High-throughput differential scanning fluorimetry (DSF) and cellular thermal shift assays (CETSA): Shifting from manual to automated screening.

Biophysical affinity screening is increasingly being adopted as a high-throughput hit finding technique in drug discovery. Automation is highly beneficial to high-throughput screening (HTS) since a large number of compounds need to be reproducibly tested against a biological target. Herein, we describe how we have automated two biophysical affinity screening methods that rely on a thermal shift in protein melting temperature upon small molecule binding: differential scanning fluorimetry (DSF) and the cellular thermal shift assay (CETSA).

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.

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.

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.

Development of a High-Throughput Cytometric Screen to Identify Anti- Wolbachia Compounds: The Power of Public-Private Partnership.

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.

Industrial scale high-throughput screening delivers multiple fast acting macrofilaricides.

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.

Techniques to Enable 1536-Well Phenotypic Screening.

Adaptation of phenotypic cell assays to 1536-well format brings major challenges in liquid handling for high-content assays requiring washing steps and coating of plates. In addition, problematic edge effects and reduced assay quality are frequently encountered. In this chapter, we describe the novel application of a centrifugal plate washer to facilitate miniaturization of 1536-well cell assays and a combination of techniques to reduce edge effects, all of which improved throughput and data quality. Cell assays currently limited in throughput because of cost and complex protocols may be enabled by the techniques presented in this chapter.

Identification and Characterization of Dual Inhibitors of the USP25/28 Deubiquitinating Enzyme Subfamily.

The ubiquitin proteasome system is widely postulated to be a new and important field of drug discovery for the future, with the ubiquitin specific proteases (USPs) representing one of the more attractive target classes within the area. Many USPs have been linked to critical axes for therapeutic intervention, and the finding that USP28 is required for c-Myc stability suggests that USP28 inhibition may represent a novel approach to targeting this so far undruggable oncogene. Here, we describe the discovery of the first reported inhibitors of USP28, which we demonstrate are able to bind to and inhibit USP28, and while displaying a dual activity against the closest homologue USP25, these inhibitors show a high degree of selectivity over other deubiquitinases (DUBs). The utility of these compounds as valuable probes to investigate and further explore cellular DUB biology is highlighted by the demonstration of target engagement against both USP25 and USP28 in cells. Furthermore, we demonstrate that these inhibitors are able to elicit modulation of both the total levels and the half-life of the c-Myc oncoprotein in cells and also induce apoptosis and loss of cell viability in a range of cancer cell lines. We however observed a narrow therapeutic index compared to a panel of tissue-matched normal cell lines. Thus, it is hoped that these probes and data presented herein will further advance our understanding of the biology and tractability of DUBs as potential future therapeutic targets.

Discovery of Imidazo[1,2-a]pyridine Ethers and Squaramides as Selective and Potent Inhibitors of Mycobacterial Adenosine Triphosphate (ATP) Synthesis.

The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis (Mtb). Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP synthesis. Through medicinal chemistry exploration, we established a robust structure-activity relationship of these two scaffolds, resulting in nanomolar potencies in an ATP synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be noncytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.

Enabling 1536-Well High-Throughput Cell-Based Screening through the Application of Novel Centrifugal Plate Washing.

Cell-based assays have long been important within hit discovery paradigms; however, improving the disease relevance of the assay system can positively affect the translation of small-molecule drug discovery, especially if adopted in the initial hit identification assay. Consequently, there is an increasing need for disease-relevant assay systems capable of running at large scale, including the use of induced pluripotent stem cells and donor-derived primary cells. Major hurdles to adopting these assays for high-throughput screening are the cost, availability of cells, and complex protocols. Miniaturization of such assays to 1536-well format is an approach that can reduce costs and increase throughput. Adaptation of these complex cell assays to 1536-well format brings major challenges in liquid handling for high-content assays requiring washing steps and coating of plates. In addition, problematic edge effects and reduced assay quality are frequently encountered. In this study, we describe the novel application of a centrifugal plate washer to facilitate miniaturization of a range of 1536-well cell assays and techniques to reduce edge effects, all of which improved throughput and data quality. Cell assays currently limited in throughput because of cost and complex protocols may be enabled by the techniques presented in this study.

High-Throughput Hit Screening Cascade to Identify Respiratory Syncytial Virus (RSV) Inhibitors.

Respiratory syncytial virus (RSV) infects 99% of children by age 2 years and is a leading cause of serious lower respiratory tract infection (LRTI) and infant hospitalization in the United Kingdom. Identification of efficacious RSV therapeutics has been hindered by the lack of a robust and appropriate primary assay for high-throughput screening (HTS). Here we report an HTS cascade that identified inhibitors of RSV replication using a robust RSV replicon luminescence-reporter assay for the primary campaign. The performance of the assay was consistent and reliable at scale, with Z' of 0.55 ± 0.08 across 150 assay plates and signal-to-background ratios >40. The HTS assay was used to screen the AstraZeneca compound library of 1 million compounds at a single concentration of 10 µM. Hits specifically targeting the RSV replicon were determined using a series of hit generation assays. Compounds nonspecifically causing cell toxicity were removed, and hits were confirmed in live viral inhibition assays exhibiting greater physiological relevance than the primary assay. In summary, we developed a robust screening cascade that identified hit molecules that specifically targeted RSV replication.

A novel high-throughput cell-based assay aimed at identifying inhibitors of DNA metabolism in bacteria.

Bacterial biosensor strains can be useful tools for the discovery and characterization of antibacterial compounds. A plasmid-based reporter vector containing a transcriptional fusion between the recA promoter and green fluorescence protein gene was introduced into an Escherichia coli ΔtolC strain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the AstraZeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening.

