U.S. patent application number 10/303446 was filed with the patent office on 2003-10-02 for apparatus and method for lead profiling assay.
This patent application is currently assigned to Xerox Corporation. Invention is credited to Bruce, Richard H., Rosenberg, Steven.
Application Number | 20030186454 10/303446 |
Document ID | / |
Family ID | 28041047 |
Filed Date | 2003-10-02 |
United States Patent
Application |
20030186454 |
Kind Code |
A1 |
Bruce, Richard H. ; et
al. |
October 2, 2003 |
Apparatus and method for lead profiling assay
Abstract
A method is disclosed for performing drug lead profiling assays
for drug discovery utilizing a nanocalorimeter. The method includes
depositing not less than one drop containing a profile molecule
solution to be screened and not less than one drop containing a
drug library compound on a test substrate. After the drops are
merged, a determination is made as to whether a reaction has
occurred.
Inventors: |
Bruce, Richard H.; (Los
Altos, CA) ; Rosenberg, Steven; (Oakland,
CA) |
Correspondence
Address: |
Xerox Corporation
Patent Documentation Center
Xerox Square 20th Floor
100 Clinton Ave. S.
Rochester
NY
14644
US
|
Assignee: |
Xerox Corporation
|
Family ID: |
28041047 |
Appl. No.: |
10/303446 |
Filed: |
November 22, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10303446 |
Nov 22, 2002 |
|
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10114611 |
Apr 1, 2002 |
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Current U.S.
Class: |
436/147 ;
422/82.12; 436/77 |
Current CPC
Class: |
G01N 25/4866 20130101;
Y10T 436/25 20150115; B82Y 15/00 20130101; Y10T 436/25625 20150115;
G01N 25/4846 20130101; Y10S 977/957 20130101; Y10T 436/2575
20150115 |
Class at
Publication: |
436/147 ; 436/77;
422/82.12 |
International
Class: |
G01N 025/20 |
Claims
What is claimed is:
1. A method for performing lead profiling assays for drug discovery
utilizing a nanocalorimeter, the method comprising: depositing not
less than one drop containing profile molecules to be screened upon
a test substrate; depositing not less than one drop containing a
drug library compound upon a test substrate; merging said not less
than one drop containing profile molecules to be screened and said
not less than one drop containing a drug library compound; and
detecting whether a reaction has occurred between said not less
than one drop containing profile molecules to be screened and said
not less than one drop containing a drug library compound.
2. The method for performing lead profiling assays for drug
discovery according to claim 1, wherein said not less than one drop
containing profile molecules to be screened comprises protein
molecules.
3. The method for performing lead profiling assays for drug
discovery according to claim 1, wherein said drug library compound
comprises a plurality of drug library compounds.
4. The method for performing lead profiling assays for drug
discovery according to claim 1, wherein said drug library compound
is known to react with a profile molecule.
5. The method for performing lead profiling assays for drug
discovery according to claim 1, wherein detecting whether a
reaction has occurred comprises detecting an increase in signal
proportional to the reaction.
6. The method for performing lead profiling assays for drug
discovery according to claim 1, wherein detecting whether a
reaction has occurred comprises detecting a thermal change.
7. The method for performing lead profiling assays for drug
discovery according to claim 1, further comprising identifying
those drug library compounds that cause a reaction with said
profile molecules.
8. The method for performing lead profiling assays for drug
discovery according to claim 1, further comprising providing a
controlled environment.
9. The method for performing lead profiling assays for drug
discovery according to claim 8, wherein said controlled environment
comprises a thermally controlled environment.
10. The method for performing lead profiling assays for drug
discovery according to claim 1, wherein merging said not less than
one drop containing profile molecules and said not less than one
drop of said drug library compound comprises utilizing
electrostatic force.
11. The method for performing lead profiling assays for drug
discovery according to claim 1, wherein the nanocalorimeter
includes a test substrate having at least one reference region and
at least one measurement region.
12. The method for performing lead profiling assays for drug
discovery according to claim 11, wherein depositing said not less
than one drop containing a profile molecule comprises depositing
not less than one drop containing said profile molecule within each
of said measurement region and said reference region.
