In-depth Exploration of Novel Hit Finding Approaches Using HTS and FBS
This blog post was written in partnership with Eurofins Discovery, a global leader in scientific testing and laboratory services for advancement of drug discovery and human health.
Identifying the most promising hits and leads with “drug-like” properties in the shortest amount of time is the primary goal in drug discovery. Successful preclinical candidates are selected using robust hit-finding strategies. In order to perform an effective drug discovery campaign, it is essential to understand the benefits that can be drawn from each step of a comprehensive hit identification cascade. With the goal of clearly demonstrating these advantages, Eurofins Discovery performed an in-depth exploration of the PIM3 kinase using both conventional High Throughput Screening (HTS) as well as Fragment-based Screening (FBS).
The PIM3 kinase was chosen for this case study, as the drug discovery of kinase inhibitors is particularly challenging due to the conservation of active sites and the high level of structural homology between kinases that can lead to safety liabilities. Using a comprehensive screening cascade designed by Eurofins Discovery’s HTS and biophysics experts, the team was able to rapidly identify selective PIM3 kinase inhibitors from the company’s proprietary libraries. The positive results of this case study provide a solid foundation showing that the techniques can be employed to quickly identify innovative and tractable hit series for other Hit-to-Lead programs.
The first step in this research was to implement an HTS cascade that integrated all phases of assay development for primary screening (ADP-Glo™ assays on a recombinant active PIM3 kinase produced by Eurofins DiscoverX®). The team then performed biophysics, MST-TRIC Spectral Shift biophysics assays and cellular NanoBRET™ target-engagement (TE) based assays to confirm and rank the identified PIM3 kinase hits. Numerous off-the-shelf assays were combined in order to quickly investigate hit selectivity for PIM3 kinase vs. PIM1 and PIM2 kinases and the whole kinome, as well as early ADME characteristics, i.e., solubility and human/mouse microsome stability.
PIM3 Kinase High Throughput Screening and Fragment-Based Screening
Recombinant active PIM3 kinase was produced by DiscoverX® to develop a robust high-throughput ADP-Glo™ primary assay used to screen a subset of the PlatinumDiv+ Eurofins Discovery library (71,000 compounds). High-quality data sets were obtained (Z’ = 0.86 ± 0.05) with a hit rate of 1.54%, enabling the selection of 1,000 hits (946 confirmed in ADP-Glo™ dose response curves (DRC), exhibiting pIC50 from 3.42 to 7.72).
The DRC phase confirmed the robustness and high quality (95.6% hit confirmation) of the data obtained from the ADP-Glo™ primary screen. At this stage, it was essential to identify the most promising compounds through an orthogonal approach, such as biophysics assays, as well as using a cellular-target engagement assay with the PIM3 NanoBRET™ assay. Prior to the DRC phase, the structure and chemical properties of the 946 confirmed hits were reviewed by Eurofins Discovery’s medicinal chemistry team in order to select the 140 most tractable hits for further testing using orthogonal assays.
After validation of the assay development, Spectral Shift technology enabled the ranking of compounds by affinity (KD values). Of the 946 confirmed hits, 140 selected molecules were then tested in a dose-response experiment, two-fold dilutions series from 30 µM. From 140 compounds, 128 were identified as binders for PIM3 kinase (92% hit rate); 22 compounds showed sub-micromolar-range affinity (Figure 3-A.) To confirm their binding potency and their stabilizing effect, the 19 tighter PIM3 binders were then tested by TSA (Figure 3). All compounds showed a drastic stabilization of PIM3 with a gain from 3 to 12°C compared to the apo-PIM3 kinase.
In addition to MST-TRIC confirmation, the 140 selected compounds were confirmed using PIM3 kinase cellular NanoBRET™ TE assay (Z’ = 0.83 ± 0.06). Out of the 140 hits identified, 128 binders were confirmed in MST-TRIC (pKD from 3.63 to 8.03) and 67 hits in a NanoBRET™ assay (pIC50 from 5.55 to 6.71). After triaging, 16 hits distributed in 6 clusters were retained and characterized in Eurofins Discovery’s proprietary KinaseProfiler™ (kinase activity) and KINOMEscan® (active site-directed competition binding assay) platforms. KinaseProfiler™ and KINOMEscan® were used to investigate the hits’ selectivity against PIM1 & PIM2 kinases (IC50 & KD) and help identify two clusters that showed reasonable selectivity for the PIM3 kinase. In parallel, the 16 hits were characterized for their solubility and stability, leading to the selection of three of the six clusters to initiate Hit-to-Lead program.
In parallel to the HTS & Biophysics exploration on the PIM3 kinase, Eurofins Discovery executed a second hit-identification strategy: a FBS cascade with the Eurofins Discovery Fragment ProbeSet library. The team used the MST-TRIC Spectral Shift as a primary assay and the thermal shift assay (TSA) as a confirmation assay.
From a selection of 826 fragments, 91 potential binders (11% hit rate) were identified in the single-dose screening, and 69 fragments were confirmed as PIM3 binders with KD values from 6.31 mM to 25.1 µM. The top 33 fragment hits were tested in TSA, and six were identified as stabilizers (∆Tm >1°C). Through the investigation of the mechanism of action (MoA), Eurofins Discovery identified three allosteric binders, promising fragment hits for future PIM3 structural biology work.
As was highlighted by Eurofins Discovery’s case study, two complementary, robust screening cascades were successfully implemented on the PIM3 kinase. The HTS campaign enabled the identification of highly selective and tractable kinase inhibitors within weeks, providing a solid basis for subsequent Hit-to-Lead and Lead Optimization programs. The FBS strategy enabled the identification of a short list of promising fragments, along with highly valuable information including their binding strength, indications about their binding site, as well as their ability to stabilize PIM3.
The HTS approach can rapidly and efficiently generate reliable preclinical candidates, and with the FBS approach, novel binding sites can be isolated. Moreover, fragment hits can be used to create small molecules by performing a fragment growing or a fragment linkage approach.
These screening strategies demonstrate the importance of having access to high-quality compound libraries, purified proteins and diverse technologies used for HTS and FBS. Combined with efficient project management and scientific skills, they lead to successful high-performance hit-finding campaigns.