Cytoptics Corporation — Department of Health and Human Services SBIR Phase II: DESCRIPTION (provided by applicant): Ion channel drug discovery has been hampered by the l

Description

DESCRIPTION (provided by applicant): Ion channel drug discovery has been hampered by the lack of cell-based high throughput screening (HTS) assay to physiologically and pharmacologically characterize compound activities. Cytoptics Corporation (Cytoptics) proposes to develop a new generation of chloride ion and pH sensing luminous quantum dots (QD) indicators to measure intracellular chloride ion concentrations, [Cl-]i, and pHi for fluorescence microscopy and cell-based high throughput screening (HTS) targeting GI drug discovery. These fluorescence microscopy and cell-based HTS assays utilize the size-tunable, non- overlapping emission wavelengths, ion-selective QD to measure [Cl-]i and pHi simultaneously in T84WT /T84C1C2 as well as in HEK293WT/HEK293C1C-2 cells. Using the quantum dots (QD) as Forester Resonance Energy Transfer (FRET) donors and the respective Cl- and H+ chemical sensors conjugated to the QD as acceptors, these two nanosensors, Cl-QD and pH-QD, quench their fluorescence intensities of the QDaccording to the concentrations of the Cl- and H+ bound to their respective Cl- and H+ sensors. We have accomplished all the proposed Phase I objectives, namely, 1) synthesis and spectral characterization of chloride ion sensing luminous QD, Cl-QD; 2) development of a loading protocol of the Cl-QD into T84 cells; and 3) measurements of the physio-pharmacological [Cl-]i responses in T84WT and T84C1C-2 cells using the Cl- QDTM. In Phase II, Cytoptics will incorporate and extend the accomplishments of Phase Ito produce six assays to target the cell-based GI drug target discovery market. They are (1) the Cl-QDTM assay for fluorescence microscopy; (2) the Cl-QDTM assay for cell- based HTS; (3) the pH-QD assay for fluorescence microscopy; (4) the pH-QD assay forcell-based HTS; (5) the Cl-QDTM and pH-QD integrated assay for cell-based HTS; and (6) the Cl-QDTM and pH-QD integrated assay targeting C1C-2 channels using the stable T84C1C-2 and HEK293C1C-2 cells for GI drug discovery. These novel assays address the critical needs of cell-based HTS drug discovery and will also broadly impact drug discovery research in ionic signal transduction. PUBLIC HEALTH RELEVANCE: Chloride channels and proton transporters are the two predominant drug discovery gastrointestinal (GI) target sites for the treatments of diarrhea, constipation and gastric reflux. Cytoptics Corporation proposes to develop a new generation of chloride ion and pH sensing indicators (Cl-QD and pH-QD, respectively) to simultaneously measure the dynamicsof the intracellular chloride ion concentrations ([Cl-]i) and pH (pHi) in epithelial cells for the first time. These nanosensors will be utilized to interrogate the Cl-channels and H+-transporters as direct physiological and pharmacological assays for GIdrug discovery. Using the quantum dots (QD) with different emission wavelengths for these two nanosensors, Cl-QD and pH-QD emit separate colors with their non-interfering fluorescence intensities varied according to their respective concentrations of Cl- and H+, Cytoptics will produce six assays utilizing these two novel QD-based nanosensors to target the cell-based GI HTS drug discovery market. These novel assays will also provide unparallel insights in the ionic cellular signal transduction mechanisms that heretofore not been possible.