High-Throughput Screening Assay for Detecting Drug-Induced Changes in Synchronized Neuronal Oscillations and Potential Seizure Risk Based on Ca2+ Fluorescence Measurements in Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neuronal 2D and 3D Cultures.

Drug-induced seizure liability is a significant safety issue and the basis for attrition in drug development. Occurrence in late development results in increased costs, human risk, and delayed market availability of novel therapeutics. Therefore, there is an urgent need for biologically relevant, in vitro high-throughput screening assays (HTS) to predict potential risks for drug-induced seizure early in drug discovery. We investigated drug-induced changes in neural Ca2+ oscillations, using fluorescent dyes as a potential indicator of seizure risk, in hiPSC-derived neurons co-cultured with human primary astrocytes in both 2D and 3D forms. The dynamics of synchronized neuronal calcium oscillations were measured with an FDSS kinetics reader. Drug responses in synchronized Ca2+ oscillations were recorded in both 2D and 3D hiPSC-derived neuron/primary astrocyte co-cultures using positive controls (4-aminopyridine and kainic acid) and negative control (acetaminophen). Subsequently, blinded tests were carried out for 25 drugs with known clinical seizure incidence. Positive predictive value (accuracy) based on significant changes in the peak number of Ca2+ oscillations among 25 reference drugs was 91% in 2D vs. 45% in 3D hiPSC-neuron/primary astrocyte co-cultures. These data suggest that drugs that alter neuronal activity and may have potential risk for seizures can be identified with high accuracy using an HTS approach using the measurements of Ca2+ oscillations in hiPSC-derived neurons co-cultured with primary astrocytes in 2D.

Engineering of ligand specificity of periplasmic binding protein for glucose sensing.

A novel glucose-sensing molecule was created based on galactose/glucose-binding protein (GGBP). GGBP mutants at Asp14, a residue interacting with the 4th hydroxyl group of the sugar molecule, were constructed by mutagenesis to improve the ligand specificity of GGBP. The autofluorescence-based analysis of the binding abilities of these engineered GGBPs showed that the GGBP mutants Asp14Asn and Asp14Glu bound only to glucose in a concentration-dependent manner, without being affected by the presence of galactose. The Phe16Ala mutation, which leads to an increase in the K (d) value toward glucose, was then introduced into these two glucose-specific mutant GGBPs. One of the constructed GGBP double-mutants, Asp14Glu/Phe16Ala, had a glucose specificity with a K(d) value of 3.9 mM, which makes it suitable for use in the measurement of the physiological glucose concentration. Our results demonstrate that it is possible to construct a GGBP which specifically recognizes glucose and has a higher K(d) value and use it as a molecular recognition element of blood glucose monitoring systems by combining two different mutations based on the 3D structure of GGBP.