U.S. patent application number 11/100262 was filed with the patent office on 2006-10-05 for system and method for performing g protein coupled receptor (gpcr) cell assays using waveguide-grating sensors.
Invention is credited to Ye Fang, Ann M. Ferrie, Norman H. Fontaine, Joydeep Lahiri, Po Ki Yuen.
Application Number | 20060223051 11/100262 |
Document ID | / |
Family ID | 36691636 |
Filed Date | 2006-10-05 |
United States Patent
Application |
20060223051 |
Kind Code |
A1 |
Fang; Ye ; et al. |
October 5, 2006 |
System and method for performing G protein coupled receptor (GPCR)
cell assays using waveguide-grating sensors
Abstract
The present invention includes a system and method that uses
optical LID biosensors to monitor in real time agonist-induced GPCR
signaling events within living cells. Particularly, the present
invention includes a system and method for using an optical LID
biosensor to screen compounds against a target GPCR within living
cells based on the mass redistribution due to agonist-induced GPCR
activation. In an extended embodiment, the present invention
discloses different ways for self-referencing the optical LID
biosensor to eliminate unwanted sensitivity to ambient temperature,
pressure fluctuations, and other environmental changes. In yet
another extended embodiment, the present invention discloses
different ways for screening multiple GPCRs in a single type of
cell or multiple GPCRs in multiple types of cells within a single
medium solution. In still yet another extended embodiment, the
present invention discloses different ways to confirm the
physiological or pharmacological effect of a compound against a
specific GPCR within living cells.
Inventors: |
Fang; Ye; (Painted Post,
NY) ; Ferrie; Ann M.; (Painted Post, NY) ;
Fontaine; Norman H.; (Painted Post, NY) ; Lahiri;
Joydeep; (Painted Post, NY) ; Yuen; Po Ki;
(Corning, NY) |
Correspondence
Address: |
CORNING INCORPORATED
SP-TI-3-1
CORNING
NY
14831
US
|
Family ID: |
36691636 |
Appl. No.: |
11/100262 |
Filed: |
April 5, 2005 |
Current U.S.
Class: |
435/4 ;
435/7.1 |
Current CPC
Class: |
G01N 2500/10 20130101;
G01N 21/553 20130101; G01N 2333/726 20130101; G01N 21/4788
20130101; G01N 33/5041 20130101 |
Class at
Publication: |
435/004 ;
435/007.1 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C12Q 1/00 20060101 C12Q001/00 |
Claims
1. A method for performing a living cell-based assay, said method
comprising the step of: using an optical label independent
detection (LID) biosensor to monitor mass redistribution within
living cells adherent on a surface of the optical LID
biosensor.
2. The method of claim 1, wherein said step of using enables one to
monitor mass redistribution due to an agonist-induced G-protein
coupled receptor (GPCR) activation within living cells by
performing the following steps: providing the optical LID
biosensor; placing the living cells in a cell medium to cover the
optical LID biosensor so the living cells are able to attach to the
surface of the optical LID biosensor; applying a solution
containing a compound into the cell medium located on the surface
of the optical LID biosensor; and interrogating the optical LID
biosensor to obtain a time dependent optical response which
indicates the mass redistribution within the living cells that
enables one to monitor an agonist-induced GPCR translocation within
the living cells.
3. The method of claim 2, wherein prior to the step of applying the
solution containing the compound into the cell medium located on
the surface of the optical LID biosensor the following step is
performed: applying a buffer solution at least once into the cell
medium located on the surface of the optical LID biosensor
solution.
4. The method of claim 1, wherein said step of using enables one to
screen an agonist against a target G-protein coupled receptor
(GPCR) based on mass redistribution within living cells by
performing the following steps: providing the optical LID
biosensor; placing the living cells in a cell medium to cover the
optical LID biosensor so the living cells are able to attach to the
surface of the optical LID biosensor; applying a solution
containing an antagonist with a known affinity at a certain
concentration into the cell medium to stabilize the optical LID
biosensor; applying a solution containing a compound into the cell
medium located on the surface of the optical LID biosensor, wherein
a concentration of the compound is sufficiently high to compete off
the antagonist; and interrogating the optical LID biosensor to
obtain a time dependent optical response which indicates the mass
redistribution within the living cells that enables one to screen
an agonist against a target GPCR based on mass redistribution
within the living cells.
5. The method of claim 1, wherein said step of using enables one to
screen an antagonist against a target G-protein coupled receptor
(GPCR) based on mass redistribution within living cells by
performing the following steps: providing the optical LID
biosensor; placing the living cells in a cell medium to cover the
optical LID biosensor so the living cells are able to attach to the
surface of the optical LID biosensor; applying a solution
containing an agonist with a known affinity at a certain
concentration into the cell medium located on the surface of the
optical LID biosensor; applying, before translocation happens due
to the presence of the agonist, a solution containing a compound
into the cell medium located on the surface of the optical LID
biosensor; and interrogating the optical LID biosensor to obtain a
time dependent optical response which indicates the mass
redistribution within the living cells that enables one to screen
an antagonist against a target GPCR based on mass redistribution
within living cells.
6. The method of claim 1, wherein said optical LID biosensor is
formed into a self-referencing optical LID biosensor prior to
performing the step of using by: blocking a portion of the surface
of the optical LID biosensor using a stamp that prevents attachment
of the living cells; placing the living cells in a cell medium to
cover an unblocked portion of the surface of the optical LID
biosensor so the living cells are able to attach to the unblocked
portion of the surface of the optical LID biosensor; and removing
the stamp from the surface of the optical LID biosensor.
7. The method of claim 1, wherein said optical LID biosensor is
formed into a self-referencing optical LID biosensor prior to
performing the step of using by: blocking a portion of the surface
of the optical LID biosensor using a stamp that prevents attachment
of the living cells; placing the living cells in a cell medium to
cover an unblocked portion of the surface of the optical LID
biosensor so the living cells are able to attach to the unblocked
portion of the surface of the optical LID biosensor; removing the
stamp from the surface of the optical LID biosensor, and and
placing a second type of cells in the same cell medium to cover the
blocked portion of the surface of the optical LID biosensor so the
second type of cells are able to attach to the blocked portion of
the surface of the optical LID biosensor.
8. The method of claim 1, wherein said step of using optical LID
sensor enables one to monitor the mass redistribution due to an
agonist-induced G-protein coupled receptor (GPCR) activation within
multiple types of the living cells by performing the following
steps: providing a chamber containing an array of the optical LID
biosensors; placing a first type of the living cells in a cell
medium to cover at least one of the optical LID biosensors so the
first type of the living cells is able to attach to the surface of
the at least one optical LID biosensor; placing a second type of
the living cells in a cell medium to cover at least one of the
remaining uncovered optical LID biosensors so the second type of
the living cells is able to attach to the surface of the at least
one remaining uncovered optical LID biosensor; applying a solution
containing a compound into the cell mediums located on the surfaces
of the optical LID biosensors; and interrogating the optical LID
biosensors to obtain time dependent optical responses which
indicates the mass redistributions within the living cells on each
of the optical LID biosensors that enables one to monitor the
agonist-induced GPCR activation within multiple types of the living
cells.
