ABSTRACT
G protein-coupled receptor (GPCR) signaling is the primary method eukaryotes use to respond to specific cues in their environment. However, the relationship between stimulus and response for each GPCR is difficult to predict due to diversity in natural signal transduction architecture and expression. Using genome engineering in yeast, we constructed an insulated, modular GPCR signal transduction system to study how the response to stimuli can be predictably tuned using synthetic tools. We delineated the contributions of a minimal set of key components via computational and experimental refactoring, identifying simple design principles for rationally tuning the dose response. Using five different GPCRs, we demonstrate how this enables cells and consortia to be engineered to respond to desired concentrations of peptides, metabolites, and hormones relevant to human health. This work enables rational tuning of cell sensing while providing a framework to guide reprogramming of GPCR-based signaling in other systems.
Subject(s)
Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Gene Expression/drug effects , Genetic Engineering , Humans , Pheromones/pharmacology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction/drug effects , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
BACKGROUND: Whole-genome sequencing (WGS) is the gold standard diagnostic tool to identify and genetically characterise emerging pathogen mutations (variants), but cost, capacity, and timeliness limit its use when large populations need rapidly assessing. We assessed the potential of genotyping assays to provide accurate and timely variant information at scale by retrospectively examining surveillance for SARS-CoV-2 variants in England between March and September, 2021, when genotyping assays were used widely for variant detection. METHODS: We chose a panel of four RT-PCR genotyping assays to detect circulating variants of SARS-COV-2 in England and developed a decision algorithm to assign a probable SARS-CoV-2 variant to samples using the assay results. We extracted surveillance data from the UK Health Security Agency databases for 115 934 SARS-CoV-2-positive samples (March 1-Sept 6, 2021) when variant information was available from both genotyping and WGS. By comparing the genotyping and WGS variant result, we calculated accuracy metrics (ie, sensitivity, specificity, and positive predictive value [PPV]) and the time difference between the sample collection date and the availability of variant information. We assessed the number of samples with a variant assigned from genotyping or WGS, or both, over time. FINDINGS: Genotyping and an initial decision algorithm (April 10-May 11, 2021 data) were accurate for key variant assignment: sensitivities and PPVs were 0·99 (95% CI 0·99-0·99) for the alpha, 1·00 (1·00-1·00) for the beta, and 0·91 (0·80-1·00) for the gamma variants; specificities were 0·97 (0·96-0·98), 1·00 (1·00-1·00), and 1·00 (1·00-1·00), respectively. A subsequent decision algorithm over a longer time period (May 27-Sept 6, 2021 data) remained accurate for key variant assignment: sensitivities were 0·91 (95% CI 0·74-1·00) for the beta, 0·98 (0·98-0·99) for the delta, and 0·93 (0·81-1·00) for the gamma variants; specificities were 1·00 (1·00-1·00), 0·96 (0·96-0·97), and 1·00 (1·00-1·00), respectively; and PPVs were 0·83 (0·62-1·00), 1·00 (1·00-1·00), and 0·78 (0·59-0·97), respectively. Genotyping produced variant information a median of 3 days (IQR 2-4) after the sample collection date, which was faster than with WGS (9 days [8-11]). The flexibility of genotyping enabled a nine-times increase in the quantity of samples tested for variants by this method (from 5000 to 45 000). INTERPRETATION: RT-PCR genotyping assays are suitable for high-throughput variant surveillance and could complement WGS, enabling larger scale testing for known variants and timelier results, with important implications for effective public health responses and disease control globally, especially in settings with low WGS capacity. However, the choice of panels of RT-PCR assays is highly dependent on database information on circulating variants generated by WGS, which could limit the use of genotyping assays when new variants are emerging and spreading rapidly. FUNDING: UK Health Security Agency and National Institute for Health Research Health Protection Research Unit in Emergency Preparedness and Response.
