How to optimize your hit identification strategy 

What is hit identification and why is it important?

Hit identification (Hit ID) is an important step in the development of new medicines. It is the process of identifying molecules with desirable biological activity, such as the ability to bind to the target and modify its function. As hit ID is one of the first steps in drug discovery, optimizing this process is essential to provide the best possible chemical starting points for your drug discovery and development process. A successful hit ID campaign will maximize the output in terms of the number of high-quality hits that enter the hit-to-lead and lead optimization stages, saving you precious time and resources further down the line. 

However, identifying high-quality, validated hits is no easy feat. There are several components of hit ID campaigns that need to be considered, including the selection of the compound library, and the development of pharmacologically sensitive and robust assays for screening, triage, and validation. Hit ID also requires a broad interplay of disciplines, like reagent production, in vitro biology, medicinal chemistry, and statistical data analysis. These interact across several approaches, such as target-directed, structure-based, in silico, and phenotypic hit ID. With this in mind, extensive multi-disciplinary expertise is applied to design and implement the right hit ID campaign for your target.

To help simplify this pivotal stage in the drug discovery process and ensure that you get the most high-quality hits for your target, here are our top considerations for hit ID campaigns. Across those, we explore how an integrated end-to-end platform is key to supporting all stages of the hit ID process, and beyond.

Considerations to help you optimize your hit identification campaign

Screening strategies

There are several well-established screening approaches for hit ID, including target-directed, structure-based, in silico, or phenotypic high-throughput screening routes. Different approaches can be run in parallel or individually, depending on your target.  

Choosing the right screening strategy is one of the most important considerations for the hit ID process, and will largely determine the success of your campaign. To learn more about the different screening approaches, visit our hit identification webpage.  

Assay development

Primary assays are used for the detection of on-target activity or binding in high-throughput screens. There are different types of primary assays, such as cell-based, or biochemical assay systems. While cell-based assays provide a more physiologically relevant context, biochemical assays can give you deeper insight into target binding or modulation of the target’s function in a cell-free environment. Therefore, the type of assay you choose depends on your target, and the specific goals and requirements of your campaign. Visit our in vitro biology webpage to learn more about the different phenotypic and cellular-based assay technologies. 

When optimizing primary assays, it is important to verify the relevance of the assay to your specific disease state and target. Additional factors such as robustness, pharmacological sensitivity, reproducibility, scalability, and cost efficiency are taken into account. Development of such primary assays can be a complex and lengthy process that involves many steps, from initial setup, optimization, and pharmacological characterization, through to the adaptation to the screening system and pre-screening.

Further to the primary assay, the development of an appropriate readout counter assay is strongly recommended. Such assay applied at later stages of the screening process enables the identification of potential readout-interfering compounds that could result in false positive hits.

Compound libraries

Compound libraries are collections of small molecules used to identify hits in high-throughput screening assays. The success of your hit ID campaign relies heavily on your chosen compound library. To maximize your chances of success, such compound libraries should consist of highly attractive, chemically diverse compounds with proven lead-like properties, but also good solubility and stability. Thus, quality, size, and diversity of the compound collection will impact the success of your hit ID campaign.  

In high-throughput screens, large libraries of several hundreds of thousands of compounds are screened to cover as much of the available chemical space as possible. However, in specific cases, a more tailored screening approach might be more appropriate, using a smaller, either diverse, or focused compound library.  

Screening and hit triaging

The screening process involves several steps, starting with a pilot screen using a representative subset of the screening collection. Upon definition of the final screening conditions including the compound concentration, the primary screen is then performed on the selected screening deck. The primary hits identified are confirmed by testing them again in replicates before the final step of concentration-response profiling is started. 

The concentration-response relationship of confirmed hits is tested against the primary assay and the readout counter, as well as against relevant selectivity targets, if applicable. A diligent, data-driven analysis of the results, involving a medicinal chemistry review and assessment, enables the prioritization of compound series with both a desired biological profile and attractive chemistry.  

Figure 1. A typical hit ID workflow, illustrating the various steps involved in high throughput screening and hit triaging. 

Hit validation 

Following hit triage, prioritized hit series are validated to confirm their biological activity through the application of secondary assays. These are carried out using orthogonal readouts like biophysical methods to confirm on-target activity, or are conducted in more physiologically relevant systems like cell-based assays. Secondary assays are used to assess several crucial properties of the hits, including functional response.  

Medicinal chemistry efforts during hit validation focus on the analysis of the hit’s structure-activity relationship (SAR). Such analysis aims to identify the structural elements that are associated with its biological activity. To further the assessment of on-target activity and selectivity, additional in vitro assays are commonly conducted at this stage. These evaluate the absorption, distribution, metabolism, and excretion (ADME) properties of the hits. You can visit our DMPK and ADME-Tox webpage to read more about secondary ADME-Tox assays for hit validation. 

