U.S. patent application number 11/540883 was filed with the patent office on 2007-02-01 for methods for rapidly identifying small organic molecule ligands for binding to biological target molecules.
This patent application is currently assigned to SUNESIS PHARMACEUTICALS, INC.. Invention is credited to Andrew C. Braisted, Dan Erlanson, Jim Wells.
Application Number | 20070026451 11/540883 |
Document ID | / |
Family ID | 22305449 |
Filed Date | 2007-02-01 |
United States Patent
Application |
20070026451 |
Kind Code |
A1 |
Wells; Jim ; et al. |
February 1, 2007 |
Methods for rapidly identifying small organic molecule ligands for
binding to biological target molecules
Abstract
The present invention is directed to novel methods for rapidly
and unambiguously identifying small organic molecule ligands for
binding to biological target molecules. Small organic molecule
ligands identified according to the methods of the present
invention may find use, for example, as novel therapeutic drug lead
compounds, enzyme inhibitors, labeling compounds, diagnostic
reagents, affinity reagents for protein purification, and the like.
Also presented are novel methods for identifying high affinity
binding ligands for a biological target molecule of interest,
wherein those methods comprise linking two or more small organic
molecule ligands previously identified as being capable of binding
to the biological target molecule of interest. Biological target
molecules include, for example, polypeptides, nucleic acids,
carbohydrates, nucleoproteins, glycoproteins, glycolipids and
lipoproteins.
Inventors: |
Wells; Jim; (Burlingame,
CA) ; Erlanson; Dan; (San Francisco, CA) ;
Braisted; Andrew C.; (San Francisco, CA) |
Correspondence
Address: |
HELLER EHRMAN LLP
275 MIDDLEFIELD ROAD
MENLO PARK
CA
94025-3506
US
|
Assignee: |
SUNESIS PHARMACEUTICALS,
INC.
|
Family ID: |
22305449 |
Appl. No.: |
11/540883 |
Filed: |
September 28, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09981547 |
Oct 17, 2001 |
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11540883 |
Sep 28, 2006 |
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09105372 |
Jun 26, 1998 |
6335155 |
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09981547 |
Oct 17, 2001 |
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Current U.S.
Class: |
435/6.16 ;
435/7.1 |
Current CPC
Class: |
Y10T 436/24 20150115;
G01N 33/566 20130101; G01N 33/53 20130101 |
Class at
Publication: |
435/006 ;
435/007.1 |
International
Class: |
C40B 30/06 20070101
C40B030/06; C40B 40/08 20070101 C40B040/08; C40B 40/10 20070101
C40B040/10 |
Claims
1. A method for identifying an organic molecule ligand that binds
to a site of interest on a biological target molecule, said method
comprising: (a) obtaining a biological target molecule that
comprises or has been modified to comprise a chemically reactive
group, wherein said site of interest on said target molecule
comprises said chemically reactive group; (b) combining said target
molecule with one or more members of a library of organic compounds
that are capable of covalently bonding to said chemically reactive
group, wherein at least one member of said library binds to said
site of interest and forms a covalent bond with said chemically
reactive group to form a target molecule/organic compound
conjugate; and (c) identifying the organic compound that forms a
covalent bond with said chemically reactive group.
2. The method according to claim 1, wherein said biological target
molecule is selected from the group consisting of a polypeptide, a
nucleic acid, a carbohydrate, a nucleoprotein, a glycopeptide, a
glycolipid and a lipoprotein.
3. The method according to claim 2, wherein said biological target
molecule is a polypeptide.
4. The method according to claim 3, wherein said polypeptide is
selected from the group consisting of an enzyme, a hormone, a
transcription factor, a receptor, a ligand for a receptor, a growth
factor and an immunoglobulin.
5. The method according to claim 1, wherein said biological target
molecule comprises said chemically reactive group without prior
modification of said target molecule.
6. The method according to claim 1, wherein said biological target
molecule obtained in step (a) has been modified to comprise said
chemically reactive group.
7. The method according to claim 6, wherein said modification
comprises bonding to said target molecule a compound that comprises
said chemically reactive group.
8. The method according to claim 1, wherein said library of organic
compounds comprises aldehydes, ketones, oximes, hydrazones,
semicarbazones, carbazides, primary amines, secondary amines,
tertiary amines, N-substituted hydrazines, hydrazides, alcohols,
ethers, thiols, thioethers, thioesters, disulfides, carboxylic
acids, esters, amides, ureas, carbamates, carbonates, ketals,
thioketals, acetals, thioacetals, aryl halides, aryl sulfonates,
alkyl halides, alkyl sulfonates, aromatic compounds, heterocyclic
compounds, anilines, alkenes, alkynes, diols, amino alcohols,
oxazolidines, oxazolines, thiazolidines, thiazolines, enamines,
sulfonamides, epoxides, aziridines, isocyanates, sulfonyl
chlorides, diazo compounds and acid chlorides.
9. The method according to claim 1, wherein said library of organic
compounds comprises primary amines, secondary amines, aldehydes or
ketones.
10. The method according to claim 1, wherein said chemically
reactive group is a primary amine group, a secondary amine group,
an aldehyde group or a ketone group.
11. The method according to claim 1, wherein step (c) is
accomplished by a process that employs mass spectrometry.
12. The method according to claim 1, wherein step (c) comprises
fragmenting said target molecule/organic compound conjugate into
two or more fragments.
13. The method according to claim 1, wherein subsequent to step (b)
and prior to step (c) said target molecule/organic compound
conjugate is combined with one or more members of a library of
organic molecules that are capable of covalently bonding to the
organic compound previously bound to said target molecule, wherein
at least one member of said library of organic molecules binds to
said target molecule/organic compound conjugate.
14. A method for identifying an organic molecule ligand that binds
to a biological target molecule of interest, said method
comprising: (a) obtaining a biological target molecule that
comprises or has been modified to comprise a first reactive
functionality, (b) reacting said target molecule with a compound
that comprises (1) a second reactive functionality and (2) a
chemically reactive group, wherein said second reactive
functionality reacts with said first reactive functionality of said
target molecule to form a covalent bond, thereby resulting in said
chemically reactive group being linked to said target molecule
through a covalent bond; (c) combining said target molecule with
one or more members of a library of organic compounds that are
capable of covalently bonding to said chemically reactive group,
wherein at least one member of said library forms a covalent bond
with said chemically reactive group to form a target
molecule/organic compound conjugate; and (d) identifying the
organic compound that forms a covalent bond with said chemically
reactive group.
