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

2019 ◽  
Vol 24 (5) ◽  
pp. 537-547
Author(s):  
Rachel H. Clare ◽  
Roger Clark ◽  
Catherine Bardelle ◽  
Paul Harper ◽  
Matthew Collier ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Neglected Tropical Diseases ◽  
Bacterial Symbiont ◽  
Tropical Diseases ◽  
Validation Process ◽  
Host Insect ◽  
Filarial Nematodes ◽  
Development And Validation ◽  
High Throughput Assay

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.

scholarly journals High-Throughput Screening Platform for Natural Product–Based Drug Discovery Against 3 Neglected Tropical Diseases

2014 ◽  
Vol 20 (1) ◽  
pp. 82-91 ◽  
Author(s):  
F. Annang ◽  
G. Pérez-Moreno ◽  
R. García-Hernández ◽  
C. Cordon-Obras ◽  
J. Martín ◽  
...  
Keyword(s):  
Natural Products ◽  
Drug Discovery ◽  
Natural Product ◽  
High Throughput ◽  
High Throughput Screening ◽  
Neglected Tropical Diseases ◽  
Chemical Diversity ◽  
Tropical Diseases ◽  
Isolation And Characterization ◽  
Microbial Extracts

African trypanosomiasis, leishmaniasis, and Chagas disease are 3 neglected tropical diseases for which current therapeutic interventions are inadequate or toxic. There is an urgent need to find new lead compounds against these diseases. Most drug discovery strategies rely on high-throughput screening (HTS) of synthetic chemical libraries using phenotypic and target-based approaches. Combinatorial chemistry libraries contain hundreds of thousands of compounds; however, they lack the structural diversity required to find entirely novel chemotypes. Natural products, in contrast, are a highly underexplored pool of unique chemical diversity that can serve as excellent templates for the synthesis of novel, biologically active molecules. We report here a validated HTS platform for the screening of microbial extracts against the 3 diseases. We have used this platform in a pilot project to screen a subset (5976) of microbial extracts from the MEDINA Natural Products library. Tandem liquid chromatography–mass spectrometry showed that 48 extracts contain potentially new compounds that are currently undergoing de-replication for future isolation and characterization. Known active components included actinomycin D, bafilomycin B1, chromomycin A3, echinomycin, hygrolidin, and nonactins, among others. The report here is, to our knowledge, the first HTS of microbial natural product extracts against the above-mentioned kinetoplastid parasites.

scholarly journals The utility of yeast as a tool for cell-based, target-directed high-throughput screening

Parasitology ◽  
2013 ◽  
Vol 141 (1) ◽  
pp. 8-16 ◽  
Author(s):  
J. L. NORCLIFFE ◽  
E. ALVAREZ-RUIZ ◽  
J. J. MARTIN-PLAZA ◽  
P. G. STEEL ◽  
P. W. DENNY
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Neglected Tropical Diseases ◽  
Cost Effective ◽  
Tropical Diseases ◽  
Protein Targets ◽  
Biochemical Assays ◽  
Cost Effective Approach ◽  
Cellular Context

SUMMARYMany Neglected Tropical Diseases (NTDs) have recently been subject of increased focus, particularly with relation to high-throughput screening (HTS) initiatives. These vital endeavours largely rely of two approaches, in vitro target-directed screening using biochemical assays or cell-based screening which takes no account of the target or targets being hit. Despite their successes both of these approaches have limitations; for example, the production of soluble protein and a lack of cellular context or the problems and expense of parasite cell culture. In addition, both can be challenging to miniaturize for ultra (u)HTS and expensive to utilize. Yeast-based systems offer a cost-effective approach to study and screen protein targets in a direct-directed manner within a eukaryotic cellular context. In this review, we examine the utility and limitations of yeast cell-based, target-directed screening. In particular we focus on the currently under-explored possibility of using such formats in uHTS screening campaigns for NTDs.

scholarly journals Development of a High-Throughput Screening Assay to Identify Inhibitors of the SARS-CoV-2 Guanine-N7-Methyltransferase Using RapidFire Mass Spectrometry

2021 ◽  
pp. 247255522110006
Author(s):  
Lesley-Anne Pearson ◽  
Charlotte J. Green ◽  
De Lin ◽  
Alain-Pierre Petit ◽  
David W. Gray ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Mutational Analysis ◽  
Nonstructural Protein ◽  
Translation Efficiency ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Starting Point ◽  
Approved Drugs ◽  
High Throughput Assay

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5′ end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3′-5′ exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.

scholarly journals AWZ1066S, a highly specific anti-Wolbachia drug candidate for a short-course treatment of filariasis

2019 ◽  
Vol 116 (4) ◽  
pp. 1414-1419 ◽  
Author(s):  
W. David Hong ◽  
Farid Benayoud ◽  
Gemma L. Nixon ◽  
Louise Ford ◽  
Kelly L. Johnston ◽  
...  
Keyword(s):  
Neglected Tropical Diseases ◽  
Drug Candidate ◽  
Phenotypic Screening ◽  
Tropical Diseases ◽  
Proof Of Concept ◽  
Preclinical Models ◽  
Superior Efficacy ◽  
Short Course ◽  
Filarial Nematodes ◽  
Candidate Molecule

