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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