15. The method according to claim 14, wherein said first and second
chemically reactive functionalities are activated thiol groups that
react to form a disulfide bond.
16. The method according to claim 15, which further comprises
subsequent to step (c) and prior to step (d) the step of liberating
the covalently-bonded organic compound from said target
molecule/organic compound conjugate by treatment with an agent that
disrupts said disulfide bond.
17. The method according to claim 16, wherein said agent that
disrupts said disulfide bond is dithiothreitol, dithioerythritol,
.beta.-mercaptoethanol, sodium borohydride or a phosphine.
18. The method according to claim 14, wherein said biological
target molecule is selected from the group consisting of a
polypeptide, a nucleic acid, a carbohydrate, a nucleoprotein, a
glycopeptide, a glycolipid and a lipoprotein.
19. The method according to claim 18, wherein said biological
target molecule is a polypeptide.
20. The method according to claim 19, wherein said polypeptide is
selected from the group consisting of an enzyme, a hormone, a
transcription factor, a receptor, a ligand for a receptor, a growth
factor and an immunoglobulin.
21. The method according to claim 19, wherein said polypeptide
comprises or has been modified to comprise only a single cysteine
residue.
22. The method according to claim 19, wherein said polypeptide is
obtained as a recombinant expression product.
23. The method according to claim 19, wherein said polypeptide is
synthetically derived.
24. The method according to claim 14, wherein said target molecule
comprises or has been modified to comprise less than about 2 free
thiol groups.
25. The method according to claim 14, wherein said library of
organic compounds comprises aldehydes, ketones, oximes, hydrazones,
semicarbazones, carbazides, primary amines, secondary amines,
tertiary amines, N-substituted hydrazines, hydrazides, alcohols,
ethers, thiols, thioethers, thioesters, disulfides, carboxylic
acids, esters, amides, ureas, carbamates, carbonates, ketals,
thioketals, acetals, thioacetals, aryl halides, aryl sulfonates,
alkyl halides, alkyl sulfonates, aromatic compounds, heterocyclic
compounds, anilines, alkenes, alkynes, diols, amino alcohols,
oxazolidines, oxazolines, thiazolidines, thiazolines, enamines,
sulfonamides, epoxides, aziridines, isocyanates, sulfonyl
chlorides, diazo compounds and acid chlorides.
26. The method according to claim 14, wherein said chemically
reactive group is selected from the group consisting of an aldehyde
group and a ketone group and said library of organic compounds
comprises primary amines and/or secondary amines.
27. The method according to claim 14, wherein said chemically
reactive group is selected from the group consisting of a primary
amine group and a secondary amine group and said library of organic
compounds comprises aldehydes and/or ketones.
28. The method according to claim 14, wherein in step (c) one
member of said library of organic compounds reacts with said
chemically reactive group to form a Schiff base adduct.
29. The method according to claim 28, wherein subsequent to step
(c) and prior to step (d), said Schiff base adduct is reduced by
addition of a reducing agent.
30. The method according to claim 29, wherein said reducing agent
is selected from the group consisting of sodium cyanoborohydride,
sodium triacetoxyborohydride and cyanide.
31. The method according to claim 14, wherein said step (d) is
accomplished by a process that employs mass spectrometry.
32. A method for identifying a ligand that binds to a biological
target molecule of interest, said method comprising: (a)
identifying a first organic molecule ligand that binds to said
biological target molecule by the method of claim 1; (b)
identifying a second organic molecule ligand that binds to said
biological target molecule by the method of claim 1; and (c)
linking said first and second organic molecule ligands through a
linker element to form a conjugate molecule that binds to said
biological target molecule.
33. The method according to claim 32, wherein said biological
target molecule is selected from the group consisting of a
polypeptide, a nucleic acid, a carbohydrate, a nucleoprotein, a
glycopeptide, a glycolipid and a lipoprotein.
34. The method according to claim 32, wherein said biological
target molecule is a polypeptide.
35. The method according to claim 34, wherein said first and said
second organic molecule ligands bind to the same site on said
polypeptide.
36. The method according to claim 34, wherein said first and said
second organic molecule ligands bind to different sites on said
polypeptide.
37. The method according to claim 32, wherein said first and second
organic molecule ligands are from the same chemical class.
38. The method according to claim 32, wherein said first and second
organic molecule ligands are from different chemical classes.
39. The method according to claim 34, wherein said conjugate
molecule binds to said polypeptide with a lower dissociation
constant than either of said first and second organic molecule
ligands.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to novel molecular methods
useful for quickly and unambiguously identifying small organic
molecule ligands for binding to specific sites on target biological
molecules. Small organic molecule ligands identified according to
the methods of the present invention find use, for example, as
novel therapeutic drug lead compounds, enzyme inhibitors, labeling
compounds, diagnostic reagents, affinity reagents for protein
purification, and the like.
BACKGROUND OF THE INVENTION
[0002] The primary task in the initial phase of generating novel
biological effector molecules is to identify and characterize one
or more tightly binding ligand(s) for a given biological target
molecule. In this regard, many molecular techniques have been
developed and are currently being employed for identifying novel
ligands that bind to specific sites on biomolecular targets, such
as proteins, nucleic acids, carbohydrates, nucleoproteins,
glycoproteins and glycolipids. Many of these techniques exploit the
inherent advantages of molecular diversity by employing
combinatorial libraries of potential ligand compounds in an effort
to speed up the identification of functional ligands. For example,
combinatorial synthesis of both oligomeric and non-oligomeric
libraries of diverse compounds combined with high-throughput
screening assays has already provided a useful format for the
identification of new lead compounds for binding to chosen
molecular targets.
[0003] While combinatorial approaches for identifying biological
effector molecules have proven useful in certain applications,
these approaches also have some significant disadvantages. For
example, current synthetic technology is limited in that it allows
one to synthesize only a relatively small fraction of the total
number of possible library members for any given molecule type. As
such, even when appropriate high-throughput screening assays are
available for screening a library, only a small fraction of the
total number of possible members of any molecule type will be
represented in the library and, therefore, screened for the ability
to bind to the chosen target. Thus, combinatorial approaches often
do not allow one to identify the "best" ligand for a target
molecule of interest.
