POLARISED views and FRETting about probe modulation assays: Learning from High Throughput Screening.

Fluorescent probe modulation assays are a widely used approach to monitor displacement or stabilisation of fluorescently labelled tool ligands by test compounds. These assays allow an optical read-out of probe-receptor binding and can be used to detect compounds that compete with the labelled ligand, either directly or indirectly. Probes for both orthosteric and allosteric sites are often employed. The method can also be used to identify test compounds that may stabilise the ternary complex, offering an opportunity to discover novel molecular glues. The utility of these fluorescence-based assays within high-throughput screening has been facilitated by the use of streptavidin labelled terbium as a donor and access to a range of different acceptor fluorophores. During 2023, the High-throughput Screening group at AstraZeneca carried out 8 high-throughput screens using these approaches. In this manuscript we will present the types of assays used, an overview of the timelines for assay development and screening, the application of orthogonal artefact methods to aid hit finding and the results of the screens in terms of hit rate and the number of compounds identified with IC50 values of better than 30 µM. Learning across the development, execution and analysis of these screens will be presented.

Screening for molecular glues - Challenges and opportunities.

Molecular glues are small molecules, typically smaller than PROTACs, and usually with improved physicochemical properties that aim to stabilise the interaction between two proteins. Most often this approach is used to improve or induce an interaction between the target and an E3 ligase, but other interactions which stabilise interactions to increase activity or to inhibit binding to a natural effector have also been demonstrated. This review will describe the effects of induced proximity, discuss current methods used to identify molecular glues and introduce approaches that could be adapted for molecular glue screening.

A perspective on the changing landscape of HTS.

Recently, there has been a change in the types of drug target entering early drug discovery portfolios. A significant increase in the number of challenging targets, or which would have historically been classed as intractable, has been observed. Such targets often have shallow or non-existent ligand-binding sites, can have disordered structures or domains or can be involved in protein-protein or protein-DNA interactions. The nature of the screens required to identify useful hits has, by necessity, also changed. The range of drug modalities explored has also increased and the chemistry required to design and optimise these molecules has adapted. In this review, we discuss this changing landscape and provide insights into the future requirements for small-molecule hit and lead generation.

High throughput screening of 0.5 million compounds against CRAF using Alpha CETSAⓇ.

The cellular thermal shift assay (CETSA®) has increasingly been used in early drug discovery to provide a measure of cellular target engagement. Traditionally, CETSA has been employed for bespoke questions with small to medium throughput and has predominantly been applied during hit validation rather than in hit identification. Using a CETSA screen versus the kinase CRAF, we assessed 3 key questions: (1) technical feasibility - could the CETSA methodology technically be applied at truly high throughput scale? (2) relevance - could hits suitable for further optimisation be identified? (3) reliability - would the approach identify known chemical equity. Here, we describe the first large scale AlphaLISA SureFire based CETSA (Alpha CETSA) approach allowing us to screen a large library of almost 0.5 million compounds. We discuss the issues overcome in automating and executing the screen and describe the resulting screen output.

Thermal unfolding methods in drug discovery.

Thermal unfolding methods, applied in both isolated protein and cell-based settings, are increasingly used to identify and characterize hits during early drug discovery. Technical developments over recent years have facilitated their application in high-throughput approaches, and they now are used more frequently for primary screening. Widespread access to instrumentation and automation, the ability to miniaturize, as well as the capability and capacity to generate the appropriate scale and quality of protein and cell reagents have all played a part in these advances. As the nature of drug targets and approaches to their modulation have evolved, these methods have broadened our ability to provide useful chemical start points. Target proteins without catalytic function, or those that may be difficult to express and purify, are amenable to these methods. Here, we provide a review of the applications of thermal unfolding methods applied in hit finding during early drug discovery.

Bromodomain Interactions with Acetylated Histone 4 Peptides in the BRD4 Tandem Domain: Effects on Domain Dynamics and Internal Flexibility.

