# Patent

## Title
Screening test drugs to identify their effects on cell membrane voltage-gated ion channel

## Abstract

A variety of applications, systems, methods and constructs are implemented for use in connection with screening of ion-channel modulators. Consistent with one such system, drug candidates are screened to identify their effects on cell membrane ion channels and pumps. The system includes screening cells having light responsive membrane ion switches, voltage-gated ion switches and fluorescence producing voltage sensors. A chemical delivery device introduces the drug candidates to be screened. An optical delivery device activates the light responsive ion switches. An optical sensor monitors fluorescence produced by the voltage sensors. A processor processes data received from the optical sensor. A memory stores the data received from the optical sensor.

## Background
RELATED PATENT DOCUMENTS
This patent document claims the benefit, under 35 U.S.C. §119(e), of U.S. Provisional Patent Application Ser. No. 60/955,116, entitled Cell Line, System and Method for Optical-Based Screening of Ion-Channel Modulators and filed on Aug. 10, 2007; this patent document is fully incorporated herein by reference.
This patent document also claims priority, as a CIP under 35 U.S.C. §120, to the following patent documents which are also individually incorporated by reference: U.S. patent application Ser. No. 11/651,422 filed on Jan. 9, 2007 now U.S. Pat. No. 8,926,959 and entitled, System for Optical Stimulation of Target Cells), which is a CIP of U.S. patent application Ser. No. 11/459,636 filed on Jul. 24, 2006 now U.S. Pat. No. 8,906,360 and entitled, Light-Activated Cation Channel and Uses Thereof, which claims the benefit of U.S. Provisional Application No. 60/701,799 filed Jul. 22, 2005.


FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with Government support under contract OD000616 awarded by the National Institutes of Health. The Government has certain rights in this invention.


FIELD OF THE INVENTION
The present invention relates generally to systems and approaches for screening drug candidates and more particularly to a cell line, system and method for optically-based screening of the drug candidates with respect to their effect on cellular ion channels.
BACKGROUND
Ion channels and ion pumps are cell-membrane proteins that control the transport of positively or negatively charged ions (e.g., sodium, potassium and chloride) across the cell membrane. Ion channels play an important part of various animal and human functions including signaling and metabolism. Ion-channel dysfunctions are associated with a wide variety of illnesses. For instance, diseases resulting from ion-channel dysfunctions in the central nervous system include anxiety, depression, epilepsy, insomnia, memory problems and chronic pain. Other diseases resulting from ion-channel dysfunctions include cardiac arrhythmia, and type II diabetes. Researchers are continually discovering diseases associated with ion-channel functionality.
Several drugs have been discovered to modify ion-channel functionality; however, the number of clinically approved drugs for restoring ion-channel functionality is limited. A major bottleneck in the discovery and development of new ion-channel drugs lies in the technical challenge of quickly, efficiently and cheaply screening drug candidates to identify structures that affect ion-channel functionality. Common screening techniques use patch clamping to measure the voltage and/or current in a cell. Micropipettes affixed to the cell membrane obtain the measurement. For example, whole-cell configuration can be used to monitor the functionality of the ion channels throughout the cell. In this manner, changes in voltage or current due to an introduced drug can be monitored. Such methods require contact between the micropipette and the cell. For this and other reasons, such techniques leave room for improvement in their ability to screen drugs quickly, efficiently and cheaply.
These and other issues have presented challenges to screening of drug candidates, including those affecting ion-channel function.



BRIEF DESCRIPTION OF THE DRAWINGS
The invention may be more completely understood in consideration of the detailed description of various embodiments of the invention that follows in connection with the accompanying drawings, in which:
FIG. 1A shows a block diagram of a system for optical drug screening, according to an example embodiment of the present invention;
FIG. 1B shows a specific system diagram of a large-format, quasi-automated system for drug screening in accordance with the present methodology, according to an example embodiment of the present invention;
FIG. 2 shows a system diagram of a small-format, fully automated drug screening system which operates in accordance with the invented methodology, according to an example embodiment of the present invention;
FIG. 3A depicts the workings of an example of emitter/detector units, according to an example embodiment of the present invention;
FIG. 3B depicts the workings of another embodiment of emitter/detector units, according to an example embodiment of the present invention;
FIG. 4A depicts an electronic circuit mechanism for activating the LED emitters used within the emitter/detector units, according to an example embodiment of the present invention;
FIG. 4B depicts an electronic circuit mechanism for light detection by the emitter/detector units, according to an example embodiment of the present invention;
FIG. 5 shows a timeline for a sequence of events in the context of an example screening process, according to an example embodiment of the present invention;
FIG. 6 illustrates an example of a layout of cell and drug samples within the wells of a well-plate, according to an example embodiment of the present invention; and
FIG. 7 illustrates the context in which the disclosed invention may be employed within a larger system that facilitates high-throughput drug screening, according to an example embodiment of the present invention.