[0004] Additionally, even when appropriate screening assays are
available, in many cases techniques which allow identification of
the actual library member(s) which bind most effectively to the
target are not available or provide ambiguous results, making the
actual identification and characterization of functional ligand
molecules difficult or impossible. Furthermore, many approaches
currently employed to identify novel ligands are dependent upon
only a single specific chemistry, thereby limiting the usefulness
of such approaches to only a narrow range of applications. Finally,
many of the approaches currently employed are expensive and
extremely time-consuming. Thus, there is a significant interest in
developing new methods which allow rapid, efficient and unambiguous
identification of small organic molecule ligands for selected
biomolecular targets. It is also desired that such techniques are
adaptable to a variety of different chemistries, thereby being
useful for a wide range of applications.
[0005] Schiff base adduct formation involves the reaction of an
available aldehyde or ketone functionality with a primary amine to
form an imine-bonded complex. While the Schiff base adduct is
relatively unstable, numerous groups have employed aldehyde or
ketone compounds for bonding to primary amine functionalities on
proteins of interest for a variety of purposes (see, e.g., Pollack
et al., Science 242:1038-1040 (1988), Abraham et al., Biochemistry
34:15006-15020 (1995) and Boyiri et al., Biochemistry
34:15021-15036 (1995)). We herein describe novel techniques useful
for rapidly and efficiently identifying organic molecule ligands
that bind to specific sites on biomolecular targets, wherein those
techniques are adaptable to a variety of different chemistries,
preferably Schiff base adduct formation between a target
polypeptide and one or more members of a library of potential
organic molecule ligands. These methods allow one to unambiguously
identify and characterize the organic molecule ligand that binds
most efficiently to the chosen target. Additionally, the herein
described methods are quick, easy to perform and inexpensive as
compared to other currently employed methods.
SUMMARY OF THE INVENTION
[0006] Applicants herein describe a molecular approach for rapidly
and efficiently identifying small organic molecule ligands that are
capable of interacting with and binding to specific sites on
biological target molecules, wherein ligand compounds identified by
the subject methods may find use, for example, as new small
molecule drug leads, enzyme inhibitors, labeling compounds,
diagnostic reagents, affinity reagents for protein purification,
and the like. The herein described approaches allow one to quickly
screen a library of small organic compounds to unambiguously
identify those that have affinity for a particular site on a
biomolecular target. Those exhibiting affinity for interacting with
a particular site are capable of forming a covalent bond with a
chemically reactive group at that site, whereby small organic
compounds capable of covalent bond formation may be readily
identified and characterized.
[0007] Such methods may be performed quickly, easily and
inexpensively and provide for unambiguous results. The small
organic molecule ligands identified by the methods described herein
may themselves be employed for numerous applications, or may be
coupled together in a variety of different combinations using one
or more linker elements to provide novel binding molecules.
[0008] With regard to the above, one embodiment of the present
invention is directed to a method for identifying an organic
molecule ligand that binds to a site of interest on a biological
target molecule, wherein the method comprises:
[0009] (a) obtaining a biological target molecule that comprises or
has been modified to comprise a chemically reactive group, wherein
the site of interest on the target molecule comprises the
chemically reactive group;
[0010] (b) combining the target molecule with one or more members
of a library of organic compounds that are capable of covalently
bonding to the chemically reactive group, wherein at least one
member of the library binds to the site of interest to form a
covalent bond with the chemically reactive group to form a target
molecule/organic compound conjugate; and
[0011] (c) identifying the organic compound that forms a covalent
bond with the chemically reactive group.
[0012] In particular embodiments, the biological target molecule is
a polypeptide, a nucleic acid, a carbohydrate, a nucleoprotein, a
glycopeptide or a glycolipid, preferably a polypeptide, which may
be, for example, an enzyme, a hormone, a transcription factor, a
receptor, a ligand for a receptor, a growth factor, an
immunoglobulin, a steroid receptor, a nuclear protein, a signal
transduction component, an allosteric enzyme regulator, and the
like. The target molecule may comprise the chemically reactive
group without prior modification of the target molecule or may be
modified to comprise the chemically reactive group, for example,
when a compound comprising the chemically reactive group is bound
to the target molecule.
[0013] Other embodiments of the above described methods employ
libraries of organic compounds which comprise aldehydes, ketones,
oximes, hydrazones, semicarbazones, carbazides, primary amines,
secondary amines, tertiary amines, N-substituted hydrazines,
hydrazides, alcohols, ethers, thiols, thioethers, thioesters,
disulfides, carboxylic acids, esters, amides, ureas, carbamates,
carbonates, ketals, thioketals, acetals, thioacetals, aryl halides,
aryl sulfonates, alkyl halides, alkyl sulfonates, aromatic
compounds, heterocyclic compounds, anilines, alkenes, alkynes,
diols, amino alcohols, oxazolidines, oxazolines, thiazolidines,
thiazolines, enamines, sulfonamides, epoxides, aziridines,
isocyanates, sulfonyl chlorides, diazo compounds and/or acid
chlorides, preferably aldehydes, ketones, primary amines, secondary
amines, alcohols, thioesters, disulfides, carboxylic acids,
acetals, anilines, diols, amino alcohols and/or epoxides, most
preferably aldehydes, ketones, primary amines, secondary amines
and/or disulfides.
[0014] Methods for identifying the organic compound that forms a
covalent bond with the chemically reactive group on the target
molecule may optionally include processes that employ mass
spectrometry, high performance liquid chromatography and/or
fragmenting the target protein/organic compound conjugate into two
or more fragments.
[0015] A particularly preferred embodiment of the present invention
is directed to a method for identifying an organic molecule ligand
that binds to a biological target molecule of interest, wherein the
method comprises:
[0016] (a) obtaining a biological target molecule that comprises or
has been modified to comprise a first reactive functionality,
[0017] (b) reacting the target molecule with a compound that
comprises (1) a second reactive functionality and (2) a chemically
reactive group, wherein the second reactive functionality reacts
with the first reactive functionality of the target molecule to
form a covalent bond, thereby providing a target molecule
comprising the chemically reactive group linked to the target
molecule through a covalent bond;
[0018] (c) combining the target molecule with one or more members
of a library of organic compounds that are capable of covalently
bonding to the chemically reactive group, wherein at least one
member of the library forms a covalent bond with the chemically
reactive group to form a target molecule/organic compound
conjugate; and
[0019] (d) identifying the organic compound that forms a covalent
bond with the chemically reactive group.