Subject(s)
COVID-19 , Humans , COVID-19/diagnosis , COVID-19/epidemiology , Genotype , Retrospective Studies , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , England/epidemiology , COVID-19 TestingABSTRACT
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology, with its ability to target a specific DNA locus using guide RNAs (gRNAs), is particularly suited for targeted mutagenesis. The targeted diversification of nucleotides in Saccharomyces cerevisiae using a CRISPR-guided error-prone DNA polymeraseâcalled yEvolvRâwas recently reported. Here, we investigate the effect of multiplexed expression of gRNAs flanking a short stretch of DNA on reversion and mutation frequencies using yEvolvR. Phenotypic assays demonstrate that higher reversion frequencies are observed when expressing multiple gRNAs simultaneously. Next generation sequencing reveals a synergistic effect of multiple gRNAs on mutation frequencies, which is more pronounced in a mutant with a partially defective DNA mismatch repair system. Additionally, we characterize a galactose-inducible yEvolvR, which enables temporal control of mutagenesis. This study demonstrates that multiplex expression of gRNAs and induction of mutagenesis greatly improves the capabilities of yEvolvR for generation of genetic libraries in vivo.
Subject(s)
Mutation Rate , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , CRISPR-Cas Systems/genetics , DNA , DNA-Directed DNA Polymerase/genetics , RNA , MutationABSTRACT
Signalling of the calcitonin-like receptor (CLR) is multifaceted, due to its interaction with receptor activity modifying proteins (RAMPs), and three endogenous peptide agonists. Previous studies have focused on the bias of G protein signalling mediated by the receptor and receptor internalisation of the CLR-RAMP complex has been assumed to follow the same pattern as other Class B1 G Protein-Coupled Receptors (GPCRs). Here we sought to measure desensitisation of the three CLR-RAMP complexes in response to the three peptide agonists, through the measurement of ß-arrestin recruitment and internalisation. We then delved further into the mechanism of desensitisation through modulation of ß-arrestin activity and the expression of GPCR kinases (GRKs), a key component of homologous GPCR desensitisation. First, we have shown that CLR-RAMP1 is capable of potently recruiting ß-arrestin1 and 2, subsequently undergoing rapid endocytosis, and that CLR-RAMP2 and -RAMP3 also utilise these pathways, although to a lesser extent. Following this we have shown that agonist-dependent internalisation of CLR is ß-arrestin dependent, but not required for full agonism. Overexpression of GRK2-6 was then found to decrease receptor signalling, due to an agonist-independent reduction in surface expression of the CLR-RAMP complex. These results represent the first systematic analysis of the importance of ß-arrestins and GRKs in CLR-RAMP signal transduction and pave the way for further investigation regarding other Class B1 GPCRs.
ABSTRACT
The heterodimeric transcription factor, hypoxia inducible factor-1 (HIF-1), is an important anticancer target as it supports the adaptation and response of tumors to hypoxia. Here, we optimized the repressed transactivator yeast two-hybrid system to further develop it as part of a versatile yeast-based drug discovery platform and validated it using HIF-1. We demonstrate both fluorescence-based and auxotrophy-based selections that could detect HIF-1α/HIF-1ß dimerization inhibition. The engineered genetic selection is tunable and able to differentiate between strong and weak interactions, shows a large dynamic range, and is stable over different growth phases. Furthermore, we engineered mechanisms to control for cellular activity and off-target drug effects. We thoroughly characterized all parts of the biosensor system and argue this tool will be generally applicable to a wide array of protein-protein interaction targets. We anticipate this biosensor will be useful as part of a drug discovery platform, particularly when screening DNA-encoded new modality drugs.
Subject(s)
Biosensing Techniques , Hypoxia-Inducible Factor 1 , Humans , Hypoxia , Drug Discovery , Trans-ActivatorsABSTRACT
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.