9. The method of claim 1, wherein said step of using optical LID
sensor enables one to monitor mass redistribution due to an
agonist-induced G-protein coupled receptor (GPCR) activation within
multiple types of the living cells by performing the following
steps: providing the optical LID biosensor; blocking a portion of
the surface of the optical LID biosensor using a stamp that
prevents attachment of the living cells to that portion of the
optical LID biosensor; placing a first type of the living cells in
a cell medium to cover an unblocked portion of the surface of the
optical LID biosensor so the first type of the living cells are
able to attach to the unblocked portion of the surface of the
optical LID biosensor; removing the stamp from the surface of the
optical LID biosensor; placing a second type of the living cells in
a cell medium to cover the optical LID biosensor so the second type
of the living cells are able to attach to the uncovered portion of
the surface on the optical LID biosensor; applying a solution
containing a compound into the cell medium located on the surface
of the optical LID biosensor; and interrogating the optical LID
biosensors to obtain time dependent optical responses which
indicates the mass redistributions within the two types of living
cells on the optical LID biosensor that enables one to monitor the
agonist-induced GPCR activation within multiple types of the living
cells.
10. The method of claim 1, wherein said step of using optical LID
sensor enables one to screen agonists against multiple G-protein
coupled receptors (GPCRs) within a single type of living cells by
performing the following steps: providing the optical LID
biosensor; placing the living cells in a cell medium to cover the
optical LID biosensor so the living cells are able to attach to the
surface of the optical LID biosensor; applying a solution
containing a cocktail solution of antagonists; applying a solution
containing a compound into the cell medium located on the surface
of the optical LID biosensor; and interrogating the optical LID
biosensor to obtain a time dependent optical response which
indicates the mass redistribution within the living cells that
enables one to screen agonists against multiple GPCRs within the
living cells.
11. A system, comprising: an interrogation system; and an optical
label independent detection (LID) biosensor, wherein said
interrogation system emits an optical beam to said optical LID
biosensor and receives an optical beam from said optical LID
biosensor which enables said interrogation system to monitor mass
redistribution within living cells located on a surface of the
optical LID biosensor.
12. The system of claim 11, wherein said interrogation system is
further capable of monitoring an agonist-induced G protein coupled
receptor (GPCR) activation within the living cells after the
following steps are performed: providing the optical LID biosensor;
placing the living cells in a cell medium to cover the optical LID
biosensor so the living cells are able to attach to the surface of
the optical LID biosensor; applying a solution containing a
compound into the cell medium located on the surface of the optical
LID biosensor; and interrogating the optical LID biosensor to
obtain a time dependent optical response which indicates the mass
redistribution within the living cells that enables one to monitor
the mass redistribution due to an agonist-induced GPCR activation
within the living cells.
13. The system of claim 12, wherein prior to the step of applying
the solution containing the compound into the cell medium located
on the surface of the optical LID biosensor the following step is
performed: applying a buffer solution at least once into the cell
medium located on the surface of the optical LID biosensor
solution.
14. The system of claim 11, wherein said interrogation system is
further capable of screening an agonist against a target G-protein
coupled receptor (GPCR) based on mass redistribution after the
following steps are performed: providing the optical LID biosensor;
placing the living cells in a cell medium to cover the optical LID
biosensor so the living cells are able to attach to the surface of
the optical LID biosensor; applying a solution containing an
antagonist with a known affinity at a certain concentration into
the cell medium to stabilize the optical LID biosensor; applying a
solution containing a compound into the cell medium located on the
surface of the optical LID biosensor, wherein a concentration of
the compound is sufficiently high to compete off the antagonist;
and interrogating the optical LID biosensor to obtain a time
dependent optical response which indicates the mass redistribution
within the living cells that enables one to screen an agonist
against a target GPCR based on mass redistribution within the
living cells.
15. The system of claim 11, wherein said interrogation system is
further capable of screening an antagonist against a target
G-protein coupled receptor (GPCR) based on mass redistribution
after the following steps are performed: providing the optical LID
biosensor; placing the living cells in a cell medium to cover the
optical LID biosensor so the living cells are able to attach to the
surface of the optical LID biosensor; applying a solution
containing an agonist with a known affinity at a certain
concentration into the cell medium located on the surface of the
optical LID biosensor; applying, before translocation happens due
to the presence of the agonist, a solution containing a compound
into the cell medium located on the surface of the optical LID
biosensor; and interrogating the optical LID biosensor to obtain a
time dependent optical response which indicates the mass
redistribution within the living cells that enables one to screen
an antagonist against a target GPCR based on mass redistribution
within living cells.
16. The system of claim 11, wherein said optical LID biosensor is
formed into a self-referencing optical LID biosensor by: blocking a
portion of the surface of the optical LID biosensor using a stamp
that prevents attachment of the living cells; placing the living
cells in a cell medium to cover an unblocked portion of the surface
of the optical LID biosensor so the living cells are able to attach
to the unblocked portion of the surface of the optical LID
biosensor; and removing the stamp from the surface of the optical
LID biosensor.
17. The system of claim 11, wherein said optical LID biosensor is
formed into a self-referencing optical LID biosensor prior to
performing the step of using by: blocking a portion of the surface
of the optical LID biosensor using a stamp that prevents attachment
of the living cells; placing the living cells in a cell medium to
cover an unblocked portion of the surface of the optical LID
biosensor so the living cells are able to attach to the unblock
portion of the surface of the optical LID biosensor; removing the
stamp from the surface of the optical LID biosensor, and and
placing a second type of cells in the same cell medium to cover the
blocked portion of the surface of the optical LID biosensor so the
second type of cells are able to attach to the blocked portion of
the surface of the optical LID biosensor.
18. The system of claim 11, wherein said interrogation system is
further capable of monitoring the mass redistribution due to an
agonist-induced G-protein coupled receptor (GPCR) activation within
multiple types of the living cells after the following steps are
performed: providing a chamber containing an array of the optical
LID biosensors; placing a first type of the living cells in a cell
medium to cover at least one of the optical LID biosensors so the
first type of the living cells is able to attach to the surface of
the at least one optical LID biosensor; placing a second type of
the living cells in a cell medium to cover at least one of the
remaining uncovered optical LID biosensors so the second type of
the living cells is able to attach to the surface of the at least
one remaining uncovered optical LID biosensor; applying a solution
containing a compound into the cell mediums located on the surfaces
of the optical LID biosensors; and interrogating the optical LID
biosensors to obtain time dependent optical responses which
indicates the mass redistributions within the living cells on each
of the optical LID biosensors that enables one to monitor the
agonist-induced GPCR translocation within multiple types of the
living cells.