[0020] Preferably, the covalent bond formed from reaction of the
first and second reactive functionalities is a disulfide bond
formed between two thiol groups and optionally, subsequent to step
(c) and prior to step (d) one may liberate the covalently-bonded
organic compounds from the conjugate by treatment with an agent
that disrupts the disulfide bond, wherein that agent may comprise,
for example, dithiothreitol, dithioerythritol,
.beta.-mercaptoethanol, sodium borohydride or a phosphine, such as
tris-(2-carboxyethyl)-phosphine (TCEP). In various embodiments, the
biological target molecule is as described above, preferably a
polypeptide that may be obtained, for example, as a recombinant
expression product or synthetically. The thiol group and thiol
functionality may be masked or activated
[0021] In particularly preferred embodiments, the chemically
reactive group is a primary or secondary amine group and the
library of organic compounds comprises aldehydes and/or ketones,
preferably aldehydes, or the chemically reactive group is an
aldehyde or ketone group, preferably an aldehyde, and the library
of organic compounds comprises primary and/or secondary amines,
thereby allowing Schiff base adduct formation between the
chemically reactive group and members of the library. Subsequent to
Schiff base adduct formation but prior to identifying the
covalently-bound organic compound, a reducing agent may optionally
be employed to reduce the imine bond of the Schiff base adduct.
[0022] Yet another embodiment of the present invention is directed
to a method for identifying a ligand that binds to a biological
target molecule of interest, wherein the method comprises:
[0023] (a) identifying a first organic molecule ligand that binds
to the biological target molecule by at least one of the methods
described above;
[0024] (b) identifying a second organic molecule ligand that binds
to the biological target molecule by at least one of the methods
described above; and
[0025] (c) linking the first and second identified organic molecule
ligands through a linker element to form a conjugate molecule that
binds to the target molecule.
[0026] Preferably, the biological target molecule is a polypeptide.
In certain embodiments, the first and second organic molecule
ligands may bind to the same site on the target molecule or to
different sites thereon. The first and second organic molecule
ligands may also be from the same or from different chemical
classes.
[0027] Additional embodiments of the present invention will become
evident to the ordinarily skilled artisan upon review of the
present specification.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The present invention provides a rapid and efficient method
for identifying small organic molecule ligands that are capable of
binding to selected sites on biological target molecules of
interest. The organic molecule ligands themselves identified by the
subject methods find use, for example, as lead compounds for the
development of novel therapeutic drugs, enzyme inhibitors, labeling
compounds, diagnostic reagents, affinity reagents for protein
purification, and the like, or two or more of the identified
organic molecule ligands may be coupled together through one or
more linker elements to provide novel biomolecule-binding conjugate
molecules.
[0029] One embodiment of the subject invention is directed to a
method for identifying an organic molecule ligand that binds to a
site of interest on a biological target molecule. As an initial
step in the herein described method, a biological target molecule
is obtained as a target for binding to the small organic molecule
ligands screened during the process. Biological target molecules
that find use in the present invention include all biological
molecules to which a small organic molecule may bind and preferably
include, for example, polypeptides, nucleic acids, including both
DNA and RNA, carbohydrates, nucleoproteins, glycoproteins,
glycolipids, and the like. The biological target molecules that
find use herein may be obtained in a variety of ways, including but
not limited to commercially, synthetically, recombinantly, from
purification from a natural source of the biological target
molecule, etc.
[0030] In a particularly preferred embodiment, the biological
target molecule is a polypeptide. Polypeptides that find use herein
as targets for binding to organic molecule ligands include
virtually any peptide or protein that comprises two or more amino
acids and which possesses or is capable of being modified to
possess a chemically reactive group for binding to a small organic
molecule. Polypeptides of interest finding use herein may be
obtained commercially, recombinantly, synthetically, by
purification from a natural source, or otherwise and, for the most
part are proteins, particularly proteins associated with a specific
human disease condition, such as cell surface and soluble receptor
proteins, such as lymphocyte cell surface receptors, enzymes, such
as proteases and thymidylate synthetase, steroid receptors, nuclear
proteins, allosteric enzyme inhibitors, clotting factors,
serine/threonine kinases and dephosphorylases, threonine kinases
and dephosphorylases, bacterial enzymes, fungal enzymes and viral
enzymes, signal transduction molecules, transcription factors,
proteins associated with DNA and/or RNA synthesis or degradation,
immunoglobulins, hormones, receptors for various cytokines
including, for example, erythropoietin/EPO, granulocyte colony
stimulating receptor, granulocyte macrophage colony stimulating
receptor, thrombopoietin (TPO), IL-2, IL-3, IL-4, IL-5, IL-6,
IL-10, IL-11, IL-12, growth hormone, prolactin, human placental
lactogen (LPL), CNTF, octostatin, various chemokines and their
receptors such as RANTES, MIP1-.alpha., IL-8, various ligands and
receptors for tyrosine kinase such as insulin, insulin-like growth
factor 1 (IGF-1), epidermal growth factor (EGF), heregulin-.alpha.
and heregulin-.beta., vascular endothelial growth factor (VEGF),
placental growth factor (PLGF), tissue growth factors (TGF-.alpha.
and TGF-.beta.), other hormones and receptors such as bone
morphogenic factors, folical stimulating hormone (FSH), and
leutinizing hormone (LH), tissue necrosis factor (TNF), apoptosis
factor-1 and -2 (AP-1 and AP-2), mdm2, and proteins and receptors
that share 20% or more sequence identity to these.
[0031] The biological target molecule of interest will be chosen
such that it possesses or is modified to possess a chemically
reactive group which is capable of forming a covalent bond with
members of a library of small organic molecules. For example, many
biological target molecules naturally possess chemically reactive
groups (for example, amine groups, thiol groups, aldehyde groups,
ketone groups, alcohol groups and a host of other chemically
reactive groups; see below) to which members of an organic molecule
library may interact and covalently bond. In this regard, it is
noted that polypeptides often have amino acids with chemically
reactive side chains (e.g., cysteine, lysine, arginine, and the
like). Additionally, synthetic technology presently allows the
synthesis of biological target molecules using, for example,
automated peptide or nucleic acid synthesizers, which possess
chemically reactive groups at predetermined sites of interest. As
such, a chemically reactive group may be synthetically introduced
into the biological target molecule during automated synthesis.
