U.S. patent application number 09/190128 was filed with the patent office on 2001-06-14 for high throughput method for functionally classifying proteins identified using a genomics approach.
Invention is credited to CARVER, THEODORE E. JR., PANTOLIANO, MICHAEL W., SALEMME, F. RAYMOND.
Application Number | 20010003648 09/190128 |
Document ID | / |
Family ID | 22060528 |
Filed Date | 2001-06-14 |
United States Patent
Application |
20010003648 |
Kind Code |
A1 |
PANTOLIANO, MICHAEL W. ; et
al. |
June 14, 2001 |
HIGH THROUGHPUT METHOD FOR FUNCTIONALLY CLASSIFYING PROTEINS
IDENTIFIED USING A GENOMICS APPROACH
Abstract
The present invention provides a method for functionally
classifying a protein that is capable of unfolding due to a thermal
change. The method comprises screening one or more of a
multiplicity of different molecules for their ability to shift the
thermal unfolding curve of the protein, wherein a shift in the
thermal unfolding curve indicates that the molecule binds to the
protein or affects the stability in a measurable way; generating an
activity spectrum for the protein wherein the activity spectrum
reflects a set of molecules, from the multiplicity of molecules,
that shift the thermal unfolding curve, of the protein and
therefore are ligands that bind to the protein, comparing the
activity spectrum for the protein to one or more functional
reference spectrum lists; and classifying the protein according to
the set of molecules in the multiplicity of different molecules
that shift the thermal unfolding curve of the protein.
Inventors: |
PANTOLIANO, MICHAEL W.;
(AVONDALE, PA) ; SALEMME, F. RAYMOND; (YARDLEY,
PA) ; CARVER, THEODORE E. JR.; (THORNDALE,
PA) |
Correspondence
Address: |
STERNE KESSLER GOLDSTEIN AND FOX
SUITE 600
1100 NEW YORK AVENUE NW
WASHINGTON
DC
200053934
|
Family ID: |
22060528 |
Appl. No.: |
09/190128 |
Filed: |
November 12, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60065129 |
Nov 12, 1997 |
|
|
|
Current U.S.
Class: |
435/4 ; 435/7.1;
530/350 |
Current CPC
Class: |
C40B 30/04 20130101;
G01N 33/5302 20130101; G01N 33/68 20130101; G01N 2400/40 20130101;
G01N 33/542 20130101; G01N 2333/96444 20130101; G01N 33/6845
20130101 |
Class at
Publication: |
435/4 ; 530/350;
435/7.1 |
International
Class: |
C07K 001/00; C07K
014/00; C07K 017/00 |
Claims
What is claimed is:
1. A method for functionally classifying a protein, said method
comprising: (a) screening one or more of a multiplicity of
different molecules for their ability to modify the stability of
said protein, wherein modification of the stability of the protein
indicates that the molecule binds to the protein; (b) generating an
activity spectrum for said protein from said screening of step (a),
wherein said activity spectrum reflects a subset of molecules, from
said multiplicity of different molecules, that modify the stability
of said protein and therefore are ligands that bind to the protein;
(c) comparing said activity spectrum for said protein to one or
more functional reference spectrum lists; and (d) classifying said
protein according to the set of molecules in said multiplicity of
different molecules that modify the stability of said protein.
2. The method of claim 1, wherein said screening step (a)
comprises: (a1) contacting said protein with one or more of said
multiplicity of different molecules in each of a multiplicity of
containers; (a2) treating said protein in each of said multiplicity
of containers to cause said protein to unfold; (a3) measuring in
each of said containers a physical change associated with the
unfolding of said target molecule; (a4) generating an unfolding
curve for said target molecule for each of said containers; and
(a5) comparing each of said unfolding curves in step (d) to (1)
each of said other unfolding curves and to (2) the unfolding curve
obtained for said protein in the absence of any of said
multiplicity of different molecules; and (a6) determining whether
any of said multiplicity of different molecules modifies the
stability of said protein, wherein a modification in stability is
indicated by a change in said unfolding curve.
3. A method for functionally classifying a protein, said method
comprising: (a) screening one or more of a multiplicity of
different molecules known to bind to a particular class of proteins
for their ability to modify the stability of said protein, wherein
modification of the stability of the protein indicates that the
molecule binds to the protein; (b) generating an activity spectrum
for said protein from said screening of step (a), wherein said
activity spectrum reflects a subset of molecules, from said
multiplicity of different molecules, that modify the stability of
said protein and therefore are ligands that bind to the protein;
and (c) classifying said protein as a member of said class of
proteins if said one or more of said multiplicity of different
molecules modify the stability of said protein.
4. The method of claim 3, wherein said screening step (a)
comprises: (a1) contacting said protein with one or more of said
multiplicity of different molecules in each of a multiplicity of
containers; (a2) treating said protein in each of said multiplicity
of containers to cause said protein to unfold; (a3) measuring in
each of said containers a physical change associated with the
unfolding of said target molecule; (a4) generating an unfolding
curve for said target molecule for each of said containers; and
(a5) comparing each of said unfolding curves in step (d) to (1)
each of said other unfolding curves and to (2) the unfolding curve
obtained for said protein in the absence of any of said
multiplicity of different molecules; and (a6) determining whether
any of said multiplicity of different molecules modifies the
stability of said protein, wherein a modification in stability is
indicated by a change in said unfolding curve.
5. A method for functionally classifying a protein, said method
comprising: classifying said protein according to the set of
molecules in a multiplicity of different molecules that modify the
stability of said protein.
6. A method for functionally classifying a protein that is capable
of unfolding due to a thermal change, said method comprising: (a)
screening one or more of a multiplicity of different molecules for
their ability to shift the thermal unfolding curve of said protein,
wherein a shift in the thermal unfolding curve ofthe protein
indicates that the molecule binds to the protein; (b) generating an
activity spectrum for said protein from said screening of step (a),
wherein said activity spectrum reflects a subset of molecules, from
said multiplicity of different molecules, that shift the thermal
unfolding curve of said protein and therefore are ligands that bind
to the protein; (c) comparing said activity spectrum for said
protein to one or more functional reference spectrum lists; and (d)
classifying said protein according to the set of molecules in said
multiplicity of different molecules that shift the thermal
unfolding curve of said protein.
7. The method of claim 6, wherein said screening step (a)
comprises: (a1) contacting said protein with one or more of said
multiplicity of different molecules in each of a multiplicity of
containers; (a2) heating said multiplicity of containers from step
(a1); (a3) measuring in each of said containers a physical change
associated with the thermal unfolding of said target molecule
resulting from said heating; (a4) generating a thermal unfolding
curve for said target molecule as a function of temperature for
each of said containers; and (a5) comparing each of said unfolding
curves in step (a4) to (1) each of said other thermal unfolding
curves and to (2) the thermal unfolding curve obtained for said
protein in the absence of any of said multiplicity of different
molecules; and (a6) determining whether any of said multiplicity of
different molecules shift the thermal unfolding curve of said
protein.
8. The method of claim 7, wherein said comparing step (a5)
comprises ranking said molecules in said multiplicity of different
molecules for said protein according to the ability of each of said
multiplicity of different molecules to shift the thermal unfolding
curve of said protein.
9. The method of claim 7, wherein in said heating step (a2), said
multiplicity of containers is heated simultaneously.
10. The method of claim 7, wherein said step (a4) further comprises
determining a midpoint temperature (T.sub.m) from the thermal
unfolding curve; and wherein said step (a5) further comprises
comparing the T.sub.m of each of said unfolding curves in step (a4)
to (1) the T.sub.m of each of said other thermal unfolding curves
and to (2) the T.sub.m of the thermal unfolding curve obtained for
said target protein in the absence of any of said different
molecules.
11. The method of claim 7, wherein said step (a3) comprises
measuring the absorbance of light by said contents of each of said
containers.
12. The method of claim 7, wherein said step (a1) comprises
contacting said protein with a fluorescence probe molecule present
in each of said multiplicity of containers and wherein said step
(a3) comprises (i) exciting said fluorescence probe molecule, in
each of said multiplicity of containers, with light; and (ii)
measuring the fluorescence from each of said multiplicity of
containers.
13. The method of claim 12, wherein said step (a3)(ii) further
comprises measuring the fluorescence from each of said multiplicity
of containers one container at a time.
14. The method of claim 12, wherein said step (a3)(ii) further
comprises measuring the fluorescence from a subset of said
multiplicity of containers simultaneously.
15. The method of claim 12, wherein said step (a3)(ii) further
comprises measuring the fluorescence from each of said multiplicity
of containers Simultaneously.
16. The method of claim 7, wherein said step (a3) comprises (i)
exciting tryptophan residues in said protein, in each of said
multiplicity of containers, with light; and (ii) measuring the
fluorescence from each of said multiplicity of containers.
17. The method of claim 7, wherein said multiplicity of containers
in step (a1) comprises a multiplicity of wells in a microplate.
18. A method for functionally classifying a protein that is capable
of unfolding due to a thermal change, said method comprising: (a)
screening one or more of a multiplicity of different molecules
known to bind to a particular class of proteins for their ability
to shift the thermal unfolding curve of said protein, wherein a
shift in the thermal unfolding curve of the protein indicates that
the molecule binds to the protein; (b) generating an activity
spectrum for said protein from said screening of step (a), wherein
said activity spectrum reflects a subset of molecules, from said
multiplicity of different molecules, that shift the thermal
unfolding curve of said protein and therefore are ligands that bind
to the protein; and (c) classifying said protein as a member of
said class of proteins if said one or more of said multiplicity of
different molecules shift the thermal unfolding curve of said
protein.
19. The method of claim 18, wherein said screening step (a)
comprises: (a1) contacting said protein with one or more of said
multiplicity of different molecules in each of a multiplicity of
containers; (a2) heating said multiplicity of containers from step
(al); (a3) measuring in each of said containers a physical change
associated with the thermal unfolding of said target molecule
resulting from said heating; (a4) generating a thermal unfolding
curve for said target molecule as a function of temperature for
each of said containers; and (a5) comparing each of said unfolding
curves in step (a4) to (1) each of said other thermal unfolding
curves and to (2) the thermal unfolding curve obtained for said
protein in the absence of any of said multiplicity of different
molecules; and (a6) determining whether any of said multiplicity of
different molecules shift the thermal unfolding curve of said
protein.
20. The method of claim 19, wherein said comparing step (a5)
comprises ranking said molecules in said multiplicity of different
molecules for said protein according to the ability of each of said
multiplicity of different molecules to shift the thermal unfolding
curve of said protein.
21. The method of claim 19, wherein in said heating step (a2), said
multiplicity of containers is heated simultaneously.
22. The method of claim 19, wherein said step (a4) further
comprises determining a midpoint temperature (T.sub.m) from the
thermal unfolding curve; and wherein said step (a5) further
comprises comparing the T.sub.m of each of said unfolding curves in
step (a4) to (1) the T.sub.m of each of said other thermal
unfolding curves and to (2) the T.sub.m of the thermal unfolding
curve obtained for said target protein in the absence of any of
said different molecules.
23. The method of claim 19, wherein said step (a3) comprises
measuring the absorbance of light by said contents of each of said
containers.
24. The method of claim 19, wherein said step (a1) comprises
contacting said protein with a fluorescence probe molecule present
in each of said multiplicity of containers and wherein said step
(a3) comprises (i) exciting said fluorescence probe molecule, in
each of said multiplicity of containers, with light; and (ii)
measuring the fluorescence from each of said multiplicity of
containers.
25. The method of claim 24, wherein said step (a3)(ii) further
comprises measuring the fluorescence from each of said multiplicity
of containers one container at a time.
26. The method of claim 24, wherein said step (a3)(ii) further
comprises measuring the fluorescence from a subset of said
multiplicity of containers simultaneously.
27. The method of claim 24, wherein said step (a3)(ii) further
comprises measuring the fluorescence from each of said multiplicity
of containers simultaneously.
28. The method of claim 19, wherein said step (a3) comprises (i)
exciting tryptophan residues in said protein, in each of said
multiplicity of containers, with light; and (ii) measuring the
fluorescence from each of said multiplicity of containers.
29. The method of claim 18, wherein said multiplicity of containers
in step (a1) comprises a multiplicity of wells in a microplate.
30. A method for functionally classifying a protein capable of
unfolding due to a thermal change, said method comprising:
classifying said protein according to the set of molecules in a
multiplicity of different molecules that shift the thermal
unfolding curve of said protein.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present invention claims priority benefit of U.S.
provisional Appl No. 60/065,129, filed Nov. 12, 1997, which is
hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to a method of
classifying a protein based on the ability of one or more ligands
to modify the stability, and particularly the thermal stability, of
the protein, such that the modification of the stability denotes an
interaction between the ligand and the protein.
[0004] 2. Related Art
[0005] The .about.3.times.10.sup.9 nucleotide base pairs contained
within the human genome code for approximately 60,000 to 100,000
essential proteins (Alberts, et al., In: "Molecular Biology of the
Cell", 3rd Ed., Alberts, B. D. et al., Eds. (1994); Rowen, L. et
al., Science 278:605 (1997)). Human Genome Project researchers are
rapidly identifying all the genes in the 23 pairs of human
chromosomes. The products of these genes are widely recognized as
the future pool of therapeutic targets for development of
pharmaceuticals in the coming decades. While the sequencing of the
human genome will be largely completed within a few years,
elucidation of the function of these genes will lag far behind.
Therefore, new technologies are required to understand the
functional organization of the human genome and make the transition
from "structural genomics," or sequence information, to "functional
genomics," or gene function, and the association with normal and
pathological phenotypes (Hieter & Boguski, Science 2 78:601
(1997)).
[0006] The difficulty of this task has been clearly illustrated by
the recent discovery that of the 4288 genes in the elementary E.
coli genome, the function of about 40% of the proteins encoded by
these genes are completely unknown (Blattner et al., Science
277:1453 (1997)). Indeed, of the 12 simple organisms for which
complete genomic information is available, with S. cerevisiae being
the largest at 12.1 megabases (6034 genes), only 44% to 69% of the
genes have been identified using current state-of-the-art
computational sequence comparisons (Pennisi, E., Science 277:1433
(1997)). Moreover, the spirochete that causes syphilis has 1,014
genes, 45% of which have no known function (Fraser et al., Science
281: 375-388 (1998)). As a result, there is a functional
information gap that presents a challenge to traditional
methodologies, and at the same time an opportunity for discovery of
new targets for therapeutic intervention.
[0007] However, classification of proteins of unknown function
based on nucleotide or amino acid homology with proteins of known
function is inaccurate and unreliable. Proteins that have
structural homology can have dissimilar functions. For example,
lysozyme and .alpha.-lactalbumin have 40% sequence homology, but
divergent functions. Lysozyme is a hydrolase and
.alpha.-lactalbumin is a calcium binding protein involved in
lactose synthesis for secretion into milk of lactating mammals
(Qasba and Kumar, Crit. Rev. Biochem. Mol. Biol. 32: 255-306
(1997)).
[0008] Some proteins have similar function, yet have no sequence
homology. For example, the serine proteases trypsin and subtilisin
exhibit similar function, but exhibit neither sequence homology nor
structural homology (Tong et al., Nature Structural Biology 9:
819-826 (1998)). Cyclic AMP-dependent protein kinases from the
kinase fold family, and D-Ala:D-Ala ligase, from the "ATP Grasp"
fold family, have no sequence homology, yet share common structural
elements for ATP recognition and are both ATP-dependent enzymes
(Denessiouk et al., Protein Science 7: 1768-1771 (1998)). Some
proteins exhibit no sequence homology, exhibit some structural
homology, yet have dissimilar functions. Examples of such proteins
are bleomycin resistance protein, biphenyl 1,2-dioxygenase, and
human glyoxalase (Bergdoll et al., Protein Science 7: 1661-1670
(1998)).
[0009] Thus, there is a need for an accurate, reliable technology
that facilitates the rapid, high-throughput classification of
proteins of unknown function.
SUMMARY OF THE INVENTION
[0010] The present invention provides methods for functionally
classifying a protein. The methods are related to the ability of
molecules in a multiplicity of different molecules to modify the
stability of a protein, and therefore bind to the protein. Three of
the methods do not involve a determination of whether the molecules
that bind to the protein shift the thermal unfolding curve of the
protein. Three alternative and distinct methods involve determining
whether molecules that bind to a protein shift the thermal
unfolding curve of the protein.
[0011] A. Methods that do not involve determining whether molecules
that bind shift the thermal unfolding curve of the protein
[0012] The present invention provides a method for functionally
classifying a protein, the method comprising screening one or more
of a multiplicity of different molecules for their ability to
modify the stability of the protein, wherein modification of the
stability of the protein indicates that the molecule binds to the
protein; generating an activity spectrum for the protein from the
screening, wherein the activity spectrum reflects a subset of
molecules, from the multiplicity of different molecules, that
modify the stability of the protein and therefore are ligands that
bind to the protein; comparing the activity spectrum for the
protein to one or more functional reference spectrum lists; and
classifying the protein according to the set of molecules in the
multiplicity of different molecules that modify the stability of
the protein.
