High Throughput Discovery: Search and Interpretation on the Path To New Drugs

2001 ◽  
pp. 301-322
Keyword(s):  
High Throughput ◽  
New Drugs

scholarly journals Accelerating High-Throughput Screening for Structural Materials with Production Management Methods

Materials ◽  
2018 ◽  
Vol 11 (8) ◽  
pp. 1330 ◽  
Author(s):  
Alexander Bader ◽  
Finn Meiners ◽  
Kirsten Tracht
Keyword(s):  
High Throughput ◽  
Material Flow ◽  
High Throughput Screening ◽  
Production Management ◽  
Waiting Times ◽  
Complex Structure ◽  
New Drugs ◽  
Order Processing ◽  
Speed Up ◽  
Process Trial

High-throughput screenings are widely accepted for pharmaceutical developments for new substances and the development of new drugs with required characteristics by evolutionary studies. Current research projects transfer this principle of high-throughput testing to the development of metallic materials. In addition to new generating and testing methods, these types of high-throughput systems need a logistical control and handling method to reduce throughput time to get test results faster. Instead of the direct material flow found in classical high-throughput screenings, these systems have a very complex structure of material flow. The result is a highly dynamic system that includes short-term changes such as rerun stations, partial tests, and temporarily paced sequences between working systems. This paper presents a framework that divides the actions for system acceleration into three main sections. First, methods for special applications in high-throughput systems are designed or adapted to speed up the generation, treatment, and testing processes. Second, methods are needed to process trial plans and to control test orders, which can efficiently reduce waiting times. The third part of the framework describes procedures for handling samples. This reduces non-productive times and reduces order processing in individual lots.

scholarly journals A Homogeneous, High-Throughput Fluorescence Anisotropy-Based DNA Supercoiling Assay

2010 ◽  
Vol 15 (9) ◽  
pp. 1088-1098 ◽  
Author(s):  
Adam Shapiro ◽  
Haris Jahic ◽  
Swati Prasad ◽  
David Ehmann ◽  
Jason Thresher ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescence Anisotropy ◽  
High Throughput Screening ◽  
Topoisomerase I ◽  
Dna Supercoiling ◽  
New Drugs ◽  
E Coli ◽  
Cellular Processes ◽  
The Difference ◽  
Cellular Replication

The degree of supercoiling of DNA is vital for cellular processes, such as replication and transcription. DNA topology is controlled by the action of DNA topoisomerase enzymes. Topoisomerases, because of their importance in cellular replication, are the targets of several anticancer and antibacterial drugs. In the search for new drugs targeting topoisomerases, a biochemical assay compatible with automated high-throughput screening (HTS) would be valuable. Gel electrophoresis is the standard method for measuring changes in the extent of supercoiling of plasmid DNA when acted upon by topoisomerases, but this is a low-throughput and laborious method. A medium-throughput method was described previously that quantitatively distinguishes relaxed and supercoiled plasmids by the difference in their abilities to form triplex structures with an immobilized oligonucleotide. In this article, the authors describe a homogeneous supercoiling assay based on triplex formation in which the oligonucleotide strand is labeled with a fluorescent dye and the readout is fluorescence anisotropy. The new assay requires no immobilization, filtration, or plate washing steps and is therefore well suited to HTS for inhibitors of topoisomerases. The utility of this assay is demonstrated with relaxation of supercoiled plasmid by Escherichia coli topoisomerase I, supercoiling of relaxed plasmid by E. coli DNA gyrase, and inhibition of gyrase by fluoroquinolones and nalidixic acid.

scholarly journals Development of a High-Throughput Scintillation Proximity Assay for the Identification of C-Domain Translational Initiation Factor 2 Inhibitors

2002 ◽  
Vol 7 (6) ◽  
pp. 541-546 ◽  
Author(s):  
Sonia Delle Fratte ◽  
Chiara Piubelli ◽  
Enrico Domenici
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Ribosomal Subunit ◽  
New Drugs ◽  
Initiation Factor ◽  
Translational Initiation ◽  
Scintillation Proximity Assay ◽  
Initiation Factor 2 ◽  
Subsequent Capture ◽  
Translational Initiation Factor

