scholarly journals Upscaling of hiPS Cell–Derived Neurons for High-Throughput Screening

2016 ◽  
Vol 22 (3) ◽  
pp. 274-286
Author(s):  
Stefanie Traub ◽  
Heiko Stahl ◽  
Holger Rosenbrock ◽  
Eric Simon ◽  
Ralf Heilker
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Detection Sensitivity ◽  
Neuronal Markers ◽  
Downstream Target ◽  
Electrophysiological Recordings ◽  
Downstream Target Gene ◽  
The Central Nervous System ◽  
Induced Pluripotent

The advent of human-induced pluripotent stem (hiPS) cell–derived neurons promised to provide better model cells for drug discovery in the context of the central nervous system. This work demonstrates both the upscaling of cellular expansion and the acceleration of neuronal differentiation to accommodate the immense material needs of a high-throughput screening (HTS) approach. Using GRowth factor–driven expansion and INhibition of NotCH (GRINCH) during maturation, the derived cells are here referred to as GRINCH neurons. GRINCH cells displayed neuronal markers, and their functional activity could be demonstrated by electrophysiological recordings. In an application of GRINCH neurons, the brain-derived neurotrophic factor (BDNF)–mediated activation of tropomyosin receptor kinase (TrkB) was investigated as a promising drug target to treat synaptic dysfunctions. To assess the phosphorylation of endogenous TrkB in the GRINCH cells, the highly sensitive amplified luminescent proximity homogeneous assay LISA (AlphaLISA) format was established as a primary screen. A high-throughput reverse transcription (RT)–PCR format was employed as a secondary assay to analyze TrkB-mediated downstream target gene expression. In summary, an optimized differentiation protocol, highly efficient cell upscaling, and advanced assay miniaturization, combined with increased detection sensitivity, pave the way for a new generation of predictive cell-based drug discovery.

scholarly journals High-Throughput Screening Platforms in the Discovery of Novel Drugs for Neurodegenerative Diseases

2021 ◽  
Vol 8 (2) ◽  
pp. 30 ◽  
Author(s):  
Hasan Aldewachi ◽  
Radhwan N. Al-Zidan ◽  
Matthew T. Conner ◽  
Mootaz M. Salman
Keyword(s):  
Drug Discovery ◽  
Neurodegenerative Diseases ◽  
High Throughput ◽  
Computer Aided Design ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Attrition Rate ◽  
Screening Efficiency ◽  
The Central Nervous System ◽  
New Treatments

Neurodegenerative diseases (NDDs) are incurable and debilitating conditions that result in progressive degeneration and/or death of nerve cells in the central nervous system (CNS). Identification of viable therapeutic targets and new treatments for CNS disorders and in particular, for NDDs is a major challenge in the field of drug discovery. These difficulties can be attributed to the diversity of cells involved, extreme complexity of the neural circuits, the limited capacity for tissue regeneration, and our incomplete understanding of the underlying pathological processes. Drug discovery is a complex and multidisciplinary process. The screening attrition rate in current drug discovery protocols mean that only one viable drug may arise from millions of screened compounds resulting in the need to improve discovery technologies and protocols to address the multiple causes of attrition. This has identified the need to screen larger libraries where the use of efficient high-throughput screening (HTS) becomes key in the discovery process. HTS can investigate hundreds of thousands of compounds per day. However, if fewer compounds could be screened without compromising the probability of success, the cost and time would be largely reduced. To that end, recent advances in computer-aided design, in silico libraries, and molecular docking software combined with the upscaling of cell-based platforms have evolved to improve screening efficiency with higher predictability and clinical applicability. We review, here, the increasing role of HTS in contemporary drug discovery processes, in particular for NDDs, and evaluate the criteria underlying its successful application. We also discuss the requirement of HTS for novel NDD therapies and examine the major current challenges in validating new drug targets and developing new treatments for NDDs.

Upscaling and Automation of Electrophysiology: Toward High Throughput Screening in Ion Channel Drug Discovery

2003 ◽  
Vol 9 (1) ◽  
pp. 49-58
Author(s):  
Margit Asmild ◽  
Nicholas Oswald ◽  
Karen M. Krzywkowski ◽  
Søren Friis ◽  
Rasmus B. Jacobsen ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Ion Channel ◽  
High Throughput ◽  
High Throughput Screening

High-Throughput Affinity Selection Mass Spectrometry Using SAMDI-MS to Identify Small-Molecule Binders of the Human Rhinovirus 3C Protease