[0013] The present invention also provides a method for
functionally classifying a protein, the method comprising screening
one or more of a multiplicity of different molecules known to bind
to a particular class of proteins for their ability to modify the
stability of the protein, wherein modification of the stability of
the protein indicates that the molecule binds to the protein;
generating an activity spectrum for the protein from the screening,
wherein the activity spectrum reflects a subset of molecules, from
the multiplicity of different molecules, that modify the stability
of the protein and therefore are ligands that bind to the protein;
and classifying the protein as a member of the class of proteins if
the one or more of the multiplicity of different molecules modify
the stability of the protein.
[0014] The present invention also provides a method for
functionally classifying a protein, the method comprising
classifying the protein according to the set of molecules in a
multiplicity of different molecules that modify the stability of
the protein.
[0015] B. Alternative and distinct methods that involve determining
whether molecules that bind shift the thermal unfolding curve of
the protein
[0016] The present invention provides a method for functionally
classifying a protein that is capable of unfolding due to a thermal
change, the method comprising screening one or more of a
multiplicity of different molecules for their ability to shift the
thermal unfolding curve of the protein, wherein a shift in the
thermal unfolding curve of the protein indicates that the molecule
binds to the protein; generating an activity spectrum for the
protein from the screening, wherein the activity spectrum reflects
a subset of molecules, from the multiplicity of different
molecules, that shift the thermal unfolding curve of the protein
and therefore are ligands that bind to the protein; comparing the
activity spectrum for the protein to one or more functional
reference spectrum lists; and classifying the protein according to
the set of molecules in the multiplicity of different molecules
that shift the thermal unfolding curve of the protein.
[0017] The present invention also provides a method for
functionally classifying a protein that is capable of unfolding due
to a thermal change, the method to bind to a particular class of
proteins for their ability to shift the thermal unfolding curve of
the protein, wherein a shift in the thermal unfolding curve of the
protein indicates that the molecule binds to the protein;
generating an activity spectrum for the protein from the screening,
wherein the activity spectrum reflects a subset of molecules, from
the multiplicity of different molecules, that shift the thermal
unfolding curve of the protein and therefore are ligands that bind
to the protein; and classifying the protein as a member of the
class of proteins if the one or more of the multiplicity of
different molecules shift the thermal unfolding curve of the
protein.
[0018] The present invention also provides a method for
functionally classifying a protein capable of unfolding due to a
thermal change, the method comprising classifying the protein
according to the set of molecules in a multiplicity of different
molecules that shift the thermal unfolding curve of the
protein.
[0019] There are several advantages of methods of the present
invention for the drug discovery process, especially with regard to
functional genomics. For example, the methods of the present
invention afford widespread cross-target utility because it is
based on thermodynamic properties common to all ligand/receptor
complexes. Further, the methods of the present invention facilitate
the direct evaluation of protein targets derived from genomic
studies because no knowledge of specific target function is
necessary.
[0020] A further advantage provided by the methods of the present
invention is that it can be applied universally to any receptor
that is a drug target. It is not necessary to invent a new assay
every time a new receptor becomes available for testing. Thus,
screening of compound libraries begin immediately upon the
preparation of the protein target. When the receptor under study is
an enzyme, researchers can determine the rank order of affinity of
a series of compounds more quickly and more easily than they can
using conventional kinetic methods. In addition, researchers can
detect ligand binding to an enzyme, regardless of whether binding
occurs at the active site, at an allosteric cofactor binding site,
or at a receptor subunit interface. The present invention is
equally applicable to non-enzyme receptors.
[0021] Yet a further advantage provided by the methods of the
present invention is that the methods can be practiced using
miniaturized assay volumes (e.g., 1-5 .mu.L), which facilitates the
use of high density microplate assay arrays of 16.times.24 (384
well), 32.times.48 (1536 well), or further customized arrays. Only
about 5 to 40 picomole of protein are required (0.1 .mu.g to 1.0
.mu.g for a 25 kDa protein) per assay well, for a final protein
concentration of about 1 to 4 .mu.M. Thus, 1.0 mg of protein can be
used to conduct 10.sup.3 to 1.0.sup.4 assays in the miniaturized
format.
[0022] Yet a further advantage provided by the present invention is
that the methods of the present invention facilitate the ultra high
throughput screening of compound libraries (e.g., functional probe
libraries). Thus the methods of the present invention make it
possible to screen 10,000 to 30,000 compounds per day per
workstation. At that rate, at least 2.5 to 6 target proteins can be
screened per day, per workstation, against a functional probe
library of 4000 compounds. At least 500 to 1200 therapeutic targets
can be screened per year, per workstation, against a 4000 compound
functional probe library. In five years, one could sample about 3
to 7.5% of the proteins encoded by the human genome per
workstation.
[0023] Yet a further advantage provided by the methods of the
present invention is that the wide dynamic range of binding
affinities that can be assayed in the single well assay spans
twelve orders of magnitude (i.e., from femtomolar (10.sup.15 M) to
millimolar (10.sup.3 M) affinities).
[0024] Yet a further advantage provided by the methods of the
present invention is that multi-ligand binding interactions can be
monitored through the near additivity of the free energy of ligand
binding for individual ligands.
[0025] Moreover, the methods of the present invention provide
information that is more accurate and reliable than information
provided by conventional sequence homology methodologies, such as
those reported in Tatusov, R. L. et al., Science 278: 631-637
(1997); and Heiter, P. and M. Boguski, Science 278: 601-602
(1997).
[0026] Moreover, different enzyme classes may be identified and
differentiated based on binding of different sets of transition
state analogs. For example, benzeneboronic acid derivatives (BBA)
have been found to reversibly bind to diverse serine proteases such
as subtilisins, from bacterial sources, and .alpha.-chymotrypsin,
from eukaryotic sources (Nakatani, H., et al., J. Biochem. (Tokyo)
77:905-8 (1975)). Similarly, boroarginine transition state analogs,
which have an arginine group in the P1 position for this synthetic
peptide mimic, were found to be more specific inhibitors for the
serine proteases, thrombin, trypsin, and plasmin (Tapparelli et
al., J. Biol. Chem. 268:4734-41 (1993)) with the observed
specificity: K.sub.d.about.10 nM (thrombin), K.sub.d.about.1,000 nm
(trypsin), K.sub.d.about.10,000 nM (plasmin). This illustrates an
important advantage that the methods of the present invention
provide, relative to the sequence comparison approach to
classifying proteins: the .alpha.T.sub.m shift expected from the
binding of a boronic acid transition state analog should be much
more characteristic of a serine protease (regardless of bacterial
or eukaryotic source) than the information provided by sequence
comparisons alone. Serine proteases from bacterial and eukaryotic
sources are textbook examples of convergent evolution, and
therefore have very little sequence homology, despite the fact that
they share catalytic function.
[0027] Further features and advantages of the present invention are
described in detail below with reference to the accompanying
drawings.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIG. 1A shows a flow diagram illustrating a method of the
present invention. FIG. 1B shows another flow diagram illustrating
a method of the present invention.
[0029] FIG. 2 is a schematic diagram illustrating a top view of an
assay apparatus that can be used to practice the microplate thermal
shift assay.
[0030] FIG. 3 shows the results of microplate thermal shift assays
of single ligand binding interactions to three different classes of
binding sites for human .alpha.-thrombin.
[0031] FIG. 4 shows the results of microplate thermal shift assays
of multi-ligand binding interactions for human
.alpha.-thrombin.
[0032] FIG. 5 shows the compounds present in plate 1 of the
functional probe library.
[0033] FIG. 6 shows the activity spectrum for Factor Xa that was
generated using the compounds in plate 1 of the functional probe
library.
[0034] FIG. 7 shows the activity spectrum for fibroblast growth
factor receptor 1 (FGFR1) that was generated using the compounds in
plate 1 of the functional probe library.
[0035] FIG. 8 shows the result of a microplate thermal shift assay
of the recombinant dimeric lac repressor binding to a synthetic 21
-mer palindromic lac operator sequence.
[0036] FIG. 9 shows the result of a microplate thermal shift assay
of bovine muscle myosin binding to adenosine triphosphate
(ATP).
[0037] FIG. 10 shows the result of a microplate thermal shift assay
of bovine heart 3', 5'-cAMP-dependent protein kinase binding to
adenosine triphosphate-.gamma.-sulphate (ATP-.gamma.-S).
[0038] FIG. 11 shows the result of a microplate thermal shift assay
of bovine dihydrofolate reductase (DHFR) binding to
methotrexate.
[0039] FIG. 12 shows the result of amicroplate thermal shift assay
of bovine dihydrofolate reductase (DHFR) binding to NADPH.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0040] In the following description, reference will be made to
various terms and methodologies known to those of skill in the
biochemical and pharmacological arts. Publications and other
materials setting forth such known terms and methodologies are
incorporated herein by reference in their entireties as though set
forth in full.
[0041] The present invention provides methods for functionally
classifying a protein, which is capable of unfolding, according to
the set of molecules in a multiplicity of different molecules that
modify the stability of the protein. A protein can be caused to
unfold by treatment with a denaturing agent (such as urea,
guanidinium hydrochloride, guanidinium thiosuccinate, etc.), a
detergent, by treating the protein with pressure, by heating the
protein, etc.
[0042] The present invention provides methods for functionally
classifying a protein that involve determining whether the thermal
unfolding curve of the protein is shifted. Only molecules that
shift the thermal unfolding curve are deemed to be ligands that
bind to the protein. Preferably, the microplate thermal shift assay
is used to determine whether the thermal unfolding curve of the
protein is shifted. The microplate thermal shift assay involves
determining whether molecules that are tested for binding shift the
thermal unfolding curve. The microplate thermal shift assay is
described in international patent Appl. No. PCT/US97/08154
(published Nov. 13, 1997 as publication no. WO 97/42500); U.S.
patent application No. 08/853,464, filed May 9, 1997; and U.S.
patent application No. 08/853,459, filed May 9, 1997.
[0043] In a preferred embodiment, the present invention provides a
method for classifying a target protein that is capable of
unfolding due to a thermal change. In one this embodiment, the
target protein is contacted with one molecule of a multiplicity of
different molecules in each of a multiplicity of containers. The
containers are then heated, in intervals, over a range of
temperatures. Preferably, the multiplicity of containers is heated
simultaneously. After each heating interval, a physical change
associated with the thermal unfolding of the target molecule is
measured. In an alternate embodiment of this method, the containers
are heated in a continuous fashion. A thermal unfolding curve is
plotted as a function of temperature for the target molecule in
each of the containers. Preferably, the temperature midpoint,
T.sub.m, of each thermal unfolding curve is identified and is then
compared to the T.sub.m of the thermal unfolding curve obtained for
the target molecule in the absence of any of the molecules in the
containers. Alternatively, an entire thermal unfolding curve can be
compared to other entire thermal unfolding curves using computer
analytical tools.
[0044] The methods of the present invention that involve
determining whether molecules shift the thermal unfolding curve of
a protein are distinct from methods that do not involve determining
whether molecules shift the thermal unfolding curve of a protein,
such as assays of susceptibility to proteolysis, surface binding by
protein, antibody binding by protein, molecular chaperone binding
of protein, differential binding to immobilized ligand, and protein
aggregation. Such assays are well-known to those of ordinary skill
in the art. For example, see U.S. Pat. No. 5,585,277; and U.S. Pat.
No. 5,679,582. These approaches disclosed in U.S. Pat. Nos.
5,585,277 and 5,679,582 involve comparing the extent of folding
and/or unfolding of the protein in the presence and in the absence
of a molecule being tested for binding. These approaches do not
involve a determination of whether any of the molecules that bind
to the protein shift the thermal unfolding curve of the
protein.
[0045] The term "functionally classifying proteins" refers to
classifying a protein according to a biological, biochemical,
physical or chemical function, such as the ability to hydrolyze a
phosphate moiety (a phosphatase), to add a phosphate moiety (a
kinase), etc. Proteins can be classified as having one or more of
numerous different functions, and the methods of the present
invention are not limited to classifying proteins as phosphatases,
kinases, or other types of enzymes.
[0046] The terms "multiplicity of molecules," "multiplicity of
compounds," or "multiplicity of containers" refer to at least two
molecules, compounds, or containers.
[0047] The term "subset of molecules" in a multiplicity of
different molecules refers to a set of molecules smaller than the
multiplicity of different molecules.
[0048] The term "multi-variable" refers to more than one
experimental variable.
[0049] The term "screening" refers to the testing of a multiplicity
of molecules or compounds for their ability to bind to a target
molecule which is capable of unfolding when heated. The screening
process is arepetitive, or iterative, process, in which molecules
are tested for binding to a protein in an assay of unfolding, and
particularly in a thermal shift assay. For example, if a subset of
molecules within a functional probe library that is screened for
binding to a protein do not bind, then the screening is repeated
with another subset of molecules. If the entire library fails to
contain any molecules that bind to the protein, then the screening
is repeated using molecules from another functional probe
library.
[0050] As used herein, a "functional probe screen" is an assessment
(e.g., an assay) of the ability of a multiplicity of different
molecules in a functional probe library to bind to the target
protein and modify the stability of the target protein.
[0051] As used herein, a "functional probe library" refers to one
or more different molecules that are tested for their ability to
bind to a target protein and modify the stability, and particularly
the thermal stability, of the protein in response to unfolding
(e.g., thermal unfolding). By performing a stability test, and
preferably a using the microplate thermal shift assay technology,
on the protein in the presence of each member of the functional
probe library, compounds may be incubated with the target protein
individually and/or in groups to determine which ligands
individually or in combination bind tightly and specifically to the
target protein.
[0052] A functional probe library can be any kind of library of
molecules, including a library of proteins, a library of protein
subunits, a library of peptides, a library of vitamins &
co-factors, an enzyme inhibitor library, a nucleic acid library, a
carbohydrate library, a generic drug library, a natural product
library, or a combinatorial library. For molecules in the
functional probe library that bind to the target protein, the
biological effect can be assessed in in vitro and in vivo
assays.
[0053] If the functional probe library is a combinatorial library,
then preferably the it is a combinatorial library created using the
DirectedDiversity.RTM. system. The DirectedDiversity.RTM. system is
disclosed in U.S. Pat. No. 5,463,564.
[0054] As used herein, the term "activity spectrum" refers to the
list of compounds (i.e., ligands) that bind to the target protein
and modify the stability (e.g., the thermal stability) of the
target protein, and the respective affinities of the ligands for
the target protein. The terms "functional probe binding profile"
and "activity spectrum" are synonymous. A decrease in T.sub.m
suggests that the compound or molecule blocks the binding of
another molecule that would stabilize the protein. For example, if
a metal chelator decreases the T.sub.m, that suggests that the
protein binds to a metal (e.g., an interaction between calcium and
.alpha.-lactalbumin). If a reducing agent decreases the T.sub.m,
that suggests that the protein contains one or more dissulfide
bonds.
[0055] As used herein, the "functional reference spectrum list"
refers to a list of target protein classes (including references to
appropriate electronic databases), associated ligands, and
corresponding binding constants, that can be used to functionally
classify a target protein. Alternatively, the functional reference
spectrum list can be a set of one or more activity spectra for one
or more known proteins. Thus, an activity spectrum for a given
protein can serve as a "fingerprint" for that protein and for the
functional class of proteins to which the protein belongs.
[0056] A "functional reference list" is a list of proteins that
share one or more common features, such as binding to a particular
ligand, or exhibiting a common activity.
[0057] As used herein, an "activity spectrum comparator" is either
a computational or a graphical means by which one can compare the
activity spectrum, derived from observing the effects ofthe
functional probe library on the target protein, with the functional
reference spectrum list. For example, the activity spectrum
comparator can be spreadsheet software that is readily available to
those of ordinary skill in the art. For example, MicroSoft Excel
(MicroSoft Inc., Redmond, Wash.) can be used.
[0058] In may cases, a function of a gene may be tentatively
assigned through homology to sequences of known function (a
"functional hypothesis" derived from sequence homology). The
thermal shift assay can be employed to validate such a functional
hypothesis, or to identify the correct function from a list of
possible functions implied by sequence homology. For example, there
are proteins that hydrolyze ATP and convert the energy of
hydrolysis into mechanical energy, known as "molecular motors."
These proteins include DNA and RNA helicases, kinesins, chaperonins
for refolding proteins, and the protein complexes in the base of
bacterial flagella. These proteins all share sequence homology in
the ATP-hydrolyzing domain, whereas their other functions are
different. In one application of the methods of the present
invention, the known sequence homology for a portion of a protein
target (e.g., an ATPase domain) may be used to design thermal shift
assays using special functional probe libraries directed at
different possible functions of the target protein (e.g., libraries
containing molecules for probing the special activities of
chaperonins, helicases, kinesins, and other molecular motors).
Alternatively, a target protein may be identified via sequence
homology as a tyrosine kinase, and the present invention could then
be used to screen this target against a peptide library containing
many possible substrate phosphorylation sites. These examples
illustrate that the present invention is highly complementary to
the process of assigning function using sequence homology, because
the present invention can be used to confirm, reject, or elaborate
the hypothetical functions indicated by sequence homology.