Using all this information together, the hit series that will progress to the hit-to-lead phase are identified. The number of series that are taken forward will depend on resource availability, although around two to three hit series are usually recommended. 

Mastering hit identification  

When optimizing your hit ID campaign, there are many factors to consider, including the quality of your compound library, and the strategies for primary screening, triage, and validation. The most successful hit ID campaigns implement a data-driven approach that utilizes techniques across a broad range of disciplines, including biochemistry, computational chemistry, in vitro biology, and medicinal chemistry.  

Figure 2. A checklist for successful hit ID campaigns, from screening strategy through to hit validation, to help you increase the number of high-quality hits obtained. 

View our webinar to learn more about the essential considerations for successful hit identification by high-throughput screening, including planning, compound collections, infrastructure, assay formats, and the benefits of an interdisciplinary approach. 

Externalizing your hit identification 

At Evotec, we provide industry-leading hit ID services through our decades of experience, state-of-the-art screening technologies, and a diverse, high-quality compound collection. This utilization of a broad range of expertise, facilities, and technologies takes away the stress of hit ID, minimizing the risks of attrition, and even allowing you to hit previously undruggable targets.  

When partnering with Evotec, our expert screening team will help you design and conduct a bespoke hit ID campaign that is optimized for your target, giving you the best possible chemical starting points. And once the best hit series for your target have been identified, our integrated, end-to-end R&D platform can support you from drug discovery, right through to drug development and manufacture, helping you get your drug to market in the simplest, most time, and cost-effective manner. 

For more information on our hit ID services, and to learn how we can support your projects from concept to clinic, visit our website.

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Also, join our ongoing webinar series on accelerated hit identification through innovative high throughput screening approaches.

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Thermostability assays (DSF) are one of the workhorses of biochemistry and structural biology. Thanks to the simplicity of the assay setup they found wide applications in many fields. To meet the high demand in DSF assays from our clients, Evotec established DSF pipelines in all five of the sites that offer biophysics services. We find that thermostability assays are highly useful for hit validation & expansion, however, can be also used to reveal the mode of action of the candidate molecule.

In this presentation we:

• Describe how we use thermal unfolding assays (DSF) to support drug discovery
• Discuss the cases when model-based analysis reveals in-depth information about the drug/target interaction
• Share the perspectives on combing high-throughput and high-content approaches

This talk was presented at MOSBRI Workshop "Pushing the limits with Differential Scanning Fluorimetry" on 16 November 2022.

Novel phosphodiesterase 10A inhibitor drugs, through a distinct mechanism on striatal dopamine receptors, could raise the possibility of producing an augmented pharmacological antipsychotic effects by a combination therapy with the standard antipsychotic drug.

TAK-063 is a novel phosphodiesterase 10A inhibitor which has been evaluated for this hypothesis. Results show that the use of TAK-063 can produce augmented antipsychotic-like activities in combination with antipsychotics without alteration of plasma prolactin levels and catalepsy

In this collaborative paper with Takeda, we demonstrate that:

• Combined treatment with TAK-063 and either haloperidol or olanzapine leads to a significant increase in phosphorylation of glutamate receptor subunit 1 in the rat striatum.
• TAK-063 enhances N-methyl-D-aspartic acid receptor mediated synaptic responses in rat cortical striatal slices in both direct and indirect pathway MSNs to a similar extent.
• Co-administration of TAK-063 with haloperidol or olanzapine additively activated the indirect pathway, but not the direct pathway.
• Combined treatment with TAK-063 and either haloperidol or olanzapine at subeffective doses produced significant effects on methamphetamine- or MK-801-induced hyperactivity in rats and MK-801-induced deficits in prepulse inhibition in mice.
• TAK-063 did not affect plasma prolactin levels and cataleptic response from antipsychotics in rats.

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In this poster, presented at SfN 2018, Sessolo et al. present 3Brain high-density multi electrode array (HD-MEA) as a system to monitor and characterize seizure-like activity in hippo-cortical slices induced by different compounds.

The high system resolution allows to monitor in detail the entire slice and through the software showing the activity map (in real-time) the sign of compounds' action is easily found.

The technology allows to acquire Local Field Potential (LFP), Multi Unit Activity (MUA) and Single-Unit Activity.

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This poster includes information about:

• Functional brain slice electrophysiology by HD-MEA platform
• Combined neuronal circuitry studies through functional brain tissue imaging

Initially presented at FENS 2018 by Ugolini et al., 3Brain high-density multi electrode array (HD-MEA) as a system for long lasting monitor and characterize spiking activity of hundreds Purkinje cells simultaneously by using different positive and negative Ca++-activated K+ channels. Responses can be evaluated though different analysis. It is a useful tool for compounds validation on cerebellar slices.

