An efficient mRNA display protocol yields potent bicyclic peptide inhibitors for FGFR3c: outperforming linear and monocyclic formats in affinity and stability.

Macrocyclization has positioned itself as a powerful method for engineering potent peptide drug candidates. Introducing one or multiple cyclizations is a common strategy to improve properties such as affinity, bioavailability and proteolytic stability. Consequently, methodologies to create large libraries of polycyclic peptides by phage or mRNA display have emerged, allowing the rapid identification of binders to virtually any target. Yet, within those libraries, the performance of linear vs. mono- or bicyclic peptides has rarely been studied. Indeed, a key parameter to perform such a comparison is to use a display protocol and cyclization chemistry that enables the formation of all 3 formats in equal quality and diversity. Here, we developed a simple, efficient and fast mRNA display protocol which meets these criteria and can be used to generate highly diverse libraries of thioether cyclized polycyclic peptides. As a proof of concept, we selected peptides against fibroblast growth factor receptor 3c (FGFR3c) and compared the different formats regarding affinity, specificity, and human plasma stability. The peptides with the best KD's and stability were identified among bicyclic peptide hits, further strengthening the body of evidence pointing at the superiority of this class of molecules and providing functional and selective inhibitors of FGFR3c.

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.

An integrated platform approach enables discovery of potent, selective and ligand-competitive cyclic peptides targeting the GIP receptor.

In any drug discovery effort, the identification of hits for further optimisation is of crucial importance. For peptide therapeutics, display technologies such as mRNA display have emerged as powerful methodologies to identify these desired de novo hit ligands against targets of interest. The diverse peptide libraries are genetically encoded in these technologies, allowing for next-generation sequencing to be used to efficiently identify the binding ligands. Despite the vast datasets that can be generated, current downstream methodologies, however, are limited by low throughput validation processes, including hit prioritisation, peptide synthesis, biochemical and biophysical assays. In this work we report a highly efficient strategy that combines bioinformatic analysis with state-of-the-art high throughput peptide synthesis to identify nanomolar cyclic peptide (CP) ligands of the human glucose-dependent insulinotropic peptide receptor (hGIP-R). Furthermore, our workflow is able to discriminate between functional and remote binding non-functional ligands. Efficient structure-activity relationship analysis (SAR) combined with advanced in silico structural studies allow deduction of a thorough and holistic binding model which informs further chemical optimisation, including efficient half-life extension. We report the identification and design of the first de novo, GIP-competitive, incretin receptor family-selective CPs, which exhibit an in vivo half-life up to 10.7 h in rats. The workflow should be generally applicable to any selection target, improving and accelerating hit identification, validation, characterisation, and prioritisation for therapeutic development.

Molecular Engineering of Efficacious Mono-Valent Ultra-Long Acting Two-Chain Insulin-Fc Conjugates.

Here, we describe molecular engineering of monovalent ultra-long acting two-chain insulin-Fc conjugates. Insulin-Fc conjugates were synthesized using trifunctional linkers with one amino reactive group for reaction with a lysine residue of insulin and two thiol reactive groups used for re-bridging of a disulfide bond within the Fc molecule. The ultra-long pharmacokinetic profile of the insulin-Fc conjugates was the result of concertedly slowing insulin receptor-mediated clearance by (1) introduction of amino acid substitutions that lowered the insulin receptor affinity and (2) conjugating insulin to the Fc element. Fc conjugation leads to recycling by the neonatal Fc receptor and increase in the molecular size, both contributing to the ultra-long pharmacokinetic and pharmacodynamic profiles.

Engineering of Orally Available, Ultralong-Acting Insulin Analogues: Discovery of OI338 and OI320.

Recently, the first basal oral insulin (OI338) was shown to provide similar treatment outcomes to insulin glargine in a phase 2a clinical trial. Here, we report the engineering of a novel class of basal oral insulin analogues of which OI338, 10, in this publication, was successfully tested in the phase 2a clinical trial. We found that the introduction of two insulin substitutions, A14E and B25H, was needed to provide increased stability toward proteolysis. Ultralong pharmacokinetic profiles were obtained by attaching an albumin-binding side chain derived from octadecanedioic (C18) or icosanedioic acid (C20) to the lysine in position B29. Crucial for obtaining the ultralong PK profile was also a significant reduction of insulin receptor affinity. Oral bioavailability in dogs indicated that C18-based analogues were superior to C20-based analogues. These studies led to the identification of the two clinical candidates OI338 and OI320 (10 and 24, respectively).

Molecular engineering of safe and efficacious oral basal insulin.

