Drug discovery is a complex and multidisciplinary process. Screening attrition rates in current drug discovery programs mean that there may be only one viable drug from millions of screened compounds, and therefore discovery techniques and programs need to be improved to address the multiple causes of attrition. This has identified the need to screen larger libraries, where the use of efficient HTS becomes key to the discovery process.

Stroke is the second leading cause of death worldwide and the leading cause of disability. The only currently approved treatment for ischemic stroke is reperfusion therapy. However, this therapy is only applicable to a small percentage of patients. Therefore, there is an urgent need to develop neuroprotective therapies. One promising approach to accelerating the development of new therapies is the use of high-throughput screening (HTS). HTS can study hundreds of thousands of compounds every day. To this end, recent advances in computer-aided design, computer simulation libraries, and molecular docking software, as well as upgrades to cell-based platforms and in vivo models of ischemic stroke, have been developed to increase screening efficiency and improve predictability and clinical applicability.

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• Small Molecule Drug Development for Stroke
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Formats and Major Considerations for HTS Platforms

HTS involves in vitro, cellular, or whole organism assays. The most common readouts for biochemical assays in HTS are optical, including absorbance, fluorescence, luminescence, and scintillation. The efficiency of data generation and the cost per screen are the main determinants in selecting the most appropriate read for a particular screen. Fluorescence technology is favored for its high sensitivity, wide range of available fluorophores, and multiplexing capabilities. This allows for miniaturized assays and real-time tracking of multiple events.

When developing assays, it is crucial to avoid short wavelength excitation (under 400 nm) to minimize interference from test compounds. HTS techniques have been effectively used to identify inhibitors for various targets, including thrombin and HIV protease. Understanding the kinetics of compound binding can provide insights into binding mechanisms and the effects of structural variations, leading to the rapid development of high-affinity leads.

Data from HTS can be managed using specialized information systems or Excel spreadsheets. Hits are classified based on thresholds, typically set at three standard deviations above the mean of control wells, which helps maintain a low false-positive rate. To enhance hit processing, "cherry picking" can be employed, allowing for the selection of several hundred compounds for further evaluation. Using the median for hit assessment, especially in triplicate screenings, can mitigate the impact of outliers and improve data reliability.

Main Types of HTS Assays

Cell-Based Assays

Cell-based assays allow for the investigation of entire biological pathways rather than focusing on specific steps, offering insights that biochemical assays may miss, such as pharmacological activity at particular receptors or intracellular targets. These assays are particularly useful for studying cell growth, differentiation, and the effects of small molecules on cell function, making them valuable for understanding complex conditions like ischemic stroke.

HTS often employs scaled-down cell-based methods, utilizing cellular microarrays on 96- or 384-well microtiter plates with 2D cell cultures. These microarrays enable the multiplexed examination of living cells by presenting small volumes of diverse biomolecules and cells. Automated spotting technology or soft lithography is used to array various molecules, including antibodies and small compounds.

In signaling pathway-focused assays, flexible readouts are available, such as protein phosphorylation or changes in abundance, depending on the antibodies used. Cell-based screening can assess multiple targets simultaneously, with the readout reflecting the outcome of entire cellular pathways or networks. Using a cell-based high-throughput oxygen-glucose deprivation (OGD) model, neuroprotective drugs can be evaluated for use in stroke treatment.

Biochemical Assays

Biochemical assays focus on purified target proteins to measure ligand binding or enzymatic inhibition in vitro, typically using a competition format where a test compound displaces a known ligand. These assays are usually performed in 384-well plates, balancing screening volume (20-50 µL), throughput, and equipment costs. Common readouts include optical methods such as absorbance, fluorescence, and luminescence.

