Your Way to Validated Hits

Hit Identification

Evotec is a leading expert in hit identification, providing diverse support for screening campaigns through virtual, biochemical, biophysical, cell based, and phenotypic platforms with high-throughput capabilities. Our flexibility allows screening of large client compound collections and our own lead-like compound collections, each comprising 400,000 compounds. Additionally, we offer 25,000 fragments, 5,000 bio-annotated compounds, 2,000 macrocycles, or 30,000 natural products and semi-synthetic derivatives.

Notably, we recently added a covalent library of ~5,000 compounds, aiding the identification of covalent inhibitor hits.

Our state-of-the-art platforms employ a wide array of detection technologies and assay development, enabling sensitivity screening and detection of valuable hits. Stringent review processes ensure the selected compounds have the required lead-like profiles. By understanding each client's project requirements, our experienced screening team designs tailored hit identification campaigns, maximizing the chances of success.

Hit Identification-Expansion_Supported_With_REACT-EU_Grant

Learn more about the EFRE/REACT-EU-Grant.

Screening Collections

Discover a treasure trove of screening potential in Evotec's extensive collection of compounds, carefully curated to fuel groundbreaking discoveries.

The Evotec Collection

Evotec’s library contains 400,000 lead-like compounds consisting of a set of 370,000 maximally diverse “islands of similarity” from commercial sources and 30,000 proprietary Evotec compounds, 25,000 fragments, 5,000 compounds with known bio-annotation, 30,000 natural products, and natural product derived semi-synthetic compounds from the Analyticon collection and 2,000 macrocycles. The recently added covalent library of ~5,000 compounds support the identification of covalent inhibitor hits.

Evotec’s screening library is differentiated through its quality, diversity and novelty and has a confirmed track record of delivering potent and attractive hit compounds representing suitable starting points for medicinal chemistry optimisation. Our compound libraries are available as single compound screening but also in pools, optimised for affinity selection mass spectrometry screening (ASMS).

For structure-based approaches, Evotec has a 20,000-fragment set available for biochemical high concentration and biophysical SPR screening. In addition, up to 4,500 fragments, specifically selected for NMR and SPR screening can be chosen. Finally, a 500-member fragment collection is available for X-ray crystallography screening by crystal soaking.

Evotec constantly invests into its libraries to maintain highest quality, expand the diversity and provide access to new chemical modalities.

The Aptuit Collection

Aptuit’s library contains 400,000 compounds which are complementary to the Evotec collection. It consists of 350,000 maximally diverse lead-like “islands of similarity” with low lipophilicity plus a 50,000 “ligand efficiency” collection.

Third Party Collection

Evotec can also screen customer’s libraries of any size, virtual screening derived hits and smaller, more target directed libraries.

High-Throughput Screening

Evotec has a long history in high-throughput screening utilizing multiple compound collections and has run more than 750 biochemical, cellular or micro-organism based HTS campaigns. Leveraging this expertise, a state-of-the-art screening platform, a strong experience in assay development and miniaturization (>1,500 assays developed), and an excellent disease biology knowledge, Evotec selects the most suitable screening strategy for your target.

High Throughput Screening at Evotec in a Nutshell

Evotec High Throughput Screening Track Record

Compound Screening Process

The integration and connection of the different stages of the screening process from assay development, automation, compound management and tracking to data acquisition and processing are essential. The Evotec screening team manages the complexity of these processes daily to deliver high-quality data to its clients in the most flexible manner to meet customers’ needs in data format for upload in different environment.

Assay Systems

Evotec has more than twenty years of experience in assay development, High-Throughput Screening (HTS) and validation of hits with orthogonal methods like biophysical techniques.

