The Nucleosomal Epigenetic Assay Technology (NEAT™) platform, which is proprietary to Proteros, enables the discovery of otherwise hard-to-access epigenetic targets through, for example, functional activity screening based on nucleosomal assays.
Our epigenetic lead discovery approach offers a three-fold value proposition comprising the identification and validation of novel oncology targets, the NEATTM discovery platform and our Discovery Service platform technologies.
Despite the fact that over 90% of all cancers involve complex epigenetic mechanisms, the most promising potential targets are hard to access by conventional screening methodologies. Proteros has an outstanding reputation for deciphering these complex mechanisms by applying its highly adaptable NEATTM platform for target discovery and nucleosome level functional activity screening.
Our NEATTM platform is the only discovery technology able to harness selective and sensitive high-throughput screening in the presence of fully assembled nucleosomes. (see also: epigenetic lead discovery)
The NEAT™ platform offers a three-fold value proposition, allowing the precise targeting of potential drug molecules against disease relevant epigenetic processes as well as predicting and monitoring of functional target biology. As a result, we design better in vivo active lead compounds for disease-tailored epigenetic drug candidates to become the well-differentiated oncology medicines of tomorrow.
1. Target identification and assessment
Our profound epigenetics knowledge and experience enables the identification of promising oncology targets with superior efficacy potential that conform to all relevant industrial requirements. For more information on the identification of oncology targets with superior efficacy potential, please visit Oncology Targets.
2. Nucleosomal Epigenetic Assay Technologies (NEATTM)
2.1 Disease relevant screening – Targeting the nucleosome
Proteros’ approach addressing epigenetic targets is based on biologically relevant fully assembled nucleosomes as assay- and high-throughput-screening (HTS) substrates. Our technology driven screening approach closely mimics the biology of the disease state. We ensure that our drug molecules interfere specifically with disease relevant pathways by applying stringent cellular epigenetic assays. This approach also elucidates early translational biomarker development, including prognostic and diagnostic markers.
2.2 The choice of the right substrate
The choice of the right substrate is crucial in epigenetic discovery. The biological target of most epigenetic enzymes in the cell are nucleosomes, not free histones. Therefore, we employ natural epigenetic substrates composed of specifically modified nucleosomes and full-length epigenetic holoenzymes, containing all relevant subunits and/ or co-factors, for screening. This strategy is universally applicable to all epigenetic target families and substantially increases the likelihood of success in your projects.
In case of large, multi-protein epigenetic complexes with multiple interaction domains, fully assembled nucleosomal substrates are required to address binding sites.
2.3 The nucleosome design process
Epigenetic mechanisms ultimately alter gene expression programs through functional interactions with nucleosomes, the fundamental regulatory scaffolds of our genome. Each epigenetic enzyme has its specific functionality, for example regularly spacing the nucleosomes along the DNA to enable the formation of condensed chromatin or sliding the nucleosomes along the DNA to make a gene of interest accessible for expression.
Therefore, the nucleosomal substrate design process is determined by the target’s functionality. In case the general functionality of the target is unknown, our approach entails a prior functionality investigation. This will feature either using a single nucleosome (mononucleosome) with DNA overhangs or a nucleosomal chain with multiple nucleosomes (oligonucleosome). In a next step, the need for marks or detection reagents is addressed.
2.4 The screening substrate production
Our NEATTM platform ensures the highly reproducible and scalable production of homogenous and pure histone variants. The chemical ligation of the detection dye is based on a genetic modification of the histone. After the ligation, the assembly of all eight histones to an octamer and the subsequent DNA assembly finally lead to a homogeneously modified nucleosome. This process is based on our present knowledge and experience deriving from the NEATTM platform which features unique and patent-protected technologies to introduce nucleosomal modifications. An assay-specific substrate can therefore be produced according to the target’s disease biology.
2.5 Disease relevant assay examples
The nucleosome sliding assay, the methyl transferase assay and the reader domain assay are examples of our screening cascade (Link to point 2.8). They are all based on Fluorescence Resonance Energy Transfer Technology (FRET) or Time Resolved Fluorescence (TR-FRET).
In the nucleosome sliding assay, one histone of the mononucleosome is dye-labeled with a donor chromophore as well as one DNA overhang with an acceptor chromophore. When the epigenetic enzyme slides the nucleosome along the DNA to the overhang end the two different dyes are broad in close proximity, an energy transfer from the donor to the acceptor chromophore takes place. In consequence, the reduction of the donor´s fluorescence intensity is measured and reflects the sliding process. This assay is applicable for the investigation of all types of inhibitors.
In the methyl transferase assay, the substrate is an ubiquitinated mononucleosome carrying a dye-labeled biotin. The ubiquitination of the biotin-labeled nucleosome leads to the methylation of one histone by the methyltransferase (i.e. Dot1L). In consequence, a dye-labeled antibody will bind to the methyl group. The reduction of the donor´s fluorescence intensity is measured and reflects the methylation process of Dot1L. With this assay, allosteric enzymes can be identified.
The reader domain assay is useful in the context of investigating reader domain inhibitors. The substrate is an oligonucleosome multiply dye-labeled, the target of interest contains a bromodomain. In case of close proximity of target and substrate, the fluorescence intensity of the donor chromophore will be reduced and reflects the enzymatic binding process.
All these assays have already been performed on our NEATTM platform as HTS screens to identify in vivo active lead compounds.
2.6 The NEATTM nucleosome library
The NEAT platform offers recombinant variants and mutants of histones, which can be assembled to 28 different histone octamers. These nucleosome variants can be combined with 34 DNAs. All these substrates are part of our NEATTM nucleosome library and available for the direct use of customized assay development and HTS upscale.
2.7 The delivery of the best possible HTS hits
The NEAT platform provides HTS technology to identify in vivo active lead compounds. Different assays are implemented for specific scientific questions, depending on the mode of action of the potential inhibitor and the functionality of the target.
For example, the ATPase Assay can only identify a subset of inhibitors, whereas the sliding assay is sensitive to all types of relevant inhibitors.
Therefore, a larger number of hits are identified while minimizing inherent false positives.
2.8 The NEATTM approach covers the full screening cascade
Our NEATTM platform comprises the full screening cascade, starting with a primary screen in HTS mode for profiling, followed by a secondary sensitive screen for detailed information.
The primary screen consists either of a nucleosomal sliding or displacement assay to determine if the compound influences the chromatin remodeling activity. The secondary assay consists of an ATPase activity or consumption assay, which indicates the mode of action of the compound. The drug candidate inhibits the enzyme activity either by competing with the ATP molecules for their binding side or via binding to a catalytic or allosteric domain of the target. In case of inhibition, off-targets also have to be tested.
In the next step, the assay is transferred to a biologically similar environment, the cellular assay. The diverse cellular assays offered within the NEAT platform, indicate what additional impact a potential drug candidate might have in a multi-cellular context. Furthermore, the results of cellular assays enable an integrated evaluation of the epigenetic efficacy and the disease modification potential. The in vivo validation represents the end of the screening cascade, where the most promising compounds are tested on xenograft models.
3. Discovery technologies for demonstration of in vivo Poof-of-Concept
The sophisticated NEATTM platform comprises Proteros’ complete service offerings, starting from structural biology, biophysics to protein science and medicinal chemistry while simultaneously combining these with epigenetic knowledge and experience. This unique in-house platform is a precondition for a successful hit-to-lead process, so potential epigenetic drug targets could finally become validated epigenetic lead candidates.