Development of a GABA Enzyme for Biosensor and Point-of-Care Applications

DESCRIPTION provided by applicant As the most important inhibitory neurotransmitter in the brain a detailed understanding of the implications of gamma aminobutyric acid GABA release remains elusive The measurement of GABA concentrations is a difficult process Microdialysis is the current standard for GABA sampling in the brains of freely moving animals but suffers from low temporal resolution and the need for labor intensive analysis methods By contrast the direct sensing of GABA by modalities including biosensors provides second by second temporal resolution without the need for additional post analysis However biosensors and other monitoring devices require an enzyme to process the analyte of interest The state of the art for the enzymatic conversion of GABA into a transducible signal is the sequential activity of three separate enzymes or antibodies entrapped within nanoparticles For CNS and systemic GABA sensing applications a single GABA oxidase enzyme is necessary No such oxidase enzyme for GABA is currently available To develop this enzyme Pinnacle will team with an interdisciplinary group of two leading scientists at the University of Kansas Professor Mark Richter is an expert in protein engineering and protein folding and Dr Philip Gao is the Director of the Protein Production Core Facility This team has already cloned expressed purified and characterized an oxidase enzyme wt pUUB Ox with some GABA activity During Phase I we will use this oxidase enzyme as a starting scaffold to evolve a true GABA oxidase enzyme At the end of Phase we will have an oxidase enzyme with a x x improvement in GABA activity relative to wt pUUB Ox and a clear path for Phase II to oxidase activity and stability suitable for the specific measurement of physiologically relevant GABA concentrations This evolved GABA oxidase enzyme will in a single reaction step oxidize GABA to produce hydrogen peroxide as a byproduct The GABA oxidase enzyme can be used as the basis for new monitoring paradigms that would otherwise be impossible By the end of Phase II two commercially available products will be available First a GABA biosensor for real time measurement of physiologically relevant levels of GABA in the brain for preclinical models and second a GABA oxidase enzyme for use in a variety of diagnostic and point of care devices PUBLIC HEALTH RELEVANCE GABA is the major inhibitory neurotransmitter in the brain and plays an important role in disorders ranging from newborn seizures to anxiety Alzheimerandapos s Huntingtonandapos s Parkinsonandapos s diseases and a wide variety of cancers The efficacy of disease models in research is well established for the development of treatments The quality of life and economic costs of these and other illnesses in which GABA plays a role are staggering These disorders disrupt millions of lives and America spends billions of dollars each year in hospital visits nursing home stays and lost productivity