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While the public has taken note of RNA-based medicine only with the advent of mRNA-based Corona virus vaccines, biopharmaceutical research and development has been working on mRNA-based medicine for almost two decades. Evotec also expanded the druggable target space to RNA and in the last years added considerable know-how in RNA-based medicine.

RNA is used by cells in multiple ways: mRNA is conveying genetic information from DNA to the ribosomes which also are made from RNA (ribosomal RNA), where another RNA species (tRNA) is transporting amino acids to the ribosomal apparatus so that a protein can be synthesized. In addition to mRNA, there are also shorter RNA molecules being used in the cell for the regulation of genes and entire genetic cascades.

This provides for plenty of potential interventions: antisense (ASO) and short interfering RNA (siRNA) can up or down regulate an RNA target, e.g., to block the translation of an unwanted or diseased protein or to suppress or stimulate the expression of genes. RNA can be targeted with (complementary) RNA, but it is also possible to alter or block the translation, re-locate or initiate RNA, degradation, etc. by small molecules interfering with the three-dimensional structure of RNAs or protein-RNA-complexes.

During our recent Innovation Week, Evotec experts Steffen Grimm, Group Leader, Hit ID & Biophysics, and Hilary Brooks, Senior Research Scientist, In Vitro Pharmacology, hosted a session called "The early bird catches the helix: Expanding the druggable target space to RNA".

In the session, they discussed how to:

• Expand the potential for drugs targeting RNA to offer alternative solutions for diseases with otherwise undrugged targets
• Target RNA providing highly specific solutions for protein removal, alternative splicing or pathway regulation via noncoding RNA
• Use the small molecule RNA targeting platform to contribute to new opportunities for target identification and validation

RNA as Therapeutics
Using RNA as therapeutics is not trivial. Nucleic acids introduced from outside may trigger adverse reactions by the innate immune system. A lot of knowledge is necessary to ensure delivery, avoid degradation and inflammation and to fine-tune the stability and function of the molecules. RNA may also have off-target effects. To ensure efficacy and safety, monitoring these early on needs to be incorporated into the developmental workflow. High quality synthetic RNA is costly to make, therefore a scaleable process and the relevant analytics must be established early in the process to accompany both the discovery and development stages of research with quality test material; Eventually producing GMP grade RNA at a commercial scale (several hundred grams) for human administration.

Evotec already has integrated all capabilities under one roof, allowing for the complete preclinical data set, reduced transition times and efficient communication to the regulators. For antisense oligonucleotide therapy, efficient hit sequences that knock down target expression can be selected in a matter of weeks. Toxicity profiling is a priority to establishing final leads and, subsequently, project-specific dose, duration and delivery will be established using optimized backbone chemistry. Using its in-silico capabilities as well as iPSCs, animal models, transcriptomics, etc. Evotec is able to predict toxicity and efficacy, and de-risk unwanted immune stimulation as well as off-target effects. For manufacturing, Evotec is discovery-capable and already building medium-scale capacity (up to 50g) which will be ready by 2023.

For inhibiting the translational machinery, Evotec has established an RNA small molecule targeting platform and established in various case studies, molecules binding to RNA, and demonstrating a significant effect in vitro without affecting cell viability. Evotec’s capabilities also allow the creation of a representation of the 3-dimensional structure of the target complex and its interaction with the compounds.

Evotec’s experienced team of scientists with proven drug discovery and development expertise already have a track record of driving RNA targeting projects forward. Its integrated medicinal and computational chemistry capabilities, combined with bioinformatics, structural biology, pharmacology, and drug safety expertise allows for the identification and characterization of RNA target species and their modulation by different modalities. Partner projects can be driven all the way from target identification to IND and beyond. Evotec therefore is a low-risk outsourcing partner and a company continually investing in its platform to the benefit of the customer.

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Rising antimicrobial resistance challenges our ability to combat bacterial infections. The problem is acute for tuberculosis (TB), the leading cause of death from infection before COVID-19.

Here, we developed a framework for multiple pharmaceutical companies to share proprietary information and compounds with multiple laboratories in the academic and government sectors for a broad examination of the ability of β-lactams to kill Mycobacterium tuberculosis (Mtb). In the TB Drug Accelerator (TBDA), a consortium organized by the Bill & Melinda Gates Foundation, individual pharmaceutical companies collaborate with academic screening laboratories.

• In this paper we describe strategies used to prioritize thousands of β-lactams from multiple companies into a small test set for further characterization.
• In a striking contrast to historical expectation, 18% of β-lactams screened were active against Mtb, many without a β-lactamase inhibitor.
• One potent cephaloporin was active in Mtb-infected mice.

The steps outlined here can serve as a blueprint for multiparty, intra- and intersector collaboration in the development of anti-infective agents.

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With more then 15 years of screening experience in anti-infectives, including antibacterials and antivirals, Evotec's medium throughput and high throughput screening expertise extends to BSL2⁺ and BSL3 containment level. 

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