1,721,106 research outputs found
The open challenges of cognitive frailty: risk factors, neuropsychological profiles and psychometric assessment for healthy aging
No full textDevelopment of a Non-Radioactive, No-Wash Biochemical Assay for High-Throughput Screening of Small Molecule Modulators of PHF20.
No full textPlant homeodomain finger protein 20 (PHF20) is a multidomain protein mainly involved in the activation of p53 and in the prevention of its ubiquitylation. Furthermore, it uses a Tudor domain to read dimethyl lysine residues and is a known component of the MOF (male absent on the first) histone acetyltransferase protein complex, suggesting that it plays a role in the cross-talk between lysine methylation and histone acetylation.
Writer and eraser proteins have been the main focus of therapeutic development but over the past few years a relatively underexplored group of proteins, the readers, have emerged as promising targets operating at the interface of translating histone marks. While therapeutic potential is evident, there’s need to establish specific biochemical assay for drug discovery.
We describe here the development of a high-throughput, nonradioactive bead-based assay that is suitable for screening applications to identify new PHF20 ligands.
The Tudor domain of the protein was expressed in E.Coli and purified by affinity chromatography using the GST tag. Biotinylated peptide H4K20me2 was incubated with the protein, afterwards the addition of biotinilated donor beads and anti-GST acceptor beads allowed us to measure the activity of the protein. The optimization of the assay was performed varying assay buffer, reaction time, substrate and protein concentration.
Overall, the results presented demonstrate that this novel homogenous and nonradioactive PHF20 assay could represent a powerful technology for measuring readers activity
DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL G9A INHIBITORS FROM A SCAFFOLD HOPPING APPROACH
No full textThe lysine methyltransferase G9a (also named euchromatin histone methyltransferase 2, EHMT2) is primarily responsible for the dimethylation of lysine 9 on histone H3 (H3K9). Several reports have
highlighted its link to a variety of cancers. In particular, it has been shown that G9a is crucial for the oncogenic role of the repressor element (RE)-1 silencing transcription factor (REST) in the pediatric brain tumor medulloblastoma. Only a few among the selective inhibitors of G9a reported to date are useful chemical probes for cell-based and animal studies. Starting from the inhibitor UNC0638,3 we applied a scaffold hopping approach to develop novel chemical entities endowed with high affinity towards the G9a. In particular, we replaced the quinazoline core, typical of most of the reported inhibitors, with 1,4-benzodiazepine nucleus, known to be a privileged structure. We chose the 3,4-dihydro-5H-benzo[e][1,4]diazepin-5-one scaffold, that can be obtained through an efficient and gram-scale continuous-flow protocol, previously optimized by our group.4 Moreover, this scaffold could be easily decorated to provide a number of highly functionalized potential ligands. To validate our approach, we designed and synthesized a small library of UNC0638 analogues. All the compounds were tested through a peptide-based AlphaLISA, measuring the levels of H3K9 dimethylation
Biomolecular and biophysical approaches to interrogate p300: a platform for drug discovery.
No full textLysine acetylation is a protein post-translational modification which effect the relaxing of the chromatin structure, making chromosomal DNA more accessible. Among the different enzymes responsible for this transformation (KATs), p300 is one of the most studied: the dysregulation of its activity leads to many human diseases.
Nevertheless, a limited number of p300 modulators have been described so far: one of the main problem is the absence of a gold standard screening technique for this enzyme because of the intrinsic limitation of each method. We decided to develop a robust and widely usable combined screening platform to identify small molecule modulators of p300, using different biophysical and biomolecular techniques to interrogate the target and to validate the outputs.
The multiple platform was applied to two different libraries of small molecule compounds, derived from the molecular pruning of anacardic acid and garcinol, natural inhibitors of p300.
This combined approach allowed us to identify and deeply characterize the activity of new chemical probes, very useful for the study of p300-mediated lysine acetylation and its implications in physiological and/or pathological processes
Discovery of new chemical probes for the MRG15 chromo domain.
No full textMethylation of histone tails represents a crucial post-translational modification involved in transcriptional regulation. An increasing body of evidence suggests that individual histone modifications, or a combination of them, may serve as a platform to recruit specific “reader” proteins, which then determine the transcriptional outcome of the target genes.
In particular, methylated histone tails are recognized by chromatin-reading modules such as Chromo, Tudor, PWWP and MBT domains, together designated as the “Royal family” of chromatin binding domains. Among them, MRG15 (MORF Related Gene in chromosome 15, Figure 1), is a conserved chromodomain-containing nuclear protein that specifically binds to Lys36-methylated histone H3.
Although several lines of evidence suggest that MRG15 and its related proteins are involved in gene regulation and DNA-repair processes, the mechanisms are still unknown.
Being interested in the development of novel small molecule modulators of epigenetic targets, here we report the identification of the first class of inhibitors of MRG15, that might be new tools to study the function of MRG15 endogenously
Synthesis of 11-aryl-5H-imidazo[2,1-c][1,4]benzodiazepines and their benzodiazepine and A1 adenosine binding activity.
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Synthesis and antifungal properties of N-[(1,1'-biphenyl)-4-ylmethyl]-1H-imidazol-1-amine derivatives.
No full textDMSO‐Related Effects on Ligand‐Binding Properties of Lysine Methyltransferases G9a and SETD8
No full textBeing the standard solvent for preparing stock solutions of compounds for drug discovery, DMSO is always present in assay buffers in concentrations ranging from 0.1% to 5% (v/v). Even at the lowest concentrations, DMSO-containing solutions can have significant effects on individual proteins and possible pitfalls cannot be eliminated. Herein, we used two protein systems, the lysine methyltransferases G9a/KMT1C and SETD8/KMT5A, to study the effects of DMSO on protein stability and on the binding of the corresponding inhibitors, using different biophysical methods such as nano Differential Scanning Fluorimetry (nanoDSF), Differential Scanning Fluorimetry (DSF), microscale thermophoresis (MST), and surface plasmon resonance (SPR), all widely used in drug discovery screening campaigns. We demonstrated that the effects of DMSO are protein- and technique-dependent and cannot be predicted or extrapolated on the basis of previous studies using different proteins and/or different assays. Moreover, we showed that the application of orthogonal biophysical methods can lead to different binding affinity data, thus confirming the importance of using at least two different orthogonal assays in screening campaigns. This variability should be taken into account in the selection and characterization of hit compounds, in order to avoid data misinterpretation
N-Pyrrylarylsulfones with High Therapeutic Potential
Full text linkThis review illustrates the various studies made to investigate the activity of N-pyrrylarylsulfone containing compounds as potential antiviral, anticancer and SNC drugs. A number of synthetic approaches to obtain tetracyclic, tricyclic and non-cyclic compounds, and their biological activity with regard to structure-activity relationships (SARs) have been reviewed. The literature reviewed here may provide useful information on the potential of N-pyrrylarylsulfone pharmacophore as well as suggest concepts for the design and synthesis of new N-pyrrylarylsulfone based agents
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