[0059] Accordingly, the present invention also provides a method
for functionally classifying a protein, the method comprising (a)
screening one or more of a multiplicity of different molecules
known to bind to a particular class of proteins for their ability
to modify the stability of said protein, wherein modification of
the stability of the protein indicates that the molecule binds to
the protein, (b) generating an activity spectrum for the protein
from the screening step, wherein the activity spectrum reflects a
subset of molecules, from the multiplicity of different molecules,
that modify the stability of said protein, and (c) classifying the
protein as a member of said class of proteins if the one or more of
the multiplicity of different molecules modify the stability of the
protein.
[0060] It should be noted that the above process for elaborating or
specifying protein function using a thermal shift assay can also be
applied to functional hypotheses generated using other methods of
assigning protein function (e.g., three-dimensional structures of
proteins and nucleic acids, patterns of cellular expression of mRNA
or a protein encoded by a target gene, and phenotypic effects of
altering a target gene to change its function at the organismal
level).
[0061] Further, using the methods of the present invention, one can
assess the binding of more than one ligand to more than one site on
a protein, and classify the protein according to the subset of
molecules that bind to the protein. For example, a protein of
unknown function that is found to bind to DNA and to adenosine
triphosphate (ATP) can be classified as a protein that affects DNA
structure. Thus, using information concerning the binding of
multiple ligands, the large number of possible protein
classifications can be narrowed to only a few likely
classifications.
[0062] Moreover, using the methods ofthe present invention, one can
also screen a protein of known function for an additional,
previously unknown, function. Preferably, the microplate thermal
shift assay is used to screen the functional probe library of
molecules against the proteins.
[0063] The term "function" refers to the biological function of a
protein, peptide or polypeptide. For example, a kinase is a protein
for which the function is catalyzing the covalent addition of a
phosphate group to another protein.
[0064] 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, such as DNA and RNA, and peptides. More
specifically, the term "molecule" encompasses compounds in a
compound or a combinatorial library. The terms "molecule" and
"ligand" are synonymous.
[0065] The term "contacting a target protein" refers broadly to
placing the target protein 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
protein and the molecule to be screened for binding. More
specifically, contacting refers to the mixing of the target protein
with the molecule to be tested for binding. Mixing can be
accomplished, for example, by repeated uptake and discharge through
a pipette tip. Preferably, contacting refers to the equilibration
of binding between the target protein and the molecule to be tested
for binding. Contacting can occur in the container or before the
target protein and the molecule to be screened are placed in the
container.
[0066] The term "container" refers to any vessel or chamber in
which the receptor and molecule to be tested for binding can be
placed. The term "container" encompasses reaction tubes (e.g., test
tubes, microtubes, vials, etc.). Preferably, the term "container"
refers to a well in a multiwell microplate or microtiter plate.
[0067] The term "sample" refers to the contents of a container.
[0068] The terms "spectral emission," "thermal change" and
"physical change" encompass the release of energy in the form of
light or heat, the absorption of energy in the form or light or
heat, changes in turbidity and changes in the polar properties of
light. Specifically, the terms refer to fluorescent emission,
fluorescent energy transfer, absorption of ultraviolet or visible
light, changes in the polarization properties of light, changes in
the polarization properties of fluorescent emission, changes in the
rate of change of fluorescence over time (i.e., fluorescence
lifetime), changes in fluorescence anisotropy, changes in
fluorescence resonance energy transfer, changes in turbidity, and
changes in enzyme activity. Preferably, the terms refer to
fluorescence , and more preferably to fluorescence emission.
Fluorescence emission can be intrinsic to a protein or can be due
to a fluorescence reporter molecule. The use of fluorescence
techniques to monitor protein unfolding is well known to those of
ordinary skill in the art. For example, see Eftink, M. R.,
Biophysical J. 66: 482-501 (1994).
[0069] The term "unfolding" refers to the loss of structure, such
as crystalline ordering of amino acid side-chains, secondary,
tertiary, or quaternary protein structure.
[0070] The terms "folding," "refolding," and "renaturing" refer to
the acquisition of the correct amino acid side-chain ordering,
secondary, tertiary, or quaternary structure, of a protein, which
affords the full chemical and biological function of the
biomolecule.
[0071] The term "denatured protein" refers to a protein which has
been treated to remove native amino acid side-chain ordering,
secondary, tertiary, or quaternary structure. The term "native
protein" refers to a protein which possesses the degree of amino
acid side-chain ordering, secondary, tertiary or quaternary
structure that provides the protein with full chemical and
biological function. A native protein is one which has not been
heated and has not been treated with unfolding agents or chemicals
such as urea.
[0072] As used herein, the terms "protein" and "polypeptide" are
synonymous.
[0073] An "unfolding curve" is a plot of the physical change
associated with the unfolding of a protein as a function
temperature, denaturant concentration, pressure, etc. A
"denaturation curve" is a plot of the physical change associated
with the denaturation of a protein or a nucleic acid as a function
of temperature, denaturant concentration, pressure, etc
[0074] A "thermal unfolding curve" is a plot of the physical change
associated with the unfolding of a protein or a nucleic acid as a
function of temperature. A "thermal denaturation curve" is a plot
of the physical change associated with the denaturation of a
protein or a nucleic acid as a function of temperature. See, for
example, Davidson et al., Nature Structure Biology 2:859 (1995);
and Clegg, R. M. et al., Proc. Natl. Acad. Sci. U.S.A. 90:2994-2998
(1993).
[0075] The term "shift in the thermal unfolding curve" refers to a
shift in the thermal unfolding curve for a protein that is bound to
a ligand, relative to the thermal unfolding curve of the protein in
the absence of the ligand.
[0076] The term "modification of stability" refers to the change in
the amount of pressure, the amount of heat, the concentration of
detergent, or the concentration of denaturant that is required to
cause a given degree of physical change in a target protein that is
bound by one or more ligands, relative to the amount of pressure,
the amount of heat, the concentration of detergent, or the
concentration of denaturant that is required to cause the same
degree of physical change in the target protein in the absence of
any ligand. Modification of stability can be exhibited as an
increase or a decrease in stability. Modification of the stability
of a protein by a ligand indicates that the ligand binds to the
protein. Modification of the stability of a protein by more than
one ligand indicates that the ligands bind to the protein.
[0077] The term "modification of thermal stability" refers to the
change in the amount of thermal energy that is required to cause a
given degree of physical change in a target protein that is bound
by one or more ligands, relative to the amount of thermal energy
that is required to cause the same degree of physical change in the
target protein in the absence of any ligand. Modification of
thermal stability can be exhibited as an increase or a decrease in
thermal stability. Modification of the thermal stability of a
protein by a ligand indicates that the ligand binds to the protein.
Modification of the thermal stability of a protein by more than one
ligand indicates that the ligands bind to the protein.
[0078] The "midpoint temperature, T.sub.m" is the temperature
midpoint of a thermal unfolding curve. The T.sub.m can be readily
determined using methods well known to those skilled in the art.
See, for example, Weber, P. C. et al., J. Am. Chem. Soc.
116:2717-2724 (1994); and Clegg, R. M. et al., Proc. Natl. Acad.
Sci. U.S.A. 90:2994-2998 (1993).
[0079] As discussed above, it is preferable to determine the effect
of one or more molecules on the thermal stability of a target
protein according to a change in the T.sub.m of the thermal
unfolding curve for the protein. Alternatively the effect of one or
more molecules on the thermal stability of a target protein can be
determined according to the change in entire thermal unfolding
curve for the target protein.
[0080] The term "fluorescence probe molecule" refers to an
extrinsic fluorophore, which is a fluorescent molecule or a
compound which is capable of associating with an unfolded or
denatured receptor and, after excitement by light of a defined
wavelength, emits fluorescent energy. The term fluorescence probe
molecule encompasses all fluorophores. More specifically, for
proteins, the term encompasses fluorophores such as thioinosine,
and N-ethenoadenosine, formycin, dansyl, dansyl derivatives,
fluorescein derivatives, 6-propionyl-2-(dimethylamino)-napthalene
(PRODAN), 2-anilinonapthalene, and N-arylamino-naphthalene
sulfonate derivatives such as 1-anilinonaphthalene-8-sulfonate
(1,8-ANS), 2-anilinonaphthalene-6-sulfonate (2,6-ANS),
2-amino-naphthalene-6-sulfona- te,
N,N-dimethyl-2-aminonaphthalene-6-sulfonate, N-phenyl-2-
aminonaphthal-ene, N-cyclohexyl-2-aminonaphthalene-6-sulfonate,
N-phenyl-2-amino-naphthalene-6-sulfonate,
N-phenyl-N-methyl-2-aminonaphth- alene-6-sulfonate,
N-(o-toluyl)-2-amino-naphthalene-6-sulfonate, N-(m-toluyl)-
2-amino-naphthalene-6-sulfonate, N-(p-toluyl)-2-aminonaphth-
alene-6-sulfonate, 2-(p-toluidinyl)-naphthalene-6-sulfonic acid
(2,6-TNS), 4-(dicyanovinyl) julolidine (DCVJ),
6-dodecanoyl-2-dimethylaminonaphthale- ne (LAURDAN),
6-hexadecanoyl-2-(((2-(trimethylammonium)ethyl)methyl)-amino-
)naphthalene chloride (PATMAN), nile red, N-phenyl-
1-naphthylamine,
1,1-dicyano-2-[6-(dirnethylamino)naphthalen-2-yl]propene (DDNP),
4,4'-dianilino- 1,1-binaphthyl-5,5-disulfonic acid (bis-ANS), and
5-(4"-dimethylaminophenyl)-2-(4'-phenyl)oxazole derivative dyes,
sold under the trademark DAPOXYL.TM. Molecular Probes, Inc.,
Eugene, Oreg.), including the
5-(4"-dimethylaminophenyl)-2-(4'-phenyl)oxazole dyes provided in
Diwu, Z. et al., Photochemistry and Photobiology 66(4): 424-431
(1997), and in BioProbes 25: pp. 8-9, Molecular Probes, Inc.,
Eugene, Oreg. (1997).
[0081] Examples of 5-(4"-dimethylaminophenyl)-2-(4'-phenyl)oxazole
derivative dyes, and the corresponding Molecular Probes catalogue
number, include 5-(4"-dimethylaminophenyl)-2-(4'-phenyl)oxazole
butylsulfonamide (D-12801),
5-(4"-dimethylaminophenyl)-2-(4'-phenyl)oxazole-(2-aminoethyl)-
sulfonamide (D-10460),
5-(4"-dimethylaminophenyl)-2-(4'-phenyl)oxazole butylsulfonamide
(D-12801), 5-(4"-dimethylaminophenyl)-2-(4'-phenyl)oxazo-
le-3-sulfonamidophyenylboronic acid (D-10402),
5-(4"-dimethylaminophenyl)-- 2-(4'-phenyl)oxazole sulfonic acid,
sodium salt (D-12800),
5-(4"-dimethylaminophenyl)-2-(4'-phenyl)oxazole sulfonyl hydrazine
(D- 10430),
5-(4"-dimethylaminophenyl)-2-(4'-phenyl)oxazole-(2-bromoacetamido-
ethyl)sulfonamide (D-10300),
5-(4"-dimethylaminophenyl)-2-(4'-phenyl)oxazo-
le-2-(3-(2-pyridyldithio) propionamidoethyl)sulfonamide (D-10301),
5-(4"-dimethylaminophenyl)-2-(4'-phenyl)oxazole sulfonyl chloride
(D-10160),
5-(4"-dimethylaminophenyl)-2-(4'-phenyl)oxazole-3-sulfonamidop-
ropionic acid, succinimidyl ester (D-10162),
5-(4"-dimethylaminophenyl)-2-- (4'-phenyl)oxazole carboxylic acid,
succinimidyl ester (D-10161).
[0082] Preferably the term "fluorescence probe molecule" refers to
1,8-ANS or 2,6-TNS, and
5-(4"-dimethylaminophenyl)-2-(4'-phenyl)oxazole derivative dyes,
sold under the trademark DAPOXYL.TM., such as those provided in
Diwu, Z. et al., Photochemistry and Photobiology 66(4):
424-431(1997). Still more preferably, the term refers to
5-(4"-dimethylaminophenyl)-2-(4'-phenyl)oxazole derivative dyes,
sold under the trademark DAPOXYL.TM., such as those provided in
Diwu, Z. et al., Photochemistry and Photobiology 66(4): 424-431
(1997). Most preferably, the term refers to
5-(4"-dimethylaminophenyl)-2-(4'-phenyl)ox- azole sulfonic acid,
sodium salt (D-12800).
[0083] The term "carrier" encompasses a platform or other object,
of any shape, which itself is capable of supporting at least two
containers. The carrier can be made of any material, including, but
not limited to glass, plastic, or metal. Preferably, the carrier is
a multiwell microplate. The terms microplate and microtiter plate
are synonymous. The carrier can be removed from the heating
element. In the present invention, a plurality of carriers are
used. Each carrier holds a plurality of containers.
[0084] The terms "spectral measurement" and "spectrophotometric
measurement" are synonymous and refer to measurements of changes in
the absorption of light. Turbidity measurements, measurements of
visible light absorption, and measurement of ultraviolet light
absorption are examples of spectral measurements. Measurement ofthe
intrinsic fluorescence of a target protein, and the fluorescence of
an extrinsic fluorophore that is complexed with or bound to a
target protein are also examples of spectral measurement and
spectrophotometric measurement.
[0085] The term "polarimetric measurement" relates to measurements
of changes in the polarization properties of light and fluorescent
emission. Circular dichroism and optical rotation are examples of
polarization properties of light which can be measured
polarimetrically. Measurements of circular dichroism and optical
rotation are taken using a spectropolarimeter. "Nonpolarimetric"
measurements are those that are not obtained using a
spectropolarimeter.
[0086] Knowledge of the cellular and/or biological function of
proteins can be a valuable asset in drug discovery, where it can be
useful in developing a detailed understanding ofthe therapeutic
hypothesis for drug function, in designing specific strategies for
drug design, and in revealing potential drug side effects.
[0087] There are tens of thousands of different enzymes and
receptors that constitute potential drug targets, and more are
constantly being discovered through genome sequencing studies.
These proteins and cellular receptors have specific functions in
the biological system, which are practically defined by the
molecular ligands with which they form specific interactions.
Typical interactions that have functional significance include
enzyme interactions with molecular ligands like substrates or
substrate analogs, cofactors, adaptor domains, nucleic acids, etc.,
and receptor interactions with specific ligands, other receptors,
cell surface structural components, nucleic acids, polysaccharides,
etc.
[0088] While it will be broadly possible to isolate, or to clone
and express proteins that are putative drug targets, in many cases
there will be no functional knowledge base about the protein that
can assist in succeeding stages of the drug discovery process.
However, a substantial fraction of known protein molecules fall
into mechanistic classes which share important characteristics,
including their ability to bind specific types of molecular
ligands, including enzyme cofactors, enzyme substrates or substrate
analogs, etc. Consequently, it is possible to classify many
proteins of otherwise unknown function by their ability to
specifically bind various kinds of ligands, either alone or in
combination.
[0089] When a protein binds to a biological ligand in a
functionally significant way, there is an effect on the physical
state of the protein that is reflected in its stability relative to
its unliganded state. Consequently, one can classify functionally a
protein of previously unknown function by incubating it with a
probe panel of biological ligands and cofactors (a functional probe
library), and measuring which ligands have effects on the stability
of the protein. Alternatively, one can determine a previously
unknown function of a protein of previously known function by
incubating it with a probe panel of biological ligands and
cofactors (a functional probe library), and measuring which ligands
have effects on the stability of the protein.
[0090] As has been established from thermodynamic studies of
protein-ligand interactions, when two molecules associate to form a
favorable and specific interaction complex, the binding
interactions are associated with a reduction in the total free
energy of the complex and a net stabilization of the protein-ligand
complex relative to the unliganded protein. In practical terms,
this means that when an enzyme or receptor interacts with its
specific cofactors, or analogs of cofactors, the enzyme or receptor
will be stabilized by the interactions. However, it is possible
that special situations may exist in which ligand binding may
destabilize the target protein. For example, some proteins contain
more than one domains or allosteric sites to which one or more
ligands can bind.
[0091] Overview of the methods of the Present Invention
[0092] The methods of the present invention, as well as other
information, are depicted in FIGS. 1A and 1B.
[0093] A. Identification of a Putative Target Gene.
[0094] Target proteins are proteins for which binding to a drug may
have therapeutic potential and whose functional characterization
may be useful in the drug discovery process. Many genes that are
potential targets for therapeutic intervention are identified
through a phenomenological correlation that relates a genetic
defect to a disease state (e.g., when an inherited disease is
correlated with a genetic defect in a specific enzyme or receptor)
or through differences in protein expression patterns in diseased
vs. normal tissues.
[0095] In many cases it is possible to determine some "function" of
a gene product through sequence homology with a homologous protein
about which functional or structural data known. However, in a
substantial fraction of cases, sequence homology may not be
sufficient to establish functional relationships, and an
alternative means is needed to establish function in a way that can
directly facilitate the drug discovery process.
[0096] B. Clone and Express the Protein
[0097] To practice the methods of the present invention, it is
necessary to obtain the target protein in sufficient quantities for
a biological assay. Proteins that are potential new therapeutic
targets and/or require functional characterization may be isolated
directly from a natural source using a variety of established
biochemical isolation procedures.