19. The system of claim 11, wherein said interrogation system is
further capable of monitoring the mass redistribution due to an
agonist-induced G-protein coupled receptor (GPCR) activation within
multiple types of the living cells after the following steps are
performed: providing the optical LID biosensor; blocking a portion
of the surface of the optical LID biosensor using a stamp that
prevents attachment of the living cells to that portion of the
optical LID biosensor; placing a first type of the living cells in
a cell medium to cover an unblocked portion of the surface of the
optical LID biosensor so the first type of the living cells are
able to attach to the unblock portion of the surface of the optical
LID biosensor; removing the stamp from the surface of the optical
LID biosensor; placing a second type of the living cells in a cell
medium to cover the optical LID biosensor so the second type of the
living cells are able to attach to the uncovered portion of the
surface on the optical LID biosensor; applying a solution
containing a compound into the cell medium located on the surface
of the optical LID biosensor; and interrogating the optical LID
biosensors to obtain time dependent optical responses which
indicates the mass redistributions within the two types of living
cells on the optical LID biosensor that enables one to monitor the
agonist-induced GPCR translocations within multiple types of the
living cells.
20. The system of claim 11, wherein said interrogation system is
further capable of screening agonists against multiple G-protein
coupled receptors (GPCRs) within a single type of living cells
after the following steps are performed: providing the optical LID
biosensor; placing the living cells in a cell medium to cover the
optical LID biosensor so the living cells are able to attach to the
surface of the optical LID biosensor; applying a solution
containing a cocktail solution of antagonists; applying a solution
containing a compound into the cell medium located on the surface
of the optical LID biosensor; and interrogating the optical LID
biosensor to obtain a time dependent optical response which
indicates the mass redistribution within the living cells that
enables one to screen agonists against multiple GPCRs within the
living cells.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates in general to the optical
non-contact sensor field and, specifically, to a system and method
for using an optical label independent detection (LID) biosensor
(e.g., waveguide grating-based biosensor) to monitor in real time
compound-induced mass redistribution in living cells, including
agonist-induced G protein coupled receptor (GPCR) desensitization
and translocation within living cells, as well as morphological
changes of adherent cells. Particularly, the present invention
relates to a system and method for using a LID biosensor to screen
compounds against a GPCR within living cells.
[0003] 2. Description of Related Art
[0004] Today an optical-based biosensor like a surface plasmon
resonance (SPR) sensor or a waveguide grating-based sensor enables
an optical label independent detection (LID) technique to be used
to detect a biomolecular binding event at the biosensor's surface.
In particular, the optical-based biosensor enables an optical LID
technique to be used to measure changes in a refractive
index/optical response of the biosensor which in turns enables a
biomolecular binding event to be detected at the biosensor's
surface. In fact, these optical-based biosensors along with
different optical LID techniques have been used to study a variety
of biomolecular binding events including oligonucleotides
interactions, antibody-antigen interactions, hormone-receptor
interactions, and enzyme-substrate interactions (for example).
[0005] In general, the optical-based biosensor includes two
components: a highly specific recognition element and an optical
transducer that converts a molecular recognition event into a
quantifiable signal. The traditional studies performed with optical
LID techniques have been associated with direct optical methods
which include the use of: surface plasmon resonance (SPR) sensors;
grating couplers; ellipsometry devices; evanescent wave devices;
and reflectometry devices. For a detail discussion about each of
these direct optical methods reference is made to the following
documents: [0006] Jordan & Corn, "Surface Plasmon Resonance
Imaging Measurements of Electrostatic Biopolymer Adsorption onto
Chemically Modified Gold Surfaces," Anal. Chem., 1997,
69:1449-1456. [0007] Morhard et al., "Immobilization of antibodies
in micropatterns for cell detection by optical diffraction,"
Sensors and Actuators B, 2000, 70, 232-242. [0008] Jin et al., "A
biosensor concept based on imaging ellipsometry for visualization
of biomolecular interactions," Analytical Biochemistry, 1995, 232,
69-72. [0009] Clerc and Lukosz "Direct immunosensing with an
integrated-optical output grating coupler" Sensors and Actuators B
1997, 40, 53-58. [0010] Brecht & Gauglitz, "Optical probes and
transducers," Biosensors and Bioelectronics, 1995, 10, 923-936. The
contents of these documents are incorporated by reference
herein.
[0011] To date, there have been relatively few reports describing
the use of optical LID techniques to perform cell-based assays. For
example, SPR biosensors have been used to investigate the adhesion
and spreading of animal cells as described in the following
document: [0012] J. J Ramsden, S. Y. Li, J. E. Prenosil and E.
Heinzle, "Kinetics of adhension and spreading of animal cells"
Biotechnol. Bioeng. 1994, 43, 939-945.
[0013] And, SPR biosensors have been used to investigate
ligand-induced cell surface and intracellular reactions of living
cells as described in the following document: [0014] M. Hide, et
al. "Real-time analysis of ligand-induced cell surface and
intracellular reactions of living mast cells using a surface
plasmon resonance-based biosensor", Anal. Biochem. 2002, 302,
28-37.
[0015] However, to date there has been no report concerning the use
of optical LID techniques to monitor compound-induced mass
redistribution within adherent cells including agonist-induced
translocation of G protein coupled receptors (GPCRs) within living
cells. It would be desirable if this was possible, because GPCRs, a
family of cell surface receptors, are the most common targets that
new drug compounds are designed against. And, because GPCRs can
transduce exogenous signals (i.e., the presence of stimuli such as
a new drug) into intracellular response(s) which makes them
extremely valuable in the testing of new drugs.
[0016] GPCRs participate in a wide array of cell signaling
pathways. Ligand binding initiates a series of intracellular and
cellular signaling events, including receptor conformational
changes, receptor oligomerization, G protein activation (GDP-GTP
exchanges on G.sub..alpha. subunit, G.sub..alpha. and
G.sub..beta..gamma. disassociation, G protein decoupling from the
receptor, generation of G.sub..alpha.- and
G.sub..beta..gamma.-signaling complexes), and downstream signaling
activation that leads to second messenger generation (Ca.sup.2+
mobilization, inositoltriphosphate generation, and/or intracellular
cAMP level modulation) and ultimately results in changes of
specific gene expression. Ligand-mediated GPCR activation also
leads to the desensitization of GPCRs from the cell surface and
trafficking of many intracellular proteins, as well as changes in
phenotypes, morphology and physical properties of the target cells.
These changes could be static, long-lasting or dynamic (e.g.,
cycling or oscillation). Distinct signaling events exhibit
significantly different kinetics ranging from milliseconds (e.g.,
GPCR conformational changes) to tens of seconds (e.g., Ca.sup.2+
flux) to even tens of minutes (e.g., gene expression, or
morphological changes). Current GPCR assays include ligand-receptor
binding, second messenger (Ca.sup.2+, cAMP of IP3) assays, protein
interaction assays, translocation assays and reporter gene assays.
Since GPCR activation ultimately leads to protein trafficking
and/or morphological changes, methods that can study the action of
any compounds through the GPCRs on cell surface and the consequent
events (e.g., trafficking and/or morphological changes) of the
effected cells would be desired. This need and other needs are
addressed by the present invention.