We believe that the success of a screening project is determined by:

  • Selection of the right target species
  • Right strategy for target expression to address specific sites of interest
  • Robust and pharmacologically sensitive assay systems
  • Well-selected, curated compound collections for screening
  • Reliable screening processes including counter and orthogonal assays
  • Careful characterization of screening hits by medicinal chemistry assessment
  • Continuous and tight interaction with our clients during the screening project

Our assay system capabilities and experience include:

  • A broad portfolio of assays addressing the major therapeutically relevant target classes: our team has developed more than 1,500 assays to support drug discovery programs
  • Assay miniaturization for over 750 cell-based, micro-organism based or biochemical HTS campaigns
  • New target classes in the field of inflammation, transporter biology, protein-protein interactions, and small molecule RNA modulators
  • Anti-infective screens ran on multiple pathogenic strains and bacterial or viral targets
  • High number of unique assays developed as no literature precedence was available
  • Assay read-outs covering a wide range of technologies including traditional fluorescence and luminescence-based assays, radiometric assays, high content imaging, high throughput electrophysiology, mass spectrometry and RT-qPCR
  • Assays using label-free and biophysical methods

Assay Overview

Biochemical assays

  • Fluorescence polarization (catalytic activity and binding)
  • Fluorescence intensity (catalytic activity and binding)
  • TR-FRET (catalytic activity and binding)
  • AlphaScreen® (catalytic activity and binding)
  • Luminescence (enzymatic and binding assays)
  • Mass spectrometry (catalytic activity)
  • Mass spectrometry (binding)
  • Mass spectrometry intact mass (covalent binding)
  • Absorbance based read-outs
  • Radiometric assays

Biophysical assays and orthogonal, label-free read-outs

  • Surface Plasmon Resonance (SPR)
  • Nuclear Magnetic Resonance (NMR)
  • Thermal shift (DSF)
  • Nephelometry
  • Microscale thermophoresis
  • SwitchSense technology
  • Isothermal titration calorimetry
  • Mass spectrometry (binding, intact protein MS, HDX-MS)

Cellular assays

  • Ca2+ flux (FLIPR)
  • Second messenger (cAMP, IP3)
  • Membrane potential (ion channels)
  • Reporter gene assays
    (Luciferase, SEAP, β-lactamase, β-galactosidase)
  • Whole cell fluorescence
  • ELISA, Mesoscale, HTRF
  • Automated patch-clamp
  • Primary cells & stem cells
  • Migration assays
  • Cytometry (cell cycle regulation, receptor expression studies)
  • Metabolic assays (radiometric, O2 consumption and pH change, metabolite production via LC/MS)
  • RTqPCR on endogenous genes
  • HiBiT protein detection

High-Throughput Screening Platforms

Evotec’s high-throughput screening platform is differentiated through integrating a large variety of detection technologies to state-of-the-art high-throughput capabilities.

  • Variety of automation systems available to offer high flexibility about assay plate formats, assay read-outs, readers, and dispenser/washers
  • Overall capacity of running ~80 HTS campaigns per year
  • Maximum throughput of 1,536-well plate systems up to 100,000 data points per system/day
  • Fully automated support of RapidFire/MS and ECHO MS screening
  • Infrastructure and permissions in place to run radiometric and BSL2+ / BSL3 screening campaigns
Our infrastructure and permissions enable the execution of radiometric and biosafety level 2 screening campaigns:

HT Screening of Micro-Organisms

Evotec has a very strong background in anti-infective disease; leveraging an exceptional in vitro and in vivo biology knowledge in anti-infectives and a comprehensive portfolio of assays. Our team provides full bio-organisms screening capabilities against BSL2 and BSL3 biological agents.

Micro-organism screening capabilities include:

  • Recombinant human cells or primary cells
  • Micro-organisms: bacteria, viruses, and yeast & fungi

Read-out technologies include:

  • Absorbance
  • Luminescence
  • Fluorescence
  • HTRF
  • High-content imaging
  • RT-qPCR

Screening assay principles in 384-well and 1536-well plate formats include:

  • Phenotypic assays on growth inhibition of micro-organisms and cells
  • Target-based assays: biochemical assays, cellular assays with gene reporter and cell-based assays with the incubation of cells and pathogen

