The Role of Pan-Assay Interference Compounds in HTS

Learning Objectives

1. Define the terms “PAINS”, “false positive”, and “triage” in reference to HTS  
2. Describe why PAINS exist and current measures being used to deal with them 
3. List common PAINS and technology used to identify them

Pan Assay Interference Compounds (PAINS) lead to false positives in high-throughput screening (HTS). The image shows interference, the cause of a false positive result. PAINS interfere with the target compound’s ability to bind or signal leading to an inaccurate assay readout. 

The Issue at Hand 

Many researchers have dealt with the frustration of discovering their assay result was a false positive. False positives are nonspecific inhibitors that incorrectly identify as active in an assay readout [2]. There are a number of reasons false positives can occur in high-throughput screens (HTS). Almost all of these reasons have to deal with interference in some shape or form [1]. Compounds may cause aggregate formation, which would interfere with binding and lead to false positives [1]. Protein reactive compounds are also known for signaling false positives [1]. Compound interference with assay technology is another way a false positive result can be received in HTS [1]. Knowledge and understanding of which compounds may result in a false positive when designing a HTS is crucial. Referencing libraries of compounds compiled from other research groups as well as being fastidious when selecting vendors are measures that can be taken to avoid false positives when designing a HTS [1]. However, receiving false positive results from various compounds remains a large issue that many have tried to combat. 

PAINS – History 

Awareness for pan assay interference compounds (PAINS) has increased over the past few years. One of the first publications on the topic, by Baell and Holloway, summarizes the different classes of PAINS and all the molecules that were grouped in this problematic compound class [1]. The article defines PAINS as compounds which are recognized by their substructure filters as problematic [1]. These substructural filters helped identify numerous problematic compounds based on protein-protein interaction screens, as well as their appearance in a variety of assays cited in other literature [1]. Baell and Hollowoy defined the first classes of PAINS to be rhodanines, phenolic Mannich bases, hydroxy-phenylhydrazones, alkylidene barbiturates, alkylidene hetero- cycles, 1,2,3-aralkylpyrroles, activated benzofurazans, 2-amino-3-carbonylthiophenes, catechols, and quinones [1]. Since this initial list of PAINS, further research has been done to identify problematic compounds which interfere with assays. 

Triaging Stage and Moving Forward

The term triage when referring to HTS involves sorting and prioritizing compounds into various groups based on their potential in drug development [2]. This is a crucial step in the screening process and can vary based on the resources available [2]. Even with computational identification and manual search and removal, PAINS still manage to slip past the triaging stage and blend in with other active compounds [2]. The creation and progression of structural filters to implement during the triaging stage has helped researchers identify and eliminate PAINS [4]. However, the design of triaging, as well as the screen as a whole, are crucial in accurately identifying compounds for drug discovery [4]. With current technology and information, one is able to readily identify top PAINS substructures and be sure to exclude them from their research (even though this does not fully ensure them of avoiding PAINS) [3]. With the growing knowledge of PAINS, peer reviewers and researchers also have a responsibility to carefully evaluate research and proposals which contain these compounds [3]. Increasing awareness of PAINS has also emphasized the importance of good scientific practice in drug and probe discovery [3]. These practices include actively testing for purity and identity of compounds as well as taking the necessary measures to rule out interference compounds [3]. 

Questions and Answers

1. What does PAINS stand for? Pan assay interference compounds 
2. What are common PAINS? rhodanines, phenolic Mannich bases, hydroxy-phenylhydrazones, alkylidene barbiturates, alkylidene hetero- cycles, 1,2,3-aralkylpyrroles, activated benzofurazans, 2-amino-3-carbonylthiophenes, catechols, and quinones
3. How can you avoid PAINS? It may be very hard to fully avoid PAINS. Being aware of them and excluding known PAINS in your research (or proceeding with caution when using them) are ways to reduce the likelihood of having to deal with. 

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PAINS Audio Recording

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