13. The method for performing lead profiling assays for drug
discovery according to claim 11, wherein depositing said not less
than one drop containing said drug library compound comprises
depositing not less than one drop containing said drug library
compound within said measurement region.
14. The method for performing lead profiling assays for drug
discovery according to claim 11, further comprising depositing not
less than one drop of a non-reactive chemical material within said
reference region.
15. The method for performing lead profiling assays for drug
discovery according to claim 1, wherein the nanocalorimeter
utilizes a single test chamber to perform lead profiling
assays.
16. The method for performing lead profiling assays for drug
discovery according to claim 1, wherein the nanocalorimeter
utilizes at least a loading chamber and a separate test chamber to
perform lead profiling assays.
17. The method for performing lead profiling assays for drug
discovery according to claim 1, wherein said substrate includes a
plurality of test surfaces.
Description
[0001] This application is a continuation in part of U.S.
application Ser. No. 10/114,611, filed Apr. 1, 2002, the disclosure
of which is totally incorporated by reference.
CROSS REFERENCE TO RELATED APPLICATIONS
[0002] The following copending application, Attorney Docket Number
D/A1578I1, U.S. application Ser. No. XX/XXXXX, filed Nov. 22, 2002,
titled "Apparatus and Method for Multiple Target Assay for Drug
Discovery", is assigned to the same assignee of the present
application. The entire disclosure of this copending application is
totally incorporated herein by reference in its entirety.
INCORPORATION BY REFERENCE
[0003] The following U.S. patents are fully incorporated herein by
reference: U.S. Pat. No. 5,967,659 ("Ultrasensitive Differential
Microcalorimeter with User-selected Gain Setting" to Plotnikov et
al.); U.S. Pat. No. 6,079,873 ("Micron-scale Differential Scanning
Calorimeter on a Chip" to Cavicchi et al.); U.S. Pat. No. 6,096,559
"Micromechanical Calorimetric Sensor" to Thundat et al.); and U.S.
Pat. No. 6,193,413 ("System and Method for an Improved Calorimeter
for Determining Thermodynamic Properties of Chemical and Biological
Reactions" to Lieberman).
BACKGROUND OF THE INVENTION
[0004] This invention relates generally to a method for performing
assays for drug discovery, and more specifically, to a method for
performing lead drug profiling assays.
[0005] In recent years, researchers and companies have turned to
combinatorial methods and techniques for synthesizing, discovering
and developing new compounds, materials, and chemistries. For
example, pharmaceutical researchers have turned to combinatorial
libraries as sources of new lead compounds for drug discovery. As
another example, Symyx Technologies.RTM. is applying combinatorial
techniques to materials discovery in the life sciences, chemical,
and electronics industries. Consequently, there is a need for tools
that can measure reactions and interactions of large numbers of
small samples in parallel, consistent with the needs of
combinatorial discovery techniques. Preferably, users desire that
these tools enable inexpensive measurements and minimize
contamination and cross-contamination problems. In addition there
has been an explosion in the number of potential drug targets due
to the accelerated implementation of genomics technologies and the
completion of the Human Genome sequence.
[0006] In some cases, the sample to be studied is precious, and it
might not be acceptable to use the relatively large amount of
material required by a standard microcalorimeter to perform only
one measurement. For example, one may desire to study a natural
extract or synthesized compound for biological interactions, but in
some cases the available amount of material at concentrations large
enough for calorimetry might be no more than a few milliliters.
Performing a measurement in standard microcalorimeters, such as
those sold, for example, by MicroCal.RTM. Inc. (model VP-ITC) or
Calorimetry Sciences Corporation.RTM. (model CSC-4500), requires
about 1 ml of sample, which means that one would possibly be faced
with using a majority or all of the precious material for one or a
small series of measurements. Tools that enable calorimetric
measurements with much smaller sample sizes would be helpful in
overcoming this limitation.
