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.

Genetic Incorporation of Olefin Cross-Metathesis Reaction Tags for Protein Modification.

Olefin cross-metathesis (CM) is a viable reaction for the modification of alkene-containing proteins. Although allyl sulfide or selenide side-chain motifs in proteins can critically enhance the rate of CM reactions, no efficient method for their site-selective genetic incorporation into proteins has been reported to date. Here, through the systematic evaluation of olefin-bearing unnatural amino acids for their metabolic incorporation, we have discovered S-allylhomocysteine (Ahc) as a genetically encodable Met analogue that is not only processed by translational cellular machinery but also a privileged CM substrate residue in proteins. In this way, Ahc was used for efficient Met codon reassignment in a Met-auxotrophic strain of E. coli (B834 (DE3)) as well as metabolic labeling of protein in human cells and was reactive toward CM in several representative proteins. This expands the use of CM in the toolkit for "tag-and-modify" functionalization of proteins.

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.

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.

Most human proteins made in both nucleus and cytoplasm turn over within minutes.

In bacteria, protein synthesis can be coupled to transcription, but in eukaryotes it is believed to occur solely in the cytoplasm. Using pulses as short as 5 s, we find that three analogues--L-azidohomoalanine, puromycin (detected after attaching fluors using 'click' chemistry or immuno-labeling), and amino acids tagged with 'heavy' 15N and 13C (detected using secondary ion mass spectrometry)--are incorporated into the nucleus and cytoplasm in a process sensitive to translational inhibitors. The nuclear incorporation represents a significant fraction of the total, and labels in both compartments have half-lives of less than a minute; results are consistent with most newly-made peptides being destroyed soon after they are made. As nascent RNA bearing a premature termination codon (detected by fluorescence in situ hybridization) is also eliminated by a mechanism sensitive to a translational inhibitor, the nuclear turnover of peptides is probably a by-product of proof-reading the RNA for stop codons (a process known as nonsense-mediated decay). We speculate that the apparently-wasteful turnover of this previously-hidden ('dark-matter') world of peptide is involved in regulating protein production.

QuaNCAT: quantitating proteome dynamics in primary cells.

Here we demonstrate quantitation of stimuli-induced proteome dynamics in primary cells by combining the power of bio-orthogonal noncanonical amino acid tagging (BONCAT) and stable-isotope labeling of amino acids in cell culture (SILAC). In conjunction with nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS), quantitative noncanonical amino acid tagging (QuaNCAT) allowed us to monitor the early expression changes of >600 proteins in primary resting T cells subjected to activation stimuli.