Drug discovery campaigns are prioritizing increasingly difficult targets in important and underserved therapeutic areas, which poses a challenge for organizations aiming to rapidly identify and develop novel therapeutics. As a result, the drug discovery community has increased efforts to maximize the breadth of chemical space of hit identification (Hit ID) campaigns and speed up compound screening to improve the chances of identifying novel and successful start-points for ongoing development. Direct-binding, biophysical high throughput screening (HTS) platforms, such as affinity-selection mass spectrometry (ASMS), have become popular approaches in this space due to high sensitivity screens of large libraries in a much shorter timeframe than traditional plate based HTS approaches, if a functional or cellular readout isn’t required or desirable. A key consideration in these short turnaround library screens is validating any identified hits in an equally efficient and effective manner, which is where Biacore™ surface plasmon resonance (SPR) system is particularly powerful.
A synergistic approach to hit identification
The HIT SYNERGY platform at Sygnature Discovery (1) utilizes a range of technologies and approaches to discover novel low molecular weight start-points which can be tailored to suit project requirements, for progression into target engagement assays (2). (Fig 1). Strategies forming this approach include ASMS (3), fragment screening using Biacore™ surface plasmon resonance (SPR), plate based biochemical and cellular high-throughput screening (HTS), and computational hit finding approaches (virtual screens, VS). Biacore SPR systems supports all four of these approaches, either by being the primary in-lab screening technology (fragments and VS) or by playing a key role in hit validation (ASMS and HTS) (Fig 1).
Fig 1. HIT SYNERGY platform at Sygnature Discovery leverages a range of technologies and approaches to discover novel low molecular weight starting points. (The top concept image is the property of Sygnature Discovery)
In the context of HIT SYNERGY, Biacore™ SPR systems have two key strengths The first is the capacity of the modern, multi-needle Biacore™ 8 series SPR systems (3) to rapidly screen large number of samples. The second is the SPR sensorgrams that are information-rich and offer key insights into the nature of binding interactions and the overall performance of a compound. For example, key indicators such as non-specific binding, stoichiometry, and solubility limitations can be quickly identified which positions Biacore™ SPR systems as invaluable tools for Hit ID, confirmation, and validation
Sygnature Discovery uses Biacore™ SPR systems to examine ligand-target interactions that range from weakly binding to very high affinity. The sensitivity of the system is utilized for hit finding campaigns, to follow-up ASMS hits and as a platform for fragment primary screening.
Here, we outline a recent case study using an ASMS primary screen with orthogonal validation using Biacore™ SPR systems. This cascade was successfully used to discover novel hit matter, elucidate binding mode, and prioritize hits for further testing by X-ray crystallography.
ASMS: high-throughput, solution-based screening
ASMS is a high-throughput primary screening tool for the detection of lead-like compounds, which has gained in popularity as a rapid and highly efficient HitID strategy for screening large libraries of molecules. The speed of ASMS is enabled by compound pooling, but this in turn requires a robust follow up and confirmation of hits. Any hits from the pooled screening experiments are then analyzed by ASMS as singletons before orthogonal confirmation with an alternative biophysical technology, such as Biacore™ SPR.
ASMS is a versatile, label-free screening technology that operates in solution and identifies novel ligands for a broad range of biological targets, including challenging non-protein targets like nucleic acids. As ASMS is focused on finding binders rather than functionally active molecules, it is particularly useful for modalities in the induced proximity therapeutics area, and cases where traditional activity-based HTS may fall short by offering an efficient, binding site agnostic route to tool compounds and new lead matter. Unlike DNA-encoded libraries (DELs), ASMS enables immediate downstream work from DMSO or solid-stock compounds without the delay of resynthesis, which streamlines the hit validation process. In the case of the HIT SYNERGY platform, this is performed using library stocks for the fastest possible confirmation of hits.
Practically, the ASMS platform at Sygnature Discovery uses 2-dimensional chromatography followed by time of flight (ToF) mass spectrometry (Fig 2). In the first dimension, a size exclusion column is used to separate target-bound and unbound compounds. Target-compound complexes are larger and are excluded from the pores of the column, eluting earlier than free compound. It is noteworthy that during time spent on the column, compound dissociation will occur. So, with the first dimension, we enrich for slower dissociating, and higher affinity binders with more lead-like characteristics. ASMS is therefore less sensitive to weaker interactions, which is why parallel fragment screening by SPR has proven a useful complementing strategy for Hit ID.
Following separation of target-compound complexes from free compound in the first dimension, the protein is denatured and separated from previously bound compound through reverse phase chromatography, the second dimension. Bound compounds are then identified through high-resolution mass spectrometry based on similarity to theoretical isotope clusters.
Fig 2. Affinity-selection mass spectrometry (ASMS) uses 2-dimensional chromatography followed by time of flight (ToF) mass spectrometry for compound identification.
Case study: discovering allosteric hits for an enzyme target
Finding novel hits for a known target
Our target protein was a His-tagged, epigenetic regulator of approximately 150 kDa molecular weight, with previously reported inhibitors that target the active site. The protein also undergoes a conformational change in the presence of an activating cofactor. The target was believed to behave as a homodimer in solution, and for tool molecule interaction, we assumed one binding event per one monomer unit.