[0007] One of the most popular uses of combinatorial techniques to
date has been in pharmaceutical research. Pharmaceutical
researchers have turned to combinatorial libraries as sources of
new lead compounds for drug discovery. A combinatorial library is a
collection of chemical compounds which have been generated, by
either chemical synthesis or biological synthesis, by combining a
number of chemical "building blocks" as reagents. For example, a
combinatorial polypeptide library is formed by combining a set of
amino acids in every possible way for a given compound length
(i.e., the number of amino acids in a polypeptide compound).
Millions of chemical compounds can theoretically be synthesized
through such combinatorial mixing of chemical building blocks.
[0008] Once a library has been constructed, it must be screened to
identify compounds, which possess some kind of biological or
pharmacological activity. For example, screening can be done with a
specific biological compound, often referred to as a target that
participates in a known biological pathway or is involved in some
regulatory function. The library compounds that are found to react
with the targets are candidates for affecting the biological
activity of the target, and hence a candidate for a therapeutic
agent.
[0009] Through the years, the pharmaceutical industry has
increasingly relied on high throughput screening (HTS) of libraries
of chemical compounds to find drug candidates. HTS describes a
method where many discrete compounds are tested in parallel so that
large numbers of test compounds are screened for biological
activity simultaneously or nearly simultaneously. Currently, the
most widely established techniques utilize 96-well microtitre
plates. In this format, 96 independent tests are performed
simultaneously on a single 8 cm.times.12 cm plastic plate that
contains 96 reaction wells. These wells typically require assay
volumes that range from 50 to 500 .mu.l. In addition to the plates,
many instruments, materials, pipettors, robotics, plate washers and
plate readers are commercially available to fit the 96-well format
to a wide range of homogeneous and heterogeneous assays. To achieve
faster testing, the industry is evolving to plates that contain 384
and 1536 wells.
[0010] A variety of measurement approaches has been used to screen
combinatorial libraries for lead compounds, one of which is the
competitive binding assay. In this assay, a ligand, often the
natural ligand in a biological pathway, is identified that will
bind well with the target protein molecule. The assay often
requires the chemical attachment of a fluorescent molecule to this
marker ligand such that the fluorescent molecule does not affect
the manner in which the marker ligand reacts with the target
protein. Alternatively, the ligand could be radioactively labeled
or labeled with a chemiluminescent molecule. To operate an
inhibitor assay, the target protein is exposed to a mixture of the
test ligands and marker ligands often in microtitre wells. After a
time for reaction, the wells are rinsed such that free marker
ligand is washed away. In wells where the target protein and the
test ligand have reacted, the test ligand has blocked the active
site of the target protein so the marker ligand cannot react and is
washed away, while in cells where the target protein and test
ligand have not reacted, the marker ligand has bound to the target
protein and is not washed away. By investigating the wells for the
presence of fluorescence after the washing, reactions of test
ligands and target proteins can be determined as having occurred in
wells where reduced fluorescence is observable relative to control
wells to which no test ligands have been added.
[0011] However, the competitive binding assay requires time and
expense to develop the labeled reagents and assay. The principal
components that need development are discovering a marker ligand
and attaching a fluorophore to the marker in a manner that does not
affect its reaction with the target protein. Attaching the
fluorescent marker can often take 3 months of development or more
and cost $250 k or more once the marker ligand is identified. An
assay method that avoids such assay development, such as measuring
the heat of the reaction with calorimetry, would eliminate this
cost and time delay in the discovery process.
[0012] The following disclosures may be relevant and/or helpful in
providing an understanding of some aspect of the present
invention:
[0013] In Plotnikov et al., U.S. Pat. No. 5,967,659
("Ultrasensitive Differential Microcalorimeter with User-selected
Gain Setting"), a differential calorimeter is disclosed that
includes sample and reference cells, a thermal shield surrounding
the cells, heating devices thermally coupled to the thermal shield
and the cells, a temperature monitoring system, and a control
system. The temperature monitoring system monitors the temperature
of the shield, cell temperatures, and temperature differentials
between the cells and the shield. The control system generates
output signals for control of the heating devices, with a gain
setting and scan rate selected by means of a user interface. Output
control signals are functions of input temperature signals and the
user-selected gain setting, as well as functions of input
temperature signals and the user-selected scan rate using a mapping
function stored in memory.
