Drug Discovery Through High-Throughput Screening Technique

Rapid lead compounds discovery through high-throughput screening

High-Throughput Screening Technology


High-Throughput Screening (HTS) is an approach to drug discovery that has gained widespread popularity over the past few years and expanded its applications for the pharmaceutical and biotechnology companies, university research laboratories et al. The technology includes screening of large chemical libraries for activity against biological targets via the use of automation, miniaturized assays and large-scale data analysis. Since its first advent in the early to mid-1990s, the field of HTS has seen not only a continuous change in technology and processes but has gone through various adaptations to suit industry needs which have given an edge to drug discovery. HTS has now evolved into a mature discipline that is crucial for the end to end manufacturing of drugs, namely from the raw material that is the chemical to the actual drug formation.

In recent years, the industry has witnessed a clear trend in drug discovery toward rigorous hit validation through the use of orthogonal readout technologies, label-free and biophysical methodologies. Today, many experts in the field see HTS at a crossroad with the need to decide on either higher throughput/more experimentation or a greater focus on assays of greater physiological relevance, both of which may lead to higher productivity in pharmaceutical research & development. The aim of high-throughput screening is to accelerate drug discovery by screening large libraries often composed of hundreds of thousands of compounds (drug candidates) at a rate that may exceed 20,000 compounds per week. The following article takes a look at the evolving technology and applications of the HTS process.


High-throughput screening is a method for scientific experimentation especially used in drug discovery and is relevant to biology and chemistry. This process in combination with robotics, data processing and control software, liquid handling devices and sensitive detectors allows a researcher to quickly conduct millions of chemical, genetic or pharmacological tests. High-throughput screening can rapidly identify active compounds, antibodies or genes which modulate a particular biomolecular pathway. It can be considered - a process in which batches of compounds are tested for binding activity or biological activity against target molecules. High-throughput screening is a process of screening more compounds against more targets per unit time, which should generate more hits, which in turn will generate more leads, subsequently generating more products.

Various technologies like high-throughput screening defined by the number of compounds tested to be in the range of 10,000-100,000 per day, ultra-high-throughput screening is defined by screening more than 100,000 data point generated per day. These two technologies play a vital role in drug discovery to find new chemical compounds.

HTS was invented by Dr. Gyula Takatsky in 1951, who machined 6 rows of 12 wells in Lucite to make the first microtiter plate. The microtiter plate has further grown to include standardized 96, 384, 1536 well formats, with additional 3072 well nanoplate formats available for specialized or quantitative reverse-transcription polymerase chain reaction assays.

“Twenty 384-well plates are currently run daily on the Accuri C6 HyperCyt combination. We could run up to 40 plates in a standard 8-hour workday, over 12,000 compounds”        

-Martha Larsen, Director, HTS, siRNA, HCS Laboratory


High-throughput screening in drug discovery

To screen

  • Novel biological active compounds
  • Natural products
  • Combinatorial libraries (Ex: peptides; chemicals)
  • Biological libraries
  • DNA chips
  • RNA chips
  • Protein chips

High-throughput screening’s main labware is the microtiter plate. Modern microplates for high-throughput screening assays are performed in automation-friendly microtiter plates with a 96, 384, 1536 or 3456 well format. These wells contain experimentally useful matter, often an aqueous solution of dimethyl sulfoxide (DMSO) and some other chemical compound, the latter of which is different for each well across the plate.

Fig. 1a) 96 wells microtiter plate Fig.  1b) 384 wells microtiter plate Fig.     1c) 1536 wells microtiter plate

For most drug discovery labs, the library collection has grown from 400,000 to 1 million or more compounds. The standard paradigms used to screen these libraries have evolved to automated 384 wells or higher density single compound test formats. The primary screen is designed to rapidly identify hits from compound libraries. The goals are to minimize the number of false positives and maximize the number of confirmed hits. Depending on the assay, hit rates typically range between 0.1 – 5 percent. This number also depends on the cutoff parameters set by the researchers, as well as the dynamic range of a given assay. Primary screens are run in multiplets of single compound concentrations. Results are expressed as percent activity in comparison to a positive (100 percent) and a negative (0 percent) control. Hits are then retested, usually independently from the first assay. If a compound exhibits the same activity, it is coined as the confirmed hit, which proceeds to secondary screens or lead optimization. The results of lead optimization are used to decide which substances will make it on to clinical trials.

