The invention generally relates to methods, compositions, and kits for measuring ion channel activity in a cell.
Fluorescence-based thallium flux assays have been used to measure monovalent cation channel and transporter activity, and modulators of those targets, for decades. At present, thallium flux assays are comprised of solutions and fluorescent thallium indicators with methods designed to measure thallium influx rates. Kinetics are typically rapid, lasting from 1 to 600 seconds, as cells in all groups approach the same tonic equilibrium after the addition of thallium. Short detection windows, however, limit a user's ability to use a broader array of analytical instrumentation, such as standard fluorescence plate readers, fluorescence microscopes, and flow cytometers in ion channel and transporter assays.
Described herein are methods, products, and kits that can be used to identify and/or measure the activity of an effector of an ion channel or transporter in a sample comprising cells having one or more cell types. The method comprises: loading a thallium-sensitive fluorescent indicator and thallium inside of the cells in the sample; contacting the cells with the effector; and measuring the change in fluorescence of the indicator in cells or in the medium surrounding the cells.
Generally, an effector is a compound that interacts with an ion channel receptor or transporter directly or indirectly to affect the activity of the ion channel or transporter. In an embodiment, the effector is an ion channel inhibitor or an ion channel activator. In other embodiments, the effector interacts with, and alters the activity of, an ion channel transporter. Exemplary effectors include GPCRs, protein kinases, or a protein phosphatases.
Cells used in the test described herein, generally, exhibit a basal influx of thallium. Exemplary cells include primary cells or induced pluripotent stem cells (iPSCs). Specific cell lines that can be used include Chinese hamster ovary (CHO) cells, Human embryonic kidney (HEK) cells, or HeLa cells.
In an embodiment, the thallium-sensitive fluorescent indicator is loaded into the cells prior to adding thallium to the cells. Subsequently, the addition of thallium to the cells increases the fluorescence of the thallium-sensitive fluorescent indicator. In one embodiment, the thallium-sensitive fluorescent indicator is loaded into the cells prior to adding thallium to the cells. In another embodiment, the thallium-sensitive indicator is loaded into the cells in the presence of thallium ions. The thallium-sensitive indicator may be an organic thallium chelating agent. Examples of organic thallium chelating agents include, but are not limited to, Thallos, Thallos Gold, FluxOR, BTC, or TL-520.
In an embodiment, the method further comprises adding a control composition to the cells that are loaded with a thallium-sensitive fluorescent indicator and thallium, wherein the control composition does not include an effector compound. In this embodiment, measuring the change in fluorescence of the cells after addition of the effector compound comprises comparing the change in fluorescence of the cells when an effector compound is added to the change in fluorescence of the cells when the control composition is added. In another embodiment, measuring the change in fluorescence of the cells comprises acquiring the fluorescence of the cells before adding the effector compound and comparing the fluorescence of the cells after adding the effector compound to the fluorescence of the cells before adding the effector compound.
The change in fluorescence can be determined by measuring the change in fluorescence of the indicator in cells or in the medium surrounding the cell using a fluorescence microscope. The fluorescence microscope can be a high content screening microscope.
In an embodiment, the cells are loaded into a fluorescence plate reader before loading the cells with a thallium-sensitive fluorescent indicator and thallium. Measuring the change in fluorescence of the cells can be performed with a fluorescence plate reader.
In another embodiment, the change in fluorescence of the cells is measured with a flow cytometer. The flow cytometer can be used to acquire multiple sequential fluorescent measurements. For example, the flow cytometer can be used to acquire simultaneous fluorescent measurements of multiple effectors. Alternatively, the flow cytometer can be used to acquire simultaneous fluorescent measurements of multiple cells.
In an embodiment, the method further comprises obtaining an image of the cells prior to the addition of an effector compound and obtaining an image of the cells after the addition of an effector compound.
Two or more cell types can be present in the sample. In an embodiment, one cell type is discriminated from another cell type using a fluorescent tag, fluorescently encoded protein, spatial information, cell morphology, or a combination of these features.
The methods described herein allow the use of fluorescence efflux to measure the activity of effectors easily and accurately on ion channels and ion mobilization using common laboratory fluorescence readers.
Advantages of the present invention will become apparent to those skilled in the art with the benefit of the following detailed description of embodiments and upon reference to the accompanying drawings in which:
(Right) Representative time resolved fluorescence profiles of treated wells for 40 minutes after the addition of VU551;
While the invention may be susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. The drawings may not be to scale. It should be understood, however, that the drawings and detailed description thereto are not intended to limit the invention to the particular form disclosed, but to the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention as defined by the appended claims.