[0032] Moreover, techniques well known in the art are available for
modifying biological target molecules such that they possess a
chemically reactive group at a site of interest which is capable of
forming a covalent bond with a small organic molecule. In this
regard, different biological molecules may be chemically modified
(using a variety of commercially or otherwise available chemical
reagents) or otherwise coupled, either covalently or
non-covalently, to a compound that comprises both a group capable
of linking to a site on the target molecule and a chemically
reactive group such that the modified biological target molecule
now possesses an available chemically reactive group at a site of
interest. With regard to the latter, techniques for linking a
compound comprising a chemically reactive group to a target
biomolecule are well known in the art and may be routinely employed
herein to obtain a modified biological target molecule which
comprises a chemically reactive group at a site of interest.
[0033] In one particular embodiment of the present invention, a
target molecule comprises at least a first reactive group which, if
the target is a polypeptide, may or may not be associated with a
cysteine residue of that polypeptide, preferably is associated with
a cysteine residue of the polypeptide of interest. Preferably, the
polypeptide of interest when initially obtained or subsequently
modified comprises only a limited number of free thiol groups which
may potentially serve as covalent binding sites for a compound
comprising a thiol functionality, where in certain embodiments the
polypeptide of interest comprises or has been modified to comprise
no more than about 5 free thiol groups, more preferably no more
than about 2 free thiol groups, most preferably no more than one
free thiol group, although polypeptides of interest having more
free thiol groups will also find use. The polypeptide of interest
may be initially obtained or selected such that it already
possesses the desired number of free thiol groups or may be
modified to possess the desired number of free thiol groups. With
regard to the latter, "modified to possess" means that the
initially selected polypeptide of interest has been recombinantly,
chemically, or otherwise altered such that it possesses a different
number of free thiol groups than when initially obtained.
[0034] Those skilled in the art are well aware of various
recombinant, chemical, synthetic, or other techniques that can
routinely be employed to modify a polypeptide of interest such that
it possess a different number of free thiol groups that are
available for covalent bonding to a subsequently-added compound
comprising a free thiol group. Such techniques include, for
example, site-directed mutagenesis, where a nucleic acid molecule
encoding the polypeptide of interest may be altered such that it
encodes a polypeptide with a different number of cysteine residues
(see, e.g., Gloss et al., Biochemistry 31:32-39 (1992)).
Site-directed (site-specific) mutagenesis allows the production of
variants of an initially obtained polypeptide of interest through
the use of specific oligonucleotide sequences that encode the DNA
sequence of the desired mutation, as well as a sufficient number of
adjacent nucleotides, to provide a primer sequence of sufficient
size and sequence complexity to form a stable duplex on both sides
of the deletion junction being traversed. Typically, a primer of
about 20 to 25 nucleotides in length is preferred, with about 5 to
10 residues on both sides of the junction of the sequence being
altered. In general, the techniques of site-directed mutagenesis
are well known in the art, as exemplified by publications such as
Edelman et al., DNA 2:183 (1983). As will be appreciated, the
site-directed mutagenesis technique typically employs a phage
vector that exists in both a single-stranded and double-stranded
form. Typical vectors useful in site-directed mutagenesis include
vectors such as the M13 phage, for example, as disclosed by Messing
et al., Third Cleveland Symposium on Macromolecules and Recombinant
DNA, A. Walton, ed., Elsevier, Amsterdam (1981). This and other
phage vectors are commercially available and their use is well
known to those skilled in the art. A versatile and efficient
procedure for the construction of oligodeoxyribonucleotide directed
site-specific mutations in DNA fragments using M13-derived vectors
was published by Zoller et al., Nucleic Acids Res. 10:6487-6500
(1982)). Also, plasmid vectors that contain a single-stranded phage
origin of replication (Veira et al., Meth. EnzymoL 153:3 (1987))
may be employed to obtain single-stranded DNA. Alternatively,
nucleotide substitutions are introduced by synthesizing the
appropriate DNA fragment in vitro, and amplifying it by PCR
procedures known in the art.
[0035] The PCR technique may also be used in modifying a
polypeptide of interest such that it contains a different number of
cysteine residues than when initially selected. In a specific
non-limiting example of PCR mutagenesis, template plasmid DNA
encoding the polypeptide of interest (1 .mu.g) is linearized by
digestion with a restriction endonuclease that has a unique
recognition site in the plasmid DNA outside of the region to be
amplified. Of this material, 100 ng is added to a PCR mixture
containing PCR buffer, which contains the four deoxynucleotide
triphosphates and is included in the GENEAMP.sup.R kits (obtained
from Perkin-Elmer Cetus, Norwalk, Conn. and Emeryville, Calif.),
and 25 .rho.mole of each oligonucleotide primer, to a final volume
of 50 .mu.l. The reaction mixture is overlayered with 35 .mu.l
mineral oil. The reaction is denatured for 5 minutes at 100.degree.
C., placed briefly on ice, and then 1 .mu.l Thermus aquaticus (Tag)
DNA polymerase (5 units/.mu.l), purchased from Perkin-Elmer Cetus,
Norwalk, Conn. and Emeryville, Calif.) is added below the mineral
oil layer. The reaction mixture is then inserted into a DNA Thermal
Cycler (purchased from Perkin-Elmer Cetus) which may be programmed
as follows: [0036] 2 min. 55.degree. C., [0037] 30 sec. 72.degree.
C., then 19 cycles of the following: [0038] 30 sec. 94.degree. C.,
[0039] 30 sec. 55.degree. C., and [0040] 30 sec. 72.degree. C.
[0041] At the end of the program, the reaction vial is removed from
the thermal cycler and the aqueous phase transferred to a new vial,
extracted with phenol/chloroform (50:50 vol), and ethanol
precipitated, and the DNA is recovered by standard procedures. This
material is subsequently subjected to appropriate treatments for
insertion into a vector and expression of the encoded modified
polypeptide.
[0042] Other methods for modifying a polypeptide of interest so
that it contains a different number of cysteine residues that when
originally selected include cassette mutagenesis which is based on
the technique described by Wells et al., Gene 34:315 (1985) and
phagemid display, for example, as described in U.S. Pat. No.
