In our current pharmaceutical landscape, a large proportion of disease-modifying targets are labeled as ‘undruggable’. The description ‘undruggable’ is used when a protein is not pharmacologically capable of being targeted. This includes targets for cancer, immunology but also neurodegeneration like Parkinson’s disease, and Alzheimer’s disease^1,2. Substantial progress has been made recently to turn these proteins into ‘druggable’ targets and nomenclature has been adjusted to ‘difficult to drug’ or ‘yet to be drugged’.

In addition, traditional hit identification approaches targeting druggable proteins often result in the progression of compounds with poor binding affinity, off-target effects, lack of efficacy, toxicity, or poor pharmacokinetic or physicochemical properties. The selection of poor-quality hits with unwanted mechanisms of action has been a major cause of attrition during later stages of drug discovery and development, wasting significant time and resources.

Major advances in hit finding technologies and methodologies in recent years offer a light at the end of the tunnel for pharmaceutical research. A diverse range of screening technologies and workflows have been developed, improving the efficiency, quality, and speed of hit identification.

Despite these advances, the integration of high throughput screening strategies can be a challenging process. Following on from our expert on-demand webinar on hit identification, this blog summarizes key points to help you minimize the risk of attrition and identify the best possible chemical starting points, even when dealing with challenging or ‘difficult to drug’ targets. This includes considerations when selecting the most appropriate screening methodologies, optimizing assays, and choosing a compound library.

When developing a tailored hit identification screening strategy, the most appropriate screening approach for each campaign will largely depend on the therapeutic area, the target of interest and its available knowledge. The most widely used approaches can be separated into the following categories:

• Target-directed (e.g. cellular, biochemical, and biophysical) – using knowledge from a well-defined target to screen compounds for specific interactions
• Structure-based (e.g. fragment-based screening) – using the target’s 3D structure to identify potent compounds
• In silico (e.g. virtual screening) – a virtual knowledge-based approach that uses computational models to predict binding of compounds to respective targets
• Phenotypic (e.g. high content imaging and analysis) – focusing on the overall impact of the screened compounds on cells, offering an unbiased approach

Following the selection of the most appropriate screening approach, the development of the primary assay begins. To identify the best compounds for your target and modality, the primary assay should meet several key requirements, including:

• Pharmacological sensitivity (EC₅₀/IC₅₀ assay values covering weakly active compounds)
• Low risk of detecting false positive or false negative hits or unwanted mechanisms
• Assay stability and robustness
• Generation of reproducible, high-quality data
• Use of high-quality, stable reagents, e.g. cell cultures and proteins

For challenging or hardly druggable targets and modalities with a high risk of attrition, a more complex assay development process is required to maximize the chances of success. This includes the intensive optimization of primary assay conditions, including buffer systems, additives, and incubation times.

Counter and orthogonal assays are developed to validate the hits and filter out molecules acting by an unwanted mechanism. Counter assays should detect a range of undesirable compounds, including those that interfere with assay read-outs, Redox-active compounds, or which cause cytotoxicity. From these assays, compound series showing chemical tractability, desirable activity, and no assay interference should be selected for follow-up. During hit validation, it is recommended that primary but also counter and orthogonal assays are repeated using fresh powder material, to discount any false hits that could be caused by synthesis impurities or degradation of the compound samples.

Mode of action (MoA) studies and translational assays should also be conducted during early hit identification. These are designed to further improve the understanding of the compound’s mechanism of action and ensure target engagement, lowering the risk of attrition at later stages.

Choosing a high-quality chemical library is essential to mitigate risks in drug discovery. Compound library curation should be based on maximizing chemical diversity, along with the selection of compounds with attractive physiochemical properties, QED scores (quantitative estimate of drug-likeness), and the exclusion of unwanted chemical motives. Chemical libraries should also be large (250-400K in size), with diverse “islands” of similar compounds, and covering novel, lead-like chemical spaces. Compound integrity and purity is also an important aspect, as degradation can largely influence assay accuracy and reliability. To ensure optimal compound quality, regular QC checks of such libraries should be performed, to actively maintain and improve the compound collection.

At Evotec, we have remained at the forefront of hit identification for over 25 years. We work with our partners to develop tailored hit identification strategies that provide the best possible chemical starting point for each target. We achieve this with a combination of advanced high throughput screening capabilities, cross-disciplinary expertise, data-driven approaches, and an expertly curated compound library.

Our world-class hit identification offerings allow partners to push the boundaries of modern medicine and identify novel, potentially life-changing compounds. This includes the development of custom compound screens for so-called undruggable targets, such as transcription factors, RNA, protein-protein interactions, RNA/DNA binding proteins, and emerging modalities such as degraders, molecular glues, and covalent modifiers.

For further detail on how to develop high throughput screening strategies, watch our on-demand webinar presented by Dr. Thomas Ahrens, Group Leader, High Throughput Biology at Evotec. With over 25 years of experience in the industry, Thomas describes the key components of Evotec’s integrated hit identification process and explains how these are essential to mitigating drug discovery risks and overcoming challenges. At the end of this webinar, you can also find key questions asked by the audience, in a Q&A session joined by Dr. Helene Jousset Sabroux, Group leader, High Throughput Biology & Screening at Evotec.

And to delve even further into high throughput screening, stream the other webinars that are part of our hit identification series. Find episodes deep diving into key topics, including protein sciences, biophysics, structural biology, and high-content imaging.

1.  Fang Y., Wang J., Zhao M., et al. Progress and Challenges in Targeted Protein Degradation for Neurodegenerative Disease Therapy. J Med Chem 2022;65:11454–77. https://doi.org/10.1021/acs.jm....
2. Coleman N., Rodon J. Taking Aim at the Undruggable. American Society of Clinical Oncology Educational Book 2021:e145–52. https://doi.org/10.1200/EDBK_3....