In drug development, the journey from target identification to clinical candidate selection involves a series of critical steps, including compound screening, lead optimization, and pre-clinical testing. Each stage helps narrow the pool of potential therapeutics, with the goal of identifying the most promising candidates for clinical evaluation. Given the time, cost, and complexity involved, reliable and high-precision tools are essential to support this process. Flow cytometry in compound screening has emerged as a powerful technology to accelerate early drug discovery.

So, what role does flow cytometry play in early drug discovery?    Widely accepted in pre-clinical and clinical studies, flow cytometry enables high-dimensional, reproducible data for cell phenotyping and functional evaluation.  With advancements in conventional and spectral flow cytometry, researchers can interrogate complex cellular responses with simultaneous evaluation of multiple cell parameters.  

 In addition to immunophenotyping and mechanism of action studies, due to flow cytometry’s ability to provide high content, precision data, this technology also serves as a valuable tool for compound screening and lead optimization studies.

In this blog, we will explore how flow cytometry accelerates drug discovery as a valuable tool in compound screening.  With an application for small or large molecules, we will review industry examples highlighting how flow cytometry based, target and phenotypic compound screening aids drug development.

 Flow Cytometry as a Pre-Clinical Drug Discovery Tool

Flow cytometry utilizes a fluidics-based system to evaluate cellular characteristics at the single-cell level.  Flow cytometry-based methods can simultaneously characterize surface and intracellular molecule expression profiles, offering a comprehensive, multi-dimensional view of individual cells.

Compound screening assays can be developed to evaluate either target-based or phenotypic responses using immortalized cell lines or primary cells.  Ideally, a cell type is chosen based on disease relevance to the test compound under investigation and is also suitable for assay scalability.

The cellular changes in response to compound treatment can be measured by flow cytometry either qualitatively or quantitatively: 

  • Qualitative measurement, such as changes in cell proliferation, cell death, or expression of activation markers, can provide valuable data on the biological response to compound treatment. 
  • Quantitative measurement is achieved through the incorporation of fluorescent calibration beads to enable standardized measurement of Molecules of Equivalent Soluble Fluorophores (MESF) or Antibody Binding Capacity (ABC).  Beads enable normalization across runs and facilitate comparison of data over time.  The MESF or ABC values can be used to quantify changes in the expression of selected target proteins.

However, the power of flow cytometry extends beyond generating high-dimensional data. With standard cell or particle collection rates of 10,000 events per second, sample collection is rapid and efficient.  Many flow cytometers can be easily adapted for even greater throughput using high-throughput sampling systems (HTS).   HTS enables collection from 96-, 384-, or even 1536-well plates, with rates often exceeding 25,000 events per second.  By utilizing HTS collection, many compounds can be screened relatively rapidly. This throughput supports efficient screening of focused compound libraries and is especially valuable when analyzing rare or low-abundance cell populations.

When applied to target-based or phenotypic compound screening, flow cytometry offers advantages over other approaches that are limited in dimensionality.   It enables analysis of discrete cell subpopulations and captures live-cell responses to compound treatment.  Responses can include activation, proliferation, phosphorylation, and apoptosis after treatment with compounds.

Applications in Drug Discovery

Target Based Screening: Target based compound screening assays focus on screening for receptor – ligand interactions to identify compounds that interfere with or enhance the binding of two molecules.   The assays can be cell or bead based, with the bead-based approach providing enhanced practicality for scaling and cost efficiency.   

A recent example of flow cytometry-based screening published by Phakham et al. shows how a combination of screening tools efficiently enabled selection of high-potency, chimeric anti-PD-1 monoclonal antibodies from hybridoma pools.  Here, more than 10,000 hybridoma pools underwent initial screening by ELISA.  From this primary screen, a subset of 51 high hPD-1 specific pools was screened using high throughput flow cytometry to separate hybridomas producing neutralizing antibodies from non-neutralizing antibodies.  To achieve this, PD-1 expressing Jurkat cells were incubated in the presence of recombinant hPD-L1Fc protein and individual hybridoma mini-pools.  After incubation, bound anti-PD-1 antibody (from hybridoma) was detected by fluorochrome-labeled secondary antibody against mouse-IgG, while bound hPD-L1Fc protein was detected by a separate fluorochrome-labeled secondary antibody against the human chimeric protein.  Hybridoma mini-pools that produced neutralizing antibodies were identified when displacement or blockade of hPD-L1Fc was observed (negative signal), and anti-PD-1 was positive.  Through this combination screening approach, more than 10,000 hybridoma pools were narrowed to 50 hybridoma clones and subsequently down to 5 pools that produced antibodies with high PD-1 binding and PD-1/PD-L1 blocking activities.  This narrowed candidate pool was then efficiently evaluated for binding affinity and functional potency to re-activate T cells in a mixed lymphocyte reaction (T. Phakham, 2022).