4,946,778.
[0043] Further details of the foregoing and similar mutagenesis
techniques are found in general textbooks, such as, for example,
Sambrook et al., Molecular Cloning: A Laboratory Manual (New York:
Cold Spring Harbor Laboratory Press, 1989) and Ausubel et al.,
Current Protocols in Molecular Biology, Greene Publishing
Associates and Wiley-lnterscience 1991.
[0044] In the particular embodiment which employs a biological
target molecule comprising a first reactive functionality, one may
directly screen a library of organic molecules that are capable of
forming a covalent bond with that first reactive functionality or
may covalently bond a compound to that first reactive functionality
which comprises the chemically reactive group of interest. With
regard to the latter, the target molecule comprising the first
reactive functionality may be reacted with a compound that
comprises (1) a second reactive functionality and (2) a chemically
reactive group, wherein that compound becomes covalently bound to
the polypeptide of interest. Specifically, the second reactive
functionality of the compound reacts with the first reactive
functionality of the target of interest to form a covalent bond,
thereby providing a modified target of interest. Preferably, the
first and second reactive functionalities are thiol groups,
preferably activated thiol groups, that react to form a covalent
bond. The target of interest is "modified" in that it now has
covalently bound thereto through a covalent bond the compound that
comprises the chemically reactive group. Reaction conditions useful
for covalently bonding the compound to the target of interest
through a covalent bond are known to those skilled in the art and
may employ activating groups such as thiopyridine,
thionitrobenzoate, and the like.
[0045] The compound that comprises the chemically reactive group
may also be covalently bound to the target biomolecule through a
covalent bond other than a disulfide bond as described above. Those
of skill in the art will be capable of covalently linking a
chemically reactive group-containing compound to a target
biomolecule through virtually any type of covalent bond, including
the disulfide bond as described above. In this regard, the first
and second reactive functionalities may be any chemically reactive
functionalities that are capable of reacting to form a covalent
bond. The reaction between the first and second reactive
functionalities to form a covalent bond may be the same or
different than the reaction between the chemically reactive group
and library member to form a covalent bond (see below).
[0046] For the most part, the compound that bonds to the target
biomolecule of interest through a covalent, preferably disulfide
bond will be relatively small, preferably comprising less than
about 20, more preferably less than about 10, most preferably less
than about 5 carbon atoms, although compounds with more carbon
atoms may also find use herein. Such compounds will also possess a
thiol functionality capable of forming a covalent bond with the
free thiol group of the biological target molecule and may also
possess other heteroatoms at certain sites within the compound. A
particularly preferred compound for use in this embodiment of the
invention is thioethylamine or a derivative thereof, such as
2-amino ethanethiol, which is capable of forming a disulfide bond
with the free thiol group of the biological target molecule as well
as providing a chemically reactive amine group for bonding to
members of a library of organic molecules.
[0047] The "chemically reactive group" that is either naturally or
otherwise possessed by the biological target molecule or becomes
part of the target molecule after modification thereof as described
above may be any of a number of different chemically reactive
groups and is chosen so as to be compatible with the library of
organic molecule compounds that will subsequently be screened for
bonding at that site. Specifically, the chemically reactive group
provides a site at which covalent bond formation between the
chemically reactive group and a member of the library of organic
compounds may occur. Thus, the chemically reactive group will be
chosen such that it is capable of forming a covalent bond with
members of the organic molecule library against which it is
subsequently screened. In certain specific embodiments, the
chemically reactive group is either a primary or secondary amine
group and the library of organic compounds comprises aldehydes
and/or ketones, wherein the chemically reactive group and the
library members are capable of forming covalent bonds. In another
specific embodiment, the chemically reactive group is either an
aldehyde or ketone group and the library of organic compounds
comprises primary and/or secondary amines, wherein the chemically
reactive group and the library members are capable of forming
covalent bonds. Using the techniques described above, chemically
reactive groups may be introduced into specific predetermined sites
on the biological target molecule or may be introduced
randomly.
[0048] Once a biological target molecule that comprises a
chemically reactive group of interest is obtained, the biological
target molecule is then used to screen a library of organic
compounds to identify those organic compounds that form a covalent
bond with the chemically reactive group. It is expected that those
members of the library of organic compounds that have the greatest
relative affinity for the site on the polypeptide being assayed
will be those that covalently bond to the chemically reactive
functionality most abundantly. For example, it has been
demonstrated that allosteric effects in a polypeptide can function
to determine the reactivity of an organic compound for a reactive
site on the polypeptide (see, e.g., Abraham et al., Biochemistry
34:15006-15020 (1995)). Thus, it is expected that by screening
mixtures of two or more organic compounds against a chemically
reactive group at a site of interest on a target biomolecule, those
organic compounds having the highest non-covalent affinity for the
site of interest will be capable of most efficiently forming
covalent bonds with the chemically reactive group at that site. In
this manner, one can determine which library members have the
highest relative binding affinity for the site being tested,
wherein that binding affinity is directly related to the ability of
those compounds to form covalent bonds with the chemically reactive
group at the site of interest.
[0049] As described above, the library of organic molecules and the
chemically reactive group are chosen to be "compatible", i.e.,
chosen such that they are capable of reacting with one another to
form a covalent bond. The library of organic compounds to be
screened against the modified polypeptide of interest may be
obtained in a variety of ways including, for example, through
commercial and non-commercial sources, by synthesizing such
compounds using standard chemical synthesis technology or
combinatorial synthesis technology (see Gallop et al., J. Med.
Chem. 37:1233-1251 (1994), Gordon et al., J. Med. Chem.
37:1385-1401 (1994), Czarnik and Ellman, Acc. Chem. Res. 29:112-170
(1996), Thompson and Ellman, Chem Rev. 96:555-600 (1996), and
Balkenhohl et al., Angew. Chem. Int. Ed. 35:2288-2337 (1996)) and
by obtaining such compounds as degradation products from larger
precursor compounds, e.g. known therapeutic drugs, large chemical
molecules, and the like. Often the covalent interaction between the
chemically reactive group and the library member will be
exchangeable, thereby allowing one to identify small molecules that
bind in the presence of those that do not. Also, exchangeable
covalent bonds will be capable of being made non-exchangeable,
thereby "trapping" the small organic ligand that is covalently
bound to the target.
