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Targeted protein degradation is an important mechanism carried out by the cellular machinery, one that is gaining momentum as an exploitable strategy for the development of drug-like compounds. Molecules which are able to induce proximity between elusive therapeutic targets of interest and E3 ligases which subsequently leads to proteasomal degradation of the target are beginning to decrease the percentage of the human proteome described as undruggable. Therefore, having the ability to screen for, and understand the mechanism of, such molecules is becoming an increasingly attractive scientific focus. We have established a number of cascade experiments including cell-based assays and orthogonal triage steps to provide annotation to the selectivity and mechanism of action for compounds identified as putative degraders from a primary high throughput screen against a high value oncology target. We will describe our current position, using PROTACs as proof-of-concept, on the analysis of these novel outputs and highlight challenges encountered., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. All authors are or have been previous employees of AstraZeneca. Geoffrey A. Holdgate is on the editorial board of SLAS Discovery, (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 IC 50 values of better than 30 µM. Learning across the development, execution and analysis of these screens will be presented., Competing Interests: Declaration of competing interests The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: All authors are or have been previous employees of AstraZeneca. Geoffrey A. Holdgate is on the editorial board of SLAS Discovery., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest All authors are employees of AstraZeneca. G. A. Holdgate is on the editorial board for SLAS Discovery., (Copyright © 2023. Published by Elsevier Inc.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

ATAD2 is an epigenetic bromodomain-containing target which is overexpressed in many cancers and has been suggested as a potential oncology target. While several small molecule inhibitors have been described in the literature, their cellular activity has proved to be underwhelming. In this work, we describe the identification of a novel series of ATAD2 inhibitors by high throughput screening, confirmation of the bromodomain region as the site of action, and the optimization campaign undertaken to improve the potency, selectivity, and permeability of the initial hit. The result is compound 5 (AZ13824374), a highly potent and selective ATAD2 inhibitor which shows cellular target engagement and antiproliferative activity in a range of breast cancer models.

The Anti- Wolbachia (A·WOL) consortium at the Liverpool School of Tropical Medicine (LSTM) has partnered with the Global High-Throughput Screening (HTS) Centre at AstraZeneca to create the first anthelmintic HTS for neglected tropical diseases (NTDs). The A·WOL consortium aims to identify novel macrofilaricidal drugs targeting the essential bacterial symbiont ( Wolbachia) of the filarial nematodes causing onchocerciasis and lymphatic filariasis. Working in collaboration, we have validated a robust high-throughput assay capable of identifying compounds that selectively kill Wolbachia over the host insect cell. We describe the development and validation process of this complex, phenotypic high-throughput assay and provide an overview of the primary outputs from screening the AstraZeneca library of 1.3 million compounds.

Nematodes causing lymphatic filariasis and onchocerciasis rely on their bacterial endosymbiont, Wolbachia, for survival and fecundity, making Wolbachia a promising therapeutic target. Here we perform a high-throughput screen of AstraZeneca's 1.3 million in-house compound library and identify 5 novel chemotypes with faster in vitro kill rates (<2 days) than existing anti-Wolbachia drugs that cure onchocerciasis and lymphatic filariasis. This industrial scale anthelmintic neglected tropical disease (NTD) screening campaign is the result of a partnership between the Anti-Wolbachia consortium (A∙WOL) and AstraZeneca. The campaign was informed throughout by rational prioritisation and triage of compounds using cheminformatics to balance chemical diversity and drug like properties reducing the chance of attrition from the outset. Ongoing development of these multiple chemotypes, all with superior time-kill kinetics than registered antibiotics with anti-Wolbachia activity, has the potential to improve upon the current therapeutic options and deliver improved, safer and more selective macrofilaricidal drugs.

Flow cytometry is a powerful tool providing multiparametric analysis of single cells or particles. The introduction of faster plate-based sampling technologies on flow cytometers has transformed the technology into one that has become attractive for higher throughput drug discovery screening. This article describes AstraZeneca's perspectives on the deployment and application of high-throughput flow cytometry (HTFC) platforms for small-molecule high-throughput screening (HTS), structure-activity relationship (SAR) and phenotypic screening, and antibody screening. We describe the overarching HTFC workflow, including the associated automation and data analysis, along with a high-level overview of our HTFC assay portfolio. We go on to discuss the practical challenges encountered and solutions adopted in the course of our deployment of HTFC, as well as future enhancements and expansion of the technology to new areas of drug discovery.