[0098] The availability of complete gene sequences from genome
sequence data facilitates the cloning and expression of protein
targets identified via genomic methods. For example, the known
target DNA sequence may be used to design oligonucleotide probes to
select full-length cDNA clones containing the entire cDNA coding
for the gene of interest from a representative library of many such
cDNA clones. In another example, the known target DNA sequence may
be used to design PCR primers fro selective amplification and
cloning of the gene of interest from total genomic DNA. These and
other methods for high-throughput cloning and expression are
well-known to those of ordinary skill in the art. Thus, full-length
gene sequence data automatically provides the direct means for
high-throughput, parallel production of protein targets, an
necessary first step in any molecule-based, high-throughput
functional screening strategy.
[0099] C. Thermal Stability Screen
[0100] In order to perform a microplate thermal shift assay of a
target protein, is necessary to determine assay conditions that are
optimal for carrying out the assay. Proteins are linear polymers of
amino acids that spontaneously fold into stable, highly organized
3-dimensional structures. The biological activity and functions of
a target protein, including virtually all of the specific binding
and catalytic properties that characterize the protein, depends on
its three-dimensional structure.
[0101] Virtually all folded, active protein domains behave
thermally as organic crystals that melt with a cooperative, well
defined, pseudo first order phase transition: i.e., melt into a
partially disordered, organic liquid-like state, with a well
defined melting temperature (T.sub.m) that reflects the free energy
of stabilization ofthe protein three-dimensional structure in the
experimental solvent conditions. The microplate thermal shift
technology uses environment-sensitive fluorescent dyes to
sensitively detect the thermal unfolding process and to directly
monitor effects on protein stability that arise from perturbations
ofthe solvent environment or through ligand binding to the
protein.
[0102] The stability of the three-dimensional folded state of a
protein can potentially be perturbed in several ways. One way is to
alter the aqueous solvent environment in which the protein
molecules initially fold from a disorganized polymer into the
3-dimensionally organized state. By changing the bulk solvent
properties around the protein, the stability of the folded state
can be altered relative to the stability of the unfolded state.
This can provide a useful strategy for finding optimal conditions
for measuring ligand binding and is the principle behind the
stability screen.
[0103] D. Microplate Thermal Shift Assay Optimization Screen
[0104] The assay optimization screen is a set of solvent conditions
and fluorescent dyes that are used with the target protein to
determine optimal conditions for performing the microplate thermal
shift assay. The protein is subjected to a variety of solution
conditions and or fluorescent dyes in order to evaluate the
behavior of the protein and/or the assay readout.
[0105] Examples of variations in conditions could include the
addition of organic solvents, variations in pH, salts, etc. that
have the potential to alter the relative stability of the folded
and unfolded states of the protein. Examples in variations in dyes
could include those whose differences in charge, polarity,
excitation wavelength, emission wavelength, background signal
intensity, or other properties that offer advantages in precision
of measurement, miniaturization or optimization of signal to noise
under specific assay conditions. The optimization of conditions
that facilitate the stability screen is an empirical process and
can readily be practiced by one of ordinary skill in the art.
[0106] E. Functional Probe Library
[0107] A substantial fraction of protein molecules that can serve
as potential drug targets fall into mechanistic classes which share
important characteristics. For example, many enzymes use ATP as an
energetic cofactor, others use pyridine nucleotides as cofactors,
some use both as cofactors, etc.
[0108] By examining the scientific literature or through
experimental means, it is possible to compile a set of enzyme
substrates, substrate analogs, cofactors, adaptor protein domains,
nucleic acid analogs, polysaccharides, fatty acids, nucleic acids,
effector peptides, or other molecules which have been determined to
specifically bind to a defined class of protein molecules, or where
functional significance has been attached to tight binding to a
functionally known class of molecules.
[0109] As used herein, a "functional probe library" refers to one
or more different molecules that are tested for their ability to
bind to a target protein and modify the thermal stability of the
protein in response to thermal unfolding. By performing a thermal
stability test (preferably by using the microplate thermal shift
assay technology) on the protein in the presence of each member the
functional probe library, compounds may be incubated with the
target protein individually and/or in groups to determine which
ligands individually or in combination bind tightly and
specifically to the target protein.
[0110] Examples of molecules that can comprise a functional probe
library include, but are not limited to the following.
[0111] 1. Vitamins and Coenzymes
[0112] NADH/AND, NADPH/NADP, ATP/ADP, ATP-.gamma.-S, acetyl-CoA,
biotin, S-adenosyl-methionine, thiamine pyrophosphate (TPP),
sulfated oligosaccharides, heparin-like oligosaccharides, GTP,
GTP-.gamma.-S, gamma-S, pyridoxal-5-phosphate, flavin
mononucleotide (FMN), flavin adenine dinucleotide (FAD), folic
acid, tetrahydrofolic acid, methotrexate, vitamin K.sub.1, vitamin
E succinate salt, vitamin D.sub.3, vitamin D.sub.3-25-hydroxy,
vitamin D.sub.3-1 -.alpha.-25-dihydroxy, vitamin B.sub.12, vitamin
C, vitamin B.sub.6, coenzyme A, coenzyme A-n-butyryl, transretinoic
acid, and heme.
[0113] 2. Amino acid residue functional groups and their mimics
[0114] Building Blocks:
[0115] Guanidino groups
[0116] Imidazole groups
[0117] Phenylgroups
[0118] Phenolic groups
[0119] Indole groups
[0120] Aliphatic chains
[0121] Single Amino acids and blocked derivatives
[0122] Higher order structures:
[0123] Peptide hormones
[0124] Vasopressin
[0125] Insulin
[0126] TRH
[0127] Corticotropin
[0128] Glucagon
[0129] SH.sub.2 domains, SH.sub.3 domains, plextrin domains,
etc.
[0130] Bioactive Peptides
[0131] Lectins
[0132] 3. Meta Chelators
[0133] Calcium Chelators (Calbiochem, San Diego, Calif.)
[0134] Iron chelators
[0135] 4. Metal Ions
[0136] Transition metals
[0137] Calcium, magnesium
[0138] 5. Carbohydrates
[0139] Building blocks
[0140] Glucose
[0141] Galactose
[0142] Xylose
[0143] Higher order biomolecules:
[0144] Cellulose
[0145] Starch
[0146] Fructose
[0147] Mannose
[0148] Sucrose
[0149] Lactose
[0150] Bioactive Carbohydrates (available from Sigma Chemical Co.,
St. Louis, Mo.)
[0151] 6. Nucleic acids
[0152] Building blocks:
[0153] Uracil
[0154] Thymidine
[0155] Cytosine
[0156] Adenine
[0157] Guanine
[0158] Higher order structures:
[0159] Oligonucleotides
[0160] Deoxyribonucleic acid (DNA)
[0161] Ribonucleic acid (RNA)
[0162] The methods of the present invention can also be used to
screen proteins against libraries of synthetic and
naturally-occurring nucleic acids (for example, oligonucleotides)
to probe for different classes of nucleic acid-binding proteins.
For example, there are many DNA-binding proteins that can be
identified by their ability to bind to particular classes of DNA
sequences. Large libraries containing many different nucleic acid
sequences (for example, the 4096 different possible synthetic
hexamers, can be purchased or synthesized. At high concentrations,
all or part of the cognate binding site of site-specific nucleic
acid binding proteins can be detected. In the event that a protein
appears to bind several different sequences, binding sites can be
reconstructed by synthesizing various combinations of nucleic acid
sequences, and then the microplate thermal shift assay, or another
assay, can be used to measure binding affinities.
[0163] There are many DNA-binding proteins that can be identified
by their ability to bind to particular classes of DNA sequences
with lower specificity. For example, it is well-known that some
transcription factors bind a variety of A/T-rich sequences in
preference to G/C-rich sequences. Telomerases are known to
recognize G/C-rich sequences. Helicases are known to bind short
fragments of single-stranded DNA with low specificity. A smaller,
more generic library could contain the following components for
detecting these and other DNA-binding proteins:
[0164] -AT-rich tracts:
[0165] d(T).sub.32/d(A).sub.32
[0166] d(ATAT).sub.8/d(TATA).sub.8
[0167] d(AAAT).sub.8/d(TTTA).sub.8
[0168] d(AAATT).sub.6/d(TTTAA).sub.6
[0169] d(AAATTT).sub.6/d(TTTAAA).sub.6
[0170] d(AAAATTTT).sub.4/d(TTTTAAAA).sub.4
[0171] -GC-rich tracts:
[0172] d(C).sub.32/d(G).sub.32
[0173] d(GCGC).sub.8/d(CGCG).sub.8
[0174] d(GGGCCC).sub.6/d(CCCGGG).sub.6
[0175] d(GGGGCCCC).sub.4/d(CCCCGGGG).sub.4
[0176] -other
[0177] d(CA).sub.32/d(GT).sub.32
[0178] d(CT).sub.32/d(GA).sub.32
[0179] d(AG).sub.32/d(TC).sub.32
[0180] -Single-stranded components of the above duplex
sequences.
[0181] -d(T).sub.40/d(A).sub.20 (an example of a fragment
containing both single-stranded and duplex DNA)
[0182] -Sheared human chromosomal DNA
[0183] -"whole genome amplification" applied to different human
chromosomes
[0184] -Sheared salmon-sperm DNA
[0185] -Sheared microbial DNA
[0186] -Supercoiled plasmid DNA
[0187] -PCR-amplification products from specific chromosomal
regions (e.g. telomeres and centromeres)
[0188] -Other known recognition sites for transcription, RNA
processing, transposition
[0189] 7. Lipids
[0190] Building blocks:
[0191] Choline
[0192] Phosphoric acid
[0193] Glycerol
[0194] Palmitic acid
[0195] Oleic acid
[0196] Cholesterol
[0197] Higher order structures:
[0198] Phosphatidyl choline
[0199] 8. Enzyme Inhibitors
[0200] Protease Inhibitors (Sigma Chemical Co., St. Louis, Mo.)
[0201] PMSF
[0202] Leupeptin
[0203] Pepstatin A
[0204] Bestatin
[0205] Peptide aldehyde Cystatin (Cysteine protease inhibitors)
Protein Tyrosine Kinase inhibitors (Calbiochem, San Diego, Calif.)
Protein Phosphatase inhibitors (Calbiochem, San Diego, Calif.)
Protein Kinase inhibitors (Calbiochem, San Diego, Calif.) Protein
Kinase Activators (Calbiochem, San Diego, Calif.) Phosphodiesterase
inhibitors (Calbiochem, San Diego, Calif.) Phospholipase Inhibitors
Transition State Analogs
[0206] Similarly, zinc metalloproteases, such as angiotensin
converting enzyme, and carboxypeptidase, would be identifiable (a)
by destabilization by EDTA or orthophenanthroline
(Zn.sup.2+chelation) and (b) by stabilization in the presence of
hydroxamates and phosphoramidates that mimic the transition state
for Zn.sup.2+ catalyzed peptide bond hydrolysis.
[0207] The functional probe library can also include steroid
compounds amine hormones, and alkaloid compounds.
[0208] The functional probe library can be a library of generic
drugs. Alternatively, the functional probe library can be a natural
product library. For example, see the Encyclopedia of Common
Natural Ingredients Used in Foods, Drugs and Cosmetics, 2.sup.nd
Edition, Leung and Foster, Eds., Wiley Interscience (1996).
[0209] F. Functional Probe Screen
[0210] In addition to optimizing conditions that modify protein
stability, another way to affect the stability of a folded protein
is to specifically bind molecules to either the unfolded or folded
state of the protein. Since virtually all biologically active
proteins are folded with organized three-dimensional structures,
most interest attaches to ligand molecules that bind to and
stabilize the folded state of a protein.
[0211] As discussed above, a functional probe screen is an assay of
the ability of a multiplicity of different molecules in the
functional probe library to bind to the target protein and modify
the stability of the target protein in response to thermal
unfolding. Using the technology, one can directly measure the
binding affinity of a small or large molecule ligand to a target
protein through its effect on the unfolding midpoint temperature
T.sub.m (or thermal unfolding profile) of the protein. For
molecules that bind to the folded state of the protein, which
include most ligands of biological interest, there is a
quantitative relationship between the affinity of ligand binding
and the extent to which the T.sub.m of the protein in the liganded
state is shifted relative to the T.sub.m of the protein in
unliganded state.
[0212] Most proteins have functions that are reflected by their
ability to bind either large or small molecule ligands with high
specificity and high affinity. Many proteins belong to functional
classes (e.g. kinases, phosphatases, pyridine nucleotide dependent
oxidoreductases, etc.) that bind specific cofactors or catalyze
specific reactions using a limited set of catalytic mechanisms.
Consequently, molecules in a given functional class like kinases,
which use ATP as a cofactor, will generally bind an
non-hydrolyzable ATP cofactor analog like AMPPNP, a property that
will be detectable using the methods of the present invention.
[0213] Moreover, many proteins will bind a combination of ligands
or make multiple sets of interactions with biological adaptor
domains. To the extent that these interactions are independent,
they will generally produce additive perturbations on the stability
of the unliganded form of the protein.
[0214] When the a protein has been tentatively assigned to a
particular protein class, one can rescreen the protein using a
library of compounds or molecules known to bind to that class of
proteins
[0215] G. Activity Spectrum
[0216] After performing a thermal stability test (preferably by
using the microplate thermal shift assay technology) on the protein
in the presence of each member the functional probe library, one
can determine which ligands bind tightly and specifically to the
target protein and modify the thermal stability of the target
protein. The list of compounds (i.e., ligands) that bind to the
target protein and modify the thermal stability of the target
protein, and the respective affinities of the ligands for the
target protein comprise the activity spectrum of the target
protein.
[0217] H. Functional Reference Spectrum List
[0218] As discussed above, a "functional reference spectrum list"
is a list of target protein classes (including references to
appropriate electronic databases), associated ligands, and
corresponding binding constants, that can be used to functionally
classify a target protein. Alternatively, the functional reference
spectrum list can be a set of one or more activity spectra for one
or more known proteins.
[0219] As discussed above, a "functional reference list" is a list
of proteins that share one or more common features, such as binding
to a particular ligand, or exhibiting a common activity. An example
of a functional reference list is given in Table 1. The features
shared by the proteins listed in Table 1 is that they bind AND and
exhibit dehydrogenase activity. The list of proteins in Table 1
illustrates how a functionally related class of proteins can be
discriminated according to their ability to bind different sets of
ligands. For example, a protein that binds nicotinamide adenine
dinucleotide (AND), NADPH, or NADH, and malate, as shown by the
ability of these compounds to modify the thermal stability of the
protein, could be classified as a malate dehydrogenase. As another
example, a protein for which thermal stability is modified by
ethanol and AND could be classified as an alcohol
dehydrogenase.
1 TABLE 1 Functional Reference List Class 3 Aldehyde Dehydrogenase
Human .delta. Alcohol Dehydrogenase .alpha.-hydroxysteroid
Dehydrogenase Malate Dehydrogenase Horse Liver Alcohol
Dehydrogenase Alcohol Dehydrogenase Glyceraldehyde-3-Phosphate
Dehydrogenase Human .beta.-Alcohol Dehydrogenase Dihydropteridine
Reductase D-2-Hydroxyisocaproate Dehydrogenase Brassica Napus Enoyl
Acp Reductase 7-.alpha.-hydroxysteroid Dehydrogenase
Holo-D-Glyceraldehyde-3-Phosphate Dehydrogenase Glutathione
Reductase D-Glyceraldehyde-3-Phosphate Dehydrogenase Glutathione
Reductase 3-Isopropylmalate Dehydrogenase Human .beta.-3 Alcohol
Dehydrogenase Isocitrate Dehydrogenase Horse Liver Alcohol
Dehydrogenase M4 Lactate Dehydrogenase Dihydrolipoamide
Dehydrogenase Udp-Gal 4-Epimerase D-3-Phosphoglycerate
Dehydrogenase Human Liver .chi..chi. Alcohol Dehydrogenase Alpha,
20 .beta.-hydroxysteroid Dehydrogenase L-Lactate Dehydrogenase NADH
Peroxidase
[0220] I. Activity Spectrum Comparator
[0221] As used herein, an "activity spectrum comparator" is either
a computational or a graphical means by which one can compare the
activity spectrum, derived from observing the effects ofthe
functional probe library on the target protein, with the functional
reference spectrum list. For example, the activity spectrum
comparator can be spreadsheet software that is readily available to
those of ordinary skill in the art. For example, MicroSoft Excel
(MicroSoft Inc., Redmond, Wash.) can be used.
[0222] J Functional Classification
[0223] In the methods ofthe present invention, protein function is
indicated by the pattern of ligands that bind to the protein. By
using the activity spectrum comparator to compare the observed
target activity spectrum with the functional reference spectrum
list, the target protein can be functionally classified according
to relational data obtained for known proteins. For example, the
protein can be classified according to the set of ligands that
stabilize the protein against thermal unfolding.
