May 1, 2018 (Vol. 38, No. 9)
Julie M. Rainard Ph.D. Drug Discovery Scientist European Screening Centre Newhouse, BioCity Scotland, University of Dundee
George C. Pandarakalam Ph.D. Drug Discovery Scientist European Screening Centre Newhouse, BioCity Scotland, University of Dundee
Stuart P. McElroy Ph.D. Head of Biology European Screening Centre Newhouse, BioCity Scotland, University of Dundee
European Lead Factory Combines HTS and a Biophysical Assay to Refine Early Drug Discovery
High-throughput screening (HTS) is an established method in drug discovery by which hundreds of thousands of compounds are screened in an automated fashion for activity as inhibitors or activators of a biological target. The goal is to find active compounds, or “hits,” that can serve as starting points for further compound optimization.
Although HTS is embraced by large pharma companies, it remains beyond the reach of many smaller entities due to the significant costs involved. To make HTS more accessible, the Innovative Medicines Initiative (IMI) created the European Lead Factory (ELF), a public-private partnership that aims to accelerate the discovery and development of novel chemical structures for drug discovery programs. The ELF asserts that it promotes “in-depth collaboration, honest brokerage, and enhanced communication” on behalf of the wider European life sciences community, not just ELF’s formal partners, which include established pharmaceutical companies, small- to medium-sized enterprises, and academic institutions.
Researchers and drug developers that work with the ELF gain access to a collection of up to half a million compounds, against which their submitted molecular targets can be screened. After completing a screen for a target, the ELF provides the target’s submitter with a qualified hit list (QHL), which includes up to 50 compounds. Subsequently, the submitter has exclusive rights to utilize these compounds as starting points for drug discovery programs or as chemical tools to further its research.
Considerable effort has been made by the ELF to provide well-designed screening workflows for each target that maximizes the probability of finding real target binders while minimizing the risk of selecting undesirable compounds or pan-assay interference compounds (PAINS). These reactive artifacts, which can interfere with primary activity assays or even disrupt the target’s function or structure, are often mistaken as genuine hits.
To assess whether the output of an HTS contains quality starting points, drug hunters implement a form of triage. Many researchers, including those of the ELF, find that following up hits from HTS with biophysical assays is very productive. Such assays discriminate between drug candidates without relying on the usual assessments of functional activity.
A biophysical assay called MicroScale Thermophoresis (MST) has been used by the ELF as a triage tool for HTS campaigns.1 As described here, MST has helped the ELF deliver well-characterized and well-validated hits.
MST at the ELF
MST measures the strength of the interaction between two molecules by detecting variation in a fluorescence signal of a fluorescently labeled or intrinsically fluorescent target, variation that occurs along an infrared laser–induced temperature gradient. The range of the variation in the fluorescence signal correlates with the binding of a ligand to the fluorescent target.
As Figure 1 shows, MST has served as an orthogonal assay at several stages in the ELF screening workflow. It has informed triage decisions, facilitated the progression of hits from primary screens, and provided detailed characterization of QHL compounds. It has also been used to evaluate newly synthesized analogs, to quantify the affinity of their interactions and reveal structure–activity relationships (SARs).
No single biophysical technique appears to be more reliable than any other as a hit triaging tool. Consequently, whenever possible, several techniques should be used in combination to provide a comprehensive assessment of target binding in an HTS campaign. The choice between biophysical technologies depends on factors such as ligand sensitivities and reagent requirements.
At the ELF, techniques such as isothermal titration and nuclear magnetic resonance are considered impractical because of strict reagent restrictions. Alternative techniques employed by the ELF include MST, surface plasmon resonance (SPR), and differential scanning fluorimetry (or the thermal shift assay, TSA), which have been used against a wide variety of targets with varying degrees of success (Figure 2).
MST nicely complements other biophysical assays and in some instances provides significant advantages. For instance, the MST assay does not require protein immobilization, which reduces the observation of nonspecific compound binding to immobilization surfaces, a potential issue with SPR. Also, the MST assay can detect binding interactions between very small ligands—even ions—and large proteins.
MST offers a wide detection range, from low millimolar to picomolar affinities, and can work in common buffers, in complex biological systems (such as cell lysates and serum), and in systems incorporating solubilized proteins and proteins in liposomes. It can also identify some types of false-positive compounds and the mechanisms by which they interfere, such as photobleaching, photoenhancement, autofluorescence, and aggregation.
Finally, assay development and screening is rapid and can be performed in an automated fashion. The use of NanoTemper Technologies’ Monolith NT.Automated Platform has enabled screening of up to 800 samples per day. MST is particularly advantageous because it works with very low amounts of sample and can be used with a wide range of proteins.
Ranking Protein–Protein Interaction Inhibitors
In an ELF screen against a protein–protein interaction target, MST aided in the prioritization of small-molecule hits from the HTS. The target was screened against over 318,000 compounds in a fluorescence polarization binding assay, and the resulting QHL of eight compounds was further characterized by a label-free MST assay. Of these compounds, two were validated for target binding, five did not show any binding, and one displayed an aggregator profile.
Adding increasing concentrations of a peptide mimic of the target’s binding partner resulted in increasing Kd values for both hit compounds. This finding demonstrated that both hit compounds are specific to the protein–protein interaction domain. Moreover, since both hit compounds are chiral, an interesting possibility presented itself: synthesizing and testing the opposite enantiomers. These showed no target binding in MST, providing chemical validation of the binding specificity of the hits (Figure 3).
These results were sufficiently encouraging to justify a subsequent chemistry program, leading to the synthesis of over 100 new analogs. Ligand-bound crystal structures were then obtained, providing validation of the binding mode and rationalization of SARs.
In the ELF’s experience, MST is a useful biophysical technique for triaging and characterizing HTS hits and can improve the chances of drug-development success by providing orthogonal evidence of target engagement. This tool can also help researchers identify which compounds are suitable for further investment of resources.
Currently, the ELF is looking into integrating the MST instrument with an automated liquid handling system, thereby increasing the throughput to more efficiently deal with the large numbers of hits that are identified during the screening of various targets.
1. Rainard JM, Pandarakalam GC, McElroy SP. Using Microscale Thermophoresis to Characterize Hits from High-Throughput Screening: A European Lead Factory Perspective. 2018. SLAS Discovery 23(3): 225–241.
Julie M. Rainard, Ph.D., and George C. Pandarakalam, Ph.D., are drug discovery scientists, and Stuart P. McElroy, Ph.D. (firstname.lastname@example.org), is head of biology at the European Screening Centre Newhouse, BioCity Scotland, University of Dundee. Website: nanotempertech.com.