2021 ◽  
pp. 247255522110232
Author(s):  
Michael D. Scholle ◽  
Doug McLaughlin ◽  
Zachary A. Gurard-Levin
Keyword(s):  
Mass Spectrometry ◽  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Human Rhinovirus ◽  
Binding Potential ◽  
Affinity Selection ◽  
Response Curves ◽  
Desorption Ionization ◽  
Self Assembled

Affinity selection mass spectrometry (ASMS) has emerged as a powerful high-throughput screening tool used in drug discovery to identify novel ligands against therapeutic targets. This report describes the first high-throughput screen using a novel self-assembled monolayer desorption ionization (SAMDI)–ASMS methodology to reveal ligands for the human rhinovirus 3C (HRV3C) protease. The approach combines self-assembled monolayers of alkanethiolates on gold with matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry (MS), a technique termed SAMDI-ASMS. The primary screen of more than 100,000 compounds in pools of 8 compounds per well was completed in less than 8 h, and informs on the binding potential and selectivity of each compound. Initial hits were confirmed in follow-up SAMDI-ASMS experiments in single-concentration and dose–response curves. The ligands identified by SAMDI-ASMS were further validated using differential scanning fluorimetry (DSF) and in functional protease assays against HRV3C and the related SARS-CoV-2 3CLpro enzyme. SAMDI-ASMS offers key benefits for drug discovery over traditional ASMS approaches, including the high-throughput workflow and readout, minimizing compound misbehavior by using smaller compound pools, and up to a 50-fold reduction in reagent consumption. The flexibility of this novel technology opens avenues for high-throughput ASMS assays of any target, thereby accelerating drug discovery for diverse diseases.

scholarly journals Development of a Robust High-Throughput Screening Platform for Inhibitors of the Striatal-Enriched Tyrosine Phosphatase (STEP)

2021 ◽  
Vol 22 (9) ◽  
pp. 4417
Author(s):  
Lester J Lambert ◽  
Stefan Grotegut ◽  
Maria Celeridad ◽  
Palak Gosalia ◽  
Laurent JS De Backer ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
Tyrosine Kinases ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Synaptic Function ◽  
Label Free ◽  
Protein Tyrosine

Many human diseases are the result of abnormal expression or activation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Not surprisingly, more than 30 tyrosine kinase inhibitors (TKIs) are currently in clinical use and provide unique treatment options for many patients. PTPs on the other hand have long been regarded as “undruggable” and only recently have gained increased attention in drug discovery. Striatal-enriched tyrosine phosphatase (STEP) is a neuron-specific PTP that is overactive in Alzheimer’s disease (AD) and other neurodegenerative and neuropsychiatric disorders, including Parkinson’s disease, schizophrenia, and fragile X syndrome. An emergent model suggests that the increase in STEP activity interferes with synaptic function and contributes to the characteristic cognitive and behavioral deficits present in these diseases. Prior efforts to generate STEP inhibitors with properties that warrant clinical development have largely failed. To identify novel STEP inhibitor scaffolds, we developed a biophysical, label-free high-throughput screening (HTS) platform based on the protein thermal shift (PTS) technology. In contrast to conventional HTS using STEP enzymatic assays, we found the PTS platform highly robust and capable of identifying true hits with confirmed STEP inhibitory activity and selectivity. This new platform promises to greatly advance STEP drug discovery and should be applicable to other PTP targets.

The Discovery Channel: microfluidics and microengineered systems in drug screening

2015 ◽  
Vol 7 (3) ◽  
pp. 285-288 ◽  
Author(s):  
Christopher Moraes
Keyword(s):  
Drug Discovery ◽  
Engineering Design ◽  
High Throughput ◽  
Drug Screening ◽  
High Throughput Screening ◽  
Recent Literature ◽  
Analytical Strategies ◽  
Discovery Pipeline

We highlight exciting findings and promising approaches in the recent literature in which researchers integrate advanced micro-engineering, design, and analytical strategies to improve the relevance and utility of high-throughput screening in the drug discovery pipeline.

scholarly journals Non-invasive and high-throughput interrogation of exon-specific isoform expression

2021 ◽  
Author(s):  
Dong-Jiunn Jeffery Truong ◽  
Teeradon Phlairaharn ◽  
Bianca Eßwein ◽  
Christoph Gruber ◽  
Deniz Tümen ◽  
...  
Keyword(s):  
Stem Cells ◽  
High Throughput ◽  
High Throughput Screening ◽  
Embryonic Stem ◽  
High Sensitivity ◽  
Therapeutic Interventions ◽  
Dominant Factor ◽  
Reporter System ◽  
Isoform Expression ◽  
Induced Pluripotent

AbstractExpression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.

Sign in / Sign up

Export Citation Format

Share Document