[0224] Thus, by comparing a plot ofthe degree to which each of a
multiplicity of molecules or compounds modify the thermal stability
of a protein (and therefore bind to the protein) to a plot of the
degree to which the same molecules modify the thermal stability of
a known protein (and therefore bind to the protein), the class of
proteins to which the protein belongs can be deduced.
[0225] Alternatively, the protein can be classified by comparing
the activity spectrum of the target protein to the activity spectra
of known, classified proteins. For example, one can consult
databases such as PDR online, Medline, SciFinder, STNExpress,
in-house databases, NAPRALERT Online, the Encyclopedia of Common
Natural Ingredients Used in Foods, Drugs and Cosmetics, 2.sup.nd
Edition, Leung and Foster, Eds., Wiley Interscience (1996), and the
Handbook of Enzyme Inhibitors, Part A and B, 2.sup.nd Edition,
Ellner, Ed., ECH (1990).
[0226] The Microplate Thermal Shift Assay and Apparatus
[0227] In principle, any means of measuring the effects of
incubating a protein in the presence of a panel of probe ligands to
determine which probe ligands can affect the stability of the
target protein will suffice as a means of functionally classifying
proteins. Preferably, the microplate thermal shift assay is used to
determine the effect of one or more molecules or ligands on the
thermal stability of a target protein. The microplate thermal shift
assay is a direct and quantitative technology for assaying the
effect of one or more molecules on the thermal stability of a
target protein.
[0228] The thermal shift assay is based on the ligand-dependent
change in the thermal unfolding curve of a receptor, such as a
protein or a nucleic acid. When heated over a range of
temperatures, a receptor will unfold. By plotting the degree of
unfolding as a function of temperature, one obtains a thermal
unfolding curve for the receptor. A useful point of reference in
the thermal unfolding curve is the temperature midpoint (T.sub.m),
the temperature at which half of the receptor molecules are
unfolded.
[0229] Thermal shift assays are based on the ligand-dependent
change in the midpoint for thermally induced unfolding curves,
.DELTA.T.sub.m, for the ligand-receptor complex (relative to the
un-complexed receptor) as an experimental observable that directly
relates to the ligand binding affinity, K.sub.d, due to the
coupling of the ligand binding and receptor unfolding free energy
functions (Schellman, J. A., Biopolymers 15: 999-1000 (1976);
Brandts, J. F., Biochemistry 29:6927-6940 (1990)). This thermal
physical screening strategy utilizes the thermal stability of
ligand-receptor mixtures as an indicator of the binding affinity
for the ligand-receptor interactions. These assays have been
traditionally carried out one at a time in differential scanning
calorimeters (DSC) that monitor the change in heat capacity as
proteins undergo temperature induced unfolding transitions (Brandts
et al., Biochemistry 29:6927-6940 (1990); and Weber, P. et al, J.
Am. Chem. Soc. 116:2717-2724 (1994)). Alternatively, thermal shift
assays can be performed, again one at a time, by employing
temperature-regulated optical instruments that monitor the
absorbance (Chavan, A. J. et al., Biochemistry 33:7193-7202
(1994)); fluorescence (Chavan, A. J. et al., Biochemistry
33:7193-7202 (1994); or circular dichroism (Bouvier, M. et al.,
Science 265:398-402 (1994); Morton, A. et al. Biochemistry
34:8564-8575 (1995)) changes that occur for the thermally induced
unfolding transitions of proteins.
[0230] There are many advantages to using the thermal shift assay
since it does not require radioactively labeled compounds, nor
fluorescent or other chromophobic labels to assist in monitoring
binding. The assay takes advantage of thermal unfolding of
biomolecules, a general physical chemical process intrinsic to
many, if not all, drug target biomolecules. General applicability
is an important aspect of this assay since it obviates the
necessity to invent a new assay every time a new therapeutic
receptor protein becomes available. The assay is particularly well
suited for measuring the binding of ligands to non-enzymatic
targets, for example growth factor/receptor interactions, where no
spectrophotometric assay is usually possible. However, the single
assay configuration of the thermal shift methods, as conventionally
performed, has limited the utility of this technique, especially
for the high throughput screening of compound libraries.
[0231] We have been able to greatly accelerate the protein/ligand
screening process by developing a generally applicable high
throughput ligand-receptor screening strategy in a 96 well plate
(or higher density) format that will identify and rank lead
compounds based on the thermodynamic stabilization of
ligand-receptor complexes.
[0232] Ligand binding stabilizes the receptor (Schellman, J.,
Biopolymers 14:999-1018 (1975)). The extent of binding and the free
energy of interaction follow parallel courses as a function of
ligand concentration (Schellman, J., Biophysical Chemistry
45:273-279 (1993); Barcelo, F. et al., Chem. Biol. Interactions
74:315-324 (1990)). As a result of stabilization by ligand, more
energy (heat) is required to unfold the receptor. Thus, ligand
binding shifts the thermal unfolding curve. That is, ligand binding
increases the thermal stability ofthe protein. This property can be
exploited to determine whether a ligand binds to a receptor: a
change, or "shift", in the thermal unfolding curve, and thus in the
T.sub.m, suggests that a ligand binds to the receptor.
[0233] The thermodynamic basis for the thermal shift assay has been
described by Schellman, J. A. (Biopolymers 15:999-1000 (1976)), and
also by Brandts et al. (Biochemistry 29:6927-6940 (1990)).
Differential scanning calorimetry studies by Brandts et al.
(Biochemistry 29:6927-6940 (1990)) have shown that for tight
binding systems of 1:1 stoichiometry, in which there is one
unfolding transition, one can estimate the binding affinity at
T.sub.m from the following expression: 1 K L T m = exp { - H u T 0
R [ 1 T m - 1 T 0 ] + C pu R [ ln ( T m T 0 ) + T 0 T m - 1 ] } L T
m ( equation 1 )
[0234] where K.sub.m.sup.T.sup..sub.m=the ligand association
constant at T.sub.m;
[0235] T.sub.m=the midpoint for the protein unfolding transition in
the presence of ligand;
[0236] T.sub.0=the midpoint for the unfolding transition in the
absence of ligand;
[0237] .DELTA.H.sub.u.sup.T.sup..sub.0=the enthalpy of protein
unfolding in the absence of ligand at T.sub.0;
[0238] .DELTA.C.sub.pu=the change in heat capacity upon protein
unfolding in the absence of ligand;
[0239] .sub.[L.sub.T.sub..sub.m]=the free ligand concentration at
T.sub.m; and
[0240] R=the gas constant.
[0241] This expression was found to be useful for the structure
based design of azobenzene ligands for streptavidin where DSC scans
of various ligand/streptavidin mixtures facilitated the measurement
of binding affinity at T.sub.m (Weber, P. et al., J. Am. Chem. Soc.
116:2717-2724 (1994)). These measurements were checked further by
performing mixing or isothermal titrating calorimetry experiments
which yielded binding affinities consistent with those determined
by DSC. The ease and reproducibility of using protein thermal
unfolding to estimate ligand binding affinity impressed upon us the
potential of further extending this approach for becoming a more
general drug discovery tool.
[0242] The parameters .DELTA.H.sub.u and .DELTA.C.sub.pu are
usually observed from DSC experiments and are specific for each
protein. Calorimetric measurements of .DELTA.H.sub.u and
.DELTA.C.sub.pu are the most accurate estimates of these parameters
because calorimeters typically collect unfolding data every
0.1.degree.C. However, the parameters, .DELTA.H.sub.u and
.DELTA.C.sub.pu, can also be estimated in the microplate thermal
shift assay, in which case the .DELTA.H.sub.u will not be a
calorimetric enthalpy but a comparable van't Hoff enthalpy based on
unfolding data collected at every 2.0.degree.C. using the current
protocol. Moreover, even in the absence of optimum data for
.DELTA.H.sub.u and .DELTA.C.sub.pu, these parameters are constants
specific to the protein involved in the compound screening and will
therefore be unchanged from well to well, resulting in no influence
on calculations of the relative values of binding affinities, i.e.,
K.sub.L at T.sub.m.
[0243] Besides the parameters .DELTA.H.sub.u and .DELTA.C.sub.pu,
it is also necessary to obtain estimates of T.sub.m and T.sub.0 to
solve for K.sub.L.sup.T.sup..sub.m in equation 1. This is
accomplished through the use of non-linear least squares computer
fits of the unfolding data for each individual well using the
following equation: 2 y ( T ) = y u + ( y f - y u ) ( 1 + exp ( [ -
H u R ] [ 1 T - 1 T m ] + [ C pu R ] [ ( T m T - 1 ) + ln ( T T m )
] ) Equation 2
[0244] Equation 2 employs five fitting parameters, .DELTA.H.sub.u,
.DELTA.C.sub.pu, T.sub.m, y.sub.f and y.sub.u, where y.sub.f and
y.sub.u are the pre-transitional and post-transitional fluorescence
levels, respectively. The computer fits are determined by floating
these parameters to arrive at the minimum of the sum of the squares
of the residuals by employing the Levenberg-Marquardt algorithm.
The T.sub.0 values are obtained for wells that contain no added
ligand and are set as the reference. Commercially available
curve-fitting software is readily available to one of ordinary
skill in the art. For example, Kaleidograph 3.0 (Synergy, Reading,
Pa.) can be used.
[0245] It is also possible to calculate the ligand association
equilibrium constant at any temperature, K.sub.L at T, the ligand
association equilibrium constant at T.sub.m, using equation 3, if
mixing calorimetry data for the binding enthalpy at T,
.DELTA.H.sub.L, and the change in heat capacity upon ligand
binding, .DELTA.C.sub.pL, are known (Brandts & Lin, 1990). 3 K
L T = K L T m exp { - H L T R [ 1 T - 1 T m ] + C pL R [ ln ( T T m
) - T T m + 1 ] } Equation 3
[0246] where K=the ligand association constant at any temperature,
T.
[0247] K.sub.L.sup.T.sup..sub.m=the ligand association constant at
T.sub.m.
[0248] T.sub.m=the midpoint for the protein unfolding transition in
the presence of ligand.
[0249] .DELTA.H.sub.L.sup.T=the enthalpy of ligand binding at
temperature, T.
[0250] .DELTA.C.sub.pL=the change in heat capacity upon binding of
ligand.
[0251] R=gas constant
[0252] The second exponential term of equation 3 is usually small
enough to be ignored so that approximate values of K.sub.L at T can
be obtained using just the first exponential term, and equation 3
reduces to equation 4: 4 K L T = K L T m exp [ - H L T R [ 1 T - 1
T m ] ] Equation 4
[0253] The parameter .DELTA.H.sub.L.sup.T can be measured using a
isothermal titrating calorimetry, using a calorimetric device such
as the Omega (MicroCal; Northampton, Mass.). When calorimetric data
are not available, .DELTA.H.sub.L.sup.T can be estimated to be
about -10.0 kcal/mol, which is an average binding enthalpy (Wiseman
et al., Anal. Biochem. 179:131-137 (1989)).
[0254] Preferably, fluorescence spectrometry is used to monitor
thermal unfolding. The fluorescence methodology is more sensitive
than the absorption methodology. The use of intrinsic protein
fluorescence and fluorescence probe molecules in fluorescence
spectroscopy experiments is well known to those skilled in the art.
See, for example, Bashford, C. L. et al., Spectrophotometry and
Spectrofluorometry: A Practical Approach, IRL Press Ltd., pub., pp.
91-114 (1987); Bell, J. E., Spectroscopy in Biochemistry, Vol. I,
CRC Press, pub., pp. 155-194 (1981); Brandts, L. et al., Ann. Rev.
Biochem. 41:843 (1972).
[0255] The microplate thermal shift assay is further described in
U. S. patent application No. 08/853,464, filed May 9, 1997, and in
international patent Appl. No. PCT/US97/08154 (published Nov. 13,
1997 as publication no. WO 97/42500), which are hereby incorporated
by reference in their entirety.
[0256] Spectral readings, preferably fluorescence readings, can be
taken on all of the samples on a carrier simultaneously.
Alternatively, readings can be taken on samples in groups of at
least two at a time.
[0257] A fluorescence imaging system, for example, a fluorescence
emission imaging system, can be used to monitor the thermal
unfolding of a target molecule or a receptor. Fluorescence imaging
systems are well known to those skilled in the art For example, the
ALPHAIMAGER.TM. Gel Documentation and Analysis System (Alpha
Innotech, San Leandro, Calif.) employs a high performance charge
coupled device (CCD) camera with 768.times.494 pixel resolution.
The charge coupled device camera is interfaced with a computer and
images are analyzed with Image analysis software.TM.. The
CHEMIIMAGER.TM. (Alpha Innotech) is a cooled charge coupled device
that performs all of the functions of the ALPHAIMAGER.TM. and in
addition captures images of chemiluminescent samples and other low
intensity samples. The CHEMIIMAGER.TM. charge coupled device
includes a Pentium processor (1.2 Gb hard drive, 16 Mb RAM),
AlphaEase.TM. analysis software, a light tight cabinet, and a UV
and white light trans-illuminator. For example, the MRC-1024
UVlVisible Laser Confocal Imaging System (BioRad, Richmond, Calif.)
facilitates the simultaneous imaging of more than one fluorophore
across a wide range of illumination wavelengths (350 to 700 nm).
The Gel Doc 1000 Fluorescent Gel Documentation System (BioRad,
Richmond, Calif.) can clearly display sample areas as large as
20.times.20 cm, or as small as 5.times.4 cm. At least two 96 well
microplates can fit into a 20.times.20 cm area. The Gel Doc 1000
system also facilitates the performance of time-based
experiments.
[0258] A fluorescence imaging system, for example, a fluorescence
emission imaging system, can be used to monitor receptor unfolding
in a microplate thermal shift assay In this embodiment, a plurality
of samples is heated simultaneously between 25 to 110.degree.C. A
fluorescence emission reading is taken for each of the plurality of
samples simultaneously For example, the fluorescence in each well
of a 96 or a 384 well microplate can be monitored simultaneously.
Alternatively, fluorescence readings can be taken continuously and
simultaneously for each sample. At lower temperatures, all samples
display a low level of fluorescence. As the temperature is
increased, the fluorescence in each sample increases. Wells which
contain ligands which bind to the target molecule with high
affinity shift the thermal unfolding curve to higher temperatures.
As a result, wells which contain ligands which bind to the target
molecule with high affinity fluoresce less, at a given temperature
above the T.sub.m of the target molecule in the absence of any
ligands, than wells which do not contain high-affinity ligands. If
the samples are heated in incremental steps, the fluorescence of
all of the plurality of samples is simultaneously imaged at each
heating step. If the samples are heated continuously, the
fluorescent emission of all of the plurality of samples is
simultaneously imaged during heating.
[0259] A thermal shift assay can be performed in a volume of 100
.mu.L volumes. For the following reasons, however, it is preferable
to perform a thermal shift assay in a volume of 1-10 .mu.L. First,
approximately 10- to 100-fold less protein is required for the
miniaturized assay. Thus, only .about.4 to 40 pmole of protein are
required (0.1 .mu.g to 1.0 .mu.g for a 25 kDa protein) for the
assay (i.e. 1 to 10 .mu.L working volume with a target molecule
concentration of about 1 to about 4 .mu.M). Thus, 1 .mu.g of
protein can be used to conduct 1,000 to 10,000 assays in the
miniaturized format. This is particularly advantageous when the
target molecule is available in minute quantities.
[0260] Second, approximately 10- to 100-fold less ligand is
required for the miniaturized assay. This advantage is very
important to researchers when screening valuable combinatorial
libraries for which library compounds are synthesized in minute
quantities. In the case of human .alpha.-thrombin, the ideal ligand
concentration is about 50 .mu.M, which translates into 25-250
pmoles of ligand, or 10-100 ng (assuming a MW of 500 Da) of ligand
per assay in the miniaturized format.
[0261] Third, the smaller working volume allows the potential of
using larger arrays of assays because the miniaturized assay can
fit into a much smaller area. For example, 384 well (16.times.24
array) or 864 well (24.times.36 array) plates have the same
dimensions as the 96 well plates (8.5.times.12.5 cm). The 384 well
plate and the 864 well plate allows the user to perform 4 and 9
times as many assays, respectively, as can be performed using a 96
well plate. Alternatively, plates with more wells, such as 1536
well plates (32.times.48 arrays; Matrix Technologies Corp.), can be
used. A 1536 well plate will facilitate sixteen times the
throughput afforded by a 96 well plate.
[0262] Thus, using the 1536 well plate configuration, assay speed
can be increased by about 16 times, relative to the speed at which
the assay can be performed using the 96 well format. The 8.times.12
assay array arrangement (in a 96-well plate) facilitates the
performance of 96 assays/hr, or about 2300 assays/24 hours. The
32.times.48 array assay arrangement facilitates the performance of
about 1536 assays hr., or about 37,000 assays/24 hours can be
performed using a 32.times.48 assay array configuration.
[0263] The assay volume can be 1-100 .mu.L. Preferably, the assay
volume is 1-50 .mu.L. More preferably, the assay volume is 1-25
.mu.L. More preferably still, the assay volume is 1-10 .mu.L. More
preferably still, the assay volume is 1-5 .mu.L. More preferably
still, the assay volume is 5 .mu.L. Most preferably, the assay
volume is 1 .mu.L or 2 .mu.L.