The bromodomain and extra-terminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Like other BET proteins, BRD4 contains two bromodomains, BD1 and BD2, that can interact cooperatively with target proteins and designed ligands, with important implications for drug discovery. Here, we used nuclear magnetic resonance (NMR) spectroscopy to study the dynamics and interactions of the isolated bromodomains, as well as the tandem construct including both domains and the intervening linker, and investigated the effects of binding a tetra-acetylated peptide corresponding to the tail of histone 4. The peptide affinity is lower for both domains in the tandem construct than for the isolated domains. Using 15N spin relaxation, we determined the global rotational correlation times and residue-specific order parameters for BD1 and BD2. Isolated BD1 is monomeric in the apo state but apparently dimerizes upon binding the tetra-acetylated peptide. Isolated BD2 partially dimerizes in both the apo and peptide-bound states. The backbone order parameters reveal marked differences between BD1 and BD2, primarily in the acetyl-lysine binding site where the ZA loop is more flexible in BD2. Peptide binding reduces the order parameters of the ZA loop in BD1 and the ZA and BC loops in BD2. The AB loop, located distally from the binding site, shows variable dynamics that reflect the different dimerization propensities of the domains. These results provide a basis for understanding target recognition by BRD4.

A perspective on the discovery of enzyme activators.

Enzyme activation remains a largely under-represented and poorly exploited area of drug discovery despite some key literature examples of the successful application of enzyme activators by various mechanisms and their importance in a wide range of therapeutic interventions. Here we describe the background nomenclature, present the current position of this field of drug discovery and discuss the challenges of hit identification for enzyme activation, as well as our perspectives on the approaches needed to overcome these challenges in early drug discovery.

Utilising acoustic mist ionisation mass spectrometry to identify redox cycling compounds in high throughput screening outputs.

Rapid triage of compounds acting via undesired mechanisms is a crucial stage in a high-throughput screening (HTS) cascade to ensure time and resource is efficiently assigned to the most propitious hits. Redox cycling compounds (RCCs) produce reactive oxygen species, such as hydrogen peroxide, which can impair protein function and appear as hits against liable targets. Direct measurement of tris(2-carboxyethyl)phosphine (TCEP) oxidation has been demonstrated as a sensitive and accurate measure of redox cycling [1]. However, the current nuclear magnetic resonance (NMR) based detection method is not compatible with the throughput required for triage of a HTS campaign. Here we employ Acoustic Mist Ionisation Mass Spectrometry (AMI-MS) [2] to rapidly measure oxidation of TCEP and accurately identify redox cyclers in a high throughput manner.

High-throughput detection of metal contamination in HTS outputs.

Large compound libraries utilised for HTS often include metal contaminated compounds which can interfere with assay signal or target biology, and therefore appear as hits. Pursuit of these compounds can divert considerable time and resource away from more propitious hits, yet there is currently no established method of detecting metal impurities in a rapid and effective manner. Here we describe the development and application of a high-throughput method to identify metal contaminants using acoustic mist ionisation mass spectrometry (AMI-MS). Although metals species by themselves are not detectable by AMI-MS, we have identified two compounds that chelate metal ions and enable their detection. 6-(diethylamino)-1,3,5-triazine-2,4(1H,3H)-dithione (DMT) and 1-(3-{[4-(4-cyanophenyl)-1-piperidinyl]carbonyl}-4-methylphenyl)-3-ethylthiourea (TU) can form complexes with a range of metal ions. Using a collection of metal catalysts, we have developed two metal chelator assays that collectively allow for the detection of Ag, Au, Co, Cu, Fe, Pd, Pt and Zn. We employed these assays to profile the hit outputs of a Zn liable target, and a Pd liable target, and identified significant quantities of metal contaminated compounds in the HTS outputs. This work provides a method of rapidly identifying metal impurities in hit compounds and has become part of an established workflow in triaging HTS outputs at AstraZeneca, facilitating faster identification of robust lead series.

Advancing automation in high-throughput screening: Modular unguarded systems enable adaptable drug discovery.

Challenged by ageing infrastructure and increasingly demanding screening cascades, AstraZeneca High Throughput Screening department has developed advanced automation systems that can support both current needs and future strategies in drug discovery. Through collaboration with HighRes Biosolutions and other third-party vendors, highly versatile automated modular platforms have been designed. Safety features such as collaborative robots allow enhanced system accessibility, and adaptive scheduling software has improved protocol design and system recovery. These innovations have led to significant improvements in system flexibility while maintaining screening productivity.

Drug discovery for epigenetics targets.

Dysregulation of the epigenome is associated with the onset and progression of several diseases, including cancer, autoimmune, cardiovascular, and neurological disorders. Members from the three families of epigenetic proteins (readers, writers, and erasers) have been shown to be druggable using small-molecule inhibitors. Increasing knowledge of the role of epigenetics in disease and the reversibility of these modifications explain why pharmacological intervention is an attractive strategy for tackling epigenetic-based disease. In this review, we provide an overview of epigenetics drug targets, focus on approaches used for initial hit identification, and describe the subsequent role of structure-guided chemistry optimisation of initial hits to clinical candidates. We also highlight current challenges and future potential for epigenetics-based therapies.

