scholarly journals A novel live cell assay to measure diacylglycerol lipase α activity

2016 ◽  
Vol 36 (3) ◽  
Author(s):  
Praveen K. Singh ◽  
Rachel Markwick ◽  
Fiona V. Howell ◽  
Gareth Williams ◽  
Patrick Doherty
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
Human Cell Line ◽  
Live Cell ◽  
Diacylglycerol Lipase ◽  
Cell Assay ◽  
Retrograde Signalling ◽  
Cell Assays ◽  
The Central Nervous System ◽  
Rate Limiting

Diacylglycerol lipase α (DAGLα) hydrolyses DAG to generate the principal endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα dependent cannabinoid (CB) signalling has been implicated in numerous processes including axonal growth and guidance, adult neurogenesis and retrograde signalling at the synapse. Recent studies have implicated DAGLα as an emerging drug target for several conditions including pain and obesity. Activity assays are critical to the drug discovery process; however, measurement of diacylglycerol lipase (DAGL) activity using its native substrate generally involves low-throughput MS techniques. Some relatively high-throughput membrane based assays utilizing surrogate substrates have been reported, but these do not take into account the rate-limiting effects often associated with the ability of a drug to cross the cell membrane. In the present study, we report the development of a live cell assay to measure DAGLα activity. Two previously reported DAGLα surrogate substrates, p-nitrophenyl butyrate (PNPB) and 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMUO), were evaluated for their ability to detect DAGLα activity in live cell assays using a human cell line stably expressing the human DAGLα transgene. Following optimization, the small molecule chromogenic substrate PNPB proved to be superior by providing lower background activity along with a larger signal window between transfected and parental cells when compared with the fluorogenic substrate DiFMUO. The assay was further validated using established DAGL inhibitors. In summary, the live cell DAGLα assay reported here offers an economical and convenient format to screen for novel inhibitors as part of drug discovery programmes and compliments previously reported high-throughput membrane based DAGL assays.

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.

scholarly journals A live-cell screen for altered Erk dynamics reveals principles of proliferative control

2019 ◽  
Author(s):  
Alexander G. Goglia ◽  
Maxwell Z. Wilson ◽  
Jillian Silbert ◽  
Lena P. Basta ◽  
Siddhartha G. Jena ◽  
...  
Keyword(s):  
High Throughput ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Pulse Frequency ◽  
Live Cell ◽  
Live Cells ◽  
Drug Induced ◽  
Primary Mouse ◽  
Cell Assays ◽  
High Throughput Screens

Complex, time-varying responses have been observed widely in cell signaling, but how specific dynamics are generated or regulated is largely unknown. One major obstacle has been that high-throughput screens for identifying pathway components are typically incompatible with the live-cell assays used to monitor dynamics. Here, we address this challenge by performing a drug screen for altered Erk signaling dynamics in primary mouse keratinocytes. We screened a library of 429 kinase inhibitors, monitoring Erk activity over 5 h in more than 80,000 single live cells. The screen revealed both known and uncharacterized modulators of Erk dynamics, including inhibitors of non-EGFR receptor tyrosine kinases (RTKs) that increased Erk pulse frequency and overall activity. Using drug treatment and direct optogenetic control, we demonstrate that drug-induced changes to Erk dynamics alter the conditions under which cells proliferate. Our work opens the door to high-throughput screens using live-cell biosensors and reveals that cell proliferation integrates information from Erk dynamics as well as additional permissive cues.

scholarly journals High-content live cell imaging with RNA probes: advancements in high-throughput antimalarial drug discovery

2009 ◽  
Vol 10 (1) ◽  
pp. 45 ◽  
Author(s):  
Serena Cervantes ◽  
Jacques Prudhomme ◽  
David Carter ◽  
Krishna G Gopi ◽  
Qian Li ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
Antimalarial Drug ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Rna Probes

High-Throughput Drug Screening of ECM Deposition Inhibitors for Antifibrotic Drug Discovery

2019 ◽  
Author(s):  
Michael Gerckens ◽  
Hani Alsafadi ◽  
Darcy Wagner ◽  
Katharina Heinzelmann ◽  
Kenji Schorpp ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
Drug Screening ◽  
High Throughput Drug Screening

Development of an Easy and High-Throughput Cell Assay System with a Culture Chip and an Assay Chip

2010 ◽  
Vol 130 (10) ◽  
pp. 471-475 ◽  
Author(s):  
Kanako Sugiura ◽  
Noritada Kaji ◽  
Yukihiro Okamoto ◽  
Manabu Tokeshi ◽  
Yoshinobu Baba
Keyword(s):  
High Throughput ◽  
Assay System ◽  
Cell Assay

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
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