A Chemical Probe for the ATAD2 Bromodomain

ATAD2 is a cancer‐associated protein whose bromodomain has been described as among the least druggable of that target class. Starting from a potent lead, permeability and selectivity were improved through a dual approach: 1) using CF₂ as a sulfone bio‐isostere to exploit the unique properties of fluorine, and 2) using 1,3‐interactions to control the conformation of a piperidine ring. This resulted in the first reported low‐nanomolar, selective and cell permeable chemical probe for ATAD2.     

Tetracyanoresorcin[4]arene selectively recognises trimethyllysine and inhibits its enzyme-catalysed demethylation

Abstract

Nε-methylation of lysine within proteins is a critical biological process that, among other roles, is involved in the control of gene expression. Compounds that recognise Nε-methylated lysine may therefore be useful probes for the study of the associated biological mechanisms and have therapeutic potential. Here, we show that tetracyanoresorcin[4]arene (1) selectively recognises Nε-trimethyllysine and binds to Nε-trimethyllysine within the context of a short peptide. Its binding properties compare favourably to a previously characterised Nε-trimethyllysine binder, p-sulfonatocalix[4]arene (2). We also show that both 1 and 2 inhibit the demethylation of Nε-trimethyllysine within a histone-derived peptide by the histone demethylase KDM4A.     

Sequencing 5‐Hydroxymethyluracil at Single‐Base Resolution

5‐hydroxymethyluracil (5hmU) is formed through oxidation of thymine both enzymatically and non‐enzymatically in various biological systems. Although 5hmU has been reported to affect biological processes such as protein–DNA interactions, the consequences of 5hmU formation in genomes have not been yet fully explored. Herein, we report a method to sequence 5hmU at single‐base resolution. We employ chemical oxidation to transform 5hmU to 5‐formyluracil (5fU), followed by the polymerase extension to induce T‐to‐C base changes owing to the inherent ability of 5fU to form 5fU:G base pairing. In combination with the Illumina next generation sequencing technology, we developed polymerase chain reaction (PCR) conditions to amplify the T‐to‐C base changes and demonstrate the method in three different synthetic oligonucleotide models as well as part of the genome of a 5hmU‐rich eukaryotic pathogen. Our method has the potential capability to map 5hmU in genomic DNA and thus will contribute to promote the understanding of this modified base.     

Multiprotein Dynamic Combinatorial Chemistry: A Strategy for the Simultaneous Discovery of Subfamily‐Selective Inhibitors for Nucleic Acid Demethylases FTO and ALKBH3

Dynamic combinatorial chemistry (DCC) is a powerful supramolecular approach for discovering ligands for biomolecules. To date, most, if not all, biologically templated DCC systems employ only a single biomolecule to direct the self‐assembly process. To expand the scope of DCC, herein, a novel multiprotein DCC strategy has been developed that combines the discriminatory power of a zwitterionic “thermal tag” with the sensitivity of differential scanning fluorimetry. This strategy is highly sensitive and could differentiate the binding of ligands to structurally similar subfamily members. Through this strategy, it was possible to simultaneously identify subfamily‐selective probes against two clinically important epigenetic enzymes: FTO ( 7 ; IC₅₀=2.6 μm ) and ALKBH3 ( 8 ; IC₅₀=3.7 μm ). To date, this is the first report of a subfamily‐selective ALKBH3 inhibitor. The developed strategy could, in principle, be adapted to a broad range of proteins; thus it is of broad scientific interest.     

Nucleobase Modifiers Identify TET Enzymes as Bifunctional DNA Dioxygenases Capable of Direct N-Demethylation

TET family enzymes are known for oxidation of the 5-methyl substituent on 5-methylcytosine (5mC) in DNA. 5mC oxidation generates the stable base 5-hydroxymethylcytosine (5hmC), starting an indirect, multi-step process that ends with reversion of 5mC to unmodified cytosine. While probing the nucleobase determinants of 5mC recognition, we discovered that TET enzymes are also proficient as direct N-demethylases of cytosine bases. We find that N-demethylase activity can be readily observed on substrates lacking a 5-methyl group and, remarkably, TET enzymes can be similarly proficient in either oxidation of 5mC or demethylation of N4-methyl substituents. Our results indicate that TET enzymes can act as both direct and indirect demethylases, highlight the active-site plasticity of these Fe^II/α-ketoglutarate-dependent dioxygenases, and suggest activity on unexplored substrates that could reveal new TET biology.     

基于化学衍生-质谱技术的生物与临床样本中核酸修饰分析

除了经典碱基外,核酸(DNA和RNA)中还包含许多化学修饰。迄今为止,已经在核酸中鉴定了超过150多种化学修饰。这些化学修饰不会改变核酸的序列,但会改变它们的结构和生化特性,最终调节基因的时空表达。阐明这些修饰的功能可以促进对生命体生理调控机制的深入认识和理解。然而,核酸修饰在体内的丰度通常很低。因此,高灵敏和特异的检测方法对破译这些修饰的功能至关重要。化学衍生与质谱技术相结合对内源性低丰度核酸修饰展现出很好的分析能力。在过去几年中,研究者建立了多种基于化学衍生-质谱分析的分析方法,用于灵敏、高效地分析核酸修饰。该文总结了通过化学衍生-质谱分析方法来破译核酸修饰的最新进展,希望能促进未来对核酸修饰功能的深入研究。