[0014] In Cavicchi et al., U.S. Pat. No. 6,079,873 ("Micron-scale
Differential Scanning Calorimeter on a Chip"), a differential
scanning microcalorimeter produced on a silicon chip enables
microscopic scanning calorimetry measurements of small samples and
thin films. The chip, fabricated using standard CMOS processes,
includes a reference zone and a sample zone. The reference and
sample zones may be at opposite ends of a suspended platform or may
reside on separate platforms. Each zone is heated with an
integrated polysilicon heater. A thermopile consisting of a
succession of thermocouple junctions generates a voltage
representing the temperature difference between the reference and
sample zones.
[0015] In Thundat et al., U.S. Pat. No. 6,096,559 ("Micromechanical
Calorimetric Sensor"), a calorimeter sensor apparatus utilizes
microcantilevered spring elements for detecting thermal changes
within a sample containing biomolecules which undergo chemical and
biochemical reactions. The spring element includes a bimaterial
layer of chemicals on a coated region on at least one surface of
the microcantilever. The chemicals generate a differential thermal
stress across the surface upon reaction of the chemicals with an
analyte or biomolecules within the sample due to the heat of
chemical reactions in the sample placed on the coated region. The
thermal stress across the spring element surface creates mechanical
bending of the microcantilever. The spring element has a low
thermal mass to allow detection and measuring of heat transfers
associated with chemical and biochemical reactions within a sample
place on or near the coated region. Deflections of the cantilever
are detected by a variety of detection techniques.
[0016] In Lieberman, U.S. Pat. No. 6,193,413 ("System and Method
for an Improved Calorimeter for Determining Thermodynamic
Properties of Chemical and Biological Reactions") a
microcalorimeter includes a thin amorphous membrane anchored to a
frame within an environmental chamber. Thermometers and heaters are
placed on one side of a thermal conduction layer mounted on the
central portion of the membrane. Samples are placed on two such
membranes; each sample is heated and its individual heat capacity
determined. The samples are then mixed by sandwiching the two
microcalorimeters together to cause a binding reaction to occur.
The amount of heat liberated during the reaction is measured to
determine the enthalpy of binding.
SUMMARY OF THE INVENTION
[0017] Briefly stated, and in accordance with one aspect of the
present invention, there is disclosed a method for performing drug
lead profiling assays for drug discovery utilizing a
nanocalorimeter. The method includes depositing not less than one
drop containing a target solution to be screened and not less than
one drop containing a drug library compound on a test substrate.
After the drops are merged, a determination is made as to whether a
reaction has occurred.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The foregoing and other features of the instant invention
will be apparent and easily understood from a further reading of
the specification, claims and by reference to the accompanying
drawings in which:
[0019] FIG. 1 is a flow chart illustrating a prior art method for
performing drug lead profiling; and
[0020] FIG. 2 is an embodiment of the method for performing lead
profiling assays in accordance with the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0021] As used herein, the term "ligand" refers to an agent that
binds a target molecule. According to the present invention, a
ligand is not limited to an agent that binds a recognized
functional region of the target protein e.g. the active site of an
enzyme, the antigen-combining site of an antibody, the
hormone-binding site of a receptor, a cofactor-binding site, and
the like. In practicing the present invention, a ligand can also be
an agent that binds any surface or conformational domains of the
target protein. Therefore, the ligands of the present invention
encompass agents that in and of themselves may have no apparent or
known biological function, beyond their ability to bind to the
target protein in the manner described above.
[0022] As used herein, the term "test ligand" refers to an agent,
comprising a compound, molecule or complex, which is being tested
for its ability to bind to a target molecule. Test ligands can be
virtually any agent, including without limitation metals, peptides,
proteins, lipids, polysaccharides, nucleic acids, small organic
molecules, and combinations thereof. Complex mixtures of substances
such as natural product extracts, which may include more than one
test ligand, can also be tested, and the component that binds the
target molecule can be purified from the mixture in a subsequent
step.