Validation of ion mobility spectrometry - mass spectrometry as a screening tool to identify type II kinase inhibitors of FGFR1 kinase.

RATIONALE: The protein kinase FGFR1 regulates cellular processes in human development. As over-activity of FGFR1 is implicated with cancer, effective inhibitors are in demand. Type I inhibitors, which bind to the active form of FGFR1, are less effective than type II inhibitors, which bind to the inactive form. Screening to distinguish between type I and type II inhibitors is required. METHODS: X-ray crystallography was used to indicate whether a range of potential inhibitors bind to the active or inactive FGFR1 kinase conformation. The binding affinity of each ligand to FGFR1 was measured using biochemical methods. Electrospray ionisation - ion mobility spectrometry - mass spectrometry (ESI-IMS-MS) in conjunction with collision-induced protein unfolding generated a conformational profile of each FGFR1-ligand complex. The results indicate that the protein's conformational profile depends on whether the inhibitor is type I or type II. RESULTS: X-ray crystallography confirmed which of the kinase inhibitors bind to the active or inactive form of FGFR1 kinase. Collision-induced unfolding combined with ESI-IMS-MS showed distinct differences in the FGFR1 folding landscape for type I and type II inhibitors. Biochemical studies indicated a similar range of FGFR1 affinities for both types of inhibitors, thus providing confidence that the conformational variations detected using ESI-IMS-MS can be interpretated unequivocally and that this is an effective screening method. CONCLUSIONS: A robust ESI-IMS-MS method has been implemented to distinguish between the binding mode of type I and type II inhibitors by monitoring the conformational unfolding profile of FGFR1. This rapid method requires low sample concentrations and could be used as a high-throughput screening technique for the characterisation of novel kinase inhibitors.

The Use of Acoustic Mist Ionization Mass Spectrometry for High-Throughput Screening.

It is clear from the analysis of the distribution of approved drug targets that enzymes continue to be a major target class for the pharmaceutical industry. The application of high-throughput screens designed to monitor the activity of these enzyme targets, and the ability of test compounds to modulate this activity, is still the predominant hit finding approach in the industry. The widespread use of enzyme activity-based screens has led to the development of several useful guidelines for the development and validation of robust and reliable assays. Key learnings for the development, validation, and implementation of acoustic mist ionization mass spectrometry for high-throughput enzyme assays are described.

Ligand Discovery: High-Throughput Binding: Fluorescence Polarization (Anisotropy).

High-throughput assays based on fluorescence polarization (or fluorescence anisotropy) technology have often been employed for primary hit-finding in drug discovery. These binding assays provide a homogeneous format and consistent performance and offer advantages over some other optical methods. Developments in assay design and improvements in fluorescent probes have enabled the application of the technique to even complex biological systems. Here we describe the practical considerations for development of FP assays applied in high-throughput screening, including fluorophore selection, assay design, data analysis, and approaches for detecting compound interference.

Small-Molecule Degraders beyond PROTACs-Challenges and Opportunities.

Targeted protein degradation (TPD) is a recent strategy, utilizing the cell's proteostasis machinery to deplete specific proteins. This represents a paradigm shift in early drug discovery, away from occupancy-driven to event-driven mechanisms.Recent efforts have focused on the development of proteolysis-targeting chimeras (PROTACs). These heterobifunctional molecules combine a target-specific binding moiety linked to an E3 ligase ligand and trigger selective ubiquitination of the target protein, marking it for proteasomal degradation. While these molecules can be highly efficacious, they generally have unfavorable physicochemical properties due to their large size.In contrast, smaller molecules that induce degradation could represent an attractive, simple option to overcoming the limitations of both traditional modulators and PROTACs. These molecules may have a range of mechanisms: recruitment of an E3 ligase (molecular glues), introduction of hydrophobic areas, or inducing local unfolding, each of which triggers degradation.We recently completed a high-throughput screen of 111,000 compounds in a cellular HiBiT assay in an effort to identify such molecules. Preliminary analysis indicates that we have been able to identify alternative small-molecule degraders. We highlight methods for triage, characterization, selectivity, and mode of action. In summary, we believe that these types of small-molecule degraders, which may possibly have more acceptable physicochemical properties than the inherently larger heterobifunctional molecules, are an exciting approach for inducing TPD, and we illustrate that a general screening approach can be successful in identifying useful start points for developing such molecules.