While the invention is amenable to various modifications and alternative forms, specifics thereof have been shown by way of example in the drawings and will be described in detail. It should be understood, however, that the intention is not to limit the invention to the particular embodiments described. On the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
DETAILED DESCRIPTION
The present invention is believed to be useful for enabling practical application of a variety of optical-based screening systems, and the invention has been found to be particularly suited for use in systems and methods dealing with identification of ion-channel modulating drugs. While the present invention is not necessarily limited to such applications, various aspects of the invention may be appreciated through a discussion of various examples using this context.
Recently discovered techniques allow for stimulation of cells resulting in the rapid depolarization of cells (e.g., in the millisecond range). Such techniques can be used to control the depolarization of cells such as neurons. Neurons use rapid depolarization to transmit signals throughout the body and for various purposes, such as motor control (e.g., muscle contractions), sensory responses (e.g., touch, hearing, and other senses) and computational functions (e.g., brain functions). Thus, the control of the depolarization of cells can be beneficial for a number of different biological applications, among others including psychological therapy, muscle control and sensory functions. For further details on specific implementations of photosensitive bio-molecular structures and methods, reference can be made to one or more of the above-listed patent documents (by Karl Deisseroth et al.) which are fully incorporated herein by reference. These references discuss use of blue-light-activated ion-channel channelrhodopsin-2 (ChR2) to cause calcium (Ca++)-mediated neural depolarization. Also discussed in one or more of these references are other applicable light-activated ion channels including, for example, halorhodopsin (NpHR) in which amber light affects chloride (Cl−) ion flow so as to hyperpolarize neuronal membrane, and make it resistant to firing. Collectively, these light-sensitive proteins, serving to regulate membrane voltage using ion switches that, when activated (or deactivated) in response to light, function as channels or pumps, are referred to herein as light-responsive ion switches or light-activated membrane potential switches (LAMPS).
Consistent with one example embodiment of the present invention, a system screens for ion-channel and ion-pump affecting compounds. The system introduces one or more drug candidates that could either block or enhance the activity of ion-channels or ion-pumps to cells that were made optically responsive by the addition of the above mentioned proteins (ChR2 and NpHR), for the purpose of screening the drug candidates. Light triggers optically responsive ion channels in the cells causing a change in the voltage seen across the cell membrane. The voltage change stimulates voltage-gated ion channels in the cells which will then cause a change in ion concentrations that can be read as optical outputs. These optical signals are detected and used to determine what effect, if any, the drug candidates have on the voltage-gated ion channels.
In addition to NpHR and ChR2, there are a number of channelrhodopsins, halorhodopsins, and microbial opsins that can be engineered to optically regulate ion flux or second messengers within cells. Various embodiments of the invention include codon-optimized, mutated, truncated, fusion proteins, targeted versions, or otherwise modified versions of such ion optical regulators. Thus, ChR2 and NpHR (e.g., GenBank accession number is EF474018 for the ‘mammalianized’ NpHR sequence and EF474017 for the ‘mammalianized’ ChR2(1-315) sequence) are used as representative of a number of different embodiments. Discussions specifically identifying ChR2 and NpHR are not meant to limit the invention to such specific examples of optical regulators. For further details regarding the above mentioned sequences reference can be made to “Multimodal fast optical interrogation of neural circuitry” by Feng Zhang, et al, Nature (Apr. 5, 2007) Vol. 446: 633-639, which is fully incorporated herein by reference.
In one instance, the system allows for different drug candidates to be screened without necessitating extensive setup between screenings. For example, an assay may be performed using optics both to stimulate the optically responsive cells and to detect the effectiveness of the drug. The use of optics instead of manual contacts, e.g., using a whole-cell patch clamp, can be particularly useful in increasing the throughput of the assay screening process. For instance, the time between screenings can be reduced by minimizing or eliminating physical manipulations otherwise necessary to stimulate or detect ion flow in the target cells. The cells can also be prepared prior to the screening process because the test equipment need only be optically coupled to the prepared cells. In another instance, throughput may be increased by screening a number of different drugs simultaneously using, for example, an array of photo detectors and a corresponding array of modified cells exposed to different drugs.
Consistent with another embodiment of the present invention, an optically-responsive cell line is created to screen for drugs that affect the functionality of ion channels. The cell line includes cells that co-express optically responsive ion switches of Channelrhodopsin-2 (ChR2) or NpHR, a voltage-gated Ca2+ channel and a hyperpolarizing channel/pump (e.g., hERG or TASK1, that can lower the membrane voltage to a point where the voltage-gated Ca2+ channel will be in a closed state). The system measures the concentration of Ca2+ using an indicator dye (e.g., Fura-2) or genetically encoded activity sensor. The above mentioned components are introduced to the cell line by standard liposomal transfection methods and the ChR2 related channel is stimulated using (blue) light; for further information in the regard, reference may be made to the patent documents cited herein and to the articles cited supra. Time lapse images of light from the Ca2+ sensitive portion of the system are taken and stored as data. A processor analyzes the data to identify potential channel-affecting drugs. For instance, the processor may identify all chemicals that have concentrations of Ca2+ that do not fall within expected parameters (e.g., concentrations that exceed or are less than an expected range of concentrations).
In a specific instance, the cell line is derived from 293T cells by co-expressing ChR2 and a voltage-gated Ca2+ channel. The 293T cells (and 293T cell line) are a variant of Human Embryonic Kidney (HEK) cells that include the Simian vacuolating virus 40 (SV40) T antigen (see, e.g., N. Louis, C. Evelegh F. L. Graham, Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line, Virology, 233(2):423-9, Jul. 7, 1997; see also U.S. Pat. No. 5,939,320 to Littman, et al. filed Jun. 19, 1996, U.S. Pat. No. 6,790,657 to Arya filed Jun. 28, 2001 and U.S. Pat. No. 6,489,115 to Lahue, et al. filed Dec. 3, 2002). Expression of the light-responsive ion channels, the voltage-gated ion channels and the hyperpolarizing channels by the 293T cells may be accomplished using appropriate transfection vectors.
More specifically, the cell lines may be derived from a stable homogeneous cell line such as HEK293, NIH3T3, or CHO. Several genes responsible for making different subunits of calcium channels have been introduced into the cell lines to provide functional calcium channel activity. In addition to the calcium channel genes, an inward-rectifying potassium channel may be expressed to mimic the natural state of calcium channels by maintaining a more hyperpolarized membrane potential (compared to the default resting membrane potential of HEK293, NIH3T3, or CHO cell lines). Also, a light-activated cation channel channelrhodopsin-2 (ChR2) may be expressed to facilitate optical depolarization and subsequent activation of the calcium channels. Another option includes the expression of a light-activated chloride pump Natronomonas pharonis halorhodopsin (NpHR) to enable rapid optical hyperpolarization of the cell membrane potential.
This cell line based approach is not limited to voltage-gated calcium channels. For example, similar cell lines can be created for voltage-gated sodium (e.g., Nav1.1 through Nav1.9), potassium (e.g., Kv such as hERG, TASK1, Shaker, or KvLQT1), or chloride conducting channels/pumps (e.g., members of the CLC family of chloride channels). The methods of introducing such genes into the cell line are known in the art and may include, for example liposomal tranfection, or viral gene transfer. For further information in this regard, reference may be made to one or more of the following references:

    Warren Pear, Transient Transfection Methods for Preparation of High-Titer Retroviral Supernatants, Supplement 68, Current Protocols in Molecular Biology, 9.11.1-9.11.18, John Wiley & Sons, Inc. (1996).
    R. E. Kingston C. A. Chen, H. Okayama, and J. K. Rose, Transfection of DNA into Eukarotic Cells. Supplement 63, Current Protocols in Molecular Biology, 9.1.1-9.1.11, John Wiley & Sons, Inc. (1996).
    R. Mortensen, J. D. Chesnut, J. P. Hoeffler, and R. E. Kingston, Selection of Transfected Mammalian Cells, Supplement 62, Current Protocols in Molecular Biology, 9.5.1-09.5.19, John Wiley & Sons, Inc. (1997).
    H. Potter, Transfection by Electroporation, Supplement 62, Current Protocols in Molecular Biology, 9.3.1-9.3.6, John Wiley & Sons, Inc. (1996).
    T. Gulick, Transfection using DEAE-Dextran, Supplement 40, Current Protocols in Molecular Biology, 9.2.1-9.2.10, John Wiley & Sons, Inc. (1997).
    R. E. Kingston, C. A. Chen, H. Okayama, Transfection and Expression of Cloned DNA, Supplement 31, Current Protocols in Immunology (CPI), 10.13.1-10.13.9, John Wiley & Sons, Inc.

Each of the above references is incorporated by reference.