BRIEF DESCRIPTION OF THE INVENTION
[0017] The present invention includes a system and method that uses
optical LID biosensors to monitor in real time compound-induced
mass redistribution including agonist-induced GPCR signaling events
within living cells. Particularly, the present invention includes a
system and method for using an optical LID biosensor to screen
compounds against a target GPCR within living cells based on the
morphological changes of the cell and/or desensitization and/or
translocation of the GPCR. In an extended embodiment, the present
invention discloses methods for self-referencing the optical LID
biosensor to eliminate unwanted sensitivity to ambient temperature,
pressure fluctuations, and other environmental changes, and also
methods to provide confirmative information of the compound action
on a particular pre-selected target through comparison of the
responses of two types of cells spatially separated but located on
the same sensor. In yet another extended embodiment, the present
invention discloses different ways for screening multiple GPCRs in
a single type of cell or multiple GPCRs in multiple types of cells
within a single medium solution. In still yet another extended
embodiment, the present invention discloses different ways to
confirm the physiological or pharmacological effect of a compound
against a specific GPCR within living cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] A more complete understanding of the present invention may
be had by reference to the following detailed description when
taken in conjunction with the accompanying drawings wherein:
[0019] FIG. 1 is a diagram showing an optical LID system being used
to monitor a mass redistribution (e.g. GPCR translocation) within a
living cell in accordance with the present invention;
[0020] FIG. 2 is a diagram that shows the different states
associated with the GPCR translocation within the living cell that
can be identified by analyzing the time dependent optical response
output from the optical LID system shown in FIG. 1 in accordance
with the present invention;
[0021] FIG. 3 is a flowchart illustrating the basic steps of a
method for monitoring in real time an agonist-induced mass
redistribution including GPCR translocation within living cells
using an optical LID biosensor in accordance with the present
invention;
[0022] FIG. 4 is a flowchart illustrating the basic steps of a
method for screening an agonist against a target GPCR based on mass
redistribution within living cells using the optical LID biosensor
in accordance with the present invention;
[0023] FIG. 5 is a flowchart illustrating the basic steps of a
method for screening an antagonist against a target GPCR based on
mass redistribution within living cells using the optical LID
biosensor in accordance with the present invention;
[0024] FIG. 6 is a flowchart illustrating the basic steps of a
method for creating a self-referencing optical LID biosensor that
can be used in any one of the methods shown in FIGS. 3-5 in
accordance with the present invention;
[0025] FIG. 7 is a flowchart illustrating the basic steps of
another method for creating a self-referencing optical LID
biosensor that hosts two types of cells adherent at spatially
separated regions within the same sensor and can be used in any one
of the methods shown in FIGS. 3-5 in accordance with the present
invention;
[0026] FIG. 8 is a flowchart illustrating the basic steps of a
method for monitoring an agonist-induced GPCR mass redistribution
within multiple types of living cells using the optical LID
biosensor in accordance with the present invention;
[0027] FIG. 9 is a flowchart illustrating the basic steps of a
method for screening agonists against multiple GPCRs within a
single type of living cell based on mass redistribution using the
optical LID biosensor in accordance with the present invention;
and
[0028] FIGS. 10-17 are various graphs and charts indicating the
results of several different experiments that were conducted to
show that the optical LID system can be used to monitor mass
redistributions like GPCR translocations within living cells that
are located on a surface of the optical LID biosensor in accordance
with the present invention. This data was obtained using an optical
waveguide grating sensor system and LID microplates
(Nb.sub.2O.sub.5 plates), manufactured by Corning Incorporated.
[0029] FIG. 10 is a time-dependent LID response of Chinese Hamster
Ovary (CHO) cells before and after compound addition.
[0030] FIG. 11 is the different kinetics of the mass redistribution
due to agonist-induced GPCR activation.
[0031] FIG. 12 is the compound-dependant total responses of
agonist-induced mass changes in the Stage 3 as highlighted in FIG.
2.
[0032] FIG. 13 is a time-dependent LID response of Chinese Hamster
Oyary (CHO) cells before and after compound addition. The compound
concentration used is 10 .mu.M for all compounds.
[0033] FIG. 14 a time-dependent LID response of engineered Chinese
Hamster Ovary (CHO) cells with over-expressed rat muscarnic
receptor subtype 1 (thus this cell line is termed as M1 CHO) before
and after compound addition. The compound concentration used is 10
.mu.M for all compounds.
[0034] FIG. 15 compares the compound-dependant total responses in
the Stage 3 as highlighted in FIG. 2 for two distinct cell
lines.
[0035] FIG. 16 is a time-dependent LID response of two types of
cells (CHO and M1 CHO) before and after addition of oxotremorine M
(10 .mu.M). Before the compound addition, the cells are
pre-incubated either HBSS buffer (Invitrogen) (referred to "without
DIP") or with dynamin inhibitory peptide (DIP) at a concentration
of 50 .mu.M for 45 minutes.
[0036] FIG. 17 is a time-dependent LID response of two types of
cells (CHO and M1 CHO) before and after addition of clonidine (10
.mu.M). Before the compound addition, the cells are pre-incubated
either HBSS buffer (Invitrogen) (referred to "without DIP") or with
dynamin inhibitory peptide (DIP) at a concentration of 50 .mu.M for
45 minutes.
[0037] FIG. 18 is a time-dependent LID response of two types of
cells (CHO and M1 CHO) before and after addition of NECA (10
.mu.M). Before the compound addition, the cells are pre-incubated
either HBSS buffer (Invitrogen) (referred to "without DIP") or with
dynamin inhibitory peptide (DIP) at a concentration of 50 .mu.M for
45 minutes.
DETAILED DESCRIPTION OF THE DRAWINGS
[0038] Referring to FIG. 1, there is a diagram that shows the basic
components of an optical LID system 100 which includes an
interrogation system 102 and an optical LID biosensor 104 that are
used to detect and monitor a mass redistribution (e.g., the
translocation of a GPCR 202, as seen in the arrow numbered 106)
within a living cell 108 (only one shown) located on a surface 110
of the optical LID biosensor 104. In the preferred embodiment, the
interrogation system 102 interrogates the optical LID biosensor 104
(e.g., SPR sensor 104, waveguide grating sensor 104) so it can
detect and monitor the mass redistribution within the living cell
108. This is done by emitting an optical beam 112 which has the
appropriate spectral or angular content towards the optical LID
biosensor 104 such that when the optical beam 112 is reflected by
the sensing surface 110, the resonant angle or wavelength response
which identifies the mass redistribution becomes dominant in the
reflected beam 114. Thus, when there is a detectable mass
redistribution within the living cell 108, the optical LID
biosensor 104 can sense a response change which is observed as an
angular or wavelength change in the reflected beam 114. The optical
response may be recorded as a function of time. In this way, the
kinetics of any event that leads to a mass redistribution within
the living cell 108 can be analyzed. Prior to discussing several
different types of living cell-based assays that can be conducted
and monitored by the optical LID system 100 (see FIGS. 2-18) a
detailed discussion is provided about some of the various
components within the living cell 108.