While biochemical assays provide clear targets for hits, they often require extensive and costly investigations into molecular mechanisms. The therapeutic potential of hits can be inconsistent, particularly given the variability in drug behavior in complex cellular environments, including issues with permeability, metabolism, toxicity, selectivity, and off-target effects. Conversely, cell-based assays can detect phenotypic changes in a more complex context but may lack clarity on target mechanisms and are generally more expensive and challenging to scale for HTS.

Fig.1. General classification of HTS assays. (Aldewach, et al., 2021)

The Need for HTS in the Discovery of Drugs for Stroke

HTS is now increasingly being used in the development of promising neuroprotective drugs for stroke, replacing the traditional "iterative" method of identifying therapeutic targets and validating biological effects. HTS involves the detection and screening of a large number of biological effectors and modulators against designated and exclusive targets. Therefore, HTS is often preferred when little is known about the target and structure-based drug design is not possible, but it can also be used in conjunction with other strategies such as computational techniques and fragment-based drug design. HTS consists of several steps, including target identification, compound management, reagent preparation, assay development, and the screening itself. High-throughput screening of small molecules can rapidly analyze the effects of thousands or even millions of small molecules. Therefore, utilizing high-throughput screening in the field of stroke drug development may be lucrative.

Modelling of Stroke for HTS

The ethical, financial, and logistical challenges of animal testing have limited the use of HTS in preliminary drug screening for stroke. Failure of clinical trials has also raised questions about the relevance of animal models, highlighting the need for improved research tools. Despite these concerns, models such as zebrafish, Drosophila melanogaster, and Caenorhabditis elegans have been successfully used in stroke research.

Zebrafish are valuable for their rapid development, genetic manipulability, and transparency during growth, making them suitable for high-throughput screening (HTS) and safety assessment. Hidradenitis elegans nematodes offer advantages such as rapid growth and cost-effectiveness for screening compounds that prevent misfolded proteins associated with diseases such as cerebral ischemia. Drosophila melanogaster has similar benefits, including a rapid life cycle and the ability to be genetically modified, but also has limitations in modeling human stroke complexity.

Fig.2. Hypoxia using oxygen absorber induced trunk and head edema and decreased survival rate. (Matsumoto, et al., 2021)

All of these models have a significant drawback: they cannot accurately replicate the complex pathophysiologic environment of human stroke, in part because of their short lifespan and evolutionary distance from humans. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have emerged as powerful alternatives for generating tissue-specific cell types that link phenotype to genotype, and the emergence of CRISPR-Cas9 technology has further enhanced the utility of iPSCs in HTS, enabling the creation of transgenic cells and humanized animal models for diseases such as ischemic stroke.

Advanced 3D culture models using hydrogel and organic chip technologies provide a more physiologically relevant environment than traditional 2D models. These models enable multicellular interactions, better mimic human organ function, and facilitate drug testing and disease modeling. Organ-on-a-chip platforms, in particular, allow for long-term culture of primary cells, preserving key cellular interactions and enabling accurate drug effect prediction.

Overall, the development of these innovative models represents a significant advancement in ischemic stroke research, providing new avenues for drug discovery and understanding disease mechanisms.

Conclusion

HTS is indispensable in the field of stroke drug discovery. However, the ability of HTS is limited not only by the availability of human-associated stroke models but also by the quality and size of the compound libraries screened in HTS. Therefore, increasing the number of small molecules available for HTS could increase the odds of discovering drugs effective in improving stroke. Advances in dynamic combinatorial chemistry (DCC), the introduction of cheminformatics in the pharmaceutical industry, and the widespread use of artificial intelligence (AI) in drug discovery are expected to generate a large number of compounds that may yield more active targeted drugs in the initial HTS assay.

1. Aldewachi, H., et al. (2021). High-throughput screening platforms in the discovery of novel drugs for neurodegenerative diseases. Bioengineering, 8(2), 30.
2. Matsumoto, M., et al. (2021). Establishing a high throughput drug screening system for cerebral ischemia using zebrafish larvae. Journal of Pharmacological Sciences, 147(1), 138-142.