These and other transfer vectors may be generated using various genetic engineering techniques. For instance, the transfer vectors may be derived from a provirus clone of a retrovirus, such as an immunodeficiency virus (e.g., HIV-1 or HIV-2, or SIV). For further details on the use of 293T cells and transfection thereof, reference can be made to U.S. Pat. No. 6,790,657 (entitled, Lentivirus Vector System, to Arya), which is fully incorporated herein by reference.
In one embodiment of the invention, optical stimulation of the modified cells may be altered to determine specific properties of an introduced drug candidate. For example, the intensity of the optical stimulus may be modified to change the corresponding level of depolarization. The level of desired depolarization can be tuned to further characterize the effectiveness of the drug under test. In another example, the optical stimulus may include rapid pulsing of the light. By correlating the temporal relationship between the optical stimulus and the resultant detected fluorescence, the drug may be further characterized in terms of a kinetic response. Thus, the drug may be characterized for a variety of different aspects including, but not limited to, the steady state effect on ion concentrations, a change in the level of depolarization necessary to trigger the voltage gated ion channels and the effect on repeated depolarization.
In one embodiment, the system allows for simple calibration of the optical stimulation and/or detection. The modified cells may be optically stimulated prior to introduction of the drug candidate. The ion channel responsiveness is detected and recorded. The recorded values may be used as a baseline for comparison to the ion channel responsiveness of the same modified cells after the introduction of the drug under test. The recorded values may also be used to modify the optical stimulus or the sensitivity of the optical detector. Such modifications may be applied to an individual test sample or an array of test samples. For such an array of test samples, each test sample may be individually calibrated by adjusting the corresponding optical stimulus. Similarly, each corresponding photo detector may be individually adjusted.
FIG. 1A shows a basic block diagram of a system for screening for ion-channel affecting drugs, according to an embodiment of the invention. Optical control 104 communicates with database 102, optical source 106 and optical detector 109. Optical source 106 provides optical stimulus to test sample 108. Test sample 108 includes the drug under test, cells with optically responsive ion channels, and a voltage/ion indicator. In one instance, the indicator fluoresces in response to light from optical source 106. Optical control 104 may also include a reconfigurable readout, so that as different LAMPS and different LEIAs are used, the same control system can be readily adapted to each paradigm. Optical detector 109 produces a signal responsive to such florescence, and optical control 104 receives the produced signal. The optical control 104 stores data obtained from the signal in database 102. The information stored may include factors such as the intensity, duration and wavelength of the detected light. In a particular instance, the stored data can be compared against baseline data, where the baseline data corresponds to data recorded prior to the introduction of the drug to the test sample 108. In another instance, optical source 106 may vary the intensity, duration or other parameters related to the control of optical source 106. These and other parameters may be stored in database 102.
It should be apparent that optical source 106 may be implemented using a single light source, such as a light-emitting diode (LED), or using several light sources. Similarly, optical detector 109 may use one or more detectors and database 102 may be implemented using any number of suitable storage devices.
FIG. 1B shows a system diagram of a large-format, quasi-automated system for drug screening in accordance with a specific embodiment of the invention. Control device 101 (e.g., a computer or control logic) controls various processes, and serves as the central point of system input/output functions. The environment may be maintained at an appropriate temperature, humidity, carbon dioxide level and ambient light level within the walls of the climate control chamber 105, with the help of one or more sensors 114 (e.g., thermostat, carbon dioxide sensor and humidity sensor), carbon dioxide and humidifier apparatus 112, and heater 110. Multi-well tray 141 contains test wells 140 for holding cultured cells, drugs, and other ingredients needed for each test. Tray 141 rests upon X-Y-Z table 125, the movement of which is carried out by table actuators 120, under control of computer 101. Xenon lamp 155 emits high-intensity white light 156, which is passed through color filter 160. In the case that ChR2 is used for stimulating the cells within wells 140, color filter 160 is blue, causing blue light 161 to exit the filter, and strike dichroic mirror 170. Blue light 161 then passes upward, through microscope objective lens apparatus 130, and through bottom of transparent tray 141. In this fashion, the contents of wells 140, with their transparent undersides, are illuminated. When a separate wavelength of light is required to stimulate a fluorescent light-emitting indicator of cellular activity, a filter of the appropriate specification may be substituted for the previous filter 160, causing light of the proper wavelength for this latter task to be piped toward well 140. If the cells within well 140 have been light-sensitized, and if the drug being tested in each of these wells does not suppress the process, a light-emitting indicator of cellular activity (LEIA), which has also been added to each well or expressed by the cells via genetic modification, will emit light in accordance with the voltage change caused by the effect of the light. This second wavelength of light, which may be much smaller in magnitude than the stimulation light, is collected by microscope turret 135, and will also be passed through dichroic mirror 175, onto the lens of (CCD) camera 180.