Translational initiation factor 2 (IF2) is the largest of the 3 factors required for translation initiation in prokaryotes and has been shown to be essential in Escherichia coli. It stimulates the binding of fMet-tRNAfMet to the 30S ribosomal subunit in the presence of GTP. The selectivity is achieved through specific recognition of the tRNAfMet blocked α-amino group. IF2 is composed of 3 structural domains: N-domain, whose function is not known; G-domain, which contains the GTP/GDP binding site and the GTPase catalytic center; and C-domain, which recognizes and binds fMet-tRNAfMet. Its activity is strictly bacteria specific and highly conserved among prokaryotes. So far, antibiotics targeting IF2 function are not known, and this makes it an ideal target for new drugs with mechanisms of resistance not yet developed. A few assays have been developed in the past, which allow the detection of IF2 activity either directly or indirectly. In both instances, the assays are based on radioactive detection and do not allow for high throughput because of the need for separation or solvent extraction steps. The authors describe a novel biochemical assay for IF2 that exploits the molecular recognition of fMet-tRNAfMet by the C-domain. The assay is based on the incubation of biotinyl-IF2 with fMet-tRNAfMet and the subsequent capture of the radiolabeled complex by streptavidin-coated beads, exploiting the scintillation proximity assay (SPA) technology. The assay has been designed in an automatable, homogeneous, miniaturized fashion suitable for high-throughput screening and is rapid, sensitive, and robust to dimethyl sulfoxide (DMSO) up to 10% v/v. The assay, used to screen a limited chemical collection of about 5000 compounds and a subset of compounds originated by a 2-D substructural search, has shown to be able to detect potential IF2 inhibitors.

scholarly journals Nano-biofilm Arrays as a Novel Universal Platform for Microscale Microbial Culture and High-Throughput Downstream Applications

2019 ◽  
Vol 26 (14) ◽  
pp. 2529-2535 ◽  
Author(s):  
Anand Srinivasan ◽  
Anand K. Ramasubramanian ◽  
José L. Lopez-Ribot
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Antimicrobial Agents ◽  
Microarray Platform ◽  
New Drugs ◽  
Microbial Culture ◽  
Phenotypic Properties ◽  
Microbial Biofilms ◽  
Antibiotic Drug

Biofilms are the predominant mode of microbial growth and it is now fully accepted that a majority of infections in humans are associated with a biofilm etiology. Biofilms are defined as attached and structured microbial communities surrounded by a protective exopolymeric matrix. Importantly, sessile microorganisms growing within a biofilm are highly resistant to antimicrobial agents. Thus, there is an urgent need to develop new and improved anti-biofilm therapies. Unfortunately, most of the current techniques for in-vitro biofilm formation are not compatible with high throughput screening techniques that can speed up discovery of new drugs with anti-biofilm activity. To try to overcome this major impediment, our group has developed a novel technique consisting of micro-scale culture of microbial biofilms on a microarray platform. Using this technique, hundreds to thousands of microbial biofilms, each with a volume of approximately 30-50 nanolitres, can be simultaneously formed on a standard microscope slide. Despite more than three orders of magnitude of miniaturization over conventional biofilms, these nanobiofilms display similar growth, structural and phenotypic properties, including antibiotic drug resistance. These nanobiofilm chips are amenable to automation, drastically reducing assay volume and costs. This technique platform allows for true high-throughput screening in search for new anti-biofilm drugs.

High Throughput Screening for Drug Discovery and Virus Detection

2021 ◽  
Vol 24 ◽  
Author(s):  
Adetola Okea ◽  
Deniz Sahin ◽  
Xin Chen ◽  
Ying Shang
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Virus Detection ◽  
Flow Time ◽  
New Drugs ◽  
Mixed Integer ◽  
General Process ◽  
Screening Systems