It is to be understood the present invention is not limited to particular devices or methods, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include singular and plural referents unless the content clearly dictates otherwise. Furthermore, the word “may” is used throughout this application in a permissive sense (i.e., having the potential to, being able to), not in a mandatory sense (i.e., must). The term “include,” and derivations thereof, mean “including, but not limited to.” The term “coupled” means directly or indirectly connected.
The present disclosure describes a novel approach to conduct thallium flux assays to identify effectors of monovalent ion channels, transporters, and channel-linked receptors using instruments not previously accessible to thallium flux assays. These instruments include a fluorescence microscope, a basic plate reader, a high content screening instrument, and a flow cytometer.
Methods described herein include an approach to thallium flux assays that results in a detectable and sustained signal for use in drug discovery and screening applications. Prior to the addition of one or more effector(s) (which can be known or unknown), cells are incubated in one or more solutions containing a thallium-sensitive indicator and a thallium salt, which results in a detectable signal (e.g., fluorescence) inside the cells. After the addition of the effector(s), thallium may or may not be extruded from cells and a change in fluorescence or lack thereof, respectively, can be recorded.
In one embodiment, schematically depicted in
In contrast to thallium influx tests, which typically rely on hard to access fluorescent imaging plate readers, thallium efflux testing can be performed using common fluorescence imaging equipment found in most laboratories. For example, the thallium efflux testing described in the present disclosure can be performed using a fluorescence microscope or a flow cytometer. In some embodiments, the fluorescent microscope is a high content screening device.
When using a fluorescence microscope, the cells can be placed on a fluorescence plate reader before loading the cells with a thallium-sensitive fluorescent indicator and thallium. In another embodiment, a flow cytometer can be used to detect the change in fluorescence. A flow cytometer can be used to acquire fluorescent measurements sequentially.
Additionally, thallium influx tests suffer from flux of thallium through alternative ion channels and transporters that are not the intended target of the desired measurements. The influx of thallium through other sources compresses the dynamic range and detection window of an assay. In contrast to influx experiments, the signal amplitude and dynamic range of an efflux assay is sustained for an extended period of time after the addition of an effector.
Data can be analyzed to determine if any lead compound(s) were identified as hits for a specific target. In one embodiment, the ratio, F/Fo, can be used to determine if a compound has an effect on ion mobilization within the cell. Ion mobilization can be controlled by a number of biological targets, referred to herein as “ion mobilizers.” Examples of ion mobilizers include, but are not limited to, ion channels and ion transporters. As used herein the terms “effector” and “effector compounds” refer to a compound that alters the normal function of a cellular ion mobilizer, or a compound that acts as an agonist, an antagonist, an allosteric modulator, inhibitor, positive modulator, negative modulator, or a potentiator of an ion mobilizer in a cell. Effectors can be ion channel blockers, ion channel activators, or ion channel transporters. Exemplary classes of effectors include, but are not limited to, G-protein coupled receptors (GPCRs, e.g., Gi/o GPCR), protein kinases, and protein phosphatases. Other effectors include compounds that interact with an ion-channel receptor or transporter. Since the test relies on thallium efflux, it is expected that in the presence of an effector compound, thallium concentrations inside the cell will change. For example, the cellular thallium concentration becomes reduced (in the presence of an ion channel activator) or the cellular thallium concentration will remain the same (in the presence of an ion channel blocker). Control compositions are compositions that do not include an effector compound. In some embodiments, a control composition has the same composition as the composition used to deliver the effector compound but does not include any compounds that act as effectors of the cells ion channel.
A variety of cells having a basal influx of thallium can be tested. Exemplary cells that can be tested include, but are not limited to, Chinese hamster ovary (CHO) cells, Human embryonic kidney (HEK) cells, and HeLa cells. Culture systems can contain two or more cell types (co-cultures, organoids, lab-on-a-chip), and a cell or group of cells can be selectively “targeted” to monitor cell-specific effects of a compound of interest on an expressed target. One cell type is discriminated from another cell type using a fluorescent tag, fluorescently encoded protein, or some other discriminating feature, including spatial information or cell morphology, to acquire cell-specific or population specific data.
The extent to which an effector compound activates or inhibits a target of interest can be correlated to additional features of cells, including gene expression, protein expression, or other relevant biological metrics. Cells expressing a target of interest can be identified within a diverse population of cells.
