Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening

Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. In contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery

Microstructural characteristics of pure gold processed by equal-channel angular pressing

Experiments were conducted to evaluate the microstructural characteristics of pure (4N) gold processed by equal-channel angular pressing using routes A or B(c). Using atomic force microscopy and X-ray diffraction, it is shown that, although these two routes lead to similar dislocation densities of similar to 1.5-1.7 x 10(15) m(-2) and similar average grain sizes of similar to 460-490 nm, there are significant differences in the shearing patterns and in the densities of planar faults

Inhibition of histone H3K27 demethylases inactivates brachyury (TBXT) and promotes chordoma cell death

Expression of the transcription factor brachyury (TBXT) is normally restricted to the embryo and its silencing is epigenetically regulated. TBXT promotes mesenchymal transition in a subset of common carcinomas, and in chordoma, a rare cancer showing notochordal differentiation, TBXT acts as a putative oncogene. We hypothesized that TBXT expression is controlled through epigenetic inhibition to promote chordoma cell death. Screening of five human chordoma cell lines revealed that pharmacological inhibition of the histone 3 lysine 27 demethylases KDM6A (UTX) and KDM6B (JMJD3) leads to cell death. This effect was phenocopied by dual genetic inactivation of KDM6A/B using CRISPR/Cas9. Inhibition of KDM6A/B with a novel compound KDOBA67 led to a genome-wide increase in repressive H3K27me3 marks with concomitant reduction in active H3K27ac, H3K9ac, and H3K4me3 marks. TBXT was a KDM6A/B target gene, and chromatin changes at TBXT following KDOBA67 treatment were associated with a reduction in TBXT protein levels in all models tested, including primary patient-derived cultures. In all models tested, KDOBA67 treatment downregulated expression of a network of transcription factors critical for chordoma survival and upregulated pathways dominated by ATF4-driven stress and pro-apoptotic responses. Blocking the AFT4 stress response did not prevent suppression of TBXT and induction of cell death, but ectopic overexpression of TBXT increased viability, therefore implicating TBXT as a potential therapeutic target of H3K27 demethylase inhibitors in chordoma. Our work highlights how knowledge of normal processes in fetal development can provide insight into tumorigenesis and identify novel therapeutic approaches

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. Here we describe the discovery of a new class of inhibitor that is highly selective towards the histone lysine demethylases KDM2A/7A. A modular synthetic approach was used to explore chemical space and accelerate investigation of key structure-activity relationships, leading to the development of a small molecule with ≥75-fold selectivity towards KDM2A/7A vs other KDMs, as well as cellular activity at low micromolar concentrations

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. Here we describe the discovery of a new class of inhibitor that is highly selective towards the histone lysine demethylases KDM2A/7A. A modular synthetic approach was used to explore chemical space and accelerate investigation of key structure-activity relationships, leading to the development of a small molecule with ≥75-fold selectivity towards KDM2A/7A vs other KDMs, as well as cellular activity at low micromolar concentrations

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.Amodular 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

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.Amodular 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

Theoretical investigation of free-standing CoPd nanoclusters as a function of cluster size and stoichiometry in the Pd-rich phase: Geometry, chemical order, magnetism, and metallic behavior

We report on a study of the geometrical structure, magnetic properties, and metallic behavior of free-standing CoPd nanoclusters with N=7, 13, 19, 23, and 26 atoms, as a function of cluster size and stoichiometry, in the Pd-rich phase. The investigated structures were obtained using a Gupta potential in combination with a genetic algorithm energy minimization. The electronic properties of the ground state geometrical structures were determined by solving a self-consistent spd tight-binding Hamiltonian. The metallic behavior was studied using Kubo's criterion. We tested our approach against benchmark SIESTA calculations for some of the small clusters investigated in the present work. We discuss the competition between segregation and mixing effects by means of an order parameter. The magnetic moments are analyzed in the context of the local chemical and atomic environments and we propose a simple empirical expression for the average magnetic moment per atom of these binary clusters. ©