In vitro assays for inhibition of microglial activation
Inventors
Akassoglou, Katerina • Arkin, Michelle • Ang, Kean-Hooi • Meyer-Franke, Anke • Wilson, Christopher
Assignees
J David Gladstone Institutes • University of California San Diego UCSD
Publication Number
US-11099172-B2
Publication Date
2021-08-24
Expiration Date
2035-01-09
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Abstract
The present invention provides cell-based assays, including high throughput cell-based assays, for identification of candidate therapeutic agents with the ability to inhibit microglial activation in vivo in response to different ligands.
Core Innovation
The invention provides cell-based assays, including high-throughput formats, for identifying candidate therapeutic agents that inhibit microglial activation in vivo. These assays involve providing in vitro activated microglial cells, contacting them with test agents, identifying agents that inhibit microglial activation, and determining the toxicity of such agents by measuring cell death. Candidate therapeutic agents are those which inhibit microglial activation and lack toxicity as determined in these assays.
The assays measure microglial activation quantitatively, where activated microglia typically have a surface area of at least 800 μm2, and dead microglia have a surface area of 150 μm2 or less. Activation can be induced by ligands such as fibrin or LPS, which activate microglia through different receptors. The assays use morphological and biochemical endpoints for activation and cell death, enabling high-content, high-throughput screening of compounds including antibodies, peptides, and small molecules.
The problem addressed is the need for methods to identify candidate therapeutic agents capable of inhibiting microglial activation associated with pathological conditions involving inflammation and neuronal injury, such as neurodegenerative diseases. Microglia become chronically activated by stimuli like LPS or neuronal damage, producing toxic factors that drive progressive neuron damage. Due to the complex biological mechanisms involved and limited predictive value of prior assays, there is a need for physiologically relevant, high-throughput cell-based assays that can accurately identify inhibitors of microglial activation with in vivo relevance.
Claims Coverage
The patent includes one independent claim which sets forth a high-throughput cell-based assay for identifying candidate therapeutic agents that inhibit microglia activation and assess toxicity simultaneously.
Simultaneous measurement of microglia activation inhibition and toxicity
A high-throughput assay providing in vitro activated microglial cells exposed to fibrin and LPS, wherein test agents are contacted with these cells and identified for their ability to inhibit microglial activation while simultaneously measuring cell death to determine toxicity.
Use of cell surface area imaging to determine activation and death
Activation of microglial cells is determined by imaging and measurement of cell surface area, where activated microglia have a surface area of at least 800 μm2 and dead microglia have a surface area of 150 μm2 or less, enabling simultaneous assessment of activation inhibition and toxicity.
Screening with diverse classes of test agents
The assay is capable of using test agents comprising known drugs with clinical data, agents with no known targets, anti-inflammatory agents, and agents with affinity to microglial activation targets.
Use of fibrin mimicking in vivo pathological properties
The assay employs fibrin that mimics the biochemical and proinflammatory properties of fibrin matrices found in vivo during pathological conditions to provide physiologically relevant microglial activation for screening.
The claim discloses a comprehensive high-throughput cell-based assay that enables simultaneous identification of microglial activation inhibition and toxicity assessment using morphologic imaging, supports screening with diverse test agents, and uses physiologically relevant fibrin activation, thereby enabling identification of candidate therapeutic agents for microglia activation inhibition in vivo.
Stated Advantages
The assays provide the ability to identify ligand-selective inhibitors of microglial activation in a high-throughput manner.
They allow direct comparison of compound selectivity for inhibition of microglia activation.
They can screen different classes and varieties of agents, including antibodies, peptides, and small molecule libraries.
The assays utilize morphological activation characteristics as an outcome measure, which has predictive value for in vivo efficacy.
They are well suited for full automation using 96 or 384-well plate formats, enabling efficient high-throughput screening.
Documented Applications
Identification of candidate therapeutic agents for prevention and treatment of pathological conditions associated with inflammation and/or neuronal injury, including neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, ALS, multiple sclerosis, and related neurodegenerative disorders.
Screening of small molecules, monoclonal antibodies, peptides, or other test agents for inhibition of microglial activation induced by fibrin or LPS.
Primary screening to discover novel inhibitors of innate immunity affecting microglia.
Secondary screening to assess ligand specificity and predicted functional activity in in vivo therapeutic use.
Use in target-based drug discovery as functional assays to confirm compounds identified by biochemical screening in a physiologically relevant context.
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