Target ID and validation

 

Evotec has established a world-leading platform to support the discovery and validation of innovative disease modifying targets across different therapeutic areas as well as aid target deconvolution following a phenotypic screen.

Our capabilities to identify and validate targets comprises:

  • Differential gene expression profiling across cells and tissues followed by bioinformatics-driven data mining and hypothesis building
  • Gene knock-out and over-expression studies both in vitro and in vivo using relevant disease models
  • World-class ex vivo imaging technology platform using tissue sections to study cellular and molecular events
  • Phenotypic screening using complex cellular models to mimic disease
  • Proteomic platforms to aid target deconvolution

Gene expression profiling

Gene activity is regulated very precisely and highly dynamically in cells, tissues or whole organisms. Observing changes in gene expression can give valuable insight into physiology, disease states, or drug action in biological systems. From early target identification to mode of action studies and biomarker identification the analysis of gene expression data supports the drug discovery processes.

Key activities include:

  • In vitro or in vivo design and execution of transcriptomics studies, choosing the relevant technology for the question at hand. Evotec routinely uses a broad range of methods – from the monitoring of single genes in very high throughput with targeted approaches to full transcriptome methods like RNA-Seq

Transcriptomics platform

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  • Bioinformatics analysis of differential gene expression: a team of experienced bio-informaticians routinely assess differential gene expression, pathway and interaction networks by analysis of raw data from transcriptomics studies and provide expert biological insight
  • Considerable experience with the major repositories of expression data and signatures is available.  Depending on project needs we also run customised in silico workflows, e.g. meta-analyses or integration of transcriptomics and predictive pharmacology data

Data handling and analysis tools

To analyse large datasets generated from deep RNA sequencing, Evotec has developed a powerful and user-friendly data analysis platform. Based on peer reviewed algorithms the Evotec transcriptomics platform allows scientists to work interactively with gene expression data and to use state-of-the-art statistics and application tools.

In vitro target modulation capabilities

Evotec offers a variety of approaches, principally based on AAV and Lentivirus delivery systems for in vitro target modulation, including the powerful CRISPR technology. This genetic toolbox allows for target manipulation in a broad spectrum of disease related models and can be coupled to a broad range of relevant read-outs. Evotec uses CRISPR-Cas9 gene editing technology licensed under ERS Genomics and the Broad Institute.

Target identification

Evotec’s genetic screening platform applies whole-genome wide CRISPR and shRNA screening to aid target identification in a variety of cellular systems. Pooled and arrayed screening coupled to relevant read-outs aim to deconstruct complex biological phenotypes. An integrated bioinformatics pipeline enables relevant hit identification and biological annotation.

Target deconvolution

In the context of identifying relevant targets of hits from phenotypic screenings, CRISPR technology allows for assessing the contribution of each gene in the genome to compound activity. This offers a broad spectrum analysis of compound biology including identifying direct compound targets.

Target validation

CRISPR and related technologies offer straight forward solutions for target validation. Evotec offers basic loss-of function and over-expression models but also more elaborate genome editing approaches in disease relevant models including primary cells and iPS-derived systems to confirm target contribution to disease phenotypes.

In vivo target validation

Targets arising from either disease relevant animal models, in vitro genetic experiments or the public domain need careful validation and confirmation, ideally prior to initiating a small molecule discovery programme. Evotec has developed a number of in and ex vivo models across numerous therapeutic indications to help answer these questions.

Target engagement and modulation with small molecules or antibodies

Using a variety of techniques ranging from microdialysis to pharmacodynamic modelling with biomarker detection and RNA expression profiling, Evotec aims to demonstrate early physiological function of the target in disease biology. This embraces a number of end points such as monitoring secreted factors, slice electrophysiology or signalling cascade modulation.

In vivo knockdown experiments

In the absence of suitable tool compounds alternative genetic approaches embracing stereotatic delivery of AAV-shRNA delivery is routinely used to modulate in vivo expression levels and activity of the host protein, to investigate the effects this protein has on cell physiology and disease progression.

High-throughput histology

To maximise the efficiency and robustness of in vivo target modulation, Evotec has implemented an automated histology platform leveraging the expertise in image analysis and quantification.

Phenotypic screening

The ability to study small molecules, biologics or genetic targets in the context of disease biology can only be truly leveraged through the application of phenotypic screening in a disease setting.  With over 15 years of experience, Evotec offers a wide selection of phenotypic assays suitable for HTS and mechanism of action studies.

A variety of read-outs

From high-content imaging through to flow cytometry and ELISA formats the most appropriate read-out for the mechanism of interest is applied.

Phenotypic fingerprinting of human liver cells

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Disease relevant in vitro models

Using target agnostic approaches, Evotec emphasises the use of disease relevance from the perspective of target tissue and cellular model through to stimuli and read-outs.

Primary cells and iPS models

Where possible Evotec employs primary cells or cell lines appropriate for the disease of interest : via the implementation of iPS cell lines and bespoke differentiation approaches, through the patient in a dish approach, Evotec aims to address human translation as early as possible.

Genetic screening

Phenotypic screening at Evotec not only enables hit identification but, through the use of CRISPR and shRNA genome wide libraries allows:

  •  The de-novo identification of novel targets
  • Study  small molecule target(s) mediating effect

Chemical Proteomics and Target ID

Evotec scientists have pioneered chemical proteomics applications to support target deconvolution of bioactive compounds emerging from phenotypic screens. Cellular target identification is the crucial step to enable further drug optimisation and development.
Evotec Cellular Target Profiling™ is a powerful technology to identify specific cellular compound targets in lysates from any cell type or tissue.  Evotec’s chemical proteomics offering further comprises photoaffinity labelling for covalent target capture in live cells.

  • Evotec’s chemical proteomics approaches use high-end quantitative mass spectrometry to reveal and verify specific cellular targets of a drug
  • Evotec Cellular Target Profiling™ determines target-specific dissociation constants for the compound studied, ranking targets according to their likely physiological relevance

Evotec Cellular Target Profiling™ workflow

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  • Drug photoaffinity labelling supports binding site identification in protein targets and complexes
  • Unbiased target ID by proteome-wide profiling of  native, endogenously expressed, post-translationally modified proteins in the presence of cellular co-factors and native complex partners
  • Complementary chemical proteomics approaches can be performed in an integrated fashion 
  • Extensive, non-target class restricted track record in successful profiling of diverse small molecule compounds

In silico target identification and drugability assessment

Target identification

Evotec uses industry standard target interaction databases which combined with other ‘omics’ data can be used to construct target interaction maps. These networks can be significantly enhanced using our predictive pharmacology tools. Over 200 million bioassay datapoints are currently sourced for model building. Pathway analysis can be used to identify new and novel targets proposed to modulate a disease hypothesis. Such approaches are fundamental in target deconvolution and drug repurposing.

Drugability assessment

Evotec can run structure-based assessment studies for novel targets to provide an additional read on drugability when no/few druglike compounds are known. New 2D sequence based “switchability” calculations projected onto 3D structures have successfully been used for predicting allosteric sites for modulation, surfaces for PPIs and mAb binding.

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