Fig. 2) Comparison of among different micro titer plate formats

In combination with bioinformatics, it allows potential drugs to be quickly and efficiently screened to find candidates that should be explored in more detail. Initial screening of these compounds for their binding ability is the job for high-throughput screening. The key to high-throughput screening is to develop a test, or assay, in which binding between a compound and a protein causes some visible change that can be automatically read by a sensor. Typically the change is emission of light by a fluorophore in the reaction mixture. One way to make this occur is to attach the fluorophore to the target protein in such a way that its ability to fluoresce is diminished (quenched) when the protein binds to another molecule. A different system measures the difference in a particular property of light (polarization) emitted by bound versus unbound fluorophores. Bound fluorophores are more highly polarized and this can be detected by sensors.

Detection technologies used in high throughput screening include time-resolved fluorescence (TR-FRET), fluorescence resonance energy transfer (FRET), fluorescence polarization, luminescence and absorbance and require sensitive and versatile multi-mode microplate readers.

ProcedureThe sets of compounds produced by combinatorial chemistry are generally referred to as libraries, which depending on how the solid-phase is handled, may be either mixtures or individual compounds. There is a range of options for testing the libraries in a biological assay.

  • Test mixture in solution
  • Test individual compounds in solution
  • Test compounds on the beads

Test mixture in solution

All the compounds are cleaved from the beads and tested in solution. If activity in a pharmacological screen is observed, it is difficult to find out which compounds are active. To identify the most active component, it is necessary to resynthesize the compounds individually and thereby find the most potent. This iterative process of resynthesis and screening is one of the most simple and successful methods for identifying active compounds from libraries.

Test individual compounds in solution

A second method is to separate the beads manually into individual wells and cleave the compounds from the solid-phase. These compounds can now be tested as individual entities

Test compounds on the beads

A third method for screening is testing on the beads, using a colorimetric or fluorescent assay technique. If there are active compounds, the appropriate beads can be selected by color or fluorescence, picked out by micromanipulation and the product structure, if a peptide, determined by sequencing on the bead. Non-peptide structures would need to be identified by one of the tagging methods.


 High-throughput technology can also be put to use in other areas besides drug development.

  • Genomics Applications 
  • DNA Sequencing
  • Protein Analysis

Case study depicting the usage of HTS by Maxim Pharmaceuticals

Maxim Pharmaceuticals’ HTS laboratory operates a working whole-cell based screening platform. Their purpose is to screen for inhibitors and inducers of apoptosis or programmed cell death. The medical justification for inhibitors is their possible use in preventing unwanted cell death often seen in heart attacks, strokes and degenerative diseases such as Alzheimer’s and Parkinson’s Disease. The justification for inducers is primarily to kill cancer cells or cells that do not have the normal cell death machinery. 

Maxim Pharmaceuticals uses a proprietary substrate that is specific for activated caspase. The screens are performed in 384 well formats and are the whole cell based. Activated caspase indicates cell death through apoptosis specifically, not just cell death by necrosis. They screen compound libraries against numerous cancer cell lines. The research flow at Maxim is typical of cell-based screening elsewhere. It starts with compound inventory and solubilization, compound transfer and plate replication, cell addition, incubation, plate reading, and finally data analysis. 

At Maxim, the laboratory layout is a combination of track and robotic arm systems. Plates usually get reagent added with a small volume 96 channel pipettor, the compound is added with a 384 pin tool head, cells are added with a large volume 96 channel pipettor (all these steps are linked to stackers, hence this portion is considered a linear setup). The plates are manually loaded into an automated incubator. Once the incubation period is finished, the plates are moved with a robotic arm from the incubator to a large volume 96 channel pipettor, a substrate is added, and the plates are moved to a fluorescent plate reader, stopping by a barcode reader on the way to keep track of what data corresponds to what compound. The barcodes are then read and the plates are moved back (by the robotic arm) to the incubator for further incubation. 