[0264] Alternatively, the assay is performed in V-bottom
polycarbonate, polystyrene, or polyproplene plates or dimple
plates. A dimple plate is a plate that contains a plurality of
round-bottom wells that hold a total volume of 15 .mu.L.
[0265] The microplate thermal shift assay is performed by (a)
contacting a protein with one or more of a multiplicity of
different molecules in each of a multiplicity of containers; (b)
heating the multiplicity of containers from step(a), preferably
simultaneously; (c) measuring in each of the containers a physical
change associated with the thermal unfolding of the target molecule
resulting from heating; (d) generating a thermal unfolding curve
for the target molecule as a function of temperature for each of
the containers; and (e) comparing each of the unfolding curves in
step (d) to (1) each of the other thermal unfolding curves and to
(2) the thermal unfolding curve obtained for the protein in the
absence of any of the multiplicity of different molecules; and (f)
determining whether any of the multiplicity of different molecules
modifies the thermal stability of the protein, wherein a
modification in thermal stability is indicated by a shift in the
thermal unfolding curve.
[0266] Step (d) may further comprise determining a midpoint
temperature (T.sub.m) from the thermal unfolding curve. Step (e)
may further comprise comparing the T.sub.m of each of the unfolding
curves in step (d) to (1) the T.sub.m of each of the other thermal
unfolding curves and to (2) the T.sub.m of the thermal unfolding
curve obtained for the target protein in the absence of any of the
different molecules.
[0267] To practice the methods of the present invention using
fluorescence spectroscopy or imaging, step (a) comprises contacting
the target protein with a fluorescence probe molecule present in
each of the multiplicity of containers and step (c) comprises (c1):
exciting the fluorescence probe molecule, in each of the
multiplicity of containers, with light; and (c2) measuring the
fluorescence from each of the multiplicity of containers.
Fluorescence, for example, fluorescence emission, can be measured
from each of the multiplicity of containers one container at a
time, from a subset of the multiplicity of containers
simultaneously, or from each of the multiplicity of containers
simultaneously.
[0268] To generate an activity spectrum, molecules are ranked
according to the degree to which they stabilize the target protein
against thermal unfolding. After the molecules are ranked, the
activity spectrum of the target protein for the molecules in the
finctional probe library is compared to one or more functional
reference spectrum lists.
[0269] Suitable heating apparatuses for practicing the methods of
the present invention are well known to those of ordinary skill in
the art. For example, the ROBOCYCLER.TM. Gradient Temperature
Cycler (Stratagene, La Jolla, Calif.) (see U.S. Pat. No. 5,525,300)
can be used. Alternatively, a temperature gradient heat block can
be used (see U.S. Pat. No. 5,255,976). Fluorescence can be read
using any suitable fluorescence spectroscopy device. For example,
the CytoFluor II apparatus (PerSeptive Biosystems, Framingham,
Mass.) can be used.
[0270] The element upon which the sample carrier is heated can be
any element capable of heating samples rapidly and in a
reproducible fashion. In the present invention, aplurality of
samples is heated simultaneously. The plurality of samples can be
heated on a single heating element. Alternatively, the plurality of
samples can be heated to a given temperature on one heating
element, and then moved to another heating element for heating to
another temperature. Heating can be accomplished in regular or
irregular intervals. To generate a smooth unfolding curve, the
samples should be heated evenly, in intervals of 1 or 2.degree.C.
The temperature range across which the samples can be heated is
from 4 to 110.degree.C. Spectral readings, and particularly
fluorescence readings, are taken after each heating step. Samples
can be heated and read by the spectral device, for example, a
fluorescence imaging camera, in a continuous fashion.
Alternatively, after each heating step, the samples may be cooled
to a lower temperature prior to taking the spectral readings.
Preferably, the samples are heated continuously and spectral
readings are taken while the samples are being heated.
[0271] Spectral, e.g., fluorescence, readings can be taken on all
of the samples in the carrier simultaneously. Alternatively,
readings can be taken on samples in groups of at least two at a
time. Finally, the readings can be taken one sample at a time.
[0272] Preferably, the instrument used to perform the microplate
thermal shift assay consists of a scanner and a control software
system. Fluorescence, for example, fluorescence emission, can be
detected by a photomultiplier tube in a light-proof detection
chamber. The software runs on a personal computer and the action of
the scanner is controlled through the software.
[0273] An exemplary apparatus 200 is shown in FIG. 2. A precision
X-Y mechanism scans the microplate with a sensitive fiber-optic
probe to quantify the fluorescence in each well. The microplate and
samples can remain stationary during the scanning of each row of
the samples, and the fiber-optic probe is then moved to the next
row. Alternatively, the microplate and samples can be moved to
position a new row of samples under the fiber-optic probe. The
scanning system is capable of scanning 96 samples in under one
minute. The scanner is capable of holding a plurality of excitation
filters and a plurality of emission filters to measure the most
common fluorophores. Thus, fluorescence emission readings can be
taken one sample at a time, or on a subset of samples
simultaneously.
[0274] The heat conducting element or block upon which the sample
carrier is heated can be any element capable of heating samples
rapidly and reproducibly. The plurality of samples can be heated on
a single heating element. Alternatively, the plurality of samples
can be heated to a given temperature on one heating element, and
then moved to another heating element for heating to another
temperature. Heating can be accomplished in regular or irregular
intervals. To generate a smooth unfolding curve, the samples should
be heated evenly, in intervals of 1 or 2.degree.C. The temperature
range across which the samples can be heated is from 4 to
110.degree.C.
[0275] Preferably, aplurality of samples is heated simultaneously.
If samples are heated in discrete temperature intervals, in a
stairstep fashion, spectral readings are taken after each heating
step. Alternatively, after each heating step, the samples may be
cooled to a lower temperature prior to taking the spectral
readings. Alternatively, samples can be heated in a continuous
fashion and spectral readings are taken during heating.
[0276] The assay apparatus can be configured so that it contains a
single heat conducting block. Alternatively, the assay apparatus
can be configured so that it contains a plurality of heat
conducting blocks upon a movable platform. The platform may be a
translatable platform that can be translated, for example, by a
servo driven linear slide device. An exemplary linear slide device
is model SA A5M400 (IAI America, Torrance, Calif.). In this
embodiment, the sensor receives spectral emissions from each of the
samples on a given heat conducting block. The platform is then
translated to place another heat conducting block and its
accompanying samples under the sensor so that it receives spectral
emissions from each ofthe samples on that heating block. The
platform is translated until spectral emissions are received from
the samples on all heat conducting blocks
[0277] Alternatively, the platform may by a rotatable platform, as
shown in FIG. 2, that may be rotated, for example, by a servo
driven axle. In the latter embodiment, the sensor receives spectral
emissions from each of the samples on a given heat conducting
block. The platform is then rotated to place another heat
conducting block and its accompanying samples under the sensor so
that it receives spectral emissions from each of the samples on
that heating block. The platform is rotated until spectral
emissions are received from the samples on all heat conducting
blocks.
[0278] In apparatus 200, a plurality of heat conducting blocks 204,
each of which includes a plurality of wells for a plurality of
samples 210, is mounted on a rotatable platform or carousel 206.
Platform or carousel 206 can be composed of a heat conducting
material, such as the material that heat conducting block 204 is
composed of. Axle 208 is rotatably connected to base 202. Rotatable
platform 206 is axially mounted to rotate about axle 208 Rotation
of axle 208 is controlled by a servo controller 210. Servo
controller 210 is controlled by a computer controller 250 in a
manner well known to one of skill in the relevant arts. Computer
controller 250 causes servo controller 210 to rotate axle 208
thereby rotating rotatable platform 206. In this manner, heat
conducting blocks 204 are sequentially placed under fiber optic
probe 212.
[0279] Each of the plurality of heat conducting blocks 204 can be
controlled independently by temperature controller 214. Thus, the
temperature of a first heat conducting block 204 can be higher or
lower than the temperature of a second heat conducting block 204.
Similarly, the temperature of a third heat conducting block 204 can
be higher or lower than the temperature of either first or second
heat conducting block 204.
[0280] Temperature controller 214 is connected to heat conducting
block 204 by a thermoelectric connection 230. Under the action of
temperature controller 214, the temperature of heat conducting
block 204 can be increased, decreased, or held constant.
Temperature controller 214 can be configured to adjust the
temperature of rotatable platform 206. In such a configuration,
when rotatable platform 206 is heated, heat conducting blocks 204
are also heated. Alternatively, the temperature of each of heat
conducting blocks 204 can be controlled by a circulating water
system such as that noted above. Particularly, the temperature of
heat conducting block 204 can be changed by temperature controller
214 in accordance with a pre-determined temperature profile.
Preferably, temperature computer controller 214 is implemented
using a computer system.
[0281] As used herein, the term "temperature profile" refers to a
change in temperature over time. The term "temperature profile"
encompasses continuous upward or downward changes in temperature,
both linear and non-linear changes. The term also encompasses any
stepwise temperature change protocols, including protocols
characterized by incremental increases or decreases in temperature
during which temperature increases or decreases are interrupted by
periods during which temperature is maintained constant. In the
apparatus shown in FIG. 2, the temperature profile can be
pre-determined by programming temperature computer controller 214.
For example, temperature profiles can be stored in a memory device
of temperature controller 214, or input to temperature controller
214 by an operator.
[0282] Assay apparatus 200 also includes a light source 218 for
emitting an excitatory wavelength of light. Excitatory light from
light source 218 excites samples 216 with excitatory light. Any
suitable light source can be used. Excitatory light causes a
spectral emission from samples 216. The spectral emission can be
electromagnetic radiation of any wavelength in the electromagnetic
spectrum. Preferably, the spectral emission is fluorescent,
ultraviolet, or visible light. Most preferably, the spectral
emission is fluorescence emission.
[0283] A sensor is removably attached to a sensor armature 226. An
exemplary sensor is a fiber optic probe 212. Fiber optic probe 212
includes a fiber optic cable capable of transmitting excitatory
light to samples 216, and a fiber optic cable capable of receiving
a spectral emission from samples 216. Electromagnetic radiation is
transmitted from excitatory light source 218 to fiber optic probe
212 by excitatory light input fiber optic cable 228.
[0284] An excitatory light filter servo controller 258 controls the
aperture of excitatory light filter 256. Excitatory light source
218 and excitatory light filter servo controller 258 are
communicatively and operatively connected to excitatory light
computer controller 254. Computer controller 254 controls the
wavelength of excitatory light transmitted to samples 216 by
controlling excitatory light filter servo controller 258.
Excitatory light is transmitted through excitatory light input
fiber optic cable 228 to fiber optic probe 212 for transmission to
samples 216.
[0285] The spectral emission from samples 216 is received by fiber
optic probe 212 and is transmitted to a spectral emission filter
238 by output fiber optic cable 250. A spectral emission servo
controller 240 controls the aperture of spectral emission filter
238, thereby controlling the wavelength of the spectral emission
that is transmitted to photomultiplier tube 220. Spectral emission
servo controller 240 is controlled by a computer controller
242.
[0286] The spectral emission from samples 216 is transmitted from
photomultiplier tube 220. Electrical output 244 connects
photomultiplier tube 220 to electric connection 224. Electric
connection 224 connects electrical output 244 to computer 222.
Driven by suitable software, computer 222 processes the spectral
emission signal from samples 216. Exemplary software is a graphical
interface that automatically analyzes fluorescence data obtained
from samples 216. Such software is well known to those of ordinary
skill in the art. For example, the CytoFluor.TM.II fluorescence
multi-well plate reader (PerSeptive Biosystems, Framingham, Mass.)
utilizes the Cytocalc.TM. Data Analysis System (PerSeptive
Biosystems, Framingham, Mass.). Other suitable software includes,
MicroSoft Excel or any comparable software.
[0287] A sensor armature relative movement means 260 moves sensor
armature 226 in directions 234 and 236. A second relative movement
means 232 moves sensor armature 226 in directions 246 and 248 so
that fiber optic probe 212 can be moved to detect spectral
emissions from samples 216.
[0288] As discussed above, the spectral receiving means or sensor
of the assay apparatus of the present invention can comprise a
photomultiplier tube. Alternatively, the spectral receiving means
or sensor can include a charge coupled device (CCD). In still
another alternative, the spectral receiving means or sensor can
include a diode array. A CCD is made of semi-conducting silicon.
When photons of light fall on it, free electrons are released.
[0289] Further, a CCD camera can be used to image fluorescence,
such as fluorescence emission. High resolution CCD cameras can
detect very small amounts of electromagnetic energy, whether it
originates from distance stars, is diffracted by crystals, or is
emitted by fluorophores. As an electronic imaging device, a CCD
camera is particularly suitable for fluorescence emission imaging
because it can detect very faint objects, affords sensitive
detection over a broad spectrum range, affords low levels of
electromagnetic noise, and detects signals over a wide dynamic
range--that is, a charge coupled device can simultaneously detect
bright objects and faint objects. Further, the output is linear so
that the amount of electrons collected is directly proportional to
the number of photons received. This means that the image
brightness is a measure ofthe real brightness of the object, a
property not afforded by, for example, photographic emulsions.
Suitable CCD cameras are available from Alpha-Innotech (San
Leandro, Calif.), Stratagene (La Jolla, Calif.), and BioRad
(Richmond, Calif.).
[0290] Apparatuses useful for practicing the microplate thermal
shift assay are further described in U.S. patent application No.
08/853,459, filed May 9, 1997, and in international patent Appl.
No. PCT/US97/08154 (published Nov. 13, 1997 as publication no. WO
97/42500), which are hereby incorporated by reference in their
entirety.
[0291] Having now generally described the invention, the same will
become more readily understood by reference to the following
specific examples which are included herein for purposes of
illustration only and are not intended to be limiting unless
otherwise specified.
EXAMPLE 1
[0292] Wide Cross Target Utility of Microplate Thermal Shift
Assay
[0293] A number of different therapeutic protein targets have been
tested in the microplate thermal shift assay, to date, and are
listed in Table 2. They include a variety of different proteins,
with a wide diversity of in vivo function. Included here are
various serine proteases, a DNA binding protein (lac repressor),
two growth factors (basic fibroblast growth factor (bFGF) and
acidic fibroblast growth factor (aFGF)), and a growth factor
receptor (domain II of the fibroblast growth factor receptor 1
(D(II)FGFR1)).
2TABLE 2 Therapeutic Targets Analyzed by the Microplate Thermal
Shift Assay Targets MW Assays/mg (in 10 uL format) .alpha.-Thrombin
37.0 kDa 1430 0.7 ug/assay (20 pmol) Factor D 25.0 kDa 1000 1.0
ug/assay (40 pmol) Factor Xa 45.0 kDa 1667 0.6 ug/assay (7 pmol)
bFGF 17.5 kDa 2000 0.5 ug/assay (29 pmol) D(II)FGFR1 13.5 kDa 588
1.7 ug/assay 126 pmol) lac Repressor 77.0 kDa 1200 0.8 ug/assay (10
pmol) Urokinase 28.0 kDa 714 1.4 ug/assay (50 pmol) NFkB protein
65.0 kDa 3030 0.33 ug/assay (5 pmol) GLP1 receptor 26.0 kDa MHCII
45.0 kDa von Willebrand Factor 500 kDa 400 2.5 ug/assay aFGF 18.0
kDa
[0294] The molecular weights ofthe target proteins range from 13.5
kDa to about 500 kDa. On average, it was possible to conduct 1322
assays per 1.0 mg of protein using a 10 .mu.L assay volume. The
number of assays that can be conducted can be doubled if the 5
.mu.L assay format is employed.
[0295] All microplate thermal shift assays were performed in
polycarbonate V-bottom 96 well plates using 200 .mu.M 1,8-ANS as
the fluorescent probe for monitoring the thermal unfolding
transitions for the protein/ligand mixtures. Changes in
fluorescence emission at 460 nm were monitored with a CytoFluor II
(PerSeptive Biosystems) fluorescence plate reader (excitation at
360 nm), and the temperature was raised in 2.degree.C. increments
with the RoboCycler.RTM. Gradient Temperature Cycler (Stratagene,
La Jolla, Calif.).
[0296] A number of other proteins have been assayed using the
microplate thermal shift assay, including the following proteins
from the following classes: serine proteases (thrombin, Factor Xa,
Factor D, urokinase, trypsin, chymotrypsin, subtilisin); cell
surface receptors (FGF receptor 1, MHC Class II, GLP 1 receptor,
.beta.-2 adrenergic receptor, fibronectin receptor (IibIIIa));
growth factors (aFGF, bFGF); DNA binding proteins (lac repressor,
NF-K-B, helicase); motor proteins (myosin, helicase);
oxido-reductases (horseradish peroxidase, cytochrome c, lactate
dehydrogenase, lactoperoxidase, malate dehydrogenasease,
cholesterol oxidase, glyceraldehyde 3-phosphate dehydrogenasee,
phosphoenolpyruvate carboxylase, dihydrofolate reductase);
carbohydrate modifications (cellulase, .alpha.-amylase,
hyaluronidase, .beta.-glucosidase, invertase); immunoglobulins
(IgGFab, IgG Fc); DNAses (DNase I, DNase II) RNAses (RNase A);
intracellular calcium receptors (calmodulin, S100 protein);
neurotransmitter hydrolase (acetylcholinesterase); free radical
scavenger (superoxide dismutase); biotin binding protein
(streptavidin); oxygen binding protein (myoglobin); and protease
inhibitor (trypsin inhibitor).