[0050] The "organic compounds" employed in the methods of the
present invention will be, for the most part, small chemical
molecules that will generally be less than about 2000 daltons in
size, usually less than about 1500 daltons in size, more usually
less than about 750 daltons in size, preferably less than about 500
daltons in size, often less than about 250 daltons in size and more
often less than about 200 daltons in size, although organic
molecules larger than 2000 daltons in size will also find use
herein. Organic molecules that find use may be employed in the
herein described method as originally obtained from a commercial or
non-commercial source (for example, a large number of small organic
chemical compounds are readily obtainable from commercial suppliers
such as Aldrich Chemical Co., Milwaukee, Wis. and Sigma Chemical
Co., St. Louis, Mo.) or may be obtained by chemical synthesis.
[0051] Organic molecule compounds that find use in the present
invention include, for example, aldehydes, ketones, oximes, such as
O-alkyl oximes, preferably O-methyl oximes, hydrazones,
semicarbazones, carbazides, primary amines, secondary amines, such
as N-methylamines, tertiary amines, such as N,N-dimethylamines,
N-substituted hydrazines, hydrazides, alcohols, ethers, thiols,
thioethers, thioesters, disulfides, carboxylic acids, esters,
amides, ureas, carbamates, carbonates, ketals, thioketals, acetals,
thioacetals, aryl halides, aryl sulfonates, alkyl halides, alkyl
sulfonates, aromatic compounds, heterocyclic compounds, anilines,
alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines,
thiazolidines, thiazolines, enamines, sulfonamides, epoxides,
aziridines, isocyanates, sulfonyl chlorides, diazo compounds, acid
chlorides, and the like, all of which have counterpart chemically
reactive groups that allow covalent bond formation with the
modified polypeptide of interest. In fact, virtually any small
organic molecule that is capable of covalently bonding to a known
chemically reactive functionality may find use in the present
invention with the proviso that it is sufficiently soluble and
stable in aqueous solutions to be tested for its ability to bind to
the biological target molecule.
[0052] Various chemistries may be employed for forming a covalent
bond between the chemically reactive group and a member of the
organic molecule library including, for example, reductive
aminations between aldehydes and ketones and amines (March,
Advanced Organic Chemistry, John Wiley & Sons, New York, 4th
edition, 1992, pp. 898-900), alternative methods for preparing
amines (March et al., supra, p. 1276), reactions between aldehydes
and ketones and hydrazine derivatives to give hydrazones and
hydrazone derivatives such as semicarbazones (March et al., supra,
pp. 904-906), amide bond formation (March et al., supra, p. 1275),
formation of ureas (March et al., supra, p. 1299), formation of
thiocarbamates (March et al., supra, p. 892), formation of
carbamates (March et al., supra, p. 1280), formation of
sulfonamides (March et al., supra, p. 1296), formation of
thioethers (March et al., supra, p. 1297), formation of disulfides
(March et al., supra, p. 1284), formation of ethers (March et al.,
supra, p. 1285), formation of esters (March et al., supra, p.
1281), additions to epoxides (March et al., supra, p. 368),
additions to aziridines (March et al., supra, p. 368), formation of
acetals and ketals (March et al., supra, p. 1269), formation of
carbonates (March et al., supra, p. 392), formation of enamines
(March et al., supra, p. 1284), metathesis of alkenes (March et
al., supra, pp. 1146-1148 and Grubbs et al., Acc. Chem. Res.
28:446-452 (1995)), transition metal-catalyzed couplings of aryl
halides and sulfonates with alkenes and acetylenes (e.g., Heck
reactions) (March et al., supra, pp. 717-178), the reaction of aryl
halides and sulfonates with organometallic reagents (March et al.,
supra, p. 662), such as organoboron (Miyaura et al., Chem. Rev.,
95:2457 (1995)), organotin, and organozinc reagents, formation of
oxazolidines (Ede et al., Tetrahedron Letts. 38:7119-7122 (1997)),
formation of thiazolidines (Patek et al., Tetrahedron Letts.
36:2227-2230 (1995)), amines linked through amidine groups by
coupling amines through imidoesters (Davies et al., Canadian J.
Biochem. 50:416-422 (1972)), and the like.
[0053] Libraries of organic compounds which find use herein will
generally comprise at least 2 organic compounds, often at least
about 25 different organic compounds, more often at least about 100
different organic compounds, usually at least about 300 different
organic compounds, more usually at least about 500 different
organic compounds, preferably at least about 1000 different organic
compounds, more preferably least about 2500 different organic
compounds and most preferably at least about 5000 or more different
organic compounds. Populations may be selected or constructed such
that each individual molecule of the population may be spatially
separated from the other molecules of the population (e.g., each
member of the library is a separate microtiter well) or two or more
members of the population may be combined if methods for
deconvolution are readily available. The methods by which the
populations of organic compounds are prepared will not be critical
to the invention. Usually, each member of the organic molecule
library will be of the same chemical class (i.e., all library
members are aldehydes, all library members are primary amines,
etc.), however, libraries of organic compounds may also contain
molecules from two or more different chemical classes.
[0054] Reaction conditions for screening a library of organic
compounds against a chemically reactive group-containing biological
target molecule will be dependent upon the nature of the chemically
reactive group and the chemical nature of the chosen library of
organic compounds and can be determined by the skilled artisan in
an empirical manner. For the step of screening a population of
organic molecules to identify those that bind to a target
polypeptide, it will be well within the skill level in the art to
determine the concentration of the organic molecules to be employed
in the binding assay. For the most part, the screening assays will
employ concentrations of organic molecules ranging from about 0.1
.mu.M to 50 mM, preferably from about 0.01 to 10 mM, although
concentrations outside those ranges may also find use herein.