[0039] Due to the limited range (.about.hundreds nanometers) of the
eletromagnetic field propagating in the optical LID sensor 104 that
can extend into the surrounding media (e.g, adherent cell 108) as
an evanescent eletromagnetic field (the depth is referred to the
penetration depth or sensing volume), only the mass redistribution
106 in the lower portion of the adherent cells that is close to the
sensor surface 110 can be detected. Biological cells 108 are
complex structures with components ranging in size from nanometers
to tens of microns. The cell 108 has a cytoplasm (10-30 .mu.M) that
contains numerous organelles. The largest organelle is the nucleus,
whose size ranges between 3 and 10 .mu.m. The nucleus is filled
with DNA-protein complexes and proteins, the most important one
being chromatin. Mitochondria are small organelles comprised of a
series of folded membranes with sizes ranging from 0.5-1.5 .mu.m.
Other cell components include endoplasmic reticulum (ER) (0.2-1
.mu.m), lysomes (0.2-0.5 .mu.m), peroxisomes (0.2-0.5 .mu.m),
endosomes (.about.100 nm), and gogli. Living cells 108 are highly
dynamic and most organelles travel extensively within cells. For
example, microtubules can transport organelles over long distances.
A stimulus can result in the submicron movement of densely packed
organelles in the very periphery of a sensor surface 100 on which
the cells 108 are cultured; such movement leads to mass
redistribution 106 within the cell 108. The mass redistribution 106
can be detected by an optical biosensor 104; the signal relating to
mass redistribution 106 is referred to as directional mass
redistribution (DMR) signal.
[0040] Cellular trafficking could occur if secretory organelles are
to occupy their docking site beneath the plasma membrane, and if
endocytic vesicles at the plasma membrane are to reach their
processing stations in the cytosol. In either direction, organelles
must penetrate the so-called actin cortex beneath the plasma
membrane, a dense meshwork of actin filaments that is up to a few
hundred nanometers thick. To the extent that actin filaments
constantly assemble and disassemble, the meshwork is dynamic and
permeable to organelles. Control mechanisms regulating the assembly
and disassembly would also regulate the permeability of the actin
cortex.
[0041] The plasma membrane is a busy place. Exocytic vesicles
insert receptors into the plasma membrane and release ligands into
the extracellular space. Endocytic vesicles carry receptors with
bound ligand to internal processing stations. Caveolae are
plasma-membrane-associated vesicles with a presumed role in cell
signaling. Lipid rafts are thought to populate the plasma membrane
as small floating islands in which select membrane proteins meet in
private to exchange signals. Finally, there is the universe of
membrane receptors. Many are probably embedded in large molecular
complexes that continually recruit and release downstream effector
molecules.
[0042] Transport of cellular components or extracellular stimuli
not only occurs at the plasma membrane, but also occurs at multiple
intracellular compartments. These events include (1) protein target
or substrate recruitment to the nucleus, to the membrane, to the
cytosol, throughout recycling pathways, to or from other
organelles, uptake from extracellular space (ligand binding, gene
transfection, infection and protein delivery); (2) redistribution
of newly synthesized intracellular components within various
functional compartments at defined microenvironments and with
mediated release locations. These cellular events lead to
directional mass redistributions at certain times during signaling
cycles.
[0043] From hereinafter, several different types of living
cell-based assays that can be conducted and monitored by the
optical LID system 100 are described in detail below with respect
to FIGS. 2-18.
[0044] Referring to FIG. 2, there is shown a diagram where the
optical LID system 100 is used to monitor an agonist-induced
translocation of G protein coupled receptors 202 (GPCRs 202) within
a living cell 108 (only one shown) located on the top surface 110
of the optical LID biosensor 104. In particular, the diagram
illustrates an agonist induced and time-dependent optical response
201 that partly is due to the translocation of a target GPCR 202
within the living cell 108. The cell is adherent on the top surface
110 of the waveguide-based biosensor 104. For clarity, the
interrogation system 102 is not shown in the portion labeled as
"C".
[0045] As can be seen, the GPCR 202 in the resting state resides at
the cell surface 204 (plasma membrane 204), while the GPCR kinase
206 (GRK 206) and arrestin 208 are uniformly distributed inside the
living cell 108 (see diagram "A"). Upon agonist activation, the
GPCR 202 activates heterotrimeric G proteins composed of .alpha.,
.beta., and .gamma. subunits. The G.alpha. and G.beta..gamma.
subunits dissociate which causes the GRK 206 to be recruited to the
receptor 202 at the plasma membrane 204. Then, the GRK 206
phosphorylates the carboxy terminus of the GPCR 202. And,
.beta.-arrestin 208, a relatively abundant intracellular protein,
rapidly (within minutes) translocates within the cytoplasm to the
activated GPCR 202 at the plasma membrane 204, binds the
GRK-phosphorylated receptor, and uncouples the receptor from its
cognate G protein. The .beta.-arrestin 208 then binds to the
desensitized GPCR 202 and translocates to clathrin-coated pits at
the cell surface 204 where the receptor 202 is internalized in
clathrin-coated vesicles (CCV) (see diagram "B"). Finally, the
entire complex 202 and 206 is delivered to the endosome 210
(endocytic vesicle 210) (see diagram "C"). This process is known as
translocation. For more information about GPCR translocation,
reference is made to the following three articles: [0046] Drews, J.
"Drug discovery: a historical perspective." Science 2000, 287,
1960-1963; [0047] Ma, P. and Zemmel, R. "Value of novelty". Nat.
Rev. Drug Discov. 2002, 1, 571-572. [0048] Pierce, K. L. et al.
"Seven-transmembrane receptors." Nat. Rev. Mol. Cell Biol. 2002, 3,
639-650.
[0049] The contents of these documents are incorporated by
reference herein.
[0050] It should be appreciated that these translocation events
lead to directional mass distribution (e.g., towards the cell
surface or leaving the cell surface) within the living cells 108 at
a certain time, therefore resulting in different optical responses
through a prolong period of time. Another possible biological event
that can lead to directional mass distribution is the cell
morphological changes due to the GPCR activation. The cell
morphological changes involve the cytoskeleton rearrangement as
well as cell adhesion changes. Cytoskeleton is a complex network of
protein filaments that extends throughout the cytoplasm of
eucaryotic cells and is involved in executing diverse activities in
these cells. As well as providing tensile strength for the cells it
also enables muscle contraction, carries out cellular movements and
is involved in intracellular signaling and trafficking, cell
division and changes in the shape of a cell. Activation of
G-protein coupled receptors (GPCR) leads to at least two
independent events that theoretically could exert an effect on the
cytoskeleton rearrangement. The first event is the activation of
the intracellular signaling pathway, and the second is a
receptor-mediated endocytosis (i.e., translocation), which occurs
after an agonist activation of the majority of GPCR. Activation of
an intracellular signaling pathway after an agonist/GPCR binding
then leads to two further sets of connected events. Processes in
the first set lead to the activation of a secondary intracellular
signaling pathway (G protein.fwdarw.effector.fwdarw.message), while
the mechanisms of the second set regulate the degree of signaling
within the cell by affecting the events in the first set. These
mechanisms include phosphorylation/desensitization, internalization
and downregulation of membrane-bound receptors. It is assumed that
both sets of events can lead to the rearrangement of actin
filaments within the cell. For example, after the activation of
GPCR, various forms of G proteins (e.g. G.sub..alpha. and
G.sub..beta..gamma.) can bind with F-actin filaments; and those and
other signaling molecules can disassociate from actin filaments.