[0023] As used herein, the term "target protein" refers to a
peptide, protein or protein complex for which identification of a
ligand or binding partner is desired. Target proteins include
without limitation peptides or proteins known or believed to be
involved in the etiology of a given disease, condition or
pathophysiological state, or in the regulation of physiological
function. Target proteins may be derived from any living organism,
such as a vertebrate, particularly a mammal and even more
particularly a human. For use in the present invention, it is not
necessary that the protein's biochemical function be specifically
identified. Target proteins include without limitation receptors,
enzymes, oncogene products, tumor suppressor gene products, vital
proteins, and transcription factors, either in purified form or as
part of a complex mixture of proteins and other compounds.
Furthermore, target proteins may comprise wild type proteins, or,
alternatively, mutant or variant proteins, including those with
altered stability, activity, or other variant properties, or hybrid
proteins to which foreign amino acid sequences, e.g. sequences that
facilitate purification, have been added.
[0024] As used herein, "test combination" refers to the combination
of a test ligand and a target protein. "Control combination" refers
to the target protein in the absence of a test ligand.
[0025] As used herein, "screening" refers to the testing of a
multiplicity of molecules or compounds for their ability to bind to
a target molecule.
[0026] As used herein, the term "lead molecule" refers to a
molecule or compound, from a combinatorial library or other
collection, which displays relatively high affinity for a target
molecule. High affinity is detected by this invention through the
heat released in a chemical reaction. The terms "lead compound" and
"lead molecule" are synonymous.
[0027] As used herein, the term "target molecule" encompasses
peptides, proteins, nucleic-acids, protein-nucleic acid complexes
and other receptors. The term encompasses both enzymes and proteins
which are not enzymes. The term encompasses monomeric and
multimeric proteins. Multimeric proteins may be homomeric or
heteromeric. The term encompasses nucleic acids comprising at least
two nucleotides, such as oligonucleotides. Nucleic acids can be
single-stranded, double-stranded, or triple-stranded. The term
encompasses a nucleic acid which is a synthetic oligonucleotide, a
portion of a recombinant DNA molecule, or a portion of chromosomal
DNA. The term target molecule also encompasses portions of
peptides, secondary, tertiary, or quaternary structure through
folding, with substituents including, but not limited to,
cofactors, coenzymes, prosthetic groups, lipids, oligosaccharides,
or phosphate groups.
[0028] As used herein, the term "molecule" refers to the compound,
which is tested for binding affinity for the target molecule. This
term encompasses chemical compounds of any structure, including,
but not limited to nucleic acids and peptides. More specifically,
the term "molecule" encompasses compounds in a compound or a
combinatorial library. The terms "molecule" and "ligand" are
synonymous.
[0029] As used herein, the term "thermal change" encompasses the
release of energy in the form of heat or the absorption of energy
in the form of heat.
[0030] As used herein, the term "contacting a target molecule"
refers broadly to placing the target molecule in solution with the
molecule to be screened for binding. Less broadly, contacting
refers to the turning, swirling, shaking or vibrating of a solution
of the target molecule and the molecule to be screened for binding.
More specifically, contacting refers to the mixing of the target
molecule with the molecule to be tested for binding. Mixing can be
accomplished, for example, by repeated uptake and discharge through
a pipette tip or by deposition by robotic means. Preferably,
contacting refers to the equilibration of binding between the
target molecule and the molecule to be tested for binding.
[0031] As used herein, the term "biochemical conditions"
encompasses any component, thermodynamic property, or kinetic
property of a physical, chemical, or biochemical reaction.
Specifically, the term refers to conditions of temperature,
pressure, protein concentration, pH, ionic strength, salt
concentration, time, electric current, potential difference, and
concentrations of cofactor, coenzyme, oxidizing agents, reducing
agents, detergents, metal ion, ligands, buffer components,
co-solvents including DMSO (dimethyl sulfoxide), glycerol, and
related compounds, enhancers, and inhibitors.
[0032] As used here in the term "lead profiling assay" encompasses
the testing for reaction of a compound or a series of compounds
with known binding activity towards a desired target against a
plurality of other targets so as to determine a reactivity profile
for said compound of compounds.
[0033] The present invention encompasses nanocalorimeters and
nanocalorimeter arrays that enable measurement of enthalpic
changes, such as enthalpic changes arising from reactions, phase
changes, changes in molecular conformation, and the like.
