A Scalable Neuron-Based High-Throughput Screening Platform for the Discovery of Compounds that Restore Protein Expression Caused by Genetic Haploinsufficiency
A Scalable Neuron-Based High-Throughput Screening Platform for the Discovery of Compounds that Restore Protein Expression Caused by Genetic Haploinsufficiency
批准号:
9370360
负责人:
GAVIN R RUMBAUGH
金额:
$68.73万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
2017
资助国家:
美国
项目状态:
已结题
起止时间:
2017-07-01 至 2020-03-31
关键词:
AcademiaAllelesAutistic DisorderBiologicalBiological AssayBrainBrain DiseasesBudgetsChemicalsChildhoodClinicalDevelopmentDiseaseEnvironmentEpilepsyEvaluationFluorescenceGene TargetingGeneticGoalsHandHumanIndustrializationIntellectual functioning disabilityKnock-in MouseLeadLibrariesMagicMindMiniaturizationModelingMolecularMusNatureNeuronsOnline Mendelian Inheritance In ManPatientsPharmaceutical PreparationsPhenotypePlant RootsProceduresProcessProteinsReporterReportingReproducibilityResearchRoboticsSeriesSystemSystems DevelopmentTestingTherapeuticVariantWorkassay developmentbasecost effectivedesigndisease phenotypedrug discoveryexperimental studyflexibilityhigh throughput screeningimprovedminiaturizeneuropsychiatric disordernovelprotein expressionscale upscreening
中文摘要
项目总结
英文摘要
PROJECT SUMMARY
Drug discovery pipelines for neuropsychiatric disorders are dry. One approach to rejuvenating these pipelines
would be to create assays based on relevant disease phenotypes in primary neurons, something that is
currently lacking. However, a scalable assay development platform that is based on bona fide neurons,
remains cost effective, and that can support industrial level HTS does not currently exist. Over the past five
years, our collaborative group has created a flexible and scalable primary neuron assay development system
that is compatible with industrial-level HTS. Here, our goal is to optimize these procedures and workflows
to determine the limit of scalability of neuron-based HTS phenotypic assays so that they can easily
support very large campaigns of >200K compounds.
A substantial proportion of childhood brain disorders are caused by single autosomal dominant variants
resulting in genetic haploinsufficiency. The rare genetic brain disorders that arise from these variants offer the
greatest potential for discovery of robust therapeutics because the disease mechanism is often straight forward
(i.e. low protein expression). Therefore, a rationale strategy to improve conditions in these patients would be to
treat them with “magic bullet” compounds that raise expression of functional proteins from the remaining
undamaged allele (e.g. “boosting compounds”). De novo nonsense variants that cause SYNGAP1
haploinsufficiency lead to a genetically-defined form of intellectual disability with autism and epilepsy (MRD5;;
OMIM#603384) that may explain up to 1-2% of all ID cases. The accepted cause of this disorder is low
functional protein expression in neurons caused most often by truncating SYNGAP1 nonsense variants. As a
means to refine the neuron-based HTS system, and to advance treatment for ASD-related disorders, we
are seeking to scale-up and implement an assay for SynGAP expression that is compatible with
industrial-level robotics. In the first Aim, we will optimize an HTS-compatible and disease-relevant SynGAP
expression assay. This assay is based on mouse primary neurons where tdTomato fluorescence reflects
steady-state endogenous SynGAP protein levels. In the second aim, we will miniaturize the SynGAP
expression assay to the 1536-well format. This miniaturization process would enable an HTS-scale screen of
this, or any other related neuron-based phenotypic assay, of up to 400,000 culture wells using a standard
screening budget. Finally, we will implement the SynGAP expression assay in a true uHTS environment and
then validate lead compounds that emerge from a 20K compound pilot screen, including a 10K compound
repurposing screen of known “safe in human” compounds. The impact of this project that we expect to
develop procedures that will increase the scale of HTS campaigns in neurons by 10-fold or more relative to the
current state-of-the-art in academic screening centers. We also expect to validate at least one lead compound
that boosts SynGAP expression, hopefully from the repurposing library.
期刊论文(0)
专著(0)
科研奖励(0)
会议论文
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Causal Interactions between genetic risk, precise cortical connectivity, and autism-associated behaviors
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依托单位:
Impact of SynGAP1 Mutations on Synapse Maturation and Cognitive Development
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依托单位:
Impact of SynGAP1 Mutations on Synapse Maturation and Cognitive Development
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依托单位:
Impact of SynGAP1 Mutations on Synapse Maturation and Cognitive Development
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依托单位:
Impact of SynGAP1 Mutations on Synapse Maturation and Cognitive Development
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资助金额:$61.46万
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财政年份:2012
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依托单位:
Targets of NMDA Receptor Signaling that Promote the Maturation of Emotional Circu
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依托单位:
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依托单位:
Regulation of Memory Formation by the GTPase-activating Protein SynGAP
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依托单位:
Probing circuit assembly errors in a monogenic model of comorbid brain disorders
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Regulation of Memory Formation by the GTPase-activating Protein SynGAP
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海外基金