Once done incubating a second time, the plates are moved by the arm from the incubator to the scanner, then to the plate reader for a final read. Once they are done with the final read, they are essentially trash. A plate sealer has been integrated with the arm so these plates are sealed for splash-free disposal into a hazardous waste barrel. Once the assay run is complete the robotic arm uses the pager function to call one of the employees to let them know the assay run has finished without problems. 

Data analysis is done for the primary screens using a software package to handle the massive quantities of data generated. The HTS laboratory at Maxim also handles all of the secondary screens such as Dose Response and Growth Inhibition. The dilutions for these assays are done off-line using 8 channel pipettors and then the integrated automation is used for the actual assay. Data from these smaller assays is analyzed using templates in Excel.

Industry challenges while working with HTS

HTS has revolutionized compound screening. However, it has also introduced new challenges to the drug discovery and development process. Just a small fraction of primary hits generated by HTS can easily overwhelm traditional follow-up testing, therefore creating new bottlenecks in the drug discovery process. Thus, it is now time to explore the application of the high throughout technologies to those new bottlenecks in the areas of medicinal chemistry, structural biology, pharmacology, ADME (Absorption, Distribution, Metabolism, and Excretion) studies and toxicology.


HTS becomes an effective technique and competitive with the latest, upcoming related technologies in the market. The growing importance of this process is cost-effectiveness of drug discovery and development, operating processes for development of homogeneous, fluorescence-based assays in reduced formats. The usage of 384, 1536 and 3456 wells density plates and robotics made the HTS process through which compounds can be screened more than 100,000 data points per day. The number of higher density plates used in the drug discovery process is inversely proportional to the samples required for the process; thereby it reducing the initial setup costs. The combination with robotics, data processing and control software, liquid handling devices, TR-FRET, FRET, Fluorescence polarization techniques have added a significant valued to each data point generated by high throughput screens.

Fig. 3) Advantages of HTS

Overall, high throughput technologies have significant potential in future drug development and design: from their immediate promise to shorten the preclinical development timeline to eventually paving the way to make drugs prescribed or tailor-made according to the genetic makeup of an individual.


[1] Martis E.A., Radhakrishnan R., Badve R.R, High-Throughput Screening: The Hits and Leads of Drug Discovery- An Overview, Journal of Applied Pharmaceutical Science 2010, 1 (1), 02-10; Available from- http://japsonline.com/pastlist.asp?jv=1&&ji=1

[2] Lorenz M Mayr, Dejan Bojanic, Novel trends in high-throughput screening, Volume 9, Issue 5, October 2009, Pages 580-588;Avaliable from -http://www.sciencedirect.com/science/article/pii/S1471489209001283

[3] James W Noah, New developments and emerging trends in high-throughput screening methods for lead compound identification, International Journal of High Throughput Screening, August 2010 Volume 2010:1; Pages 141 -149- Dove Medical Press Ltd-http://www.dovepress.com/articles.php?article_id=4965

[4] Paper by Eric Allen, Candace Crogan-Grundy, Jessica Rivers, Tatyana Nisan and Shaoxian Su on “High throughput screening in drug discovery.” Source for Maxim Pharmaceuticals case study-http://www.esallen.com/professional/papers_presentations/bioengineering/HTS_paper_draft.pdf

Image Reference

Fig. 1a), 1b): Thermo Sciencitifc website, http://www.nuncbrand.com/en/page.aspx?ID=11978

Fig. 1c): BioTek Instruments, Inc website, http://www.biotek.com/microplate-washer-f.htm

Fig. 2):  PerkinElmer Inc, http://www.perkinelmer.com/Catalog/Product/ID/6004430

Fig.  3): From Andreas Boettcher and Dr Lorenz M. Mayr, Miniaturisation of assay development and screening, Summer 2006; Available from- http://www.ddw-online.com/s/screening/p97061/miniaturisation-of-assay-development-and-screening-summer-2006.html

Fig. 4): From Andreas Boettcher and Dr Lorenz M. Mayr, Miniaturisation of assay development and screening, Summer 2006; Available from- http://www.ddw-online.com/s/screening/p97061/miniaturisation-of-assay-development-and-screening-summer-2006.html

To contact the author email: articles@worldofchemicals.com

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