RT-QPCR Screening: Targeting the RNA

Evotec’s assay development and HTS includes high-throughput RT-qPCR capabilities. As many proteins are still considered challenging therapeutic candidates, RNA is increasingly appreciated as an alternative potential drug target. Evotec’s RT-qPCR platform allows to investigate the fluctuation of cellular mRNA, or non-coding RNA, in response to treatment with drug candidates, e.g. small molecules, antisense oligonucleotides, interfering RNAs or proteins. Inhibition of the translation of otherwise undruggable targets, directed splicing or monitoring compounds activity on viral loads can be evaluated in a high throughput manner. RT-qPCR is compatible with all cell types, adherent or in suspension, and even with the most advanced models such as induced pluripotent stem cells or organoids, allowing the development of assays in physiologically relevant models. It is faster to develop than reporter assays and can also be multiplexed to analyze the expression of different genes in a single well, which gives high information content and is especially useful during targeted splicing studies. The sensitivity, specificity, robustness, and flexibility of RT-qPCR make it a useful tool to be used at the early stages of drug discovery projects as the primary screening assay, but can also be applied to hit confirmation and hit profiling approaches as a secondary or orthogonal assay to other technologies.

RT-qPCR screening capabilities and expertise include:

  • A miniaturized, accelerated and cost-optimized RT-qPCR assay
  • A fully automated robotic structure unique to Evotec consisting of incubators, washer/dispensers, nanoliter Echo550 acoustic liquid handler and 4 BioRad CFX384 real-time PCR systems
  • A high throughput screening approach to gene expression profiling with established processes for 384-well plates. A maximum throughput of 15000 compounds per day on 4 different targets per well can be achieved
  • Screening analysis with Genedata, a powerful tool specifically developed and optimized for the treatment of HTS data flow and adapted for quantitative PCR.
  • A system well characterized over the years, with a large number of assays developed for a track record of several million compounds screened.
  • A PCR activity in continuous development with implementation of new automated processes in the platform, such as high throughput mRNA purification

High-Content Screening

Evotec has strong expertise and long-term experience in the development and implementation of high-content based assays for high-throughput screening and compound validation to probe the pathophysiological environment.

High-content screening (“HCS”) is routinely used throughout the hit identification cascade from target validation to mechanistic studies. The assays developed at Evotec encompass both a target centric as well as phenotypic approach with emphasis on tissue samples, stem cell derived or primary cells from appropriate hosts. This approach is a core part of Evotec’s efforts to identify novel targets and drugs with disease-modifying potential.

Evotec’s HCS expertise is built around the PE Opera Phenix platform. An essential understanding of the mechanics and image analysis tools, gathered over a period of more than 15 years in the development of the platform, flows into the hit identification process. Further systems like the Operetta CLS and Incucyte readers support different requirements in resolution and throughput. The platform is completed with a sophisticated data management system and powerful data analysis tools including multifactorial analysis, also allowing to perform applications like large scale cell painting screens.

Based on this expertise, Evotec has performed many high-throughput screens of up to 400,000 compounds and developed over 50 assays to study mode of action of compounds and targets in a multitude of cellular backgrounds.


  • Proliferation
  • Cell cycle analysis
  • Cell death/necrosis/apoptosis
  • Differentiation markers
  • DNA damage
  • Cytoskeleton rearrangements
  • Neurite outgrowth
  • Mitochondrial mass/-toxicity
  • Endocytosis
  • Synapse density
  • Neurodegeneration/-protection
  • Intracellular aggregation
  • Post-translational modification

Biophysical & Label Free Platform

Evotec has established a comprehensive biophysics platform. Backed by years of experience the team will design the optimal experiment to meet the demands of research budgets and timelines. The platform delivers binding information and the kinetic profiling of target engagement. Such orthogonal biophysical readouts are an essential component of the hit validation process. We provide confidence about on-target activity of identified hit series and thus help focusing on the relevant chemical matter for further progression.