High-Throughput Mechanism of Inhibition.

Enzymes represent a significant proportion of the druggable genome and constitute a rich source of drug targets. Delivery of a successful program for developing a modulator of enzyme activity requires an understanding of the enzyme's mechanism and the mode of interaction of compounds. This allows an understanding of how physiological conditions in disease-relevant cells will affect inhibitor potency. As a result, there is increasing interest in evaluating hit compounds from high-throughput screens to determine their mode of interaction with the target. This work revisits the common inhibition modalities and illustrates the impact of substrate concentration relative to Km upon the pattern of changes in IC50 that are expected for increasing substrate concentration. It proposes a new, high-throughput approach for assessing mode of inhibition, incorporating analyses based on a minimal descriptive model, to deliver a workflow that allows rapid and earlier compound classification immediately after high-throughput screening.

Evaluation of the Use of Cold Plasma for Microtiter Plate Cleaning to Reduce Plastic Biohazard Waste.

Plastic pollution is the accumulation of plastic objects in the Earth's environment and is a global problem of increasing importance. The laboratory and health care industries contribute to this problem by the widely accepted single use of plastics, including microtiter plates used for compound testing. At AstraZeneca, we predict the use of more than 45,000 384-well and more than 11,000 1536-well microtiter plates per year. IonField Systems has developed a microplate cleaning system (MCS) powered by PlasmaKnife technology that uses cold plasma to clean microtiter plates. AstraZeneca proposed the use of this system for standard ANSI (https://slas.org/resources/information/industry-standards/) microtiter plate regeneration. Here we present the results of an evaluation using a model system involving the cleaning of plates following an enzyme-based biochemical assay, as well as the software and hardware enhancements that were incorporated into the production PlasmaKnife MCS. The method involved determining the level of inhibition achieved by residual compound following different cleaning protocols and showed that cleaning achieved in about 2 min was sufficient to remove trace compound contamination. Future work will focus on assessing the number of regeneration cycles that can be reliably achieved.

Regenerable Biosensors for Small-Molecule Kinetic Characterization Using SPR.

A key activity in small-molecule drug discovery is the characterization of compound-target interactions. Surface plasmon resonance (SPR) is a flexible technique for this purpose, with a wide affinity range (micromoles to picomoles), low protein requirements, and the ability to characterize the kinetics of compound binding. However, a key requirement of SPR is the immobilization of the target protein to the surface of the sensor chip. The most commonly used immobilization techniques (covalent immobilization, streptavidin-biotin) are irreversible in nature, which can afford excellent baseline stability but impose limitations throughput for slowly dissociating compounds or unstable targets. Reversible immobilization (e.g., His-tag-Ni-NTA) is possible but typically precludes accurate quantification of slow dissociation kinetics due to baseline drift.Here we present our investigation of three immobilization strategies (dual-His-tagged target protein, His-tagged streptavidin, and switchavidin) that combine the robustness of irreversible immobilization with the flexibility of reversible immobilization. Each has its own advantages and limitations, and while a universal immobilization procedure remains to be found, these strategies add to the immobilization toolbox that enables previously out-of-scope applications. Such applications are highlighted in two examples that greatly increased throughput for the kinetic characterization of potent kinase inhibitors and kinetic profiling of covalent inhibitors.

Detection of Drug Binding to a Target Protein Using EVV 2DIR Spectroscopy.

We demonstrate that electron-vibration-vibration two-dimensional infrared spectroscopy (EVV 2DIR) can be used to detect the binding of a drug to a target protein-active site. The EVV 2DIR spectrum of the FGFR1 kinase target protein is found to have ∼200 detectable cross-peaks in the spectral region 1250-1750 cm-1/2600-3400 cm-1, with additional 63 peaks caused by the addition of a drug, SU5402. Of these 63 new peaks, it is shown that only six are due to protein-drug interactions, with the other 57 being due to vibrational coupling within the drug itself. Quantum mechanical calculations employing density functional theory are used to support assignment of the six binding-dependent peaks, with one being assigned to a known interaction between the drug and a backbone carbonyl group which forms part of the binding site. None of the 57 intramolecular coupling peaks associated with the drug molecule change substantially in either intensity or frequency when the drug binds to the target protein. This strongly suggests that the structure of the drug in the target binding site is essentially identical to that when it is not bound.