Phase II year
2011
(last award dollars: 2012)
Phase II Amount
$1,015,419
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 QD according 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 I to 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 for cell-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 dynamics of 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 GI drug 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.
Thesaurus Terms: Address;Assay;Binding;Binding (Molecular Function);Bioassay;Biologic Assays;Biological Assay;Cell Communication And Signaling;Cell Line;Cell Lines, Strains;Cell Signaling;Cellline;Cells;Chemicals;Chloride;Chloride Channels;Chloride Ion;Cl- Element;Color;Constipation;Data;Development;Diarrhea;Drug Delivery;Drug Delivery Systems;Drug Targeting;Drug Targetings;Electromagnetic, Microwave;Energy Transfer;Epithelial Cells;Fluorescence;Fluorescence Microscopy;Gastrointestinal Agents;Gastrointestinal Drugs;Generations;H+ Element;High Throughput Assay;Human;Human, General;Hydrogen Ions;Intracellular Communication And Signaling;Ion Channel;Ion Channels, Chloride;Ionic Channels;Ions;Lentiviral Vector;Lentivirus Vector;Life;Man (Taxonomy);Man, Modern;Marketing;Measurement;Measures;Membrane Channels;Microscopy, Fluorescence;Microscopy, Light, Fluorescence;Microwaves;Molecular Interaction;Phase;Physiologic;Physiological;Production;Progress Reports;Protocol;Protocols Documentation;Protons;Q-Dot;Quantum Dots;Reflux;Research;Signal Transduction;Signal Transduction Systems;Signaling;Stomach;Technology;Testing;Time;Validation;Assay Development;Base;Biological Signal Transduction;Cell Type;Commercialization;Cultured Cell Line;Drug Discovery;Fluorescence Microscopy;Gastric;Gastrointestinal;High Throughput Screening;Insight;Microwave Electromagnetic Radiation;Microwave Radiation;Nano Sensing;Nano Sensors;Nanosensing;Nanosensors;Novel;Response;Sensor;Treatment Site