Recently, the clinical proof of concept for the first ultra-long oral insulin was reported, showing efficacy and safety similar to subcutaneously administered insulin glargine. Here, we report the molecular engineering as well as biological and pharmacological properties of these insulin analogues. Molecules were designed to have ultra-long pharmacokinetic profile to minimize variability in plasma exposure. Elimination plasma half-life of ~20 h in dogs and ~70 h in man is achieved by a strong albumin binding, and by lowering the insulin receptor affinity 500-fold to slow down receptor mediated clearance. These insulin analogues still stimulate efficient glucose disposal in rats, pigs and dogs during constant intravenous infusion and euglycemic clamp conditions. The albumin binding facilitates initial high plasma exposure with a concomitant delay in distribution to peripheral tissues. This slow appearance in the periphery mediates an early transient hepato-centric insulin action and blunts hypoglycaemia in dogs in response to overdosing.

Aspartate/asparagine-ß-hydroxylase crystal structures reveal an unexpected epidermal growth factor-like domain substrate disulfide pattern.

AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1-3, 2-4, 5-6 disulfide bonding pattern; an unexpected Cys3-4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases.

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.

YcfDRM is a thermophilic oxygen-dependent ribosomal protein uL16 oxygenase.

YcfD from Escherichia coli is a homologue of the human ribosomal oxygenases NO66 and MINA53, which catalyse histidyl-hydroxylation of the 60S subunit and affect cellular proliferation (Ge et al., Nat Chem Biol 12:960-962, 2012). Bioinformatic analysis identified a potential homologue of ycfD in the thermophilic bacterium Rhodothermus marinus (ycfDRM). We describe studies on the characterization of ycfDRM, which is a functional 2OG oxygenase catalysing (2S,3R)-hydroxylation of the ribosomal protein uL16 at R82, and which is active at significantly higher temperatures than previously reported for any other 2OG oxygenase. Recombinant ycfDRM manifests high thermostability (Tm 84 °C) and activity at higher temperatures (Topt 55 °C) than ycfDEC (Tm 50.6 °C, Topt 40 °C). Mass spectrometric studies on purified R. marinus ribosomal proteins demonstrate a temperature-dependent variation in uL16 hydroxylation. Kinetic studies of oxygen dependence suggest that dioxygen availability can be a limiting factor for ycfDRM catalysis at high temperatures, consistent with incomplete uL16 hydroxylation observed in R. marinus cells. Overall, the results that extend the known range of ribosomal hydroxylation, reveal the potential for ycfD-catalysed hydroxylation to be regulated by temperature/dioxygen availability, and that thermophilic 2OG oxygenases are of interest from a biocatalytic perspective.

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.

Analysis of JmjC Demethylase-Catalyzed Demethylation Using Geometrically-Constrained Lysine Analogues.

The dynamic post-translational modifications of histones play important roles in the regulation of transcription in animals. The demethylation of N(ε)-methyl lysine residues in the N-terminal tail of histone H3 is catalyzed by demethylases, of which the largest family is the ferrous iron and 2-oxoglutarate dependent demethylases (JmjC KDMs), which catalyze demethylation via initial hydroxylation of the N-methyl groups. We report studies on the conformational requirements of the JmjC KDM substrates using N-methylated lysine analogues prepared by metathesis reactions of suitably protected N-allylglycine. The results support the proposed requirement for a positively charged N(ε)-amino group in JmjC KDM catalysis. Demethylation of a trans-C-4/C-5 dehydrolysine substrate analogue was observed with representative KDM4 subfamily members KDM4A, KDM4B and KDM4E, and KDM7B, which are predicted, based on crystallographic analyses, to bind the N(ε)-methylated lysine residue in different conformations during catalysis. This information may be useful in the design of JmjC KDM selective inhibitors.

Optimal translational termination requires C4 lysyl hydroxylation of eRF1.

Efficient stop codon recognition and peptidyl-tRNA hydrolysis are essential in order to terminate translational elongation and maintain protein sequence fidelity. Eukaryotic translational termination is mediated by a release factor complex that includes eukaryotic release factor 1 (eRF1) and eRF3. The N terminus of eRF1 contains highly conserved sequence motifs that couple stop codon recognition at the ribosomal A site to peptidyl-tRNA hydrolysis. We reveal that Jumonji domain-containing 4 (Jmjd4), a 2-oxoglutarate- and Fe(II)-dependent oxygenase, catalyzes carbon 4 (C4) lysyl hydroxylation of eRF1. This posttranslational modification takes place at an invariant lysine within the eRF1 NIKS motif and is required for optimal translational termination efficiency. These findings further highlight the role of 2-oxoglutarate/Fe(II) oxygenases in fundamental cellular processes and provide additional evidence that ensuring fidelity of protein translation is a major role of hydroxylation.

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.

Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives.