The internalization process of membrane-bound receptors that occurs
via receptor-mediated endocytosis could also be responsible for the
dynamics of actin filaments.
[0051] Referring again to FIG. 2 and in accordance with the present
invention, the different states associated with GPCR translocation
within a living cell 108 can be identified and monitored by
analyzing the optical response 201 from the optical LID system 100.
In fact, three different events can be identified when looking at
the optical response 201 shown in FIG. 2. The three major events
that can be seen include: (1) a very large and sharp decrease in
signal 201 upon the addition of agonist, due to bulk index of
refraction changes (i.e., generally the compound solution has
relatively lower refractive index than the cell medium. Thus
compound addition results in a decreased LID signal); (2) a
transition stage which has slow changes in the response signal 201
and lasts almost 20 minutes: this stage might be related to the
phosphorylation of the activated receptors 202 by GRKs 206,
arrestin binding, desensitization of the receptors 202 to
chathrin-coated pits, and/or other cellular responses; and (3) a
slow decrease of response signal 201 which lasts almost 50 minutes,
corresponding to the translocation of the GPCR complexes 202 and
208 to the endosome 202. In other cases, an additional event that
immediately followed the initial step can be evident (e.g., FIG.
11); that is an increase of response signal 201, mainly due to
diffusion of the compound in the cell medium and/or recruitment of
intracellular components to activated GPCRs at cell surface.
Details about how this test can be performed by the optical LID
system 100 are described below with respect to method 300 shown in
FIG. 3.
[0052] Referring to FIG. 3, there is shown a flowchart illustrating
the basic steps of a method 300 for monitoring in real time the
mass redistribution due to an agonist-induced GPCR activation
within living cells 108 using an optical LID biosensor 104 in
accordance with the present invention. The method 300 includes the
following steps: (a) provide an optical LID biosensor 104 (step
302); (b) place a certain number of living cells 108 in a medium
which covers the optical LID biosensor 104 such that the living
cells 108 attach onto the surface 110 of the optical LID biosensor
104 (step 304); (c) optionally apply a buffer solution at least
once into the cell medium (step 306); (d) apply a solution
containing a compound (agonist) into the cell medium (step 308);
and (e) interrogate the optical LID biosensor 104 and monitor the
time dependent optical response 201 of the living cells 108
cultured on the optical LID biosensor 104 (step 310).
[0053] It should be appreciated that if step 306 is performed and a
buffer solution (the same buffer solution that is used to formulate
the compound of interest) is applied to the living cells 108 before
applying the compound, any unwanted effect, due to the living cells
108 responding to the environmental changes, can be minimized. This
is possible because living cells 108 that are cultured on the
optical LID biosensor 104 are alive and dynamic which means that
they can sense changes in the surrounding medium compositions as
well as temperature and can respond to those changes. However, as
the living cells 108 senses changes like the addition of a buffer
then they tend to become resistant to those changes in the medium
composition assuming no additional chemical is introduced.
[0054] It should also be appreciated that the real time method 300
provides quantifiable information, and equally important, it
provides the kinetics of the mass redistribution within cells due
to GPCR activation. In contrast to traditional methods of screening
GPCRs, this method 300 is simpler to perform, more sensitive,
label-independent and is applicable to all GPCRs 202 without
requiring prior knowledge of natural ligands or how a given
receptor is coupled to downstream signaling pathways.
[0055] It should also be appreciated that in the step 304 the
number of cells should be optimized such that after a certain time
cultured under optimal conditions the cells become adherent and
reach high confluency (optionally larger than 75%) on the surface
110 of optical LID sensor 104 in order to achieve high
sensitivity.
[0056] Referring to FIG. 4, there is shown a flowchart illustrating
the basic steps of a method 400 for screening an agonist against a
target GPCR 202 based on mass redistribution within living cells
108 using the optical LID biosensor 104 in accordance with the
present invention. The method 400 includes the following steps:
[0057] (a) provide the optical LID biosensor 104 (step 402); (b)
place a certain number of living cells 108 in a medium which covers
the optical LID biosensor 104 such that the living cells 108 attach
onto the surface 110 of the biosensor 104 (step 404); (c) apply a
solution containing an antagonist with a known affinity at a
certain concentration into the cell medium for a certain time until
the optical LID biosensor 104 becomes stabilized (step 406); (d)
apply a solution containing a compound (agonist) into the cell
medium (step 408) where the concentration of the compound is
sufficiently high to compete off the receptor-bound antagonist; and
(e) interrogate the optical LID biosensor 104 and monitor the time
dependent optical response 201 of the living cells 108 cultured on
the optical LID biosensor 104 (step 410).
[0058] It should be appreciated that in this method 400 by
pre-applying the antagonist to one receptor in the living cells
108, effectively enables one to screen the compounds for their
agonism against this particular receptor. Moreover, it should be
appreciated that this method 400 is similar to the previous method
300 except for one difference in that method 400 requires
pre-knowledge about the functionality of the compound for its
cognate receptor in the living cells 108. For instance, one needs
to know whether the antagonist inhibits the activation of GPCR 202,
or whether the antagonist activates the GPCR 200 which leads to
translocation.
[0059] Referring to FIG. 5, there is shown a flowchart illustrating
the basic steps of a method 500 for screening an antagonist against
a target GPCR 202 based on mass redistribution within living cells
108 using the optical LID biosensor 104 in accordance with the
present invention. The method 500 includes the following steps:
[0060] (a) provide an optical LID biosensor 104 (step 502); (b)
place a certain number of living cells 108 in a medium which covers
the optical LID biosensor 104 such that the living cells 108 attach
onto the surface 110 of the biosensor 104 (step 504); (c) apply a
solution containing an agonist which has a known affinity at a
certain concentration into the cell medium for a short time such
that the translocation does not happen (step 506); (d) after this
short time, apply a solution containing a compound having a certain
concentration into the cell medium (step 508); and (e) interrogate
the optical LID biosensor 104 and monitor the time dependent
optical response 201 of the living cells 108 cultured on the
optical LID biosensor 104. It should be appreciated that like
method 400, this method 500 requires pre-knowledge about the target
GPCR 202 in the living cells 108 and also requires the
pre-selection of an antagonist or angonist for pre-treating the
living cell 108 against this particular GPCR 202.
[0061] It should be appreciated that the step 506 and the step 508
can be combined into one step; that is, the agonist known to the
target GPCR in the cell can be added into together with a compound.
It also should be appreciated that similar to the method 300, the
compound to be tested can be introduced first, followed by the
addition of the known of agonist.