Flow cytometry is a technology providing multiparametric analysis of single cells or other suspension particles. High-throughput (HT) flow cytometry has become an attractive screening platform for drug discovery. In this review, we highlight the recent HT flow cytometry applications, and then focus on HT flow cytometry deployment at AstraZeneca (AZ). Practical considerations for successful HT flow cytometry assay development and screening are provided based on experience from four project case studies at AZ. We provide an overview of the scientific rationale, explain why HT flow cytometry was chosen and how HT flow cytometry assays deliver new ways to support the drug discovery process., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Small molecule inhibitors of the ATM pathway could represent a promising opportunity for cancer therapy, working either by enhancing the clinical efficacy of radiotherapy and existing chemotherapies or by synthetic lethality-based mechanisms. In this chapter, we describe a high-throughput, high-content imaging assay monitoring levels of ATM phosphorylation at Serine 1981 following induction of DNA damage by ionizing radiation.

Potent and selective inhibitors of Dyrk1B kinase were developed to explore the hypothesis, based on siRNA studies, that Dyrk1B may be a resistance mechanism in cells undergoing a stress response.

Quantitative real-time polymerase chain reaction (PCR) is regarded as the gold standard for molecular profiling and target identification, but not in the context of high-throughput screening owing to limitations on workflow, cost of reagents, and miniaturization opportunities. Recent advances have moved reverse transcription quantitative PCR (RT-qPCR) forward, such as improvements in liquid handling, the launch of higher throughput platforms, and the release of one-step products. These one-step reagents enable the user to go straight from a cellular assay format to qPCR without the need for cumbersome and potentially expensive multistep RNA purification protocols. Our aim was to investigate the use of a one-step accelerated workflow to measure the levels of epidermal growth factor receptor (EGFR) and nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) gene expression using lysates generated by the RealTime ready Cell Lysis kit in downstream quantitative RT-qPCR. We present, for the first time, data from a vendor-independent one-step 1536 workflow that compares reporter gene and RT-qPCR screening approaches for oncology drug discovery. We also demonstrate a miniaturized and high-throughput workflow that could enable future application of this sensitive assay technology, with particular impact against phenotypic assays and those using rare cell types.

The genome is constantly exposed to DNA damage agents, leading up to as many as 1 million individual lesions per cell per day. Cells have developed a variety of DNA damage repair (DDR) mechanisms to respond to harmful effects of DNA damage. Failure to repair the damaged DNA causes genomic instability and, as a result, leads to cellular transformation. Indeed, deficiencies of DDR frequently occur in human cancers, thus providing a great opportunity for cancer therapy by developing anticancer agents that work by synthetic lethality-based mechanisms or enhancing the clinical efficacy of radiotherapy and existing chemotherapies. Ataxia-telangiectasia mutated (ATM) plays a key role in regulating the cellular response to DNA double-strand breaks. Ionizing radiation causes double-strand breaks and induces rapid ATM autophosphorylation on serine 1981 that initiates ATM kinase activity. Activation of ATM results in phosphorylation of many downstream targets that modulate numerous damage-response pathways, most notably cell-cycle checkpoints. We describe here the development and validation of a high-throughput imaging assay measuring levels of phospho-ATM Ser1981 in HT29 cells after exposure to ionizing radiation. We also examined activation of downstream ATM effectors and checked specificity of the endpoint using known inhibitors of DNA repair pathways.

A directed screen of a relatively small number of compounds, selected for kinase ATP pocket binding potential, yielded a novel series of hit compounds (1). Hit explosion on two binding residues identified compounds 27 and 43 as the best leads for an optimization program having reduced secondary metabolism, as measured by in vitro rat hepatocytes incubation, leading to oral bio-availability. Structure-activity relationships and molecular modeling have suggested a binding mode for the most potent inhibitor 12., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Optimization of our bis-anilino-pyrimidine series of EphB4 kinase inhibitors led to the discovery of compound 12 which incorporates a key m-hydroxymethylene group on the C4 aniline. 12 displays a good kinase selectivity profile, good physical properties and pharmacokinetic parameters, suggesting it is a suitable candidate to investigate the therapeutic potential of EphB4 kinase inhibitors., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Starting from the initial bis-anilinopyrimidine 1, good potency against EphB4 was retained when benzodioxole at C-4 was replaced by an indazole. The key interactions of the indazole with the protein were characterised by crystallographic studies. Further optimisation led to compound 20, a potent inhibitor of the EphB4 and Src kinases with good pharmacokinetics in various preclinical species and high fraction unbound in plasma. Compound 20 may be used as a tool for evaluating the potential of EphB4 kinase inhibitors in vivo., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Crystallographic studies of a range of 3-substituted anilinopyrimidine inhibitors of EphB4 have highlighted two alternative C-2 aniline conformations and this discovery has been exploited in the design of a highly potent series of 3,5-disubstituted anilinopyrimidines. The observed range of cellular activities has been rationalised on the basis of physicochemical and structural characteristics.