Furthermore, the present invention encompasses combinatorial
methods and high-throughput screening methods that use
nanocalorimeters in the study, discovery, and development of new
compounds, materials, chemistries, and chemical processes, as well
as high-throughput monitoring of compounds or materials, or
high-throughput monitoring of the processes used to synthesize or
modify compounds or materials. The present invention also relates
to compounds or materials identified by the above methods and their
therapeutic uses (for diagnostic, preventive or treatment
purposes), uses in purification and separation methods, and uses
related to their novel physical or chemical properties. For the
purposes herein, a nanocalorimeter refers to a device capable of
measuring heats of reaction in the range of nanocalories.
[0034] As an example, the present invention encompasses
high-throughput screening methods for identifying a ligand that
binds a target protein. If the target protein to which the test
ligand binds is associated with or causative of a disease or
condition, the ligand may be useful for diagnosing, preventing or
treating the disease or condition. A ligand identified by the
present method can also be one that is used in a purification or
separation method, such as a method that results in purification or
separation of the target protein from a mixture. The present
invention also relates to ligands identified by the present method
and their therapeutic uses (for diagnostic, preventive or treatment
purposes) and uses in purification and separation methods.
[0035] In practicing the present invention, the test ligand is
combined with a target molecule, and the mixture is maintained
under appropriate conditions and for a sufficient time to allow
binding of the test ligand to the target molecule. Experimental
conditions are determined empirically for each target molecule.
When testing multiple test ligands, incubation conditions are
usually chosen so that most ligand:target molecule interactions
would be expected to proceed to completion. In high-throughput
screening applications, the test ligand is usually present in molar
excess relative to the target molecule. The target molecule can be
in a soluble form, or, alternatively, can be bound to a solid phase
matrix. The matrix may comprise without limitation beads, membrane
filters, plastic surfaces, or other suitable solid supports.
[0036] Binding to a given target is a prerequisite for
pharmaceuticals intended to modify directly the action of that
target. Thus, if a test ligand is shown, through use of the present
method, to bind a target that reflects or affects the etiology of a
condition, it may indicate the potential ability of the test ligand
to alter target function and to be an effective pharmaceutical or
lead compound for the development of such a pharmaceutical.
Alternatively, the ligand may serve as the basis for the
construction of hybrid compounds containing an additional component
that has the potential to alter the target's function. For example,
a known compound that inhibits the activity of a family of related
enzymes may be rendered specific to one member of the family by
conjugation of the known compound to a ligand, identified by the
methods of the present invention, that binds specifically to that
member at a different site than that recognized by the known
compound.
[0037] The fact that the present method is based on physicochemical
properties common to most targets gives it widespread application.
The present invention can be applied to large-scale systematic
high-throughput procedures that allow a cost-effective screening of
many thousands of test ligands. Once a ligand has been identified
by the methods of the present invention, it can be further analyzed
in more detail using known methods specific to the particular
target used. Also, the ligand can be tested for its ability to
influence, either positively or negatively, a known biological
activity of the target.
[0038] In the drug discovery process, a drug target protein is
screened for reactivity against a large number (500,000) of
compounds from a drug library of compounds. Often it is desirable
to screen several different drug target proteins against the same
library of proteins if the target proteins are thought to have a
similar function such as the enzymatic function of a kinase.
[0039] Turning to FIG. 1, there is shown an embodiment of a
conventional drug screening process, a competitive screen, using a
fluorescent assay. In a competitive screen, the reaction of the
drug library compound with the target protein prevents the reaction
of a second known reactive compound that contains a detectable
label such as, in the case of a fluorescent assay, a fluorescent
label. The level of reactivity of the drug library compound is
inferred by detecting the fluorescence coming from the bound
labeled compound. Several types of fluorescent assays are currently
utilized in the art, but the two most practiced are fluorescence
intensity and fluorescence polarization.