[0062] Each of the methods 300, 400 and 500 can be further enhanced
by using a self-referencing optical LID biosensor 104. It is well
known that the performance of the optical LID biosensor 104 is
generally affected by the designs and characteristics of the
sensor, the optics, and by the environmental fluctuations including
ambient temperature and pressure. A main advantage of using the
self-referencing optical LID biosensor 104 is that the top surface
110 has both a reference region and a sample region which enables
one to use the sample region to detect the mass redistribution in
the living cells 108 and at the same time use the reference region
which does not have living cells 108 attached thereto to reference
out spurious changes that can adversely affect the detection of the
mass redistribution within the living cells 108.
[0063] In one embodiment, the self-referencing optical LID
biosensor 104 can be made in accordance with method 600 shown in
FIG. 6. This self-referencing optical LID biosensor 104 can be
created by using the following steps: (a) provide the optical LID
biosensor 104 (step 602); (b) physically block one region
(reference region) of the surface 110 of the optical LID biosensor
104 by using a soft stamp (e.g., rubber stamp) (step 604); (c)
place a certain number of living cells in a growth medium which
covers an unblocked region (sample region) of the optical LID
biosensor 104 (step 606); and (d) remove the soft stamp after the
living cells 108 have attached to the unblocked region on the
optical LID biosensor 104 (step 608). At this point, the living
cell-based assay can be performed as described in methods 300, 400
and 500. It should be appreciated that different methods can also
be applied to create the self-referencing LID sensors for cell
studies. For example, a physical barrier can be used to divide the
sensor into two portions, and cells in a medium are only applied to
cover one portion. After cell adhesion, the physical barrier can be
removed.
[0064] Referring now to another feature of the present invention,
it is well known that multiplexed cell assays have become
increasingly important, not only for increasing throughput, but
also for the rich and confirmative information available from a
single assay. As such, it is desirable if the present invention
could be further enhanced to perform multiple living cell-based
assays at the same time.
[0065] In one embodiment, the present invention can be enhanced to
perform multiple living cell-based assays at the same time by using
the method 700 shown in FIG. 7. In accordance with method 700 one
can monitor mass redistribution due to agonist-induced GPCR
activation within multiple types of the living cells 108 by: (a)
providing an optical LID biosensor 104 (step 702); (b) blocking a
portion of the top surface 110 of the optical LID biosensor 104 by
using a stamp that prevents the attachment of the living cells 108
to that portion of the optical LID biosensor 104 (step 704); (c)
placing a first type of living cells 108 in a cell medium which
covers the unblocked portion of the surface 110 of the optical LID
biosensor 104 so the living cells 108 are able to attach to the
unblock portion of the surface 110 of the optical LID biosensor 104
(step 706); (d) removing the stamp from the top surface 110 of the
optical LID biosensor 104 (step 708); (e) placing a second type of
living cells 108 in a cell medium which covers the optical LID
biosensor 104 so the second type of living cells 108 are able to
attach to the recently uncovered top surface 110 of the optical LID
biosensor 104 (step 710); (f) applying a solution containing a
compound into the cell medium located on the top surface 110 of the
optical LID biosensor 104 (step 712); and (g) interrogating the
optical LID biosensor 104 to monitor time dependent optical
responses 201 which indicate mass redistributions within the two
types of living cells 108 on the optical LID biosensors 104 (step
714).
[0066] It should be appreciated that the two types of cells can be
related; e.g., Chinese Hamster Ovary (CHO) cells versus engineered
CHO cells containing an overexpressed target receptor. This
approach not only enables multiplexed cell assays, but also provide
confirmative results regarding to the compound effect on the target
receptor by comparison of the optical responses of the same
compound acting on two different cells, since two cells are
identical except for the target receptor expression level.
[0067] In another embodiment, the present invention can be enhanced
to perform multiple living cell-based assays at the same time using
the method 800 shown in FIG. 8. In accordance with method 800 one
can monitor the mass redistribution due to agonist-induced GPCR
activation in multiple types of living cells 108 by: (a) providing
a chamber (microplate) containing an array of the optical LID
biosensors 104 (step 802); (b) placing a first type of living cells
108 in a cell medium which covers one or more of the optical LID
biosensors 104 so the first type of living cells 108 are able to
attach to the surfaces 110 of the one or more optical LID
biosensors 104 (step 804); (c) placing a second type of living
cells 108 in a cell medium which covers one or more of the
remaining uncovered optical LID biosensors so the second type of
living cells 108 are able to attach to the surfaces 110 of the one
or more remaining uncovered optical LID biosensors 104 (step 806);
(d) applying a solution containing a compound into the cell mediums
located on the top surfaces 110 of covered optical LID biosensors
104 (step 810); and (e) interrogating the covered optical LID
biosensors 110 to monitor the time dependent optical responses 201
which indicate mass redistributions within the living cells 108 on
each of the covered optical LID biosensors 104 (step 812).
[0068] It should be appreciated that arrays of different DNA
vectors containing distinct target receptor genes in combination
with transfection reagents can be deposited onto a LID sensor; a
single type of cells is placed and overlaid with such array and
uptakes the genes. Thus only cells overlaid on each spot area
become transfected and therefore forming a transfected cell cluster
array (U.S. Pat. No. 6,544,790 B1 "Reverse transfection method").
Similarly, array of functional receptor proteins in complexed with
protein delivery reagents can be used to similar transfected cell
cluster array (US2004/0023391A1 "Methods and devices for protein
delivery"). Both types of transfected cell arrays can be used for
compound screening using the current technology.
[0069] In yet another embodiment, the present invention can be
further enhanced to perform multiple target screens in a single
type of cellsat the same time by using method 900 shown in FIG. 9.
In accordance with method 900 one can screen agonists against
multiple GPCRs 202 within a single type of living cells 108 by
performing the following steps: (a) providing a optical LID
biosensor 104 (step 902); (b) placing the living cells 108 in a
cell medium which covers the optical LID biosensor 104 so the
living cells 108 are able to attach to the surface 110 of the
optical LID biosensor 104 (step 904); (c) applying a solution
containing a cocktail solution of antagonists (step 906); (d)
applying a solution containing a compound into the cell medium
located on the top surface 110 of the optical LID biosensor 104
(step 908); and (e) interrogating the optical LID biosensor 104 to
monitor a time dependent optical response 201 which indicates mass
redistributions within the living cells 108 (step 910).
[0070] It should be appreciated that similar method can be used to
screen antagonist against multiple receptors in the same cell line
by modifying the method 900. Instead of a cocktail solution of
antagonists in the step 906, one can use a solution of compounds of
interest; at the same time, a cocktail solution of agonists is used
to replace the compound solution in the step 908.
[0071] Following is a discussion about the results of several
different experiments that were conducted to show that an optical
LID system 100 can be used to monitor a mass redistribution within
living cells 108 that are located on the surface 110 of the optical
LID biosensor 104 (see FIG. 2).