The platform for biophysics includes the following techniques:

  • NMR
  • SPR
  • HDX-MS
  • GCI
  • DSF
  • Affinity mass spectroscopy
  • MST
  • SwitchSense
  • ITC

The established workflow includes a complete fragment-based drug discovery (“FBDD”) platform. Sensitive screening techniques such as NMR, SPR, DSF, and mass spectrometry (MS), combined with specifically selected fragment compound collections, identify attractive and diverse, low molecular weight fragment hits. Those provide multiple, highly efficient starting points for chemical elaboration. Routine and high-throughput crystallography of the fragment-target complex delivers efficient fragment evolution, driving optimization of potency and selectivity.

Biophysics – Screening Using Labelled & Non-labelled Methods

NMR Screening and Hit Qualification

NMR screening as part of the Evotec fragment-based design platform (FBDD) platform is an option for the identification of new starting points for structure-based drug discovery (SBDD) programs. Evotec offers both protein-observed NMR assays monitoring chemical shift perturbations (SAR-by-NMR) as well as ligand-detected NMR assays (Saturation Transfer Difference “STD” NMR and Water LOGSY) for hit identification. Hence, a broad range of target proteins amenable to NMR screening technique can be covered. The recent upgrade of the system with a 1.7 mm TXI cryo-probe head and resulting reduction of sample volume enables us to also consider the screening of hard to get or hard to produce targets.

Evotec has employed its NMR screening platform on more than 30 protein targets covering a whole range of target classes from frequently studied enzymes (like kinases or proteases) over nucleotide binding proteins or epigenetic targets, to protein-protein interaction targets, even receptor domains. Recently, RNA targets have been investigated as well.

NMR also adds considerable value to biochemical screening campaigns by validating hit compounds for direct binding to the target protein and ultimately correlating this information to structure data, thereby enabling, or facilitating SBDD programs.

Surface Plasmon Resonance ("SPR") Screening

SPR technology represents a powerful tool for studying biomolecular interactions in a sensitive and label-free manner in real-time.

While commonly used at later stages of drug discovery projects to qualify hit compounds with regards to binding kinetics, the SPR technology available at Evotec also allows for primary screening of fragment libraries. Moreover, we are using SPR in all kind of drug discovery approaches early on to characterize target-drug interactions including fragments, lead like compounds, peptides, biologics and RNA molecules.

Direct binding assays are run on the Biacore™ 4000 or Biacore 8k and 8k+ screening devices enabling the identification of fragments interacting with immobilized target proteins. As the throughput is sufficiently high, we can screen large fragment sets of up to 25,000 fragments, thus increasing the chance to find bona fide binders of the target.

Mass Spectrometry

Evotec offers affinity screening by online SEC-LC/MS and enzymatic screening by SPE-MS/MS or acoustic ejection MS (ECHO-MS) and intact MS for covalent compounds.

Affinity screening (ASMS): Through a rapid assay development, this label free technology allows to test all possible binding site at once. Mixture of compounds (up to 400 compounds per well) can be screened, hit confirmation is performed with pure compound. Up to 20,000 compounds (in mixture) can be screened per day and system.

Enzymatic screening (RFMS or ECHO-MS): This label-free technology allows a direct read-out of enzymatic reactions in biochemical or cellular assay via MS detection of substrate, products. The fully automated process allows screening of >10,000 samples per day.

Intact protein MS screening: screening for covalent modifiers of selected target proteins is enabled by the application of intact protein mass spectrometry. Covalent libraries of up to a few thousand compounds can be screening using such approach.

Hydroger-Deuterium Exchange Mass Spectrometry (HDX-MS): Binding and conformational events can be confirmed and localized by monitoring the hydrogen-deuterium exchange of a protein`s amide hydrogens in deuterated assay buffer. Typical applications are epitope/paratope mapping, protein-ligand interactions (e.g. small molecules), protein stability/folding studies, determination of flexible regions to assist construct design.

Isothermal Titration Calorimetry ("ITC"), Microscale Thermophoresis (“MST”), Diffental Scanning Fluorimetry “DSF” (Thermal Shift) and switchSENSE technology.

We typically combine SPR or NMR approaches with orthogonal assays and technologies like Isothermal Titration Calorimetry (“ITC”), Differential Scanning Fluorimetry (“DSF”), HDX-MS, Microscale Thermophoresis (“MST”) and switchSENSE.

