SBIR-STTR Award

Ion channel targeted drug discovery has been hampered by the unavailability of cell-based assays that utilize physiological and pharmacological relevant measurements to characterize compound activity. This project will develop an assay to measure intracellular chloride ion and pH as direct physiological measurements to interrogate the chloride ion channels and proton transporter(s) for GI drug discovery. The Phase I of this proposal will focus on the synthesis, characterization and validation of the chloride ion sensing indicator using T84 cells. The Phase II of this proposal will focus on the development of the pH sensing indicator, integration of the pH sensing assay with the Cl assay; validation of the joint chloride and pH assay using T84 cells by delineating the intracellular coupling dynamics of [Cl-]i and pHi for GI drug discovery; and scaling up the manufacturing of the assays for commercialization.

Public Health Relevance:
Ion channels of chloride and proton transports 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 to measure intracellular chloride ion concentrations, [Cl-]i, and pHi simultaneously for cell-based GI drug discovery. This Phase I proposal will focus on the 1) synthesis and spectral characterization of chloride ion sensing indicator; 2) development of the loading protocols of the indicator; and 3) physio-pharmacological measurements of [Cl-]i in T84 cells. This will enable a chloride ion sensing assay kit to be readily marketed for GI drug discovery and development following the completion of the Phase I. The Phase II of this proposal will focus on the development of the pH sensing indicator, integration of the pH sensing assay with the Cl assay; validation of the joint chloride and pH assay using T84 cells by delineating the intracellular coupling dynamics of [Cl-]i and pHi for GI drug discovery; and scaling up the manufacturing of the assays for commercialization.

Public Health Relevance Statement:
Ion channels of chloride and proton transports 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 to measure intracellular chloride ion concentrations, [Cl-]i, and pHi simultaneously for cell-based GI drug discovery. This Phase I proposal will focus on the 1) synthesis and spectral characterization of chloride ion sensing indicator; 2) development of the loading protocols of the indicator; and 3) physio-pharmacological measurements of [Cl-]i in T84 cells. This will enable a chloride ion sensing assay kit to be readily marketed for GI drug discovery and development following the completion of the Phase I. The Phase II of this proposal will focus on the development of the pH sensing indicator, integration of the pH sensing assay with the Cl assay; validation of the joint chloride and pH assay using T84 cells by delineating the intracellular coupling dynamics of [Cl-]i and pHi for GI drug discovery; and scaling up the manufacturing of the assays for commercialization.

NIH Spending Category:
Bioengineering; Digestive Diseases; Nanotechnology

Project Terms:
Articulation; Assay; Bioassay; Biologic Assays; Biological Assay; Cells; Characteristics; Chloride; Chloride Channels; Chloride Ion; Chlorides; Cl- element; Constipation; Coupling; Data; Development; Diarrhea; Electromagnetic, Microwave; Epithelial Cells; Exhibitions (PT); Exhibitions [Publication Type]; Gastrointestinal Agents; Gastrointestinal Drugs; Generations; H+ element; High Throughput Assay; Human; Human, General; Hydrogen Ions; Hydrogen Oxide; Ion Channel; Ion Channels, Chloride; Ionic Channels; Ions; Joints; Liposomal; Liposomes; Man (Taxonomy); Man, Modern; Marketing; Measurement; Measures; Membrane Channels; Microwaves; Phase; Physiologic; Physiological; Protocol; Protocols documentation; Protons; Q-Dot; Quantum Dots; Reflux; Reporting; Stomach; Time; Validation; Water; base; commercialization; drug discovery; exhibitions; gastric; gastrointestinal; high throughput screening; manufacturing scale-up; microwave electromagnetic radiation; microwave radiation; public health relevance; response; sensor; success; treatment site

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