Dichroic mirror 170 allows for upward reflection of both the wavelength required to stimulate the optical gating of the membrane (e.g., blue for ChR2), and the wavelength required by any LEIA used (e.g., ultraviolet for FURA-2). This dichroic mirror may be arranged to allow passage of the output spectrum of the LEIA (e.g., blue-green for FURA-2) with minimal reflection or absorption.
FIG. 2 is a system diagram of an automated-drug-screening system, according to an example embodiment of the invention. Emitter/detector units 250 make up the emitter/detector array 251. Emitter/detector array 251 matches the number, size, and layout of the wells on tray 240. Tray holding device 225 permits tray swapping mechanism 220 to rapidly move a new tray into position once testing of a given tray has been completed. The entire process may be automated, and under the control of device 201. Device 201 can be implemented using a computer, control logic, programmable logic arrays, discrete logic and the like. The introduction of the drug candidates under test can also be automated using a machine that provides a reservoir for storing the drugs and a dispensing nozzle for injecting the drugs into the tray. In a manner similar to that shown by FIG. 1, the environment within the walls of the climate control chamber 205 may be maintained at an appropriate temperature, humidity, carbon dioxide level and ambient light level, with the help of thermostat, carbon dioxide sensor and humidity sensor 214, carbon dioxide and humidifier apparatus 212, and heater 210. The use of multiple stimulator/detector elements simultaneously and in parallel, can be particularly useful for augmenting the speed of the overall process. Low cost elements may be used to make multiple parallel detectors (e.g., the components detailed below in description of FIGS. 3A and 3B), the multiple parallel emitter/detector units may also be quite economically feasible.
FIG. 3A depicts the workings of emitter/detector units, such as those shown in FIG. 2, according to an example embodiment of the invention. An LED stimulates light-sensitive ion channels of cells located within a well, and a photodiode detects the response of a LEIA. In this embodiment, device 301 includes LED 310, which produces light pulses 311, at the proper wavelength, pulse frequency and intensity, so as to stimulate light-sensitive transgenic cells 305 in culture within well 306. In the case that ChR2 is the molecular target being used, blue light of 1-10 mW/mm2 is generally appropriate. Due to the presences of an LEIA (e.g., a voltage-sensitive dye or a calcium dye), light 316 is returned from cells 305, and is detected by photodiode 315. In the case that RH 1691 being used, red light is fluoresced and detected by photodiode 315. In the absence of cellular depolarization, no fluorescence is detected by photodiode 315. Other light detecting technologies may also be used instead of a photodiode including phototransistors, and CCD elements.
The combination of photostimulation with optical imaging techniques of LEIAs may be useful for a number of different reasons. For example, photostimulation may simplify the study of excitable cells by reducing the need to use mechanical electrodes for stimulation. Several commercially available LEIAs are suitable for photogrammetrically indicating the activation of electrically excitable cells. One such LEIA is calcium dye Fura-2, which may be stimulated with violet/ultraviolet light around 340 nm, and whose fluorescent output is detectable as blue-green light around 535 nm. Another example is voltage sensitive dye RH 1691, which may be stimulated with green light at about 550 nm, and whose fluorescent output is detectable as red light at about 70 nm. Another example is voltage sensitive dye di-4-ANEPPS, which is stimulated by blue light at about 560 nm, and whose fluorescent output is detectable as red light at about 640 nm.
FIG. 3B depicts the workings of another embodiment of the emitter/detector units shown in the FIG. 2, in which multiple effects are tested within the context of a single well. For example, the cells 355 in the wells 356 may express both ChR2 and NpHR, and hence be sensitive to both the depolarizing effects of blue light, and the hyperpolarizing effects of amber light. Device 351 includes LED 360, which is used for the stimulation of the targeted ion channel or pump (e.g., ChR2) of light-sensitive transgenic cells 355. Additional LED 375 may be used to stimulate a second targeted ion channel or pump (e.g., NpHR). Yet another LED 380 may be used to stimulate a voltage sensitive dye (e.g. RH1691 or calcium dye, such as Fura-2). Each LED may be arranged to output specific wavelengths and intensities for stimulus of respective targeted compounds. In one instance, an LED may affect more than one target, depending upon the specific sensitivities of each compound used. Photodiode 365 detects the fluorescence of a selected voltage dye, while photodiode 370 is sensitive to the spectrum fluoresced by a selected calcium dye. The use of multiple LEDs for the same cell allows for the stimulation of LEIAs at different wavelengths. Multiple LEDs may also be used to detect different light wavelengths emitted by the LEIA.
FIG. 4A depicts an electronic circuit mechanism for activating the LED emitters used within the emitter/detector units, according to an example embodiment of the invention. Control device 401 generates a “light on signal” 402 to transistor base 405. This “light on signal” 402 will remain on for the duration of a light flash desired, or alternatively may turn on and off in order to produce rhythmic light flashes at a specified frequency. Light on signal 402 permits (conventional) current to flow from power source 410, through resister 411, and through transistor collector 407 and transistor emitter 412, to ground 413. Current is also thereby permitted to pass through resistor 415, and into LED 420. LED 420 emits light 421, which falls upon well 425. In a particular instance, the transistor functions as transconductance amplifier of signal 402. In this manner, light of the appropriate wavelength, intensity and frequency is delivered to cells within the well 425, so as to cause them to stimulate the particular ion channel (e.g., ChR2) or pump (e.g., NpHR), or other photoactive membrane structure being used to regulate the activity of electrically excitable cells. Various other circuits are also possible. For example, other circuits can be used in place of circuit 406 to control LED 420 including, but not limited to, replacing the transistor with an operational amplifier, a field-effect-transistor, a resistor divider network, transistor-transistor logic, push-pull driver circuits and switches.