Subject(s)
Plasma Gases , Plastics , Hazardous SubstancesABSTRACT
BACKGROUND: The rise of new SARS-CoV-2 variants worldwide requires global molecular surveillance strategies to support public health control. Early detection and evaluation of their associated risk of spreading within the population are pivotal. METHODS: Between April 2020 and February 2021, the UK Lighthouse Labs Network at Alderley Park tested more than eight million nose and throat swab samples for the presence of SARS-CoV-2, via PCR. The assay targeted three genomic regions of the virus: N, Orf1ab and S. Whole-genome next-generation sequencing was used to confirm positive PCR results. Positive results were mapped using the postal district origin of samples to allow real-time tracking of the spread of a new variant through the UK. FINDINGS: In mid-November 2020, the assay identified an increasing number of S gene negative, N and Orf1ab positive samples. Whole-genome sequencing demonstrated that the loss of S gene detection was due to the appearance of a SARS-CoV-2 lineage (B.1.1.7) designated as Variant of concern (VOC) 202012/01. By the beginning of January 2021, the new SARS-CoV-2 VOC comprised 70% of daily positive samples tested at Alderley Park and â¼98% by the end of February 2021. INTERPRETATION: The timeline view identified the rapid spread of the new SARS-CoV-2 variant across England during the first three weeks of December. Coupling high-throughput diagnostics and molecular surveillance was pivotal to the early detection of the spread of this variant. The availability of real-time tracking of an emerging variant is an important new tool to inform decision-making authorities for risk mitigation. In a respiratory pandemic, a tool for the timely response to the emergence and spread of a novel variant is vital, even more so when a variant is associated with the enhanced transmission, as has occurred with VOC 202012/01. FUNDING: None.
Subject(s)
COVID-19/virology , SARS-CoV-2/genetics , England , High-Throughput Nucleotide Sequencing/methods , Humans , Mutation/genetics , Pandemics/prevention & control , Risk AssessmentABSTRACT
The global impact of synthetic biology has been accelerating, because of the plummeting cost of DNA synthesis, advances in genetic engineering, growing understanding of genome organization, and explosion in data science. However, much of the discipline's application in the pharmaceutical industry remains enigmatic. In this review, we highlight recent examples of the impact of synthetic biology on target validation, assay development, hit finding, lead optimization, and chemical synthesis, through to the development of cellular therapeutics. We also highlight the availability of tools and technologies driving the discipline. Synthetic biology is certainly impacting all stages of drug discovery and development, and the recognition of the discipline's contribution can further enhance the opportunities for the drug discovery and development value chain.
Subject(s)
Drug Development/methods , Drug Discovery/methods , Synthetic Biology/methods , Drug Development/trends , Drug Discovery/trends , Humans , Synthetic Biology/trendsABSTRACT
Agonist bias occurs when different ligands produce distinct signalling outputs when acting at the same receptor. However, its physiological relevance is not always clear. Using primary human cells and gene editing techniques, we demonstrate endogenous agonist bias with physiological consequences for the calcitonin receptor-like receptor, CLR. By switching the receptor-activity modifying protein (RAMP) associated with CLR we can "re-route" the physiological pathways activated by endogenous agonists calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2 (AM2). AM2 promotes calcium-mediated nitric oxide signalling whereas CGRP and AM show pro-proliferative effects in cardiovascular cells, thus providing a rationale for the expression of the three peptides. CLR-based agonist bias occurs naturally in human cells and has a fundamental purpose for its existence. We anticipate this will be a starting point for more studies into RAMP function in native environments and their importance in endogenous GPCR signalling.