Onchocerciasis and lymphatic filariasis are two neglected tropical diseases that together affect ∼157 million people and inflict severe disability. Both diseases are caused by parasitic filarial nematodes with elimination efforts constrained by the lack of a safe drug that can kill the adult filaria (macrofilaricide). Previous proof-of-concept human trials have demonstrated that depleting >90% of the essential nematode endosymbiont bacterium, Wolbachia, using antibiotics, can lead to permanent sterilization of adult female parasites and a safe macrofilaricidal outcome. AWZ1066S is a highly specific anti-Wolbachia candidate selected through a lead optimization program focused on balancing efficacy, safety and drug metabolism/pharmacokinetic (DMPK) features of a thienopyrimidine/quinazoline scaffold derived from phenotypic screening. AWZ1066S shows superior efficacy to existing anti-Wolbachia therapies in validated preclinical models of infection and has DMPK characteristics that are compatible with a short therapeutic regimen of 7 days or less. This candidate molecule is well-positioned for onward development and has the potential to make a significant impact on communities affected by filariasis.

scholarly journals Identification and Characterization of a New Series of Ghrelin O-Acyl Transferase Inhibitors

2017 ◽  
Vol 23 (2) ◽  
pp. 154-163 ◽  
Author(s):  
Mariko Yoneyama-Hirozane ◽  
Kohei Deguchi ◽  
Takeshi Hirakawa ◽  
Tsuyoshi Ishii ◽  
Tomoyuki Odani ◽  
...  
Keyword(s):  
High Throughput ◽  
Inhibitory Activity ◽  
High Throughput Screening ◽  
Enzyme Linked Immunosorbent Assay ◽  
Acyl Transferase ◽  
Lead Compound ◽  
Time Resolved ◽  
Acyl Ghrelin ◽  
Antiobesity Drugs ◽  
High Throughput Assay

Ghrelin O-acyl transferase (GOAT; MBOAT4) catalyzes O-acylation at serine-3 of des-acyl ghrelin. Acyl ghrelin is secreted by stomach X/A-like cells and plays a role in appetite and metabolism. Therefore, GOAT has been expected to be a novel antiobesity target because it is responsible for acyl ghrelin production. Here, we report homogeneous time-resolved fluorescence (HTRF) and enzyme-linked immunosorbent assay (ELISA) methods utilizing human GOAT-expressing microsomes as a novel high-throughput assay system for the discovery of hit compounds and optimization of lead compounds. Hit compounds exemplified by compound A (2-[(2,4-dichlorobenzyl)sulfanyl]-1,3-benzoxazole-5-carboxylic acid) were identified by high-throughput screening using the HTRF assay and confirmed to have GOAT inhibitory activity using the ELISA. Based on the hit compound information, the novel lead compound (compound B, (4-chloro-6-{[2-methyl-6-(trifluoromethyl)pyridin-3-yl]methoxy}-1-benzothiophen-3-yl)acetic acid) was synthesized and exhibited potent GOAT inhibition with oral bioavailability. Both the hit compound and lead compound showed octanoyl-CoA competitive inhibitory activity. Moreover, these two compounds decreased acyl ghrelin production in the stomach of mice after their oral administration. These novel findings demonstrate that GOAT is a druggable target, and its inhibitors are promising antiobesity drugs.

Application of the BD ACTOne™ Technology for the High-Throughput Screening of Gs-Coupled Receptor Antagonists

2007 ◽  
Vol 12 (8) ◽  
pp. 1068-1073 ◽  
Author(s):  
András Visegrády ◽  
András Boros ◽  
Zsolt Némethy ◽  
Béla Kiss ◽  
György M. Keserű
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Calcium Influx ◽  
Live Cells ◽  
Cyclic Nucleotide Gated Channels ◽  
Novel Technology ◽  
Adenosine Cyclic Monophosphate ◽  
Compound Screening ◽  
High Throughput Assay ◽  
Act One

A novel technology for monitoring the changes of 3,′5′-adenosine cyclic monophosphate (cAMP) in live cells suitable for drug screening relies on the use of cyclic nucleotide-gated channels as biosensors coexpressed with the appropriate target receptor. The technique (termed BD ACT One™) offers measurement of cAMP-dependent calcium influx or membrane depolarization with conventional fluorescent methods both in kinetic and in endpoint modes, optimal for high-throughput and subsequent compound screening. The utility of the technique is reported here based on assay development and high-throughput screening for small-molecule antagonists of the peptide parathyroid hormone 2 receptor (PTH2R). The dual-signaling properties of the receptor were retained in the recombinant system, and the observed pharmacological profile corresponded to data from radiolabeled cAMP determination. The membrane-potential-based high-throughput assay produced reproducible actives and led to the identification of several chemical scaffolds with potential utility as PTH2R ligands. ( Journal of Biomolecular Screening 2007:1068-1073)

scholarly journals High-Throughput Screening of Psychotropic Compounds: Impacts on Swimming Behaviours in Artemia franciscana