FIG. 4B depicts an example electronic circuit mechanism for light detection by the emitter/detector units, according to one embodiment of the invention. Control device 450 may (optionally, depending upon specific implementation) provide power to photodiode 455. Photodiode 455 receives fluoresced (emitted) light 456 from the LEIA on the cells within well 457. The received light results in an output signal. This output passes through resistor 460, and is input to Schmitt triggered hex inverter 470, which conditions the signal, providing a clean “high” or “low value” to be input to computer 450.
Operation of the photodetector is shown in photovoltaic mode, but the element may also be used in the photoconductive mode of operation. Of course, many other light-detection devices and methods may also be used, including phototransistors, photothyristors, and charged-coupled device (CCD) elements, or arrays of elements.
Alternatively, the 4B circuit can be used without Schmitt-triggered hex inverter 470, permitting a continuum of signal intensities to be transmitted directly to an analog input to computer 450 or to an analog-to-digital converter. Various other signal conditioning circuits are also possible.
FIG. 5 shows a sequence of steps using the embodiment shown in FIGS. 2, 3 and 4, in the context of projected high-throughput process time course 500 and in accordance with one embodiment of the invention. In step 505, light of the appropriate wavelength and intensity for the targeted ion channel is flashed—in this case for approximately 3 seconds. Concurrently, a LEIA stimulation flash 510 may optionally be triggered, depending upon the specific voltage or calcium dye, etc. being used. This LEIA compound may have been previously added to the well, or may be (artificially) genetically imparted upon the cells such that the chemical is produced/expressed by the cells. In step 515, the light signal produced by the LEIA is detected by the photodetector element (e.g. photodiode). For example, RH1691, fluoresces red light at about 70 nm.
In step 520, the signal resulting from the impingement of light onto the photodetector element is sent back to the computer. This may be a binary (e.g. “high” versus “low” signal intensity), or may be graded to reflect a continuum of activation levels. In the case that multiple photodetectors are used to determine energies at different wavelengths, the individual readings of these photodetectors may be logged in parallel or in sequence for appropriate interpretation in a later stage of the automated process. In step 530, the system calls for the next tray to be placed by the automated system. The next tray is moved into position at step 535 and the process may be repeated until all trays in a batch have been processed.
The level of light fluoresced is typically much lower than that required to optically stimulate a cell via light-sensitive ion channels or pumps. For example, ChR2 may require blue light of 1-10 mW/mm2 or more in order to robustly depolarize cells. RH 1691 may require approximately 0.1 mW/mm2 to stimulate it. Given that RH1691 shows significant sensitivity to blue light, (peak sensitivity is at the blue-green wavelengths), RH1691 is adequately stimulated by the same pulse used to stimulate ChR2, but emits light upon depolarization at a power of only on the order of 0.001 mW/mm2. This small amount of output light would be difficult to distinguish from the comparatively massive blue pulse used to stimulate ChR2, even if efficient filters were used in front of the detectors. Fortunately, temporal differences between the ChR2 stimulation (with simultaneous LEIA stimulation), and the fluorescent output of depolarized cells can be used to distinguish the light sources. For instance, the dye-based fluorescence may continue for a few seconds after the delivery of the depolarization pulse and the resultant action potential. Thus in some instances, such as a non-fluorescent LEIA or a luminescent activity dye, a separate stimulation flash is not required.
The amount of time allotted for light delivery may vary, and depends on factors including the level of light-gated ion channel/pump expression, and the density and characteristics of other ionic channel characteristics of that cell population. The amount of time allotted for light receipt may vary, and depends upon factors including the degree of accuracy required for the screening session. The amount of time allotted for well-plate (tray) changing may vary, and depends upon factors including the mechanical speed of the automated apparatus. If fast neurons are used as the cells being tested, the cellular stimulation and LEIA detection process may be accomplished in milliseconds.
In an example process, a 293T cell line expressing TASK-1 (to simulate the natural hyperpolarized membrane potential of neurons), ChR2 (to induce depolarization of the cell membrane), and the L-type calcium channel are used. Whole-cell patch clamping experiments show that the membrane of the modified 293T cell line is hyperpolarized to the point where the L-type calcium channels are closed. The cells are stimulated for 5 seconds with continuous blue light (470 nm) to activate ChR2. ChR2-mediated depolarization opens the co-expressed voltage-gated calcium channels. Upon ChR2 illumination, a strong calcium influx is recorded using a genetically-encoded calcium dye indicator, which fluoresced light with cellular depolarization. Nimodopine, a well-known L-type calcium channel blocker, abolishes the calcium influx—and hence the fluoresced signal when applied to the cells for 10 minutes. This data demonstrates the effectiveness of the system described herein.
