1,720,988 research outputs found
Development 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
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
DMSO‐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
Development of a Microscale Thermophoresis-Based Method for Screening and Characterizing Inhibitors of the Methyl-Lysine Reader Protein MRG15
No full textMRG15 is a transcription factor containing the methyl-lysine reader chromodomain. Despite its involvement in different physiological and pathological states, to date the role of this protein has not been fully elucidated due to the lack of a specific and potent chemical probe.In this work, we report the development of a microscale thermophoresis (MST)-based assay for the study of MRG15-ligand binding interactions. After the development, the assay was validated using a small focused library and UNC1215 as the reference compound, to yield the identification of 10 MRG15 ligands with affinities ranging from 37.8 nM to 59.1 μM. Hence, our method is robust, convenient, and fast and could be applied to other methylation reader domain-containing proteins for the identification of new chemical probes
Lysine methyltransferase inhibitors: where we are now.
No full textProtein lysine methyltransferases constitute a large family of epigenetic writers which catalyse the transfer of a methyl group from the cofactor S-adenosyl-L-methionine to histone and non-histone specific substrates. Alterations in the expression and activity of these proteins have been linked to the insurgence and progress of several diseases, including cancer, neurological disorders, and growing defects, hence they represent interesting targets for new therapeutical approaches. Over the past two decades, the identification of modulators of lysine methyltransferases has increased tremendously, clarifying the role of these proteins in different physio-pathological states. The aim of this review is to furnish an updated outlook about the protein lysine methyltransferases disclosed modulators, reporting their potency, the mechanism of action and their eventual use in clinical and preclinical studies
Use of Microscale Thermophoresis (MST) for Studying binding interactions of PRSet-7/SETD8 with small molecule specific inhibitors EPI-9 and EPI-23.
No full textHistone methylation plays a key role in establishing and maintaining stable gene expression patterns during cellular differentiation and embryonic development. Considering that a number of small molecules identified as modulators of methyltransferases by high-throughput screening were dyes or derivatives, a small focused library of dye-like compounds was prepared and the small molecules synthesized were pre-screened for potential inhibition of histone lysine methyltransferases (HKMT) using in vitro HMT assays. Two compounds, EPI-
9 and EPI-23, showed low IC50 values against nucleosomal HKMT PR-Set7. Prompted by our interest in the study of small molecule modulators of these epigenetic targets, we applied MST (Microscale Thermophoresis) to the investigation of the interaction of PRset7 with small molecule ligands EPI-9 and EPI-23. Microscale thermophoresis (MST) is a new method that enables the quantitative analysis of molecular interactions in solution. MST is the directed movement of particles in a microscopic temperature gradient: any change of the hydration
shell of biomolecules due to changes in their structure/conformation results in a relative change of movement along the temperature gradient and is used to determine binding affinities, binding kinetics and activity kinetics. Events such as the binding of small molecules to a target can be monitored by this tecnique
Identification of new inhibitors of PRMTs by a multi-substrate-adduct approach
No full textThe methylation of arginine residues is a prevalent posttranslational modification found in both nuclear and cytoplasmic proteins, which is involved in a number of different cellular processes, including transcriptional regulation, RNA metabolism, and DNA damage repair. Enzymes of the protein arginine N-methyltransferase (PRMT) family catalyze the transfer of a methyl group from the donor S-adenosyl-L-methionine (SAM or AdoMet) to the guanidinium side chain of arginine residues in the target protein. Despite extensive research aimed at better understand the role of PRMTs in physiological and pathological pathways, there have been only a few publications to date describing small-molecule chemical modulators of the PRMTs. A few years ago, starting from AMI-1 (the first selective inhibitor of PRMTs)1 we identified EML108, which was characterized by an improved selectivity profile among methyltransferases and a good cellular activity.2 Moreover, docking studies clearly showed that EML108 bind SAM and arginine pocket without fully occupying them. Starting from this evidence, we herein report the design and the synthesis of new PRMTs inhibitors based on the naphthalene scaffold of EML108. Firstly, we prepared some derivatives bearing a guanidine moiety connected to the naphthalene scaffold via a variable linker. After optimization, we further functionalized this scaffold with an adenosine moiety (Figure 1). This multi-substrate-adduct approach lead to the identification of new sub-micromolar inhibitors of the arginine methyltransferase PRMT1
Inside Cover: Identification of Structural Features of 2-Alkylidene-1,3-Dicarbonyl Derivatives that Induce Inhibition and/or Activation of Histone Acetyltransferases KAT3B/p300 and KAT2B/PCAF (ChemMedChem 1/2015)
No full textThe inside cover picture shows the modulation of lysine acetyltransferase (KAT) activity by SPV106 (yellow). By manipulating the structural features of SPV106, different activity profiles—from pure PCAF (top left) activator, pan inhibitor, or mixed PCAF activator/p300 (bottom right) inhibitor—were obtained. The reported compounds represent useful chemical tools for mechanistic studies of histone H3 (lime) or H4 (orange) acetylation (Ac groups shown as purple spheres) and its implications in physiological and pathological processes. For more details, see the Full Paper by Alessandra Tosco, Gianluca Sbardella et al. on p. 144 ff
Progress in the Development of Lysine Methyltransferase SETD8 Inhibitors
No full textSETD8/SET8/Pr-SET7/KMT5A is the only known lysine methyltransferase that monomethylates lysine 20 of histone H4 (H4K20) in vivo. The methyltransferase activity of SETD8 has been implicated in many essential cellular processes, including DNA replication, DNA damage response, transcription modulation, and cell cycle regulation. In addition to H4K20, SETD8 monomethylates non-histone substrates including proliferating cell nuclear antigen and p53. During the past decade, different structural classes of inhibitors targeting various lysine methyltransferases have been designed and developed. However, the development of SETD8 inhibitors is still in its infancy. This review covers the progress made to date in inhibiting the activity of SETD8 by small molecules, with an emphasis on their discovery, selectivity over other methyltransferases, and cellular activity
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