[0040] In a fluorescence intensity assay, a labeled ligand 1130 at
a low concentration and one or more drug library compounds 1150 at
a higher concentration (5 .mu.M) are mixed with a target protein
1110. The labeled ligand 1130 is known to react strongly with the
target protein 1110 and is often the natural ligand. The receptor
of the target protein 1110 is immobilized to the container and
incubated with the mixture of labeled ligand and drug library
compounds at step 1140. The label on the ligand in this example is
a molecule that fluoresces in a particular way when stimulated by
light such as a laser or an ultraviolet light source. Radioactive
compounds can also be used as labels. Following the incubation step
1152, the free ligand is removed by washing at step 1155 and the
amount of bound, labeled ligand is measured by detecting the amount
and nature of the light emitted from the fluorescent label attached
to ligand 1130 at step 1160. If the fluorescence is reduced, then a
reaction with a drug library compound has occurred, as shown at
step 1180. The reaction will reduce the amount of labeled ligand
that reacts by a predicted amount ranging from 20% to 50% or more.
If the fluorescence is not reduced, then a reaction has not
occurred, as shown at step 1170, since the labeled ligand reaction
is not inhibited. The variation in the amount of fluorescent light
for uninhibited binding is approximately 10%, resulting in an
acceptable signal to noise ratio.
[0041] For the fluorescence polarization approach, the labeled
ligand is incubated with the receptor and the drug library
compounds but the receptor does not need to be immobilized. Here
the assay relies on the observation that fluorescence from the
labeled ligand bound to the receptor is substantially more
polarized than the fluorescence from an unbound labeled ligand.
Again in this approach, the signal produced is maximum when no the
drug library compounds bind to the target receptor and is reduced
by binding. In both straight fluorescence and fluorescence
polarization, reaction of the target protein and a labeled ligand
is required for the assay.
[0042] For competitive assays, a compound is required that strongly
interacts with the protein target. This compound is often the
ligand that reacts with the protein target in nature. In the
fluorescent assays described here, this ligand must have an
attached molecule that serves as a fluorescent label. The label
must be attached in a manner that does not affect the ligand
reaction to the protein target. Developing a competitive assay by
creating such a labeled ligand is a costly, time-consuming
effort.
[0043] Lead profiling identifies and prioritizes a portfolio of
compounds that have the best chance of success in clinical
development. Assays can range from relatively simple
ligand-receptor interaction to cell-based assays using genetically
modified cells with multiple endpoints or reporter genes. The
choice for a specific assay is determined by factors such as the
desired target profile, sensitivity, robustness, and ease of
automation. Drug leads are screened against other naturally
occurring proteins to determine the level of interaction with these
other targets. To become a drug lead, the protein must not only
interact with the desired target but must not interact with other
possibly similar proteins. Leads that do not affect proteins other
than their target protein are more likely to not be toxic.
Alternatively, a specific profile of interaction with more than one
target may be desired, and specific other targets definitely
excluded. Testing for interactions with these other proteins using
existing technology requires a unique assay for each protein. In
many cases, screening against other proteins can require many
different assay formats and reagent development and consequently be
costly and time-consuming. For competitive assays this requires a
high affinity interaction between the protein and a labeled
ligand.
[0044] However, with the device taught herein, there is no need to
develop a specific assay for each target, since a direct
measurement of the heat of interaction is made utilizing
calorimetry. Turning now to FIG. 2, there is shown an embodiment of
the nanocalorimeter as used for a lead profiling assay. In FIG. 12,
a target substance to be screened, for example a protein, 1210 is
combined at step 1220 with one or more drug library compounds 1230,
which had reacted with an original target protein. For example,
with a kinase inhibitor, the drug lead is tested for reaction with
other kinases as well as other key proteins to test activity level.
The enthalpy of this reaction is measured at step 1240. The
enthalpy of this reaction is then compared with the enthalpy for a
reaction with no inhibitor. If there is a reaction, it is
identified at step 1260; if a reaction is not present, it is
identified at step 1250.
[0045] While the present invention has been illustrated and
described with reference to specific embodiments, further
modification and improvements will occur to those skilled in the
art. It is to be understood, therefore, that this invention is not
limited to the particular forms illustrated and that it is intended
in the appended claims to embrace all alternatives, modifications,
and variations which do not depart from the spirit and scope of
this invention.
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