How are life-saving drugs discovered?
Join DNDi researcher and series host Fanny Escudié to learn more about how scientists are using the most advanced technologies to improve the lives of millions of neglected patients around the world.
Episode 2
High-throughput screening: testing millions of molecules to find drugs for neglected diseases
High-throughput screening is a cutting-edge drug discovery technique used to test large numbers of molecules against disease-causing pathogens to identify potential treatments. DNDi is using this tool with major partners around the globe, including the Institut Pasteur in Korea.
Inside the pressurized, sterile glass cube, a giant robotic arm is swiftly moving small plates from one rack to another. Each of these plates contains about 400 minuscule ‘wells’, where cells, pathogens, and molecules are mixed – extraordinarily tiny experiments that will then be photographed and analysed by software. Around the glass cube, dressed in protective equipment to avoid any contamination, researchers are busy monitoring the operations.
Welcome to the P3 high-security lab at the Institut Pasteur Korea (IPK), in the Seongnam district – Korea’s ‘Silicon Valley’ – located in the south of Seoul.
Here, approximately two million different molecules or ‘compounds’ have been tested against pathogens responsible for neglected diseases such as leishmaniasis and Chagas disease. A massive endeavour made possible by a cutting-edge technique called high-throughput screening.
DNDi is applying high-throughput screenings to find new medicines through partnerships with a wide range of prominent drug discovery research institutes around the world, including the University of Dundee in the UK and Nagasaki University in Japan. Together, DNDi and its partners tested a total of four million different molecules owned by research institutes and pharmaceutical companies that agreed to freely share their compound libraries.
The idea behind high-throughput screening is simple: mammalians cells (including human cells) infected by a pathogen are placed in tiny ‘wells’ of a few millimetres in diameter; then, a few molecules are added through a micropipette. Exactly 384 wells fit into each hand-sized plate. A few days (or hours, depending on the biology of the cells and the pathogens) later, an automated imagery analysis is performed to observe if the cells managed to prevent or clear the infection – without being killed by the molecule.
‘The challenging part is to build the best biology conditions in those little, one millimetre-diameter drops, which can take months of hard work. But once the assays have been developed and finetuned, we are then able to test 20,000 to 50,000 different compounds in a single day! We can get a full library tested in a week.’
David Shum, Drug Discovery Team Leader at IPK. Photo credit: IPK
David Shum, Drug Discovery Team Leader at IPK. Photo credit: IPK
No Joo-hwan, Head of the Chemical & Structural Biology of Pathogen Lab at IPK
No Joo-hwan, Head of the Chemical & Structural Biology of Pathogen Lab at IPK
Setting up complex experiments in a tiny drop
But setting up an experiment in such a tiny space comes with a lot of challenges.
IPK researchers point out that, as they use extremely precise machines to dose nano-litres of fluid, fluid mechanics completely change. Working on such a small scale makes moving from a basic biology experiment to a high-throughput screening setup with living organisms a very big step.
‘Biology is very sensitive. The first challenge is to create a micro-environment in a five micro-litre drop in which the host cells and the parasite behave as expected. Temperature can be very important: some diseases, like cutaneous leishmaniasis, propagate outside of the skin; for them to grow during the experiment, you need to lower the temperature to 32 or 33 degrees Celsius.’
The second challenge is to detect: many results must be analysed in a very short time. ‘In a regular lab, you would use a microscope to observe the results and count the number of infected cells,’ explains No Joo-hwan. ‘Here, we use a high-throughput, high-content imager to take images of these 384 wells on the plate. And instead of using the eye, we use an algorithm to quantify the parasites and translate the results into numbers.’
To visualize the infection, IPK researchers use different techniques, such as staining of the cells and the parasites with fluorescent dye or marker.
‘In the past, we used to quantify only two or three dimensions, such as number of parasites, number of infected cells, and total number of cells. But now, with machine learning and AI, we can extract over 5,000 features from a single cell! We can use this big data to see and find new things. This is exciting.’
This series of images show human cells infected by the T. cruzi parasite (Chagas disease) and exposed to a specific molecule. Researchers use different dyes of different colours to highlight the various compartments of the cells. This allows them to study the molecule’s effects on each part of the cells, helping them understand its mechanism of action. Image source: IPK
This series of images show human cells infected by the T. cruzi parasite (Chagas disease) and exposed to a specific molecule. Researchers use different dyes of different colours to highlight the various compartments of the cells. This allows them to study the molecule’s effects on each part of the cells, helping them understand its mechanism of action. Image source: IPK
Discovery ‘hits’
When a molecule is found to have prevented or cleared the infection, it is called a ‘hit’. But to find hits, drug discovery researchers need to test a vast number of molecules.