EXAMPLE 2
[0297] Multi-Ligand Binding Interactions With A Single Target
Protein
[0298] The near universal utility of the microplate thermal shift
assay technology is also illustrated for multi-ligand binding
interactions that many times occur within a single protein
molecule. The ability to assess the binding of many different kinds
of ligands to a single protein without re-tooling the assay is a
great advantage of this technology and easily lends itself to the
task of assigning function to a protein for which nothing is known
other than the primary sequence. Knowledge for the binding of
different ligands will help in the evaluation of the function of a
sample protein derived from genomics information.
[0299] As previously demonstrated, the microplate thermal shift
assay can be used to screen ligands for binding to single sites on
target proteins. However, based upon the near additivity ofthe free
energy of ligand binding and protein unfolding, it is also possible
to employ the microplate thermal shift assay to analyzing
multi-ligand binding interactions on a target protein. In
principle, if the free energy of binding of different ligands
binding to the same protein are nearly additive then one can
analyze multi-ligand binding systems either non-cooperative or
cooperative (positive or negative).
[0300] In this regard, human thrombin is an ideal system to test
the utility of the assay for analysis of multi-ligand binding
interactions because it has at least four different binding sites:
(1) the catalytic binding site; (2) the fibrin binding site
(exosite I); (3) the heparin binding site (exosite II); (4) the Na
binding site, located about 15 .ANG. from the catalytic site.
[0301] First, the binding of individual ligands was determined.
3DP-4660, Hirugen (hirudin 53-64) (Sigma), and heparin 5000
(CalBiochem), bind to the catalytic site, the fibrin binding site
and the heparin binding site, respectively of thrombin.
[0302] A stock thrombin solution was diluted to 1 .mu.M in 50 mM
Hepes, pH 7.5, 0.1 M NaCl, 1 mM CaCl.sub.2, and 100 .mu.M 1,8-ANS.
Each thrombin ligand was included singly and in various
combinations to 1 .mu.M thrombin solutions at final concentrations
of 50.mu.M each, except for heparin 5000, which was 200 .mu.M. 100
.mu.L of thrombin or thrombin/ligand(s) solution was dispensed into
wells of a 96-well V-bottom polycarbonate microtiter plate. The
contents were mixed by repeated uptake and discharge in a 100 .mu.L
pipette tip. Finally, one drop of mineral oil (Sigma, St. Lois,
Mo.) was added on top of each reaction well to reduce evaporation
from samples at elevated temperatures. The plate was subjected to 3
minutes of heating in a RoboCycler.RTM. Gradient Temperature Cycler
(Stratagene, La Jolla, Calif.) thermal block, with which a
temperature gradient was created across the microplate, followed by
30 seconds cooling at 25.degree.C., and subsequent reading in a
fluorescence plate reader. Data were analyzed by non-linear least
squares fitting.
[0303] The results of these individual binding reactions are shown
in FIG. 3. The rank order of binding affinity was
3DP-4660>hirugen>heparin 5000, corresponding to K.sub.d of 15
nM, 185 nM and 3434 nM, respectively, for the ligands binding at
each T.sub.m (see Equation (1)).
[0304] Next, the binding of combinations of two ligands was
studied. The data are shown in FIG. 4. The results in FIG. 4 reveal
thermal unfolding shifts that are slightly smaller than that
expected for fill additivity. For example, Hirugen alone gave a
.DELTA.T.sub.m of 5.8..degree.C., and 3DP-4660 alone gave a
.DELTA.T.sub.m of 7.7.degree.C., but together they gave a
.DELTA.T.sub.m of 12.2.degree.C., and not the 13.5.degree.C. shift
that would be expected if the binding energies were fully additive.
This result could mean that the binding affinity of one or both
ligands is diminished when both ligands are bound to thrombin, and
would be an example of negative cooperativity between the fibrin
binding site and the catalytic binding site. Such a result is
consistent with the thrombin literature, in which the kinetics of
hydrolysis of various chromogenic substrates has been found to
depend upon ligands binding to exosite I. Indeed, a 60% decrease in
Km for the hydrolysis of D-phenylalanylpipecolyl
arginyl-p-nitroanilide was observed when Hirugen was present
(Dennis et al., Eur. J. Biochem. 188:61-66 (1990)). Moreover, there
is also structural evidence for cooperativity between the catalytic
site and exosite I. A comparison of the isomorphous structures of
PPACK-bound thrombin (PPACK is a thrombin catalytic site inhibitor)
and hirugen-bound thrombin revealed conformational changes that
occur at the active site as a result of hirugen binding at the
exosite I (Vijayalakshmi et al., Protein Science 3:2254-2271
(1994)). Thus, the apparent cooperativity observed by between the
catalytic center and the exosite I are consistent with functional
and structural data in the literature.
[0305] One would expect that if the energies of binding of all
three ligands were fully additive, a .DELTA.T.sub.m of
17.7.degree.C. would be seen. However, when all three ligands were
present together, the .DELTA.T.sub.m was 12.9.degree.C. This result
implies further negative cooperativity that involves ligand binding
at all three protein binding sites. There is some evidence in the
literature that is consistent with this supposition. For example,
thrombin, in a ternary complex with heparin and fibrin monomer, has
decreased activity toward tri-peptide chromogenic substrates and
pro-thrombin (Hogg & Jackson, J. Biol. Chem. 265:248-255
(1990)), and markedly reduced reactivity with anti-thrombin (Hogg
& Jackson, Proc. Natl. Acad. Sci. USA 86:3619-3623 (1989)).
Also, recent observations by Hotchkiss et al. (Blood 84:498-503
(1994)) indicate that ternary complexes also form in plasma and
markedly compromise heparin anticoagulant activity.
[0306] A summary of the thrombin multi-ligand binding results is
shown in Table 3. From the results in FIGS. 3 and 4 and in Table 3,
the following conclusions were made. First, in the presence of
heparin 5000, hirudin 53-65 bound thrombin about 21 times less
tightly than in the absence of heparin; and in the presence of
heparin 5000, 3DP-4660 bound thrombin about 10 times less tightly
than in the absence of heparin.
[0307] Second, in the presence of hirudin 53-65, heparin bound
thrombin about 18 times less tightly than in the absence of hirudin
53-65; and in the presence of hirudin 53-65, 3DP-4660 bound
thrombin about 3 times less tightly than in the absence of hirudin
53-65.
[0308] Third, in the presence of 3DP-4660, heparin bound thrombin
about 25% more tightly than in the absence of 3DP-4660; and in the
presence of 3DP-4660, hirudin bound thrombin about 2.3 times less
tightly than in the absence of 3DP-4660.
3TABLE 3 Assay for Ligands Binding to the Active Site, Exosite, and
Heparin binding Site of Thrombin K.sub.d at [Ligand] T.sub.m
.DELTA.T.sub.m K.sub.d at T.sub.m.sup.a 298.degree. K..sup.b
Protein/Ligand (.mu.M) (.degree. K.) (.degree. K.) (nM) (nM)
Thrombin (TH) none 323.75 0.0 TH/Heparin 5000 200 327.95 4.2 3434
470 TH/Hirudin 53-65 50 329.52 5.8 185 23 TH/3dp-4660 50 331.40 7.7
29 3 TH/Heparin 5000 200 327.95 TH/Hep./Hir. 50 330.57 2.6 4254 478
TH/Heparin 5000 200 327.95 TH/Hep./3dp 4660 50 333.20 5.3 350 32
TH/Hirudin 53-65 50 329.52 TH/Hir./Hep. 200 330.57 1.1 75422 8467
TH/Hirudin 53-65 50 329.52 TH/Hir./3dp-4660 50 335.97 6.5 117 9
TH/3dp-4660 50 331.40 TH/3dp-4660/Hep. 200 333.20 1.8 38205 351
TH/dp-4660 50 331.40 TH/3dp-4660/Hir. 50 335.97 4.6 731 54 .sup.a:
Calculations for K.sub.d at T.sub.m were made using equation (1)
with .DELTA.H.sub.M.sup.To = 200.0 kcal/mole, as observed for
pre-thrombin 1 by Leintz et al., Biochemistry 33:5460-5468 (1994),
and an estimated .DELTA.c.sub.pn = 2.0 kcal/mole - .degree. K.; and
K.sub.d = 1/K.sub.a. .sup.b: Estimates for K.sub.d at T 298.degree.
K. were made using the equation (3), where .DELTA.H.sub.L.sup.T is
estimated to be -10.0 kcal/mole.
[0309] Thus, the microplate thermal shift assay offers many
advantages for analyzing multi-ligand binding interactions in
functional genomics classification studies. For example, the same
assay can simultaneously detect the binding of different kinds of
ligands that bind at multiple binding sites on a target protein.
Each ligand binding interaction identified aids the user in
assigning a function to a protein. When the functions are summed
up, one obtains a response curve that is characteristic of a
particular class of proteins.
[0310] For example, if one considers the information obtained here
for thrombin, and for the moment forget what is known about this
protein, the heparin binding data might suggest an extracellular
role for this protein since heparin and other sulfated
oligosaccharide are important components of the extracellular
matrix of the tissues of higher organisms. The catalytic binding
site ligand, 3DP-4660, is a non-peptide mimic of a peptide that has
an arginyl side chain at the P1 position, characteristic of
substrates and inhibitors of trypsin-like serine proteases.
Similarly, boroarginine transition state analogs, which have an
arginine group in the P1 position for this synthetic peptide mimic,
were found to be specific inhibitors for the serine proteases,
thrombin, trypsin, and plasmin (Tapparelli et al., J. Biol. Chem.
268:4734-4741 (1993)) with the observed specificity:
K.sub.d.about.10 nM (thrombin), K.sub.d.about.1,000 nM (trypsin),
K.sub.d.about.10,000 nM (plasmin). Thus, the combined knowledge of
heparin binding with the observed binding to boroarginine
transition state analogs would quickly focus the assignment of this
protein to an extracellular proteolytic function in the absence of
any other information.
[0311] Further, the microplate thermal shift assay can be used, in
a high throughput fashion, to detect cooperativity in ligand
binding. Information about ligand binding cooperativity can be
collected and analyzed very quickly, over a few hours, rather than
over several months, as is required when conventional methods are
used to classify protein function.
EXAMPLE 3
[0312] Functional Probe Library Screen against Human Factor Xa
[0313] A functional probe library is shown in FIG. 5. A 96 well
plate (Plate 1) contained 94 compounds (and two control wells) and
included many compounds that are considered useful for providing
information about the ligand binding preferences, and thus probable
function, of proteins. For example, cofactors such as AND and ATP
are found in wells A4 and A5, respectively. This particular plate
also contained a great many metal ion binding conditions to help
probe a target protein for metal ion cofactors.
[0314] In order to validate the functional probe screen, two known
proteins were incubated with the compounds of Plate 1 and were then
assayed using the microplate thermal shift assay. For example, the
activity spectrum obtained for Factor Xa (Enzyme Research Labs) is
shown in FIG. 6.
[0315] Factor Xa was purchased from Enzyme research Labs (South
Bend, Ind.). Reactions were prepared in 96-well polycarbonate
microtitre plate v-bottom wells. The final concentration of Factor
Xa was 1.4 .mu.M (55 ng/mL) in 200 mM Tris.HCl, pH 8. The final
concentration of 1,8-ANS was 100 .mu.M. The final concentration of
each of the molecules tested for binding is shown in FIG. 6. The
contents were mixed by repeated uptake and discharge in a 100 .mu.L
pipette tip. Finally, one drop of mineral oil (Sigma, St. Lois,
Mo.) was added on top of each reaction well to reduce evaporation
from samples at elevated temperatures.
[0316] The microplate reactions were heated simultaneously, in two
degree increments, from 40 to 70.degree.C., using a RoboCycler.RTM.
Gradient Temperature Cycler (Stratagene, La Jolla, Calif.). After
each heating step, prior to fluorescence scanning, the sample was
cooled to 25.degree.C. Fluorescence was measured using a CytoFluor
II fluorescence microplate reader (PerSeptive Biosystems,
Framingham, Mass.). 1,8-ANS was excited with light at a wavelength
of 360 nm. The fluorescence emission was measured at 460 nm.
[0317] There were found to be six conditions that stabilized this
enzyme with a .DELTA.T.sub.m of greater than 1.0.degree.C.: (1) 0.5
M (NH.sub.4).sub.2SO.sub.4, (2) 0.5 MgSO.sub.4, (3) 0.5 M
Li.sub.2SO.sub.4, (4) 0.5 M KCl (5) 0.1 M tri-polyphosphate, and
(6) 0.1 M CaCl.sub.2. The last two conditions are probably most
significant, since tri-polyphosphate is a 5 polyelectrolyte that
mimics heparin and other sulfated oligosaccharides, and its binding
to proteins suggests the presence of a heparin binding site,
something that is well known for Factor Xa. Similarly, Ca.sup.2+is
known to bind to the Gla domain of Factor Xa, which is consistent
with the stabilizing effect seen for 0.1 M CaCl.sub.2.
[0318] Some of metal ions were found to have a strong destabilizing
effect on Factor Xa. For example, [Co(NH.sub.3).sub.6]Cl.sub.3,
BaC1.sub.2, CdC1.sub.2, YC1.sub.2, and NiSO.sub.4 were observed to
destabilize Factor Xa by from 6 to 17.degree.C. The reason for this
destabilizing effect is unknown. It is possible that these metal
ions preferentially bind to the unfolded form of Factor Xa. Some
interference with the fluorescence probe is also possible.
EXAMPLE 4
[0319] Functional Probe Library Screen against Human D(II)
FGFRI.
[0320] The compounds in functional probe library Plate 1 was also
employed to generate an activity spectrum for D(II) FGFR1. D(II)
FGFR1 was cloned and expressed in E. coli. Recombinant D(II) FGFR1
was renatured from inclusion bodies essentially as described
(Wetmore, D. R. et al., Proc. Soc. Mtg., San Diego, Calif. (1994)),
except that a hexa-histidine tag was included at the N-terminus to
facilitate recovery by affinity chromatography on a
Ni.sup.2+chelate column (Janknecht, R. et al., Natl. Acad. Sci. USA
88:8972-8976 (1991)). D(II) FGFR1 was further purified on a
heparin-sepharose column (Kan, M. et al., Science 259:1918-1921
(1993); Pantoliano, M. W. et al., Biochemistry 33:10229-10248
(1994)). Purity was >95%, as judged by SDS-PAGE. The D(II) FGFR1
protein was concentrated to 12 mg/mL (.about.1 mM) and stored at
4.degree.C.
[0321] Reactions were prepared in 96-well polycarbonate microtitre
plate v-bottom wells. The final concentration of D(II) FGFR1 was 50
.mu.M in 200 mM Tris.HCl, pH 8 in each well of a 96-well
polycarbonate microtitre plate. The final concentration of 1,8-ANS
was 100 .mu.M. The final concentration of each of the molecules
tested for binding is shown in FIG. 7. The contents were mixed by
repeated uptake and discharge in a 100 .mu.L pipette tip. Finally,
one drop of mineral oil (Sigma, St. Lois, Mo.) was added on top of
each reaction well to reduce evaporation from samples at elevated
temperatures.
[0322] The microplate reactions were heated simultaneously, in two
degree increments, from 25 to 60.degree.C., using a RoboCycler.RTM.
Gradient Temperature Cycler (Stratagene, La Jolla, Calif.). After
each heating step, prior to fluorescence scanning, the sample was
cooled to 25.degree.C. Fluorescence was measured using a CytoFluor
II fluorescence microplate reader (PerSeptive Biosystems,
Framingham, Mass.). 1,8-ANS was excited with light at a wavelength
of 360 nm. The fluorescence emission was measured at 460 nm.
[0323] The resultant activity spectrum is shown in FIG. 7. A larger
number of compounds were found to stabilize D(II) FGFR1. For
example, all ofthe sugars, D(+)-glucose, D(+)-sucrose, xylitol, and
sorbitol were all found to stabilize (and presumably bind) to D(II)
FGFR1. This result may be consistent with the known heparin binding
properties of this protein. Tri-polyphosphate, a known
polyelectrolyte heparin mimic, yielded the largest shift: about
11.degree.C. This result is consistent with the heparin binding
properties of this protein (Pantoliano, M. W. et al., Biochemistry
33:10229-10248 (1994)).
[0324] Thus, in a situation where a user did not know anything
about this protein (as is typically the case when a new gene is
cloned and the function ofthe encoded protein is unknown), the
information obtained by screening just the compounds in Plate 1
would have provided a user some evidence that D(II)FGFR1 could be
classified as a heparin-binding protein.