The process above may be repeated under varying conditions. For example, a given set of cells may be tested with no drug present, and subsequently with one or more drugs present. The response of electrically-excitable cells under those conditions may be thereby documented, compared and studied. If the invention is implemented with at least one emitter/detector for each well on a tray and at least two concurrently operating devices, continuous operation may be maintained for extended periods of time.
FIG. 6 illustrates an example of a layout of cell and drug samples within the wells of a well-plate which is suitable for use within an embodiment of the invention. In this figure, well-plate 601 (also referred to herein as a “tray” contains wells 605 (examples), which are organized into columns 625, labeled with numbers 1-12 and rows 620, labeled with letters A-H. More specifically, an example column and row are defined by 610 and 615 respectively.
As an example of a functional layout of contents introduced into these wells, rows A-H of a single plate might be used for the testing of two different drugs. To represent a baseline condition, column 1 might contain optically gated cells, an endogenous or exogenous LEIA, but no drug. Columns 2-6 might be used for five different concentrations of Drug X, one concentration level per column. Likewise, columns 7-11 might be use for five different concentrations of Drug Y, one concentration per column. Column 12, while fully usable, is left unused in this particular example.
Variables in the various wells might include the type of cell being tested, the type of ion channel being tested for, the type of drug placed in the cell, the concentration of the drug placed in the well, the specific LEIA used, and the optical gating stimulation parameters (e.g. wavelength, intensity, frequency, duration) applied to the cells in that well.
FIG. 7 illustrates the context in which the disclosed invention may be employed within a larger system which facilitates high-throughput drug screening. Well-plate 706 contains wells 705. These are carried forward by conveyer 720, which may be a device such as a conveyor belt, robotic transporter or other delivery mechanism. Pipettes 710 are held in array by robotic member 715, and serve to inject the proper number of cultured cells and media into wells 705. Subsequently, well-plate 706 is moved down conveyer 720, where robotic member 725, analogous to robotic member 715 and also containing pipettes, injects the proper amount of a LEIA into wells 705. Conveyer 720 then brings well-plate 705 into screening chamber 730. An emitter/detector apparatus, such as those described in connection with FIG. 2, FIG. 3A, FIG. 3B, FIG. 4A, and FIG. 4B, is located within chamber 730. Additionally, portions of the processes described in FIG. 5 may occur within this chamber. Subsequently, well-plates 735 is moved out of screening chamber 730 by conveyor 740, and discarded at 745. In an alternative embodiment, one or more robotic devices may move pipettes 710, screening chamber 730, etc. to the locations of well-plate 706, rather than vice-versa.
Consistent with the above discussion, example screening methods could include the collection of multiple data points without having to switch samples. Because control over the samples is reversible in the same sample preparation by simply turning the activating light on and off with fast shutters, the same samples can be reused. Further, a range of patterns of stimulation can be provided to the same cell sample so that testing can be performed for the effect of drugs without concern with regards to differences across different sample preparations. By modulating the level of excitation (e.g., by ramping the level from no light to a high or maximum intensity), the effect of the drug across a range of membrane potentials can be tested. This permits for the identification of drugs that are efficacious during hyperpolarized, natural, or depolarized membrane potentials.
The cell lines described herein may be a particularly useful for detailed characterization of drug candidates in a high-throughput manner. Optical control is relatively fast, thereby allowing for the testing the drug's activity under more physiological forms of activation. For example, different frequencies of depolarization and/or hyperpolarization may be used to determine how a drug interacts with the channel under physiological forms of neural activity. In some instances, the process may be accomplished without the application of expensive chemical dyes to the cell lines.
In conjunction with the various properties discussed herein, the use of various embodiments of the invention may be particularly useful for improving screening throughput by eliminating the need for cumbersome mechanical manipulation and liquid handling. Various embodiments may also be useful for repeatable the screening assay using the same samples, reducing screening cost by eliminating the need for chemically-based fluorescence reports, producing high temporal precision and low signal artifact (due to the optical nature of the voltage manipulation), modulating the level of depolarization by attenuating the light intensity used for stimulation, and ascertaining the kinetics of the drug's modulation on the ion channel through the use of pulsed light patterns.
The various embodiments described above are provided by way of illustration only and should not be construed to limit the invention. Based on the above discussion and illustrations, those skilled in the art will readily recognize that various modifications and changes may be made to the present invention without strictly following the exemplary embodiments and applications illustrated and described herein. For instance, such changes may include the use of digital logic or microprocessors to control the emitted light. Such modifications and changes do not depart from the true spirit and scope of the present invention, which is set forth in the following claims.