Differential subcellular and extracellular localisations of proteins required for insulin-like growth factor- and extracellular matrix-induced signalling events in breast cancer progression.

BACKGROUND: Cancer metastasis is the main contributor to breast cancer fatalities as women with the metastatic disease have poorer survival outcomes than women with localised breast cancers. There is an urgent need to develop appropriate prognostic methods to stratify patients based on the propensities of their cancers to metastasise. The insulin-like growth factor (IGF)-I: IGF binding protein (IGFBP):vitronectin complexes have been shown to stimulate changes in gene expression favouring increased breast cancer cell survival and a migratory phenotype. We therefore investigated the prognostic potential of these IGF- and extracellular matrix (ECM) interaction-induced proteins in the early identification of breast cancers with a propensity to metastasise using patient-derived tissue microarrays. METHODS: Semiquantitative immunohistochemistry analyses were performed to compare the extracellular and subcellular distribution of IGF- and ECM-induced signalling proteins among matched normal, primary cancer and metastatic cancer formalin-fixed paraffin-embedded breast tissue samples. RESULTS: The IGF- and ECM-induced signalling proteins were differentially expressed between subcellular and extracellular localisations. Vitronectin and IGFBP-5 immunoreactivity was lower while ß1 integrin immunoreactivity was higher in the stroma surrounding metastatic cancer tissues, as compared to normal breast and primary cancer stromal tissues. Similarly, immunoreactive stratifin was found to be increased in the stroma of primary as well as metastatic breast tissues. Immunoreactive fibronectin and ß1 integrin was found to be highly expressed at the leading edge of tumours. Based on the immunoreactivity it was apparent that the cell signalling proteins AKT1 and ERK1/2 shuffled from the nucleus to the cytoplasm with tumour progression. CONCLUSION: This is the first in-depth, compartmentalised analysis of the distribution of IGF- and ECM-induced signalling proteins in metastatic breast cancers. This study has provided insights into the changing pattern of cellular localisation and expression of IGF- and ECM-induced signalling proteins in different stages of breast cancer. The differential distribution of these biomarkers could provide important prognostic and predictive indicators that may assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy.

Discovery of ATP-Competitive Inhibitors of tRNAIle Lysidine Synthetase (TilS) by High-Throughput Screening.

A novel, ultrahigh-throughput, fluorescence anisotropy-based assay was developed and used to screen a 1.4-million-sample library for compounds that compete with adenosine triphosphate (ATP) for binding to Escherichia coli tRNA(Ile) lysidine synthetase (TilS), an essential, conserved, ATP-dependent, tRNA-modifying enzyme of bacterial pathogens. TilS modifies a cytidine base in the anticodon loop of Ile2 tRNA by attaching lysine, thereby altering codon recognition of the CAU anticodon from AUG (methionine) to AUA (isoleucine). A scintillation proximity assay for the incorporation of lysine into Ile2 tRNA was used to eliminate false positives in the initial screen resulting from detection artifacts as well as compounds competitive with the fluorescent label instead of ATP, and to measure inhibitor potencies against E. coli and Pseudomonas aeruginosa TilS isozymes. The tRNA(Ile) substrate for P. aeruginosa TilS was identified for the first time to enable these measurements. ATP-competitive binding of inhibitors was confirmed by one-dimensional ligand-observe nuclear magnetic resonance. A preliminary structure-activity relationship is shown for two inhibitor series.

Development of a high-throughput replicon assay for the identification of respiratory syncytial virus inhibitors.

Respiratory syncytial virus (RSV) drug discovery has been hindered by the lack of good chemistry starting points and would benefit from robust and convenient assays for high-throughput screening (HTS). In this paper, we present the development and optimization of a 384-well RSV replicon assay that enabled HTS for RSV replication inhibitors with a low bio-containment requirement. The established replicon assay was successfully implemented for high-throughput screening. A validation screen was performed which demonstrated high assay performance and reproducibility. Assay quality was further confirmed via demonstration of appropriate pharmacology for different classes of RSV replication tool inhibitors. RSV replicon and cytotoxicity assays were further developed into a multiplexed format that measured both inhibition of viral replication and cytotoxicity from the same well. This provided a time and cost efficient approach to support lead optimization. In summary, we have developed a robust RSV replicon assay to help expedite the discovery of novel RSV therapeutics.

Development of a high-throughput fluorescence polarization DNA cleavage assay for the identification of FEN1 inhibitors.

Flap endonuclease-1 (FEN1) is a highly conserved metallonuclease and is the main human flap endonuclease involved in the recognition and cleavage of single-stranded 5' overhangs from DNA flap structures. The involvement of FEN1 in multiple DNA metabolism pathways and the identification of FEN1 overexpression in a variety of cancers has led to interest in FEN1 as an oncology target. In this article, we describe the development of a 1536-well high-throughput screening assay based on the change in fluorescence polarization of a FEN1 DNA substrate labeled with Atto495 dye. The assay was subsequently used to screen 850 000 compounds from the AstraZeneca compound collection, with a Z' factor of 0.66 ± 0.06. Hits were followed up by IC50 determination in both a concentration-response assay and a technology artifact assay.

Diverse heterocyclic scaffolds as allosteric inhibitors of AKT.

Wide-ranging exploration of potential replacements for a quinoline-based inhibitor of activation of AKT kinase led to number of alternative, novel scaffolds with potentially improved potency and physicochemical properties. Examples showed predictable DMPK properties, and one such compound demonstrated pharmacodynamic knockdown of phosphorylation of AKT and downstream biomarkers in vivo and inhibition of tumor growth in a breast cancer xenograft model.

The discovery of benzanilides as c-Met receptor tyrosine kinase inhibitors by a directed screening approach.

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.