[0072] FIG. 10 is a graph that shows several agonist-induced
responses within chinese hamster ovary cells 108 (CHO 108) that
were monitored by the optical LID system 100. It is known that CHO
cells 108 endogenously express beta adrenergic receptors,
alpha2-adrenergic receptors, P2Y receptors, as well as
beta-arrestin and GRKs. It is also known that muscarinic receptors
are endogenously expressed at very low level in the CHO cells 108.
In this experiment, approximately .about.5.times.10.sup.4 CHO cells
108 were placed within each well of a microplate that contained an
array of optical LID biosensors 104. The CHO cells 108 were then
cultured in 150 .mu.l serum medium for 24 hours to ensure that the
CHO cells 108 became adherent to the substrate surface 110.
[0073] The graph shows the optical responses of the CHO cells 108
to four different compounds which were examined: (1) ATP (100
.mu.M), agonist for P2Y receptors; (2) clonidine (10 .mu.M),
agonist for alpha2-adrenergic receptors; (3) epinephrine (100
.mu.M), agonist for beta adrenergic receptors; and (4) oxotremorine
M (10 .mu.M), agonist for muscarinic receptors. Since muscarinic
receptors are endogenously expressed at very low level in CHO cells
108, oxotremorine M, agonist for muscarinic receptors, served as a
control. Each of these agonists was directly applied to a different
one of the wells which contained the serum medium. The optical
responses were then collected by the optical LID system 100.
[0074] The results showed that these adherent CHO cells 108 gave
rise to similar kinetics and transitions as shown by the optical
responses after the introduction of the three agonists: ATP,
clonidine, and epinephrine. Oxotremorine M caused almost no cell
response. In FIG. 11, a kinetics analysis of the later stage of the
process revealed that all three agonists (ATP, epinephrine,
clonidine) resulted in a similar slow process. The changes for the
Stage 3 as shown in FIG. 2, caused by those agonists, are shown in
the graph in FIG. 12. The similar changes might reflect the fact
that beta-arrestin, a critical component for GPCR translocation,
could be the limiting factor in the CHO cells 108, given that the
size of clathrin-coated pits and beta-arrestin are similar.
[0075] FIG. 13 is a graph that shows the results from an experiment
which indicates the ligand- and time-dependent response of a
monolayer of living CHO cells 108 on wave-guide biosensors 104. The
agonists which were used included: (1) clonidine; (2) oxotremorine
M; (3) NECA; and an telenzepine, an antagonist for M1 receptor, is
also used.
[0076] FIG. 14 is a graph that shows the results from an experiment
which indicates the ligand- and time-dependent response of
monolayer of living CHO cells 108 with stably overexpressed rat
muscarinic receptor subtype 1 (M1) on wave-guide biosensors 104.
The agonists which were used included: (1) clonidine; (2)
oxotremorine M; and (3) NECA; and an telenzepine, an antagonist for
M1 receptor, is also used.
[0077] FIG. 15 is a graph that shows the results from an experiment
which indicates the ligand-induced total change in response of
monolayer of living CHO cells 108 without (CHO) and with stably
overexpressed rat muscarinic receptor subtype 1(M1CHO) on
wave-guide biosensors 104. Results shown in FIGS. 11 and 13-15
indicated that (1) there are alpha2 adrenergic receptors expressed
in both CHO and M1-CHO cells; and their agonist (clonidine) induced
mass redistribution signals; (2) there is relatively low or almost
no M1 receptor expressed in CHO cells, but high in M1-CHO cells
since its agonist (oxotremorine M) but not its antagonist
(telenzepine) results in significantly larger responses in M1-CHO
cells.
[0078] FIG. 16 is a graph that shows the results from an experiment
which indicates the effect of pre-incubation of a dynamin
phosphorylation inhibitor (dynamin inhibitory peptide, DIP) on
oxotremorine M-induced time-dependent response of a monolayer of
living CHO cells 108 without and with stably overexpressed rat
muscarinic receptor subtype 1 (M1CHO) on wave-guide biosensors 104.
Results show that the pre-incubation of cells with DIP almost
totally eliminates the oxotremorine M-induced mass distribution
responses in both cell lines, suggesting that oxotremorine
M-induced mass distribution is dynamin-dependent. The
dynamin-dependency is common for most of agonist-induced GPCR
translocation.
[0079] FIG. 17 is a graph that shows the results from an experiment
which indicates the effect of pre-incubation of a dynamin
phosphorylation inhibitor (dynamin inhibitory peptide, DIP) on
clonidine-induced time-dependent response of a monolayer of living
Chinese Hamster Ovary (CHO) cells without and with stably
overexpressed rat muscarinic receptor subtype 1 (M1CHO) on
wave-guide biosensors 104. Results show that the pre-incubation of
both cell lines with DIP almost totally eliminates the
clonidine-induced mass distribution response, suggesting that
clonidine-induced mass distribution is also dynamin-dependent.
[0080] FIG. 18 is a graph that shows the results from an experiment
which indicates the effect of pre-incubation of a dynamin
phosphorylation inhibitor (dynamin inhibitory peptide, DIP) on
NECA-induced time-dependent response of a monolayer of living CHO
cells 108 without and with stably overexpressed rat muscarinic
receptor subtype 1 (M1CHO) on wave-guide biosensors 104. Results
showed that the pre-incubation of both cells with DIP has little
effect on NECA-induced response, suggesting that NECA results in
mass redistribution signals in both cell lines through a
dynamin-independent pathway.
[0081] Some additional features and advantages of using the optical
LID system 100 of the present invention are as follows:
[0082] (1) The present invention discloses a real time method that
can be used to perform a label free functional GPCR cell-based
assay which enables compound screening and profiling. This method
allows one to study an endogenous but relatively highly expressed
GPCR in living cells without needing to genetically engineer the
cell to over-express a receptor of interest.
[0083] (2) The present invention discloses methods to perform
multiplexed cell-based assays using a single sensor which offers an
advantage of increased throughput.
[0084] (3) The preferred optical LID biosensor 104 is a SPR sensor
104 or a waveguide grating based sensor 104. Other optical-based
biosensors can also be used such as ellipsometry devices,
evanescent wave devices, and reflectometry devices. For a more
detailed discussion about the structure and operation of these two
types of optical LID biosensors 104 reference is made to the
following documents: [0085] U.S. Pat. No. 4,815,843 entitled
"Optical Sensor for Selective Detection of Substances and/or for
the Detection of Refractive Index Changes in Gaseous, Liquid, Solid
and Porous Samples". [0086] K. Tiefenthaler et al. "Integrated
Optical Switches and Gas Sensors" Opt. Lett. 10, No. 4, April 1984,
pp. 137-139. The contents of these documents are incorporated by
reference herein.
[0087] Although several embodiments of the present invention have
been illustrated in the accompanying Drawings and described in the
foregoing Detailed Description, it should be understood that the
invention is not limited to the embodiments disclosed, but is
capable of numerous rearrangements, modifications and substitutions
without departing from the spirit of the invention as set forth and
defined by the following claims.
* * * * *