Tet proteins oxidize 5-methylcytosine (mC) to generate 5-hydroxymethyl (hmC), 5-formyl (fC), and 5-carboxylcytosine (caC). The exact function of these oxidative cytosine bases remains elusive. We applied quantitative mass-spectrometry-based proteomics to identify readers for mC and hmC in mouse embryonic stem cells (mESC), neuronal progenitor cells (NPC), and adult mouse brain tissue. Readers for these modifications are only partially overlapping, and some readers, such as Rfx proteins, display strong specificity. Interactions are dynamic during differentiation, as for example evidenced by the mESC-specific binding of Klf4 to mC and the NPC-specific binding of Uhrf2 to hmC, suggesting specific biological roles for mC and hmC. Oxidized derivatives of mC recruit distinct transcription regulators as well as a large number of DNA repair proteins in mouse ES cells, implicating the DNA damage response as a major player in active DNA demethylation.

Low values of 5-hydroxymethylcytosine (5hmC), the "sixth base," are associated with anaplasia in human brain tumors.

5-Methylcytosine (5 mC) in genomic DNA has important epigenetic functions in embryonic development and tumor biology. 5-Hydroxymethylcytosine (5 hmC) is generated from 5 mC by the action of the TET (Ten-Eleven-Translocation) enzymes and may be an intermediate to further oxidation and finally demethylation of 5 mC. We have used immunohistochemistry (IHC) and isotope-based liquid chromatography mass spectrometry (LC-MS) to investigate the presence and distribution of 5 hmC in human brain and brain tumors. In the normal adult brain, IHC identified 61.5% 5 hmC positive cells in the cortex and 32.4% 5 hmC in white matter (WM) areas. In tumors, positive staining of cells ranged from 1.1% in glioblastomas (GBMs) (WHO Grade IV) to 8.9% in Grade I gliomas (pilocytic astrocytomas). In the normal adult human brain, LC-MS also showed highest values in cortical areas (1.17% 5 hmC/dG [deoxyguanosine]), in the cerebral WM we measured around 0.70% 5 hmC/dG. levels were related to tumor differentiation, ranging from lowest values of 0.078% 5 hmC/dG in GBMs (WHO Grade IV) to 0.24% 5 hmC/dG in WHO Grade II diffuse astrocytomas. 5 hmC measurements were unrelated to 5 mC values. We find that the number of 5 hmC positive cells and the amount of 5 hmC/dG in the genome that has been proposed to be related to pluripotency and lineage commitment in embryonic stem cells is also associated with brain tumor differentiation and anaplasia.

Improved synthesis and mutagenicity of oligonucleotides containing 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine.

5-Formylcytosine (fC or (5-CHO)dC) and 5-carboxylcytosine (caC or (5-COOH)dC) have recently been identified as constituents of mammalian DNA. The nucleosides are formed from 5-methylcytosine (mC or (5-Me)dC) via 5-hydroxymethylcytosine (hmC or (5-HOMe)dC) and are possible intermediates of an active DNA demethylation process. Here we show efficient syntheses of phosphoramidites which enable the synthesis of DNA strands containing these cytosine modifications based on Pd(0)-catalyzed functionalization of 5-iododeoxycytidine. The first crystal structure of fC reveals the existence of an intramolecular H-bond between the exocyclic amine and the formyl group, which controls the conformation of the formyl substituent. Using a newly designed in vitro mutagenicity assay we show that fC and caC are only marginally mutagenic, which is a prerequisite for the bases to function as epigenetic control units.

5-Hydroxymethylcytosine, the sixth base of the genome.

5-Hydroxymethylcytosine (hmC) was recently discovered as a new constituent of mammalian DNA. Besides 5-methylcytosine (mC), it is the only other modified base in higher organisms. The discovery is of enormous importance because it shows that the methylation of cytosines to imprint epigenetic information is not a final chemical step that leads to gene silencing but that further chemistry occurs at the methyl group that might have regulatory function. Recent progress in hmC detection--most notably LC-MS and glucosyltransferase assays--helped to decipher the precise distribution of hmC in the body. This led to the surprising finding that, in contrast to constant mC levels, the hmC levels are strongly tissue-specific. The highest values of hmC are found in the central nervous system. It was furthermore discovered that hmC is involved in regulating the pluripotency of stem cells and that it is connected to the processes of cellular development and carcinogenesis. Evidence is currently accumulating that hmC may not exclusively be an intermediate of an active demethylation process, but that it functions instead as an important epigenetic marker.

Discovery and synthesis of new UV-induced intrastrand C(4-8)G and G(8-4)C photolesions.

UV irradiation of cellular DNA leads to the formation of a number of defined mutagenic DNA lesions. Here we report the discovery of new intrastrand C(4-8)G and G(8-4)C cross-link lesions in which the C(4) amino group of the cytosine base is covalently linked to the C(8) position of an adjacent dG base. The structure of the novel lesions was clarified by HPLC-MS/MS data for UV-irradiated DNA in combination with chemical synthesis and direct comparison of the synthetic material with irradiated DNA. We also report the ability to generate the lesions directly in DNA with the help of a photoactive precursor that was site-specifically incorporated into DNA. This should enable detailed chemical and biochemical investigations of these lesions.