Background: High throughput screening systems are automated labs for the analysis of many biochemical substances in the drug discovery and virus detection process. This paper was motivated by the problem of automating testing for viruses and new drugs using high throughput screening systems. The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the turn of 2019-2020 presented extradentary challenges to public health. Existing approaches to test viruses and new drugs do not use optimal schedules and are not efficient. Objective: The scheduling of activities performed by various resources in a high throughput screening system affects its efficiency, throughput, operations cost, and quality of screening. This study aims to minimize the total screening (flow) time and ensure the consistency and quality of screening. Methods: This paper develops innovative mixed integer models that efficiently compute optimal schedules for screening many microplates to identify new drugs and determine whether samples contain viruses. The methods integrate job-shop and cyclic scheduling. Experiments are conducted for a drug discovery process of screening an enzymatic assay and a general process of detecting SARS-CoV-2. Results: The method developed in this article can reduce screening time by as much as 91.67%. Conclusion: The optimal schedules for high throughput screening systems greatly reduce the total flow time and can be computed efficiently to help discover new drugs and detect viruses.

scholarly journals High-throughput decoding of drug targets and drug resistance mechanisms in African trypanosomes

Parasitology ◽  
2013 ◽  
Vol 141 (1) ◽  
pp. 77-82 ◽  
Author(s):  
DAVID HORN
Keyword(s):  
Drug Resistance ◽  
High Throughput ◽  
Drug Targets ◽  
Sequence Data ◽  
Resistance Mechanisms ◽  
New Drugs ◽  
Drug Uptake ◽  
Microbial Pathogens ◽  
African Trypanosomes ◽  
Genome Scale

SUMMARYThe availability of genome sequence data has facilitated the development of high-throughput genetic screening approaches in microbial pathogens. In the African trypanosome, Trypanosoma brucei, genome-scale RNA interference screens have proven particularly effective in this regard. These genetic screens allow for identification of the genes that contribute to a particular pathway or mechanisms of interest. The approach has been used to assess loss-of-fitness, revealing the genes and proteins required for parasite viability and growth. The outputs from these screens predict essential and dispensable genes and facilitate drug target prioritization efforts. The approach has also been used to assess resistance to anti-trypanosomal drugs, revealing the genes and proteins that facilitate drug uptake and action. These outputs also highlight likely mechanisms underlying clinically relevant drug resistance. I first review these findings in the context of what we know about current drugs. I then describe potential contributions that these high-throughput approaches could make to the development and implementation of new drugs.

scholarly journals Investigating the mode of action of tuberculosis drugs using hypersensitive mutants of Mycobacterium smegmatis

2021 ◽  
Author(s):  
◽  
Richard Laurence Campen
Keyword(s):  
High Throughput ◽  
Mode Of Action ◽  
Drug Hypersensitivity ◽  
Resistance Mechanisms ◽  
New Drugs ◽  
Drug Resistant ◽  
Primary Target ◽  
Modes Of Action ◽  
Increased Sensitivity ◽  
High Throughput Assay