Subject(s)
Adrenomedullin/physiology , Calcitonin Gene-Related Peptide/physiology , Peptide Hormones/physiology , Receptors, G-Protein-Coupled/agonists , Calcitonin Receptor-Like Protein/physiology , Cells, Cultured , Cyclic AMP/metabolism , Endothelial Cells/physiology , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Receptors, Adrenomedullin/agonists , Receptors, Adrenomedullin/analysis , Receptors, Calcitonin Gene-Related Peptide/physiologyABSTRACT
Asthma is a multifactorial disease, in which the intricate interplay between genetic and environmental factors underlies the overall phenotype of the disease. Using a genome-wide scan for linkage in a population comprising of Danish families, we identified a novel linked locus on chromosome 1qter (LOD 3.6, asthma) and supporting evidence for this locus was identified for both asthma and atopic-asthma phenotypes in the GAIN (Genetics of Asthma International Network) families. The putative susceptibility gene was progressively localized to a 4.5 Mb region on chromosome 1q adjacent to the telomere, through a series of genotyping screens. Further screening using the pedigree-based association test (PBAT) identified polymorphisms in the OPN3 and CHML genes as being associated with asthma and atopic asthma after correcting for multiple comparisons. We observed that polymorphisms flanking the OPN3 and CHML genes wholly accounted for the original linkage in the Danish population and the genetic association was also confirmed in two separate studies involving the GAIN families. OPN3 and CHML are unique genes with no known function that are related to the pathophysiology of asthma. Significantly, analysis of gene expression at both RNA and protein levels, clearly demonstrated OPN3 expression in lung bronchial epithelia as well as immune cells, while CHML expression appeared minimal. Moreover, OPN3 down-regulation by siRNA knock-down in Jurkat cells suggested a possible role for OPN3 in modulation of T-cell responses. Collectively, these data suggest that OPN3 is an asthma susceptibility gene on 1qter, which unexpectedly may play a role in immune modulation.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Asthma/genetics , Chromosomes, Human, Pair 1/genetics , Genetic Predisposition to Disease , Rod Opsins/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adolescent , Adult , Asthma/physiopathology , Cell Line , Child , Chromosome Mapping , Female , Genetic Linkage , Humans , Lung/metabolism , Lung/physiopathology , Male , Middle Aged , Oligonucleotide Array Sequence Analysis , Polymorphism, Genetic , RNA, Small Interfering/genetics , Rod Opsins/metabolism , White People/geneticsABSTRACT
The receptor for calcitonin gene-related peptide (CGRP) has been the target for the development of novel small molecule antagonists for the treatment of migraine. Two such antagonists, BIBN4096BS and MK-0974, have shown great promise in clinical trials and hence a deeper understanding of the mechanism of their interaction with the receptor is now required. The structure of the CGRP receptor is unusual since it is comprised of a hetero-oligomeric complex between the calcitonin receptor-like receptor (CRL) and an accessory protein (RAMP1). Both the CLR and RAMP1 components have extracellular domains which interact with each other and together form part of the peptide-binding site. It seems likely that the antagonist binding site will also be located on the extracellular domains and indeed Trp-74 of RAMP1 has been shown to form part of the binding site for BIBN4096BS. However, despite a chimeric study demonstrating the role of the N-terminal domain of CLR in antagonist binding, no specific residues have been identified. Here we carry out a mutagenic screen of the extreme N-terminal domain of CLR (residues 23-63) and identify a mutant, Met-42-Ala, which displays 48-fold lower affinity for BIBN4096BS and almost 900-fold lower affinity for MK-0974. In addition, we confirm that the Trp-74-Lys mutation at human RAMP1 reduces BIBN4096BS affinity by over 300-fold and show for the first time a similar effect for MK-0974 affinity. The data suggest that the non-peptide antagonists occupy a binding site close to the interface of the N-terminal domains of CLR and RAMP1.
Subject(s)
Azepines/metabolism , Calcitonin Gene-Related Peptide Receptor Antagonists , Imidazoles/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Piperazines/metabolism , Quinazolines/metabolism , Receptors, Calcitonin/metabolism , Azepines/chemistry , Azepines/pharmacology , Calcitonin Receptor-Like Protein , Humans , Imidazoles/chemistry , Imidazoles/pharmacology , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins/genetics , Methionine/genetics , Methionine/metabolism , Piperazines/chemistry , Piperazines/pharmacology , Protein Structure, Tertiary , Quinazolines/chemistry , Quinazolines/pharmacology , Receptor Activity-Modifying Protein 1 , Receptor Activity-Modifying Proteins , Receptors, Calcitonin/genetics , Tryptophan/genetics , Tryptophan/metabolismABSTRACT
A novel imidazobenzazepine template (5a) with potent dual H(1)/5-HT(2A) antagonist activity was identified. Application of a zwitterionic approach to this poorly selective and poorly developable starting point successfully delivered a class of high quality leads, 3-[4-(3-R(1)-2-R-5H-imidazo[1,2-b][2]benzazepin-11-yl)-1-piperazinyl]-2,2-dimethylpropanoic acids (e.g., 9, 19, 20, and 21), characterized by potent and balanced H(1)/5-HT(2A) receptor antagonist activities and good developability profiles.