‘For Chagas for example, the hit rate is less than 1%,’ explains David Shum. ‘And these are just the initial hits: you must eliminate all the compounds that affect the host cell, which leads to 90% of the initial hits being eliminated.’
After this, drug discovery researchers will need to go through a complex, lab-intensive chemistry process involving chemical modifications of the hit compounds to optimize a range of important properties. They will then need to find the right dosage for optimal exposure and evaluate safety and other characteristics before finally being able to test them in human trials. Many compounds will be abandoned along the way. In the end, only two or three drug candidates will make it through this phase of drug discovery that is so challenging and uncertain it is known ‘valley of death’.
This is why DNDi and its partners need to test such an astronomical number of molecules. ‘This is really a numbers game,’ explains Jean-Robert Ioset, Discovery Leader at DNDi. ‘We need to test a lot. For example, for the parasite Trypanosoma cruzi that causes Chagas disease, we have just a 0.18% hit rate after testing 563,000 compounds!’
Jean-Robert Ioset, Discovery Leader at DNDi. Photo credit: DNDi
Jean-Robert Ioset, Discovery Leader at DNDi. Photo credit: DNDi
These compounds are owned by pharmaceutical companies, such as Sanofi, Novartis, and GSK, and by other non-profit research organizations, such as MMV, who partnered with DNDi to share them freely – agreeing in advance to make any resulting new medicines affordable and accessible in low- and middle-income countries if proven effective against a neglected disease. Some screenings were performed directly by these industrial partners.
‘Drug discovery is a very long process, and we are at the starting point. We are providing seeds that can be further developed. We have a huge responsibility in delivering potential solutions to patients.’
DNDi’s success stories with high-throughput screenings
Many promising compounds currently in development at DNDi were identified thanks to high-throughput screening. This is the case for acoziborole, an oral medicine for sleeping sickness at the advanced stages of development that was discovered screening a compound library owned by Anacor, which was later acquired by Pfizer. DNDi’s high-throughput screening partnerships have also led to the identification of the UW series of compounds for Chagas and several compounds for leishmaniasis, such as LXE408.
‘Our work with institutes such as IPK, Swiss TPH, and universities such as Dundee, Antwerp, and Nagasaki, has been instrumental in applying the most cutting-edge technologies to identify medicines for the most neglected.’
‘These organizations are true partners and bring to the table a wealth of knowledge and expertise on conducting these massive screenings. To advance the science and support other researchers in the field, we do our best to share all of our results and new learning. It’s a truly collaborative approach – all for neglected patients.’
In Seoul, the robotic arm of IPK is nowadays moving plates containing cells infected with the Trypanosoma cruzi parasite. David Shum and his team are developing a new assay model to identify promising molecules that could be effective against Chagas.
‘We are very proud and honoured to have been part of the screening campaign for leishmaniasis and Chagas disease with DNDi,’ he says. ‘We have helped build a solid pipeline for leishmaniasis, finding several promising candidates, and we hope to be able to do the same for Chagas.’
‘This is what attracted me to this job. I always worked for non-profits, and I saw the impact diseases have on people’s lives. Working with DNDi to learn as much as possible about those diseases and apply cutting-edge techniques to develop therapeutics and diagnostics is really stimulating.’
Learn more about high-throughput screening and DNDi’s work
- Next-generation phenotypic screening in early drug discovery for infectious diseases - Trends in Parasitology
- Screening strategies to identify new chemical diversity for drug development to treat kinetoplastid infections - Parasitology
- Phenotypic screening approaches for Chagas disease drug discovery - Expert Opinion on Drug Discovery
- Drug discovery for human African trypanosomiasis: identification of novel scaffolds by the newly developed HTS SYBR Green assay for Trypanosoma brucei - Journal of Biomolecular Screening
- Comparison of a high-throughput high-content intracellular Leishmania donovani assay with an axenic amastigote assay - Antimicrobial Agents and Chemotherapy
- Development and validation of a novel Leishmania donovani screening cascade for high-throughput screening using a novel axenic assay with high predictivity of Leishmanicidal intracellular activity - PLOS Neglected Tropical Diseases
- New compound sets identified from high throughput phenotypic screening against three kinetoplastid parasites: An open resource - Scientific Reports
- Institut Pasteur Korea (IPK)
- DNDi's drug discovery programme
- DNDi's work on Chagas disease
- DNDi's work on leishmaniasis
All photographs by Rosie Wills-DNDi (except where mentioned).
The Drugs for Neglected Diseases initiative (DNDi) is an international non-profit research and development organization that discovers, develops, and delivers safe, effective, and affordable treatments for neglected patients.
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