To provide novel hits for this target, which fall outside of the existing chemical space and mechanism of action, we focused on identifying binders to allosteric sites, which were agnostic to protein conformation.
Developing the assays
To understand whether an ASMS assay could be developed, we optimized target protein concentration, buffer systems, and injection parameters alongside the final detection of a known tool in the mass spectrometer. In parallel, Flow Induced Dispersion Analysis (FIDA) (4) was used to understand protein integrity through measurement of the hydrodynamic radius in solution and in the presence of a representative compound cocktail at 3% DMSO. These analyses directly informed our screening batch run times. From these development assays, we determined that the protein was stable up to 48 hours.
Biacore™ assay development followed a typical process of understanding an effective coupling strategy and monitoring coupled target protein performance. The protein was captured onto Sensor Chip NTA via the hexa-histidine tag and covalently attached to the sensor surface through amine coupling using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-Hydroxysuccinimide (NHS). Binding activity of the coupled target protein was assessed over time using low molecular weight tool molecules to determine our SPR assay time-window. These development steps allowed us to determine batch sizes for screening and orthogonal testing, and how long the surface stays sufficiently active for accurate binding determination.
Screening a HTS library by ASMS
The ASMS screen was performed using the Sygnature Discovery LeadFinder Diversity library (5), which is composed of approximately 150,000 diverse and lead-like compounds with an average molecular weight of 350 Da. This library is highly effective for hit finding as it provides a high diversity selection of lead like molecules, with highly desirable chemical properties for further development. This contrasts with other hit finding approaches, such as DEL screening, and often results in larger, less ligand-efficient molecules. In addition, ASMS has no requirement for DNA tagging which imposes constraints on molecular design.
In this case the target protein was screened against pools of 300 compounds per well, with the protein concentration higher than concentration than of the individual small molecules, in batches of approximately 30,000 compounds. The primary hit rate was 0.08%, or 132 compounds, which is a typical hit rate for a pooled ASMS screen of an unbiased library. Nominal hits were then taken forward into single compound testing at an increased concentration, resulting in 23 reconfirmed compounds from ASMS. Finally, these 23 hits were analyzed orthogonally with Biacore™ SPR in concentration-response format to determine binding affinity and behavior.
Validating hits with Biacore™ SPR systems
The ASMS screen was performed without the activating cofactor to enable detection of binders to the inactive protein conformation. To validate both the binding of the hits and their prospective mechanism of action, the 23 hits from ASMS were screened using the Biacore™ SPR system both in the presence and absence of the activating cofactor in separate experiments. If hits bound similarly in both conditions (and therefore both protein conformations), we could infer that those hits occupied an allosteric site.
All 23 compounds were tested in concentration-response format, with a dilution series prepared from 50 µM, diluted two-fold across eight concentrations and analyzed from low to high concentration. In total, ten compounds were designated as validated hits at the end of this process.
The use of Biacore™ SPR system allowed us to separate higher quality hits that demonstrated desirable sensorgram profiles, identify saturable binding responses from weak, poor target interacting compounds, and prioritize hits for X-ray crystallography screening (Fig 3). Desirable responses are typically positive sensorgrams with signal that is proportional to the amount of ligand on the sensor chip surface, and which show an expected dissociation based on the specific decay of the complex. For an ASMS screen of Sygnature Discovery LeadFinder library, initial hits in the µM range could be anticipated and thus there may be some hints of kinetics. The determination of kinetics would contrast fragment-based approaches where starting points are typically weaker, by virtue of their size and number of possible interactions, leading to faster rates of binding and decay.
Fig 3. Biacore™ SPR system separates higher quality hits that demonstrate desirable sensorgram profiles and identify saturable binding responses from weak, poor target interacting compounds.
Of the ten hits confirmed using the Biacore™ SPR systems, two demonstrated binding to the target with and without the presence of the cofactor. One hit was equipotent across conditions and was later confirmed to bind to an allosteric site by X-ray crystallography (Fig 4).
Fig 4. The Biacore™ SPR system identified two compounds that demonstrated binding to the target with (orange curve) and without (green curve) the presence of the cofactor. Indeed, one hit was equipotent across conditions and was later confirmed to bind to an allosteric site by X-ray crystallography
Accelerating hit discovery with an integrated platform
The combination of ASMS primary screening and Biacore™ SPR validation provided a rapid, efficient, and highly reliable hit finding platform to discover hits against this epigenetic regulator. Further work using this approach has confirmed that it’s also applicable to a range of other targets, with many successful Hit ID campaigns being completed at Sygnature Discovery. ASMS offers high-throughput, label-free, in-solution system to screen a large library. Validation with Biacore™ SPR system, allows Sygnature Discovery to rationalize the performance of those hits through the observation of real-time target interaction profiles and the calculation of affinity and kinetic data.
This parallel approach is key to driving success and has now been demonstrated on several projects at Sygnature Discovery, across a range of target types. It also highlights the key place that Biacore™ SPR system continues to have within the drug discovery landscape by complementing a range of technologies and hit finding approaches, from more traditional HTS, through virtual approaches, to the support of more recent screening approaches such as ASMS.
References
- Sygnature Discovery's HIT SYNERGY Platform
- Biophysical assays at Sygnature Discovery
- Biacore™ 8 series SPR system
- Flow Induced Dispersion Analysis (FIDA)