Open Access

It is proposed to install an experimental setup in the fixed-target hall of the Nuclotron with the final goal to perform a research program focused on the production of strange matter in heavyion collisions at beam energies between 2 and 6 A GeV. The basic setup will comprise a large acceptance dipole magnet with inner tracking detector modules based on double-sided Silicon micro-strip sensors and GEMs. The outer tracking will be based on the drift chambers and straw tube detector. Particle identification will be based on the time-of-flight measurements. This setup will be sufficient perform a comprehensive study of strangeness production in heavy-ion collisions, including multi-strange hyperons, multi-strange hypernuclei, and exotic multi-strange heavy objects. These pioneering measurements would provide the first data on the production of these particles in heavy-ion collisions at Nuclotron beam energies, and would open an avenue to explore the third (strangeness) axis of the nuclear chart. The extension of the experimental program is related with the study of in-medium effects for vector mesons decaying in hadronic modes. The studies of the NN and NA reactions for the reference is assumed.

Bromodomains (BRDs) are conserved protein interaction modules which recognize (read) acetyl-lysine modifications, however their role(s) in regulating cellular states and their potential as targets for the development of targeted treatment strategies is poorly understood. Here we present a set of 25 chemical probes, selective small molecule inhibitors, covering 29 human bromodomain targets. We comprehensively evaluate the selectivity of this probe-set using BROMOscan and demonstrate the utility of the set identifying roles of BRDs in cellular processes and potential translational applications. For instance, we discovered crosstalk between histone acetylation and the glycolytic pathway resulting in a vulnerability of breast cancer cell lines under conditions of glucose deprivation or GLUT1 inhibition to inhibition of BRPF2/3 BRDs. This chemical probe-set will serve as a resource for future applications in the discovery of new physiological roles of bromodomain proteins in normal and disease states, and as a toolset for bromodomain target validation.

Background: Histone lysine demethylases (KDMs) are of interest as drug targets due to their regulatory roles in chromatin organization and their tight associations with diseases including cancer and mental disorders. The first KDM inhibitors for KDM1 have entered clinical trials, and efforts are ongoing to develop potent, selective and cell-active ‘probe’ molecules for this target class. Robust cellular assays to assess the specific engagement of KDM inhibitors in cells as well as their cellular selectivity are a prerequisite for the development of high-quality inhibitors. Here we describe the use of a high-content cellular immunofluorescence assay as a method for demonstrating target engagement in cells. Results: A panel of assays for the Jumonji C subfamily of KDMs was developed to encompass all major branches of the JmjC phylogenetic tree. These assays compare compound activity against wild-type KDM proteins to a catalytically inactive version of the KDM, in which residues involved in the active-site iron coordination are mutated to inactivate the enzyme activity. These mutants are critical for assessing the specific effect of KDM inhibitors and for revealing indirect effects on histone methylation status. The reported assays make use of ectopically expressed demethylases, and we demonstrate their use to profile several recently identified classes of KDM inhibitors and their structurally matched inactive controls. The generated data correlate well with assay results assessing endogenous KDM inhibition and confirm the selectivity observed in biochemical assays with isolated enzymes. We find that both cellular permeability and competition with 2-oxoglutarate affect the translation of biochemical activity to cellular inhibition. Conclusions: High-content-based immunofluorescence assays have been established for eight KDM members of the 2-oxoglutarate-dependent oxygenases covering all major branches of the JmjC-KDM phylogenetic tree. The usage of both full-length, wild-type and catalytically inactive mutant ectopically expressed protein, as well as structure-matched inactive control compounds, allowed for detection of nonspecific effects causing changes in histone methylation as a result of compound toxicity. The developed assays offer a histone lysine demethylase family-wide tool for assessing KDM inhibitors for cell activity and on-target efficacy. In addition, the presented data may inform further studies to assess the cell-based activity of histone lysine methylation inhibitors.

Selectivity remains a challenge for ATP-mimetic kinase inhibitors, an issue that may be overcome by targeting unique residues or binding pockets. However, to date only few strategies have been developed. Here we identify that bulky residues located N-terminal to the DFG motif (DFG-1) represent an opportunity for designing highly selective inhibitors with unexpected binding modes. We demonstrate that several diverse inhibitors exerted selective, noncanonical binding modes that exclusively target large hydrophobic DFG-1 residues present in many kinases including PIM, CK1, DAPK, and CLK. By use of the CLK family as a model, structural and biochemical data revealed that the DFG-1 valine controlled a noncanonical binding mode in CLK1, providing a rationale for selectivity over the closely related CLK3 which harbors a smaller DFG-1 alanine. Our data suggest that targeting the restricted back pocket in the small fraction of kinases that harbor bulky DFG-1 residues offers a versatile selectivity filter for inhibitor design.