A series of bis-anilinopyrimidines have been identified as potent inhibitors of the tyrosine kinase EphB4. Structural information from two alternative series identified from screening efforts was combined to identify the initial leads.

Members of the Bcl-2 family are critical regulators of apoptosis. Antiapoptotic family proteins such as Bcl-2 and Bcl-x(L) function, at least in part, by binding proapoptotic members such as Bax and Bak and thereby preventing release of apoptotic proteins, including cytochrome c, from the mitochondria. "BH3-only" members of the family disrupt this interaction by binding, via their BH3 domain, to a hydrophobic pocket on the surface of the antiapoptotic members. Disruption of heterodimerizations by small-molecule inhibitors could be used to modulate cell death in both cancer (to increase apoptosis) and degenerative disorders (to decrease apoptosis), and assays are necessary to screen compound libraries. Fluorescence polarization and enzyme-linked immunosorbent assay-based methods to detect Bcl-2 protein interactions have been described. Here, two further methods that are rapid, "mix and read," homogeneous reactions, insensitive to compound autofluorescence, and amenable to high-throughput screening, are described: a scintillation proximity assay and a time-resolved fluorescence resonance energy transfer assay (HTRF). The assays are designed using tags such that different Bcl-2 family members or BH3 domain peptides can be readily applied to either format, as exemplified by the use here of histidine-tagged Bcl-x(L) and biotinylated BH3 peptides.

Cyclic nucleotides are key regulators of many cellular processes. Their immediate action is terminated through the activity of phosphodiesterases, a diverse family of enzymes. This diversity has given rise to drug discovery opportunities, and assay technology is therefore of key importance. Inhibitors of the cyclic-AMP-specific phosphodiesterases (the PDE4 family) are drug candidates for a variety of inflammatory disorders. However, PDE4 inhibitors, besides their immunomodulatory effects, also cause side effects including nausea and emesis. Recently, it has been suggested that PDE4 exists in two different conformations with respect to inhibition by the prototypical compound rolipram. Inhibition of the low-affinity conformer is thought to give rise to anti-inflammatory effects, and inhibition of the high-affinity conformer to side effects. Therefore, a selective inhibitor of the low-affinity conformer may have clinical utility. Methods are described to prepare recombinant forms of PDE4B that allow screening for compounds that could preferentially inhibit the low-affinity conformer. Furthermore, conditions for an efficient, scintillation proximity, microtiter plate-based assay are described, providing a considerable advance over previous assays in terms of throughput and automatability., (Copyright 1999 Academic Press.)

A recombinant Factor VIII (Factor VIII-delta II) consists of a unique polypeptide chain of 165 kDa deleted from the major part of the B-domain and from the cleavage site at Arg-1648-Glu-1649 found in plasma-derived Factor VIII. It was expressed in mammalian cells in serum-free medium containing von Willebrand factor and purified by a one-step immunopurification. The recombinant Factor VIII was characterized as a single active peak when subjected to f.p.l.c., in contrast with the plasma-derived molecule. Its coagulant activity was decreased in the presence of EDTA, suggesting that a bivalent ion is required, as for plasma-derived Factor VIII. The activation by thrombin and the inactivation by activated protein C were studied and the resulting molecular forms were analysed by f.p.l.c. and SDS/PAGE. The results clearly demonstrate that, despite the structural differences between plasma-derived and recombinant Factor VIII, activation and inactivation of Factor VIII-delta II generate proteolysed complexes similar to that described for plasma-derived Factor VIII. Thus this deleted recombinant Factor VIII, which is processed similarly to plasma-derived Factor VIII, should be normally integrated in the regulation system of Factor X activation in the blood-coagulation cascade.