Subject(s)
Receptor, Serotonin, 5-HT1A/drug effects , Receptor, Serotonin, 5-HT2A/drug effects , Serotonin Antagonists/therapeutic use , Sleep Wake Disorders/drug therapy , HumansABSTRACT
Receptor component protein (RCP) is a 148 amino acid intracellular peripheral membrane protein, previously identified as promoting the coupling of CGRP to cAMP production at the CGRP receptor, a heterodimer of calcitonin receptor like-receptor (CLR), a family B G protein-coupled receptor (GPCR) and receptor activity modifying protein 1 (RAMP1). We extend these observations to show that it selectively enhances CGRP receptor coupling to Gs but not Gq or pERK activation. At other family B GPCRs, it enhances cAMP production at the calcitonin, corticotrophin releasing factor type 1a and glucagon-like peptide type 2 receptors with their cognate ligands but not at the adrenomedullin type 1 (AM1), gastric inhibitory peptide and glucagon-like peptide type 1 receptors, all expressed in transfected HEK293S cells. However, there is also cell-line variability as RCP did not enhance cAMP production at the endogenous calcitonin receptor in HEK293T cells and it has previously been reported that it is active on the AM1 receptor expressed on NIH3T3 cells. RCP appears to behave as a positive allosteric modulator at coupling a number of family B GPCRs to Gs, albeit in a manner that is regulated by cell-specific factors. It may exert its effects at the interface between the 2nd intracellular loop of the GPCR and Gs, although there is likely to be some overlap between this location and that occupied by the C-terminus of RAMPs if they bind to the GPCRs.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Membrane Proteins/metabolism , Receptor Activity-Modifying Protein 1/metabolism , Adrenomedullin/metabolism , Animals , Calcitonin Gene-Related Peptide , Calcitonin Receptor-Like Protein/chemistry , Calcitonin Receptor-Like Protein/metabolism , Cyclic AMP/metabolism , HEK293 Cells , Humans , Ligands , Peptide Hormones , Receptors, G-Protein-Coupled/metabolism , Signal TransductionABSTRACT
Cytoplasmic dynein-1 (hereafter dynein) is a six-subunit motor complex that transports a variety of cellular components and pathogens along microtubules. Dynein's cellular functions are only partially understood, and potent and specific small-molecule inhibitors and activators of this motor would be valuable for addressing this issue. It has also been hypothesized that an inhibitor of dynein-based transport could be used in antiviral or antimitotic therapy, whereas an activator could alleviate age-related neurodegenerative diseases by enhancing microtubule-based transport in axons. Here, we present the first high-throughput screening (HTS) assay capable of identifying both activators and inhibitors of dynein-based transport. This project is also the first collaborative screening report from the Medical Research Council and AstraZeneca agreement to form the UK Centre for Lead Discovery. A cellular imaging assay was used, involving chemically controlled recruitment of activated dynein complexes to peroxisomes. Such a system has the potential to identify molecules that affect multiple aspects of dynein biology in vivo. Following optimization of key parameters, the assay was developed in a 384-well format with semiautomated liquid handling and image acquisition. Testing of more than 500,000 compounds identified both inhibitors and activators of dynein-based transport in multiple chemical series. Additional analysis indicated that many of the identified compounds do not affect the integrity of the microtubule cytoskeleton and are therefore candidates to directly target the transport machinery.
