Drugdiscoverypipelinesforneuropsychiatricdisordersaredry.Oneapproachtorejuvenatingthesepipelines 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,ourcollaborativegrouphascreatedaflexibleandscalableprimaryneuronassaydevelopmentsystem thatiscompatiblewithindustrial-levelHTS.Here,ourgoalistooptimizetheseproceduresandworkflows to determine the limit of scalability of neuron-based HTS phenotypic assays so that they can easily supportverylargecampaignsof>200Kcompounds. A substantial proportion of childhood brain disorders are caused by single autosomal dominant variants resultingingenetichaploinsufficiency.Theraregeneticbraindisordersthatarisefromthesevariantsofferthe greatestpotentialfordiscoveryofrobusttherapeuticsbecausethediseasemechanismisoftenstraightforward (i.e.lowproteinexpression).Therefore,arationalestrategytoimproveconditionsinthesepatientswouldbeto 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 functionalproteinexpressioninneuronscausedmostoftenbytruncatingSYNGAP1nonsensevariants.Asa meanstorefinetheneuron-basedHTSsystem,andtoadvancetreatmentforASD-relateddisorders,we are seeking to scale-up and implement an assay for SynGAP expression that is compatible with industrial-levelrobotics.
Inthe firstAim, wewilloptimizeanHTS-compatibleanddisease-relevantSynGAP 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 expressionassaytothe1536-wellformat.ThisminiaturizationprocesswouldenableanHTS-scalescreenof this, or any other related neuron-based phenotypic assay, of up to 400,000 culture wells using a standard screeningbudget.Finally,wewillimplementtheSynGAPexpressionassayinatrueuHTSenvironmentand 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 developproceduresthatwillincreasethescaleofHTScampaignsinneuronsby10-foldormorerelativetothe currentstate-of-the-artinacademicscreeningcenters.Wealsoexpecttovalidateatleastoneleadcompound thatboostsSynGAPexpression,hopefullyfromtherepurposinglibrary.
OneapproachtorejuvenatingdrugpipelinesforneuropsychiatricdisordersistocreateHTSscalableassays basedonrelevantdiseasephenotypesinprimaryneurons,thoughacosteffectiveplatformfortrueHTS- compatibleneuron-basedphenotypicassaysdoesnotcurrentlyexistinacademia.Asameanstorefineour novelneuron-basedHTSsystem,andtoadvancetreatmentforASD-relateddisorders,weareseekingto scale-upandimplementaprimaryneuron-basedphenotypicassaythatcanrevealchemicalprobescapableof reversingtherootcauseofararegeneticbraindisorder.
| Spicer, Timothy P; Hubbs, Christopher; Vaissiere, Thomas et al. (2018) Improved Scalability of Neuron-Based Phenotypic Screening Assays for Therapeutic Discovery in Neuropsychiatric Disorders. Mol Neuropsychiatry 3:141-150 |
| Kilinc, Murat; Creson, Thomas; Rojas, Camilo et al. (2018) Species-conserved SYNGAP1 phenotypes associated with neurodevelopmental disorders. Mol Cell Neurosci 91:140-150 |