Phenotypic Screening: Another approach to compound screening by flow cytometry is phenotypic screening where a cell-based assay design is used to screen compounds that interfere with or augment a cellular phenotype after compound binding.  One advantage of the phenotypic screen is that a full understanding of the molecular mechanisms of the disease is not required.  This phenotypic readout can be cytotoxicity, proliferation, surface or intracellular expression profile change, phosphorylation event, or other measurable phenotypic change (Sklar, 2015).

The broad application of flow cytometry in phenotypic drug discovery by industry has been well documented.  An example of one such phenotypic screen is a compound screen focused on identification of compounds that modulate proliferation and immunosuppressive function of T regulatory cells.  Here, primary human CD4+T cells are enriched and cultured appropriately to polarize T cells to Tregs.  This culture is conducted in the presence of vehicle only, test compounds and a Rapamycin positive.  At the conclusion of the culture, active compounds were noted as those that increased or decreased Treg proliferation more than 2-fold relative to vehicle only.    Leveraging a 384-well design, more than 250,000 test compounds were screened.  Hits were subsequently screened to confirm a dose-dependent response and then were analyzed by orthogonal assays for functional endpoints (Joslin, 2018).      

Target-based and phenotypic screening approaches both aid in screening drug candidates, with the former focusing on specific molecular interactions like receptor-ligand binding and the latter assessing broader cellular responses. These complementary strategies, as demonstrated by Phakham et al. and Joslin et al., illustrate how high-throughput flow cytometry enables efficient screening of libraries to enable drug candidate progression.

Considerations for Assay Design

To ensure success, the flow of cytometry-based compound screening assay must be carefully designed.  A few key considerations include:

  • Scale of the screening: Define the scale of the screening, including the library size and number of required replicates. The scale will dictate automation needs. Success requires consistency in cell plating, treatment, staining, washing, and acquisition. Manual workflows may suffice for hundreds of compounds; thousands of compounds may require automation.
  • Assay Controls: Assay controls should include vehicle-only wells and, if available, reference compounds with known functions.  Including a reference compound ensures that cells are responding as anticipated and serves as a benchmark for comparing test compound performance.  With a clear understanding of the types of assay controls and their placement on the plate, you can help ensure reliable and meaningful data generation.
  • Critical Reagents: Identify critical reagents and materials. Understanding nuances in cell confluency, culture requirements, cell passage, and more will be important for designing a robust assay. In addition, it is necessary to identify which stimulation and/or detection reagents are critical for the endpoint readout.  Once identified, titrate the reagents and assess lot-to-lot variability.  Establish a bridging plan to mitigate changes in the assay performance when new reagent lots are used. 
  • Data Analysis Plan: Flow cytometry generates complex and high-volume data.  Analyzing the data requires an investment of time and expertise.  When evaluating the scale of the study, consider whether manual data analysis is appropriate or if machine learning analysis is a better fit to maintain consistency and increase efficiency and throughput.

Takeaways and Support

Flow cytometry offers powerful insights at the single-cell level.  Its utility is found not only in standard phenotyping and functional evaluations but also as a key drug discovery tool. By leveraging high-throughput features, live cell methods, and multi-dimensional analysis, flow cytometry enables efficient compound screening.  When integrated with orthogonal bioanalytical platforms such as Ligand Binding Assays (LBA) by ELISA or MSD, flow cytometry unlocks a multi-modal view of compound performance. Complementary approaches support confident and data-driven decision-making across discovery and development.

KCAS Bio has a documented history of more than 45 years of providing a wide range of bioanalytical support.  With expertise in flow cytometry, ELISpot, LBA, mass spectrometry, and more, we are well-suited to partner with you on your most complex programs.  Whether you are looking to utilize flow cytometry or are interested in combining flow cytometry with other bioanalytical tools, we invite you to set up a call today to learn more about our services and how KCAS Bio can accelerate your science.

Works Cited

Joslin, J. e. (2018). A fully automated high-throughput flow cytometry screening system enabling phenotypic drug discovery. SLAS Discovery, 697-707.

Sklar, B. S. (2015). Flow Cytometry: Impact on Early Drug Discovery. Journal of Biomolecular Screening, 689-707.

T. Phakham, C. B. (2022). Highly efficient hybridoma generation and screening strategy for anti-PD-1 monoclonal antibody development. Scientific Reports.