Research scientist Cerevance CAMBRIDGE, England, United Kingdom
Abstract: Rosie Waters-Hall, Clare Bender, Jenna Harvey, Bernardino Ossola, Philip Pickford, Anna Rowland, Louise Dickson, Emery Smith, Timothy Spicer, Justin Shumate, Louis Scampavia, Daniel Barker, Victoria Mulligan, Keith Page, Xiao Xu, Roland Bürli, Nicola Brice, Lee Dawson, Mark Carlton.
Cerevance, 418 Cambridge Science Park, Milton Road, Cambridge CB4 0PZ, United Kingdom
The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, Department of Molecular Medicine, 130 Scripps Way #1A1, Jupiter, FL 33458, United States
Kir4.1 is a constitutively active, inwardly rectifying potassium (K+) channel encoded by the KCNJ10 gene. Kir4.1 is specifically expressed in brain astrocytes, where it plays a key role in regulating synaptic glutamate levels. Astrocytes are critical for maintaining glutamate levels at the synapse; dysregulation of this process leads to neuronal hyperexcitability, which is a common early pathological feature of many neurodegenerative diseases. Using Cerevance’s proprietary Nuclear Enriched Transcript Sort Sequencing (NETSseq) platform, deep RNA-seq transcriptomic profiles (expression of > 12,000 genes per cell type) from human post-mortem brain tissue for multiple glial and neuronal cell types (>65 different cell types) have been generated. These analyses revealed gene signatures associated with regional differences and disease progression and specifically down-regulation of genes involved in glutamate homeostasis, including KCNJ10. Due to the specificity of expression and the disease associated changes, we hypothesized that enhancement of Kir4.1 current using a small molecule activator could serve as a novel approach to prevent neuronal hyperexcitability in Alzheimer’s disease. To identify small molecular activators, an in vitro screening assay for Kir4.1 using the thallium flux technology was established. Kir4.1 demonstrated high constitutive activity in this assay, necessitating further modulation to establish an assay window for activator detection. We found that lowering pH was sufficient to reduce the constitutive activity of Kir4.1, thereby generating a suitable assay window for activator screening. This assay was miniaturized and used to screen 666,000 compounds for Kir4.1 activity. Despite a low hit rate in the HTS, several compounds were found to activate Kir4.1. This work highlights an in vitro screening approach, to identify small molecule activators of Kir4.1, which may ultimately have therapeutic utility in diseases such as Alzheimer’s disease.