## Claims

1. A method for screening test drugs to identify their effects on cell membrane voltage-gated ion channel, the method comprising: a) contacting a mammalian cell with a test drug, wherein the cell: is genetically modified to express a heterologous, microbial light-responsive ChR2 ion channel comprising an amino acid sequence having at least 75% amino acid sequence identity to SEQ ID NO: 1, wherein the heterologous ChR2 ion channel, when activated by light, results in a change in the voltage in the cell membrane; wherein the cell comprises: i) a voltage-gated ion channel that causes a change in the intracellular concentration of an ion in response to the light-induced change in voltage; and ii) an indicator that produces a fluorescent signal in response to a change in the intracellular concentration of the ion; b) activating the heterologous, microbial light responsive ChR2 ion channel with light; c) detecting a fluorescent signal produced by the indicator; processing data received from the indicator; and storing the processed data received from the indicator, wherein a change in the signal produced by the indicator in the presence of the test drug, compared to the signal produced in the absence of the test drug, indicates that the test drug is a candidate drug that affects the voltage-gated ion channel.

2. The method of claim 1, wherein the voltage-gated ion channel is a Ca2+channel, a K+ channel, a Cl− channel, or a Na+ channel.

3. The method of claim 1, wherein the voltage-gated ion channel is a Ca2+ channel, and wherein the fluorescence producing indicator is a calcium-sensitive dye.

4. The method of claim 1, further comprising introducing a plurality of test drugs to respective wells of an array of wells that each contain the mammalian cells and detecting light from respective wells from the array of wells using an array of optical sensors for detecting light.

5. The method of claim 1, wherein the light-responsive ChR2 ion channel comprises an amino acid sequence having at least 85% amino acid sequence identity to SEQ ID NO:1.

6. The method of claim 1, wherein the light-responsive ChR2 ion channel comprises an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO:1.

7. The method of claim 1, wherein the light-responsive ChR2 ion channel comprises an amino acid sequence having at least 95% amino acid sequence identity to SEQ ID NO:1.

8. The method of claim 1, wherein the cell is an immortalized cell line.

9. The method of claim 1, wherein the indicator is a voltage-sensitive dye.

10. A method for screening test drugs to identify their effects on cell membrane voltage-gated ion channel, the method comprising:
a) contacting a mammalian cell with a test drug, wherein the cell is genetically modified to express a heterologous, microbial light-responsive ion channel and a heterologous, microbial light-responsive ion pump that, when activated by light, results in a change in the voltage in the cell membrane; wherein the cell comprises:
i) a voltage-gated ion channel that causes a change in the intracellular concentration of an ion in response to the light-induced change in voltage; and
ii) an indicator that produces a fluorescent signal in response to a change in the intracellular concentration of the ion;

b) activating the heterologous, microbial light responsive ion channel or heterologous, microbial light-responsive ion pump with light;
c) detecting a fluorescent signal produced by the indicator;
processing data received from the indicator; and
storing the processed data received from the indicator, wherein a change in the signal produced by the indicator in the presence of the test drug, compared to the signal produced in the absence of the test drug, indicates that the test drug is a candidate drug that affects the voltage-gated ion channel.

11. The method of claim 10, wherein the light-responsive ion channel is a ChR2 ion channel comprising an amino acid sequence having at least 75% amino acid sequence identity to SEQ ID NO:1.

12. The method of claim 10, wherein the light-responsive ion pump is an NpHR ion pump.

13. The method of claim 10, wherein the voltage-gated ion channel is a Ca2+ channel, a K+ channel, a Cl− channel, or a Na+ channel.

14. The method of claim 10, wherein the voltage-gated ion channel is a Ca2+ channel, and wherein the fluorescence producing indicator is a calcium-sensitive dye.

15. The method of claim 10, wherein the light-responsive ion channel is a ChR2 ion channel comprising an amino acid sequence having at least 85% amino acid sequence identity to SEQ ID NO:1.

16. The method of claim 10, wherein the light-responsive ion channel is a ChR2 ion channel comprising an amino acid sequence having at least 90% amino acid sequence identity to SEQ ID NO:1.

17. The method of claim 10, wherein the light-responsive ion channel is a ChR2 ion channel comprising an amino acid sequence having at least 95% amino acid sequence identity to SEQ ID NO:1.

18. The method of claim 10, wherein the cell is an immortalized cell line.

19. The method of claim 10, wherein the indicator is a voltage-sensitive dye.