<p>Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), is the leading cause of death and disease by a bacterial pathogen worldwide. The growing incidence of drug resistant TB, especially multi-drug resistant TB highlights the need for new drugs with novel modes of action. Current treatment of TB involves a multi-drug regimen of four drugs including isoniazid and rifampicin, both of which were discovered over 40 years ago. Bedaquiline is one of the first novel TB drugs to enter clinical trials since the discovery of rifampicin, and has shown excellent activity against drug resistant TB. Although isoniazid and rifampicin are well established anti-TB drugs, significant gaps in knowledge regarding their modes of action exist. Furthermore, little information on the mode of action of the novel drug bedaquiline is known beyond its primary target. Characterisation of drug mode of action facilitates rational modifications of drugs to improve the treatment of TB.  The aim of this study was to identify novel aspects of the modes of action of isoniazid, rifampicin, and bedaquiline by characterising drug hypersensitive mutants of M. smegmatis mc²155. A sub-saturated M. smegmatis mc²155 transposon mutant collection with 1.1-fold genome coverage (7680 mutants) was constructed, with this collection estimated to contain mutations in 73.2% of all genes capable of maintaining a transposon insertion (non-essential genes). A high-throughput assay was developed for screening the collection, and mutants related to known drug mode of action were identified for isoniazid (ahpC and eccCa₁) and bedaquiline (atpB). Additionally, known mechanisms of drug inactivation were identified for isoniazid (nudC), rifampicin (arr and lspA), and bedaquiline (mmpL5). The finding that transposon mutants of nudC are hypersensitive to isoniazid independently validated the recent discovery of the role of NudC in basal isoniazid resistance by Wang et al. (2011). The remaining genes identified in this thesis represent potentially novel aspects of the modes of action or resistance mechanisms of these drugs.  Cross-sensitivity to other drugs indicated that the mechanism of sensitivity was drug specific for the mutants examined. Differential-sensitivity testing against drug analogues revealed that Arr is involved in resistance to the rifampicin analogue rifapentine as well, indicating that Arr can detoxify rifapentine similar to rifampicin. The nudC mutant showed increased sensitivity to a range of isoniazid analogues, indicating that it can detoxify these analogues similar to the parent compound. Interestingly six analogues were found to be less active against the nudC mutant than expected. A number of overexpression strains were tested against these six analogues; a nudC overexpression strain, and a strain overexpressing inhA, the primary target for isoniazid. Overexpression of nudC as well inhA increased the resistance of WT to isoniazid, but failed to increase resistance to three of the analogues, NSC27607, NSC33759, and NSC40350. Isoniazid is a prodrug and is activated by the peroxidase/catalase enzyme KatG. Overexpression of katG resulted in increased isoniazid sensitivity, as well as increased sensitivity to NSC27607, NSC33759, and NSC40350. Together these results suggest that NSC27607, NSC33759, and NSC40350 are activated by KatG, but that InhA is not the primary target. Additionally the inability of NudC overexpression to confer resistance suggests these analogues are not acting via a NAD adduct, the mechanism by which isoniazid inhibits InhA. These results suggest that there are other toxic metabolites being produced by KatG activation of these three analogues.  In conclusion, characterisation of mutants identified in a high-throughput assay for drug hypersensitivity identified genes involved in the modes of action or resistance mechanisms for isoniazid, rifampicin, and bedaquiline. Additionally, a number of novel genes were identified that have no known connections to the known modes of action or resistance mechanisms for these drugs. Further testing of a nudC mutant revealed three isoniazid analogues that appear to inhibit growth of M. smegmatis mc²155 independent of InhA, the primary target of isoniazid. This study has successfully demonstrated that screening for drug hypersensitivity can generate novel information on drug mode of action and resistance mechanisms. This information can ultimately be used to help drive the development of new drugs, and improve treatment of TB.</p>

scholarly journals mDrop-Seq: Massively Parallel Single-Cell RNA-Seq of Saccharomyces cerevisiae and Candida albicans

Vaccines ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 30
Author(s):  
Ryan Dohn ◽  
Bingqing Xie ◽  
Rebecca Back ◽  
Alan Selewa ◽  
Heather Eckart ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Stress Response ◽  
Heat Shock ◽  
Single Cell ◽  
High Throughput ◽  
Yeast Species ◽  
Treatment Strategies ◽  
New Drugs ◽  
Mrna Levels

Advances in high-throughput single-cell RNA sequencing (scRNA-seq) have been limited by technical challenges such as tough cell walls and low RNA quantity that prevent transcriptomic profiling of microbial species at throughput. We present microbial Drop-seq or mDrop-seq, a high-throughput scRNA-seq technique that is demonstrated on two yeast species, Saccharomyces cerevisiae, a popular model organism, and Candida albicans, a common opportunistic pathogen. We benchmarked mDrop-seq for sensitivity and specificity and used it to profile 35,109 S. cerevisiae cells to detect variation in mRNA levels between them. As a proof of concept, we quantified expression differences in heat shock S. cerevisiae using mDrop-seq. We detected differential activation of stress response genes within a seemingly homogenous population of S. cerevisiae under heat shock. We also applied mDrop-seq to C. albicans cells, a polymorphic and clinically relevant species of yeast with a thicker cell wall compared to S. cerevisiae. Single-cell transcriptomes in 39,705 C. albicans cells were characterized using mDrop-seq under different conditions, including exposure to fluconazole, a common anti-fungal drug. We noted differential regulation in stress response and drug target pathways between C. albicans cells, changes in cell cycle patterns and marked increases in histone activity when treated with fluconazole. We demonstrate mDrop-seq to be an affordable and scalable technique that can quantify the variability in gene expression in different yeast species. We hope that mDrop-seq will lead to a better understanding of genetic variation in pathogens in response to stimuli and find immediate applications in investigating drug resistance, infection outcome and developing new drugs and treatment strategies.