ITC is a label-free, in solution technique considered to be the “gold standard” for measuring a broad range of ligand interactions. It allows the full thermodynamic dissection of an interaction (Kd, stoichiometry) delivering details unavailable through other techniques.

MST is applied in two modes available, the label free as well as the labelled version of the technology. This allows for maximal flexibility on a broad variety of targets in conjunction with low affinity fragment like compounds. Through a fast assay development and a fast Kd determination, binding affinities of proteins, nucleic acids, fragments, peptides, small molecules with non-immobilized proteins can be evaluated.

DSF provides binding affinity information of proteins, nucleic acids, fragments, peptides, small molecules through stability measurement. This technology does not require immobilization as interaction can be evaluated in solution. Via a fast assay development, up to 30,000 compounds per day can be analyzed with a moderate protein consumption.

SwitchSENSE represents a novel method offering different measurement modes for multiparameter characterization of biomolecules. The target is immobilized on the chip surface via a DNA strand. Measurement of binding kinetics, affinities and several more parameters is enabled via that technology.

Virtual Screening & HIT Expansion

As part of its large portfolio of screening capabilities, Evotec offers virtual screening and hit expansion campaigns to its customers.

Virtual Screening

Leveraging a strong expertise in research informatics and in silico design, our scientists will design the most suitable in silico screening campaign to meet our clients’ requirements. For virtual screening, Evotec has access to multiple methods using both the best of-breed commercial software as well as in-house developed methods. 

The screening campaign can be run using number of compound collections:

  • commercially available collections
  • in-house screening collections
  • ad hoc prepared virtual libraries resulting from specific enumerations of reagents and reactions (up to +100M)

Chemogenomics and ortholog approaches are also available to our client and provide a good starting point for identifying novel active compounds. Evotec has licensed LeadIT and Spark for scaffold hopping but also has several in-house tools using structure merging and vector placement algorithms.

Hit Expansion

The hits identified from screening require expansion to provide evidence of SAR. This is traditionally based upon chemical similarity and involves searching databases for ‘like’ molecules using virtual screening derived approaches. Evotec also uses predictive pharmacology so the molecules that possess a similar target pathway signature can be identified. Library design, AI/ML approaches, statistical analysis and virtual chemistry tools can be used to design the most information rich set of molecules for synthesis.

Evotec's Hit Identification Virtual Screening Toolbox

Natural Products Strategy

Natural products (NPs) are widely distributed for a variety of indications in pharmaceutical industry and have a very long record of success stories from the past. In the past 30 years approximately 1,200 small molecule drugs were approved. Of those, 35% derived from synthetic libraries and 65% were either NPs, NP derivatives or drugs inspired by NP scaffolds. For infectious diseases and for hardly druggable targets in other indications the use of NPs increases the probability of success to identify novel chemistry as starting points. 

Technologies to prioritize NPs from established producer organisms have advanced in recent years and untapped producer micro-organisms have been shown to harbor novel chemistry. We utilize both resources in our platform to identify hits in screens using NP extracts, fractions, and purified compounds. To this end, we joined forces with partners in the NP discovery field to provide the best NPs on the market to our customers:

  • Early discovery processes using soil samples and untapped cultured diversity of micro-organisms
  • Dereplication for identification of novel NPs
  • Isolation and structure elucidation
  • Compound production by large scale fermentation, Mut synthesis as well as semi- or total synthesis
  • Compound profiling in active to hit projects
  • Medicinal chemistry-guided hit expansion
  • Hit-to-Lead and lead optimization programs

Why Hit Identification with Evotec

Learn more about our Hit Identification technology in the video above.

Dirk Winkler

Dirk Winkler

SVP Hit Identification - Drug Discovery Services

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Evotec has the right technologies & disease understanding to meet our partners' evolving needs: a comprehensive disease knowledge at the molecular level, cutting-edge technologies & platforms to translate this expertise into effective precision medicines.