Toxics ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 64
Author(s):  
Shanelle A. Kohler ◽  
Matthew O. Parker ◽  
Alex T. Ford
Keyword(s):  
High Throughput ◽  
Swimming Speed ◽  
High Throughput Screening ◽  
Artemia Franciscana ◽  
Animal Behaviour ◽  
Crustacean Species ◽  
Significant Difference ◽  
Increased Sensitivity ◽  
Light Side ◽  
High Throughput Assay

Animal behaviour is becoming increasingly popular as an endpoint in ecotoxicology due to its increased sensitivity and speed compared to traditional endpoints. However, the widespread use of animal behaviours in environmental risk assessment is currently hindered by a lack of optimisation and standardisation of behavioural assays for model species. In this study, assays to assess swimming speed were developed for a model crustacean species, the brine shrimp Artemia franciscana. Preliminary works were performed to determine optimal arena size for this species, and weather lux used in the experiments had an impact on the animals phototactic response. Swimming speed was significantly lower in the smallest arena, whilst no difference was observed between the two larger arenas, suggesting that the small arena was limiting swimming ability. No significant difference was observed in attraction to light between high and low light intensities. Arena size had a significant impact on phototaxis behaviours. Large arenas resulted in animals spending more time in the light side of the arena compared to medium and small, irrespective of light intensity. The swimming speed assay was then used to expose specimens to a range of psychotropic compounds with varying modes of action. Results indicate that swimming speed provides a valid measure of the impacts of behaviour modulating compounds on A. franciscana. The psychotropic compounds tested varied in their impacts on animal behaviour. Fluoxetine resulted in increased swimming speed as has been found in other crustacean species, whilst oxazepam, venlafaxine and amitriptyline had no significant impacts on the behaviours measured. The results from this study suggest a simple, fast, high throughput assay for A. franciscana and gains insight on the impacts of a range of psychotropic compounds on the swimming behaviours of a model crustacean species used in ecotoxicology studies.

scholarly journals Multidimensional Profiling of CSF1R Screening Hits and Inhibitors

2011 ◽  
Vol 16 (9) ◽  
pp. 1007-1017 ◽  
Author(s):  
Joost C. M. Uitdehaag ◽  
Cecile M. Sünnen ◽  
Antoon M. van Doornmalen ◽  
Nikki de Rouw ◽  
Arthur Oubrie ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Active Form ◽  
Dissociation Rate ◽  
The Past ◽  
Compound Libraries ◽  
Competitive Kinetics ◽  
Homogeneous Assay ◽  
High Throughput Assay ◽  
Stimulating Factor

Over the past years, improvements in high-throughput screening (HTS) technology and compound libraries have resulted in a dramatic increase in the amounts of good-quality screening hits, and there is a growing need for follow-on hit profiling assays with medium throughput to further triage hits. Here the authors present such assays for the colony-stimulating factor 1 receptor (CSF1R, Fms), including tests for cellular activity and a homogeneous assay to measure affinity for inactive CSF1R. They also present a high-throughput assay to measure target residence time, which is based on competitive binding kinetics. To better fit koff rates, they present a modified mathematical model for competitive kinetics. In all assays, they profiled eight reference inhibitors (imatinib, sorafenib, sunitinib, tandutinib, dasatinib, GW2580, Ki20227, and J&J’s pyrido[2,3-d]pyrimidin-5-one). Using the known biochemical selectivities of these inhibitors, which can be quantified using metrics such as the selectivity entropy, the authors have determined which assay readout best predicts hit selectivity. Their profiling shows surprisingly that imatinib has a preference for the active form of CSF1R and that Ki20227 has an unusually slow target dissociation rate. This confirms that follow-on hit profiling is essential to ensure that the best hits are selected for lead optimization.

scholarly journals SCAN ™—A High-Throughput Assay for Detecting Small Molecule Binding to RNA Targets

2009 ◽  
Vol 14 (3) ◽  
pp. 219-229 ◽  
Author(s):  
Chris Baugh ◽  
Shaohui Wang ◽  
Bin Li ◽  
James R. Appleman ◽  
Peggy A. Thompson
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assay ◽  
Laboratory Equipment ◽  
Rna Targets ◽  
High Throughput Screening Assay ◽  
Small Molecule Ligands ◽  
Hcv Ires ◽  
High Throughput Assay

A novel optical-based high-throughput screening technology has been developed for increasing the rate of discovering chemical leads against RNA targets. SCAN™ ( Screen for Compounds with Affinity for Nucleic Acids) is an affinity-based assay that identifies small molecules that bind and recognize structured RNA elements. This technology provides the opportunity to conduct high-throughput screening of a new class of targets—RNA. SCAN™ offers many attractive features including a simple homogeneous format, low screening costs, and the ability to use common laboratory equipment. A SCAN™ assay was developed for the HCV IRES Loop IIId RNA domain. A high-throughput screen of our entire compound library resulted in the identification of small molecule ligands that bind to Loop IIId. The Z′ values were greater than 0.8, showing this to be a robust high-throughput screening assay. A correlation between SCAN™ EC50 and KD values is reported suggesting the ability to use the assay for compound optimization. ( Journal of Biomolecular Screening 2009:219-229)

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