Biomolecular and biophysical approaches to interrogate epigenetic targets: a platform for drug discovery

2014 - 2015The term epigenetics refers to heritable changes in gene expression that do not involve changes in the DNA sequence. A large number of enzymes, which act mainly on histone tails and DNA, carries out epigenetic modifications, influencing several biological mechanisms. The interplay between epigenetic enzymes and chromatin is highly complex, and despite great progress has been made in understanding the role of these proteins in biological contexts, much remains still unknown. On the other hand, it is widely reported that specific epigenetic modifications are associated with disease states, therefore epigenetic enzymes represent potential therapeutic targets. However, the lack of specific and robust screening methods to evaluate epigenetic enzyme activity limits the identification and development of epigenetic modulators. In this scenery, this thesis is focused on the development of a robust and widely usable combined screening platform to identify small-molecule modulators of epigenetic proteins. Different biochemical and biophysical techniques were used in order to evaluate potency, selectivity, binding and mechanism of action of the modulators synthesized in the Epigenetic Medicinal Chemistry Laboratory (EMCL). As model systems, among all the epigenetic enzymes, the attention was focused on the acetyltransferase p300, the methyltransferase SETD8 and the readers Tudor domains of PHF20. By the use of a combined approach, a set of small-molecule modulators was identified. These compounds could be used as chemical probes to further investigate the biological role of these enzymes and their implications in physiological and/or pathological processes. [edited by author]XIV n.s

Development of a Non-Radioactive, No-Wash Biochemical Assay for High-Throughput Screening of Small Molecule Modulators of PHF20.

Plant 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.

Lysine 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

Lysine methyltransferase inhibitors: where we are now.

Protein 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.

Histone 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

NADPH Oxidases: From Molecular Mechanisms to Current Inhibitors

NADPH oxidases (NOXs) form a family of electron-transporting membrane enzymes whose main function is reactive oxygen species (ROS) generation. Strong evidence suggests that ROS produced by NOX enzymes are major contributors to oxidative damage under pathologic conditions. Therefore, blocking the undesirable actions of these enzymes is a therapeutic strategy for treating various pathological disorders, such as cardiovascular diseases, inflammation, and cancer. To date, identification of selective NOX inhibitors is quite challenging, precluding a pharmacologic demonstration of NOX as therapeutic targets in vivo. The aim of this Perspective is to furnish an updated outlook about the small-molecule NOX inhibitors described over the last two decades. Structures, activities, and in vitro/in vivo specificity are discussed, as well as the main biological assays used

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)

The 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

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

Dysregulation of the activity of lysine acetyltransferases (KATs) is related to a variety of diseases and/or pathological cellular states; however, their role remains unclear. Therefore, the development of selective modulators of these enzymes is of paramount importance, because these molecules could be invaluable tools for assessing the importance of KATs in several pathologies. We recently found that diethyl pentadecylidenemalonate (SPV106) possesses a previously unobserved inhibitor/activator activity profile against protein acetyltransferases. Herein, we report that manipulation of the carbonyl functions of a series of analogues of SPV106 yielded different activity profiles against KAT2B and KAT3B (pure KAT2B activator, pan-inhibitor, or mixed KAT2B activator/KAT3B inhibitor). Among the novel compounds, a few derivatives may be useful chemical tools for studying the mechanism of lysine acetylation and its implications in physiological and/or pathological processes

Identification of new inhibitors of PRMTs by a multi-substrate-adduct approach

The 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

Design, synthesis and biological evaluation of novel G9a inhibitors with improved brain permeability from a scaffold hopping approach

The lysine methyltransferase G9a (also known as EHMT2) catalyses the addition two methyl groups to lysine 9 of histone H3. Due to its central role in epigenetic control, the aberrant activity of this enzyme is associated to several diseases including cancer. In particular, recent evidences revealed G9a involvement in the progression of REST-expressing (repressor element (RE)-1 silencing transcription factor) medulloblastomas. 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 G9a. In particular, we replaced the quinazoline core, common to 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 (Figure 1). To validate our approach, we designed and synthesized a small library of UNC0638 analogues. The UNC0638 benzodiazepine analogue (EML741) showed a good activity in a peptide-based AlphaLISA, together with a promising membrane permeability profile (PAMPA-BBB)