Subject(s)
Cytoplasmic Dyneins/antagonists & inhibitors , High-Throughput Screening Assays/methods , Peroxisomes/genetics , Small Molecule Libraries/pharmacology , Biological Transport/drug effects , Cytoplasmic Dyneins/chemistry , Cytoplasmic Dyneins/genetics , Humans , Ion Transport/genetics , Microtubules/drug effectsABSTRACT
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.
Subject(s)
Anti-Bacterial Agents/pharmacology , High-Throughput Screening Assays , Host-Pathogen Interactions/drug effects , Wolbachia/drug effects , Anti-Bacterial Agents/chemistry , Cell Culture Techniques/methods , Drug Discovery , Elephantiasis, Filarial/drug therapy , Humans , Image Cytometry , Onchocerciasis/drug therapy , Wolbachia/pathogenicity , Wolbachia/ultrastructureABSTRACT
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 (AâWOL) 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.
Subject(s)
Drug Discovery , Filaricides/analysis , High-Throughput Screening Assays , Aedes , Animals , Cell Line , WolbachiaABSTRACT
Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) bind and activate the PTH/PTHrP receptor (PTH-1R). However, while the related receptor PTH-2R responds potently to PTH, it is not activated by PTHrP. Two hormone sites are known to be responsible for these different potencies. First, the absence of efficacy for PTHrP at PTH-2R is due to the presence of His-5 in PTHrP (Ile-5 in PTH), which interacts with the receptor's juxtamembrane domain. Second, PTHrP has lower affinity than PTH for PTH-2R because of the presence of Phe-23 (Trp-23 in PTH), which interacts with the receptor's N-terminal extracellular domain. We used these different receptor subtype properties to demonstrate that residue 41 in PTH-1R, when either the native Leu or substituted by Ile or Met, can accommodate either Phe or Trp at position 23 of the ligand. However, when Leu-41 is substituted by a smaller side chain, either Ala or Val (its equivalent residue in PTH-2R), the receptor becomes highly selective for those peptide ligands with Trp-23. Hence, despite the conservative nature of the substitutions found in the native ligands (Phe for Trp) and receptors (Leu for Val), they nevertheless enable a significant degree of selectivity to be achieved. Analysis of this functionally important ligand-receptor contact, within the context of the recent X-ray structure of the peptide-bound PTH-1R N domain, reveals the nature of the selectivity filter and how it is by-passed in PTH-1R.
Subject(s)
Amino Acids/metabolism , Receptors, Parathyroid Hormone/chemistry , Receptors, Parathyroid Hormone/metabolism , Amino Acid Sequence , Binding, Competitive , Cell Line , Cell Membrane/metabolism , Crystallography, X-Ray , Humans , Ligands , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Peptides/chemistry , Peptides/metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Drug Evaluation, Preclinical , High-Throughput Screening Assays , Animals , Cell Culture Techniques , Cell Line , Drug Evaluation, Preclinical/methods , Fluorescent Antibody Technique , Genes, Reporter , Humans , PhenotypeABSTRACT
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.
Subject(s)
Drug Discovery , Flow Cytometry , High-Throughput Screening Assays , Automation , Drug Discovery/methods , Drug Industry , Flow Cytometry/methods , Small Molecule Libraries , Structure-Activity Relationship , WorkflowABSTRACT
Site saturation mutagenesis (SSM) is a powerful mutagenesis strategy for protein engineering and directed evolution experiments. However, limiting factors using this method are either biased representation of variants, or limiting library size. To overcome these hurdles, we generated large scale targeted synthetic SSM libraries using massively parallel oligonucleotide synthesis and benchmarked this against an error-prone (epPCR) library. The yeast glucose activated GPCR-Gpr1 was chosen as a prototype to evolve novel glucose sensors. We demonstrate superior variant representation and several unique hits in the synthetic library compared to the PCR generated library. Application of this mutational approach further builds the possibilities of synthetic biology in tuning of a response to known ligands and in generating biosensors for novel ligands.