Because of the longer detection windows, monitoring the efflux of thallium ions offers a number of advantages over traditional methods which rely on thallium ion influx. One advantage lies in the long detection time available during efflux. Under the detection conditions set forth herein, thallium ions exit the cell through the ion channel or via a transporter. In a typical experiment, the fluorescence of the cells can be monitored from the time the effector compound (or control compound) is introduced to the cell for up to about 60 minutes. This is in contrast to thallium influx tests which are typically only capable of measurement for up to about 600 seconds (10 minutes) after introduction of the effector.
In another embodiment, schematically depicted in
In another embodiment, schematically depicted in
In some embodiments, the method includes obtaining an image of the cells prior to the addition of an effector compound and obtaining an image of the cells after the addition of an effector compound.
In some embodiments, two or more cell types are present in the sample being tested with effector compounds. In some embodiments, one cell type can be discriminated from another cell type using a fluorescent tag, fluorescently encoded protein, spatial information, cell morphology, or a combination of these features.
In a comparative test of the efflux method disclosed herein, EC50 values were determined using VU551 as the effector. Three different instruments were used to monitor the change in fluorescence: 1- kinetic imaging plate reader (Hamamatsu FDSS), 2 - fluorescent microscope; 3- fluorescent plate reader.
In another test of the method, data was acquired on a plate reader using the test method depicted in
Imaging based detection methods can also be used in the efflux method described herein. A cell targeting experiment was performed using image-based analysis in co-cultures. CHO K1 cells were stained prior to plating with cytotracker red, and images acquired using Texas Red filters were used to discriminate between stained and unstained cells. A representative subpopulation mask is shown in
In
In
In
The assays described herein can be used with sodium and potassium indicators. An exemplary sodium sensitive fluorescent indicator is ION Natrium Green-2 (ING-2, Ion Biosciences). Exemplary potassium sensitive fluorescent indicators include ION Potassium Green indicators (e.g., IPG-1, IPG-2, IPG-4, and PBFI, Ion Biosciences). In some embodiments, sodium or potassium-sensitive indicators are loaded into cells. Sodium or potassium salts, as appropriate, can be added to the cells to activate the indicator. In some embodiments, the addition of sodium salts or potassium salts is not needed as the solution used to load the indicator into the cell already contains sodium or potassium.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Table 1 below represents the primary composition of the dye loading solution that was applied to cells in this assay, prior to thallium addition (See
Table 2 below represents the primary composition of the Tl+ solution when added separately (See
CHO K1s and CHO G12 cells were passaged from separate flasks in parallel. CHO K1s were stained in suspension with cytotracker red, a cytosolic dye that is well retained, following recommended protocols. Prior to mixing the two cell populations together for seeding a 96-well plate, the cytotracker red loading solution was aspirated from the CHO K1 cells and replaced with serum-containing medium. Cells were incubated overnight in the well plate. The following day, a thallium snapshot assay was conducted using the methods described in this application. Cells were imaged on a Cytation 5—imaging plate reader—5 minutes after the addition of Tl+ (Frame 0) then imaged again 10 minutes after the addition of VU551 (Frame 1). (See
The workflow for this example is depicted in FIG. 1.
CHO K1 and HEK 293 cells were plated in coculture and loaded with a sodium indicator. HEK293 cells were stained with celltracker red prior to plating in order to discriminate between the two cell types on the same plate. Images of the cells are shown in
CHO GIRK cells were treated with HEPES-Buffered Hanks Balanced Salt Solution (HHBSS, Control) or GIRK activator, VU0466551 (VU551). Data was acquired 50 minutes after addition of 1 μM VU551 using a BD Accuri C6 Flow Cytometer. Mean cell fluorescence of the treated group is 2.5M RFUs versus 4.5M RFUs for the control, indicating that VU551 is a GIRK channel agonist.
In this patent, certain U.S. patents, U.S. patent applications, and other materials (e.g., articles) have been incorporated by reference. The text of such U.S. patents, U.S. patent applications, and other materials is, however, only incorporated by reference to the extent that no conflict exists between such text and the other statements and drawings set forth herein. In the event of such conflict, then any such conflicting text in such incorporated by reference U.S. patents, U.S. patent applications, and other materials is specifically not incorporated by reference in this patent.
Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.
This application claims priority to U.S. Provisional Application Ser. No. 63/357,764, filed Jul. 1, 2022, entitled “METHODS AND COMPOSITIONS OF STABLE THALLIUM FLUX ASSAYS FOR DETECTING MODULATORS OF ION CHANNELS”, which is incorporated herein by reference.
Number | Date | Country | |
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63357764 | Jul 2022 | US |