scholarly journals High-Throughput Plasmodium falciparum Growth Assay for Malaria Drug Discovery

2006 ◽  
Vol 51 (2) ◽  
pp. 716-723 ◽  
Author(s):  
Mary Lynn Baniecki ◽  
Dyann F. Wirth ◽  
Jon Clardy
Keyword(s):  
Plasmodium Falciparum ◽  
Small Molecules ◽  
High Throughput ◽  
Multidrug Resistant ◽  
Therapeutic Agents ◽  
New Drugs ◽  
Sub Saharan Africa ◽  
Highly Active ◽  
Growth Assay ◽  
Sub Saharan

ABSTRACT New therapeutic agents for the treatment of malaria, the world's most deadly parasitic disease, are urgently needed. Malaria afflicts 300 to 500 million people and results in 1 to 2 million deaths annually, and more than 85% of all malaria-related mortality involves young children and pregnant women in sub-Saharan Africa. The emergence of multidrug-resistant parasites, especially in Plasmodium falciparum, has eroded the efficacy of almost all currently available therapeutic agents. The discovery of new drugs, including drugs with novel cellular targets, could be accelerated with a whole-organism high-throughput screen (HTS) of structurally diverse small-molecule libraries. The standard whole-organism screen is based on incorporation of [3H]hypoxanthine and has liabilities, such as limited throughput, high cost, multiple labor-intensive steps, and disposal of radioactive waste. Recently, screens have been reported that do not use radioactive incorporation, but their reporter signal is not robust enough for HTS. We report a P. falciparum growth assay that is technically simple, robust, and compatible with the automation necessary for HTS. The assay monitors DNA content by addition of the fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI) as a reporter of blood-stage parasite growth. This DAPI P. falciparum growth assay was used to measure the 50% inhibitory concentrations (IC50s) of a diverse set of known antimalarials. The resultant IC50s compared favorably with those obtained in the [3H]hypoxanthine incorporation assay. Over 79,000 small molecules have been tested for antiplasmodial activity using the DAPI P. falciparum growth assay, and 181 small molecules were identified as highly active against multidrug-resistant parasites.

scholarly journals Adrien Albert Award: How to Mine Chemistry Space for New Drugs and Biomedical Therapies

2015 ◽  
Vol 68 (8) ◽  
pp. 1174 ◽  
Author(s):  
David Winkler
Keyword(s):  
Regenerative Medicine ◽  
High Throughput ◽  
De Novo ◽  
Molecular Design ◽  
Chemical Space ◽  
Gene Array ◽  
Computational Modelling ◽  
New Drugs ◽  
Peptide Motifs ◽  
Bioactive Agents

It is clear that the sizes of chemical, ‘drug-like’, and materials spaces are enormous. If scientists working in established therapeutic, and newly established regenerative medicine fields are to discover better molecules or materials, they must find better ways of probing these enormous spaces. There are essentially five ways that this can be achieved: combinatorial and high throughput synthesis and screening approaches; fragment-based methods; de novo molecular design, design of experiments, diversity libraries; supramolecular approaches; evolutionary approaches. These methods either synthesise materials and screen them more quickly, or constrain chemical spaces using biology or other types of ‘fitness functions’. High throughput experimental approaches cannot explore more than a minute part of chemical space. We are nevertheless entering into an era that is data dominated. High throughput experiments, robotics, automated crystallographic beam lines, combinatorial and flow synthesis, high content screening, and the ‘omics’ technologies are providing a flood of data, and efficient methods for extracting meaning from it are essential. This paper describes how new developments in mathematics have provided excellent, robust computational modelling tools for exploring large chemical spaces, for extracting meaning from large datasets, for designing new bioactive agents and materials, and for making truly predictive, quantitative models of the properties of molecules and materials for use in therapeutic and regenerative medicine. We describe these broadly applicable modelling tools and provide examples of their application to serum free stem cell culture, pathogen resistant polymers for implantable devices, new markers and biological mechanisms derived from mathematical analyses of gene array data, and pharmacokinetically important physicochemical properties of small molecules. We also discuss biologically conserved peptide motifs as a design framework for small molecule drugs and give examples of the application of this concept to drug design.

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