rapidly generated against a given target using mRNA display. In this study we harness mRNA display technology and the wealth of next generation sequencing (NGS) data generated to explore both experimental approaches and bioinformatic, statistical data analysis of peptide enrichment in cross-screen selections to rapidly generate high affinity CPs with differing intra-family protein selectivity profiles against fibroblast growth factor receptor (FGF-R) family proteins. Using these methods, CPs with distinct selectivity profiles can be generated which can serve as valuable tool compounds to decipher biological questions.

The oncometabolite 2-hydroxyglutarate inhibits histone lysine demethylases.

Mutations in isocitrate dehydrogenases (IDHs) have a gain-of-function effect leading to R(-)-2-hydroxyglutarate (R-2HG) accumulation. By using biochemical, structural and cellular assays, we show that either or both R- and S-2HG inhibit 2-oxoglutarate (2OG)-dependent oxygenases with varying potencies. Half-maximal inhibitory concentration (IC(50)) values for the R-form of 2HG varied from approximately 25 µM for the histone N(ɛ)-lysine demethylase JMJD2A to more than 5 mM for the hypoxia-inducible factor (HIF) prolyl hydroxylase. The results indicate that candidate oncogenic pathways in IDH-associated malignancy should include those that are regulated by other 2OG oxygenases than HIF hydroxylases, in particular those involving the regulation of histone methylation.

Dual-action inhibitors of HIF prolyl hydroxylases that induce binding of a second iron ion.

Inhibition of the hypoxia-inducible factor (HIF) prolyl hydroxylases (PHD or EGLN enzymes) is of interest for the treatment of anemia and ischemia-related diseases. Most PHD inhibitors work by binding to the single ferrous ion and competing with 2-oxoglutarate (2OG) co-substrate for binding at the PHD active site. Non-specific iron chelators also inhibit the PHDs, both in vitro and in cells. We report the identification of dual action PHD inhibitors, which bind to the active site iron and also induce the binding of a second iron ion at the active site. Following analysis of small-molecule iron complexes and application of non-denaturing protein mass spectrometry to assess PHD2·iron·inhibitor stoichiometry, selected diacylhydrazines were identified as PHD2 inhibitors that induce the binding of a second iron ion. Some compounds were shown to inhibit the HIF hydroxylases in human hepatoma and renal carcinoma cell lines.

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.