EXAMPLE 5
[0325] Identification of Protein Targets containing DNA Binding
Sites
[0326] The lac repressor is normally tetrameric protein, a dimer of
dimers. However, this protein has been shown to bind to DNA in its
dimeric state. Lewis et al. solved the crystal structure of Lac
repressor bound to its cognate DNA ligand (Lewis et al., 1996,
Science 271:1247-1254). A genetically altered dimer, one that is
unable to form a tetramer, and a synthetic 21 -mer oligonucleotide,
the palindromic sequence of the native lac operator, were obtained
from Dr. Mitch Lewis at the University of Pennsylvania. Binding of
the synthetic lac operator to the mutant lac repressor was assayed
using the microplate thermal shift assay.
[0327] The final concentration of lac repressor was 60 .beta.M in
200 mM Tris.HCl, pH 8. Reactions were prepared in 96-well
polycarbonate microtiter plate V-bottom wells. The final
concentration of 1,8-ANS was 100 .mu.M. The final concentration of
each of the molecules tested for binding is shown in FIG. 7. The
contents were mixed by repeated uptake and discharge in a 100 .mu.L
pipette tip. Finally, one drop of mineral oil (Sigma, St. Lois,
Mo.) was added on top of each reaction well to reduce evaporation
from samples at elevated temperatures.
[0328] The microplate reactions were heated simultaneously, in two
degree increments, from 25.degree.C. to 75.degree.C., using a
ROBOCYCLER.RTM. Gradient Temperature Cycler (Stratagene, La Jolla,
Calif.). After each heating step, prior to fluorescence scanning,
the sample was cooled to 25.degree.C. Fluorescence was measured
using a CytoFluor II fluorescence microplate reader (PerSeptive
Biosystems, Framingham, Mass.). ANS was excited with light at a
wavelength of 360 nm. The fluorescence emission was measured at 460
nm.
[0329] In the presence of 80 .mu.M synthetic operator DNA, the
T.sub.m for the unfolding transition of lac repressor was shifted
5.6.degree.C. (FIG. 8). The calculated K.sub.d at T.sub.m is 6
.mu.M. Using educated guesses for .DELTA.H.sub.L (-10.0 kcal/mol),
the calculated K.sub.d at 25.degree.C. is 1.2 .mu.M and the
calculated K.sub.d at physiological temperature (37.degree.C.) is
3.4 .mu.M. The fluorescent probe, 1,8-ANS, did not bind to DNA
alone (i.e., there was no fluorescence signal for the control
reaction in which no lac repressor was included).
[0330] These results show that the microplate thermal shift assay
can be used to assay DNA/protein interactions.
EXAMPLE 6
[0331] Assays of ATP Binding
[0332] Adenosine triphosphate (ATP) and ATP analogue binding can be
assayed using the microplate thermal shift assay. Bovine muscle
myosin (Sigma), bovine heart 3'-5'cAMP-dependent protein kinase
(Sigma), and chicken muscle pyruvate kinase (Sigma) were each
dissolved in Buffer A to generate stock solutions at a final
concentration of 2 mg/mL. Magnesium chloride (MgCl.sub.2),
adenosine triphosphate, adenosine triphosphate-.gamma.-S
(ATP-.gamma.-S), aluminum trifluoride (AlF.sub.3), and sodium
fluoride (NaF) were dissolved in Buffer A (50 mM HEPES, pH 7.5, 100
mM NaCl) to the stock concentrations used in each experiment.
Dapoxyl.TM. 12800 solution was prepared by diluting a stock of 20
mM Dapoxyl 12800.TM.
(5-(4"-dimethylaminophenyl)-2-(4'-phenyl)oxazole sulfonic acid,
sodium salt, Molecular Probes, Inc.) in dimethyl sulfoxide to the
appropriate concentration in Buffer A.
[0333] In the ATP and ATP-.gamma.-S reactions, each sample
contained 12 .mu.L of protein stock solution (2 mgs/mL), 9.6 uL of
either ATP or ATP-.gamma.-S (50 mM), 4.8 .mu.L of MgCl.sub.2 (100
mM) and 21.6 .mu.L of a solution of 222 uM of dapoxyl 12800 in
Buffer A. In the ATP, aluminum trifluoride, and sodium fluoride
reactions, each sample contained 12 .mu.L of protein stock solution
(2 mgs/mL), 9.6 uL of ATP (50 mM), 9.6 mL of aluminum trifluoride
(50 mM)+sodium fluoride (50 mM), 4.8 .mu.L of 100 mM MgCl.sub.2,
and 12 .mu.L of a solution of 400 uM Dapoxyl 12800 in Buffer A.
[0334] For the thermal shift assay, four 10 .mu.L aliquots of each
assay mixture were dispensed into four wells located in different
quadrants of an MJ Research 384-well thermocycler plate. 10 .mu.L
of mineral oil was then added to each of the four wells to prevent
evaporation. Each data point shown was collected by heating the
plate at the temperatures shown for three minutes. For example, the
plate was heated to a given temperature, and then allowed to cool
to 25.degree.C. for one minute, followed by UV illumination and
collection ofthe data. Then the plate was heated to the next higher
temperature, and so forth. UV illumination was performed using a
long wavelength illumination at 200-420 nm, having a peak at 365
nm. Fluorescence was imaged using a CCD camera having a bandpass
filter centered at 550 nm.
[0335] FIG. 9 shows the results of a microplate thermal shift assay
of ATP to bovine muscle myosin. The data is plotted as fluorescence
intensity as a function of temperature. The T.sub.m of the control
thermal unfolding curve (no ATP) was 49.3.degree.C. (microplate
well K2). The T.sub.m of the thermal unfolding curve for bovine
muscle myosin bound to ATP ((+) ATP) was 51.4.degree.C. (microplate
well K16). Thus the .DELTA.T.sub.m for ATP binding was
2.1.degree.C. The K.sub.d was 440 .mu.M.
[0336] FIG. 10 shows the result of a microplate thermal shift assay
of ATP-.gamma.-S to and 3', 5'-cAMP-dependent protein kinase. The
data is plotted as fluorescence intensity as a function of
temperature. The T.sub.m of the control thermal unfolding curve (no
ATP-.gamma.-S) was 46.2.degree.C. (microplate well E14). The
T.sub.m of the thermal unfolding curve for 3', 5'-cAMP-dependent
protein kinase bound to ATP-.gamma.-S ((+) ATP-.gamma.-S) was
51.8.degree.C. (microplate well M15). Thus the .DELTA.T.sub.m for
ATP-.gamma.-S binding was 5.6.degree.C. The K.sub.d was 200 .mu.M.
The results, including the results for pyruvate kinase, are
summarized in Table 4.
[0337] Table 4. Summary of results for enzymes that bind to ATP.
The value in parentheses is standard deviation.
4 Refe- ATP-.gamma.-S ATP ATP + AlF.sub.3 rence (10 mM) (10 mM) (10
mM) Protein T.sub.m .DELTA.T.sub.m .DELTA.T.sub.m .DELTA.T.sub.m
Myosin 49.4 0.0 (.+-.0.2) 2.2 (.+-.0.4) 2.8 (.+-.0.4) 3'-5' cAMP
44.7 5.6 (.+-.1.7) 7.5 (.+-.0.7) 8.2 (.+-.1.4) Protein kinase
Pyruvate kinase 54.5 0.8 (.+-.0.11) -0.44 (.+-.0.1) -0.27
(.+-.0.2)
EXAMPLE 7
[0338] Assay of Folic Acid Binding
[0339] Folic acid binding can be assayed using the microplate
thermal shift assay.
[0340] Bovine liver dihydrofolate reductase (DHFR, Sigma), chicken
liver dihydrofolate reductase (DHFR, Sigma), pigeon liver arylamine
acetyltransferase (ArAcT, Sigma), and porcine liver formimino
glutamic acid transferase (FGT, Sigma) were each dissolved in
Buffer A (50 mM HEPES, pH 7.5, 1 00 mM NaCl) to generate stock
solutions at a final concentration of2 mg/mL. Solutions of
dihydrofolic acid (FAH.sub.2), methotrexate, nicotinamide adenine
dinucleotide phosphate (NADP), were prepared by dissolving solid
material into Buffer A immediately prior to use. DapoXy.TM.12800
solution was prepared by diluting a stock of 20 mM DapoXy.TM.12800
in dimethyl sulfoxide to the appropriate concentration in Buffer
A.
[0341] Each assay sample contained 12 .mu.L of protein stock
solution (2 mg/mL), 4.8 .mu.L of either dihydrofolic acid
(FAH.sub.2) or methotrexate stock solution (1 mM), and 31.2 .mu.L
of a solution of 154 .mu.M Dapoxyl.TM. 12800 in Buffer A. Each
sample contained 12 .mu.L of protein stock solution (2 mgs/mL), 4.8
.mu.L of NADP stock solution (50 mM), and 31.2 .mu.L of a solution
of 154 .mu.M Dapoxyl.TM. 12800 in Buffer A.
[0342] For the thermal shift assay, four 10 .mu.L aliquots of each
assay mixture were dispensed into four wells located in different
quadrants of an MJ Research 384-well thermocycler plate. 10 .mu.L
of mineral oil was then added to each of the four wells to prevent
evaporation. Each data point shown was collected by heating the
plate at the temperature shown for three minutes, followed by
incubation at 25.degree.C. for one minute, followed by UV
illumination and collection of the data. The results are shown in
Table 5.
[0343] Table 5. Results for proteins that bind methotrexate,
FAH.sub.2 and NADP. The value in parentheses is standard
deviation.
5 Methotrexate FAH.sub.2 NADP Reference (100 .mu.M) (100 .mu.M) (5
mM) Protein T.sub.m .DELTA.T.sub.m .DELTA.T.sub.m .DELTA.T.sub.m
DHFR 52.47 7.0 (.+-.-0.1) -0.64 (.+-.0.2) 3.2 (.+-.0.13) DHFR 56.6
8.6 (.+-.0.2) 2.5 (.+-.0.2) 3.8 (.+-.0.4) Arylamine 49.8 1.0
(.+-.0.4) -1.8 (.+-.0.5) 2.8 (.+-.0.4) Acetyl transferase Formimino
47.2 0.9 (.+-.0.5) 3.62 (.+-.0.4) 0.0 (.+-.0.2) L-Glutamic acid
Transferase
EXAMPLE 8
[0344] Assay of Methotrexate/NADP(H) Binding
[0345] The ability to measure temperature shifts for the binding of
methotrexate and NADPH, both separately and simultaneously, is
another example of the utility of the present invention in
measuring milti-ligand binding interactions. In this case, the
binding sites of the two ligands are proximal, and there is
positive cooperativity in the binding of the two ligands, as shown
by the fact that thermal shift for both ligands binding
simultaneously is 2-4 degrees more than the total of shifts for
each ligand binding separately (Table 6).
[0346] Methotrexate (MTX) and NADPH binding can be assayed using
the microplate thermal shift assay. Bovine liver dihydrofolate
reductase (DHFR, Sigma) and chicken liver dihydrofolate reductase
(DHFR, Sigma were each dissolved in Buffer A (50 mM HEPES, pH 7,5,
100 mM NaCI) to generate stock solutions at a final concentration
of 2 mg/mL. All stock solutions of ligands were prepared by
dissolving solid material in Buffer A immediately prior to use.
Stock solutions of nicotinamide adenine dinucleotide
phosphate-reduced form (NADPH, 100 mM), NADP (100 mM), and
methotrexate (1 mM) were diluted further in Buffer A to twice to
the final assay concentration (2.times.stocks): methotrexate (200
.mu.M), NADP (20 mM), NADPH (20 mM), methotrexate+NADP (200
.mu.M+20 mM), methotrexate+NADPH (200 .mu.M+20 mM). Dapoxyl.TM.
12800 solutions was prepared by diluting a stock of 20 mM
Dapoxyl.TM. 12800 in dimethyl sulfoxide to the appropriate
concentration in Buffer A. 5 .mu.L of each protein stock solution
was added to 25 .mu.L of 2.times.ligand stock solution mixed with
20 .mu.L of a solution of 250 .mu.M Dapoxyl.TM. 12800 in Buffer
A.
[0347] The final ligand concentrations were 10 mM NADP; 10 mM
NADPH; and 100 .mu.M MTX.
[0348] For the thermal shift assay, four 10 .mu.L aliquots of each
assay mixture were dispensed into four wells located in different
quadrants of an MJ Research 384-well thermocycler plate. 10 .mu.L
of mineral oil was then added to each of the four wells to prevent
evaporation. Each data point shown was collected by heating the
plate at the temperature shown for three minutes, followed by
incubation at 25.degree.C. for one minute, followed by UV
illumination and collection of the data.
[0349] FIG. 11 shows the result of a microplate thermal shift assay
of methotrexate to dihydrofolate reductase. The data is plotted as
fluorescence intensity as a function of temperature. The T.sub.m of
the control thermal unfolding curve (no MTX) was 47.2.degree.C.
(microplate well M1). The T.sub.m of the thermal unfolding curve
for DHFR bound to methotrexate ((+) MTX) was 56.4.degree.C.
(microplate well G6). Thus the .DELTA.T.sub.m for methotrexate
binding was 9.2.degree.C. The K.sub.d was 24 nM.
[0350] FIG. 12 shows the result of a microplate thermal shift assay
of NADPH to dihydrofolate reductase. The data is plotted as
fluorescence intensity as a function of temperature. The T.sub.m of
the control thermal unfolding curve (no NADPH) was 50.8.degree.C.
(microplate well G8). The T.sub.m of the thermal unfolding curve
for DHFR bound to NADPH ((+) NADPH) was 53.8.degree.C. (microplate
well B20). Thus the .DELTA.T.sub.m for NADPH binding was
3.degree.C. The K.sub.d was 0.7 .mu.M.
[0351] Table 6. .DELTA.T.sub.m's of ligand complexed with DHFR. The
value in parentheses is standard deviation.
6TABLE 6 .DELTA.T.sub.m's of ligand complexed with DHFR The value
in parentheses is standard deviation NADP + NADP NADPH + Protein
NADP MTX Sum.sup.a MTX.sup.b H MTX Sum.sup.a MTX.sup.b DHFR 7.5
10.1 17.6 20.9 11.9 10.1 22 23.8 (chicken) (.+-.0.38) (.+-.0.32)
(.+-.0.4) (.+-.1.3) (.+-.0.32) (.+-.0.4) DHFR 6.3 7.7 14 18.1 9.7
7.7 17.4 24.6 (cow) (.+-.0.1) (.+-.0.3) (.+-.0.4) (.+-.0.2)
(.+-.0.3) (.+-.0.6) .sup.aThe value shown is the sum of the
individual .DELTA.T.sub.m's observed from the protein incubated
separately with each ligand. .sup.bThe value shown is the
.DELTA.T.sub.m observed when the protein was incubated
simultaneously with both ligands.
EXAMPLE 9
[0352] Dihydrofolic acid is a substrate of dihydrofolate reductase
(DHFR). Methotrexate is a folic acid analog that binds to DHFR. As
evidence that the method of the present invention is reliable, it
was shown that the method can be used to detect binding of
dihydrofolic acid to DHFR. Bovine liver DHFR was combined with 80
compounds to screen for the function of the protein, and binding to
methotrexate, but not to a number of other compounds, was
detected.
[0353] Each well of microsource compound plate #198104 contained
one of 80 different compounds at a concentration 10 mM in dimethyl
sulfoxide. Each compound solution was diluted in Buffer A (50 mM
HEPES, pH 7.5, 100 mM NaCl) to a final concentration of 200 .mu.M
in separate wells in a 384-well polystyrene plate. 5 .mu.L of the
solution contained in each well was transferred to an MJ research
polypropylene plate containing 5 .mu.L of bovine liver DHFR (at a
concentration of 0.5 mg/mL and Dapoxyl.TM. 12800 dye at a
concentration of 200 .mu.M, yielding final concentrations of 100
.mu.M ligand, 0.25 mg/mL DHFR, and 100 .mu.M dapoxyl in the 10 L
volume of each well.
[0354] 10 .mu.L of mineral oil was added to each well to prevent
evaporation. Thermal unfolding profiles were then measured for each
well from 25 to 70.degree.C., by collecting data points at each
temperature, separated by one-degree increments. Each data point
was collected by heating the plate at the temperature shown for 3
minutes, followed by incubation at 25.degree.C. for one minute,
followed by long-wavelength UV illumination and collection of the
data using a CCD camera.
[0355] The data were collected as four replicates of 80 compounds
in the quadrants of a 384-well plate. The four quadrants consist
of: wells A2 through H11 (first quadrant), wells A14 through H23
(second quadrant), wells I2 through P11 (third quadrant), and wells
I14 through 123 (fourth quadrant). Columns 1, 12, 13, and 24
consist of reference wells containing only DHFR and dimethyl
sulfoxide.
[0356] Wells F2, F14, N2, and N14 contained methotrexate. Binding
was revealed by fitting software as a red well. Methotrexate
shifted the T.sub.m by 5.13.+-.0.19 degrees (average of 4
quadrants), and the other compounds on the plate had little or no
effect (shown as near-white wells). These results which indicated
that DHFR binds methotrexate.
[0357] All publications and patents mentioned hereinabove are
hereby incorporated in their entireties by reference.
[0358] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be
appreciated by one skilled in the art from a reading of this
disclosure that various changes in form and detail can be made
without departing from the true scope ofthe invention and appended
claims.
* * * * *