[0055] In a particularly preferred embodiment, the chemically
reactive group that is linked to the biological target molecule and
the library of organic molecules to be screened against the target
molecule are chosen such that they are capable of reacting to form
a Schiff base adduct. A Schiff base adduct is formed from the
condensation of aldehydes or ketones with primary or secondary
amines. Thus, in one embodiment of the present invention, the
chemically reactive group is a primary or secondary amine group and
the library of organic compounds against which the target molecule
is screened comprises aldehyde and/or ketone compounds. In another
preferred embodiment, the chemically reactive group is either an
aldehyde or ketone group and the library of organic compounds
against which the biological target molecule is screened comprises
primary and/or secondary amines. Once a reversible Schiff base
adduct is formed between the aldehyde or ketone group and the
primary or secondary amine (an interaction that is relatively
unstable and reversible), the imine bond created may optionally be
reduced (i.e., made irreversible) by the addition of a reducing
agent so as to stabilize the covalently bonded product of the
reaction. Such allows one to identify small organic molecule
ligands that bind to the target protein in the presence of those
that do not. Reducing agents that find use for such purposes
include, for example, sodium cyanoborohydride, sodium
triacetoxyborohydride, cyanide, and the like, i.e., agents that
would not be expected to disrupt any disulfide bonds present on the
target biomolecule (see, e.g., Geoghegan et al.,J. Peptide and
Protein Res. 17(3):345-352 (1981)).
[0056] Combining the biological target molecule of interest with
one or more members of a library of organic compounds will result
in the formation of a covalent bond between the chemically reactive
group present on the target molecule and a member of the organic
compound library. Once such a covalent bond is formed, one may
identify the organic compound that bound in a number of ways. For
example, in the case where the chemically reactive group was linked
to the target biomolecule through a disulfide bond, one may
liberate the organic compound from the target molecule by treatment
of the covalently bound complex with an agent that disrupts the
disulfide bond that was formed between the free thiol group of the
target molecule of interest and the compound that comprises (1) a
thiol functionality and (2) the chemically reactive group. For the
most part, agents capable of disrupting the disulfide bond through
which the covalently bound organic compound is linked to the target
molecule of interest will be reducing agents such as, for example,
dithiothreitol, dithioerythritol, .beta.-mercaptoethanol,
phosphines, sodium borohydride, and the like, preferably
thiol-group containing reducing agents.
[0057] Once an organic compound that covalently bound to the
chemically reactive group of the target molecule has been liberated
from the complex by treatment with an agent that disrupts the
disulfide bond through which the organic compound is linked, the
identity of the actual organic compound that bound to the target
molecule of interest is determined by a variety of means. For
example, the well known technique of mass spectrometry may
preferably be employed either alone or in combination with other
means for detection for identifying the organic compound ligand
that bound to the target of interest. Techniques employing mass
spectrometry are well known in the art and have been employed for a
variety of applications (see, e.g., Fitzgerald and Siuzdak,
Chemistry & Biology 3:707-715 (1996), Chu et al., J. Am. Chem.
Soc. 118:7827-7835 (1996), Siuzdak, Proc. Natl. Acad. Sci USA
91:11290-11297 (1994), Burlingame et al., Anal. Chem. 68:599R-651R
(1996), Wu et al., Chemistry & Biology 4:653-657 (1997) and Loo
et al., Am. Reports Med. Chem. 31:319-325 (1996)).
[0058] In other embodiments, subsequent to the covalent bonding of
the library member to the chemically reactive group of the target
molecule, the target molecule/organic compound conjugate may be
directly subjected to mass spectrometry or may be fragmented and
the fragments then subjected to mass spectrometry for
identification of the organic compound that bound to the target
molecule. The success of mass spectrometry analysis of the intact
target protein/organic compound conjugate or fragments thereof will
depend upon the nature of the target molecule and can be determined
on an empirical basis.
[0059] In addition to the use of mass spectrometry, one may employ
a variety of other techniques to identify the organic compound that
covalently bound to the biological target molecule of interest. For
example, one may employ various chromatographic techniques such as
liquid chromatography, thin layer chromatography, and the like, for
separation of the components of the reaction mixture so as to
enhance the ability to identify the covalently bound organic
molecule. Such chromatographic techniques may be employed in
combination with mass spectrometry or separate from mass
spectrometry. One may optionally couple a labeled probe
(fluorescently, radioactively, or otherwise) to the liberated
organic compound so as to facilitate its identification using any
of the above techniques. Other techniques that may find use for
identifying the organic compound that bound to the target
biomolecule include, for example, nuclear magnetic resonance (NMR),
capillary electrophoresis, X-ray crystallography, and the like, all
of which will be well known by those skilled in the art.
[0060] Another embodiment of the present invention is directed to a
method for identifying a ligand that binds to a biological target
molecule of interest, wherein the method comprises employing the
above described methods to identify two or more organic molecule
ligands that bind to the target of interest and linking those two
or more organic molecule ligands through a linker element to form a
conjugate molecule that also binds to the target of interest. For
the most part, the conjugate molecule that is comprised of two or
more individual organic molecule ligands for the target molecule
will bind to the target of interest with a lower dissociation
constant than any of the individual components, although such is
not a requirement of the invention. The individual organic molecule
components of a conjugate molecule may bind to the same site or
different sites on the target of interest and may be from the same
or different chemical classes. By "same chemical class" is meant
that each component of the conjugate is of the same chemical type,
i.e., each are aldehydes, each are amines, etc.
[0061] Linker elements that find use for linking two or more
organic molecule ligands to produce a conjugate molecule will be
multifunctional, preferably bifunctional, cross-linking molecules
that can function to covalently bond at least two organic molecules
together via reactive functionalities possessed by those molecules.
Linker elements will have at least two, and preferably only two,
reactive functionalities that are available for bonding to at least
two organic molecules, wherein those functionalities may appear
anywhere on the linker, preferably at each end of the linker and
wherein those functionalities may be the same or different
depending upon whether the organic molecules to be linked have the
same or different reactive functionalities. Linker elements that
find use herein may be straight-chain, branched, aromatic, and the
like, preferably straight chain, and will generally be at least
about 2 atoms in length, more generally more than about 4 atoms in
length, and often as many as about 12 or more atoms in length.
Linker elements will generally comprise carbon atoms, either
hydrogen saturated or unsaturated, and therefore, may comprise
alkanes, alkenes or alkynes, and/or other heteroatoms including
nitrogen, sulfur, oxygen, and the like, which may be unsubstituted
or substituted, preferably with alkyl, alkoxyl, hydroxyalkyl or
hydroxy groups. Linker elements that find use will be a varying
lengths, thereby providing a means for optimizing the binding
properties of a conjugate ligand compound prepared therefrom.
[0062] In yet other embodiments of the present invention, one may
obtain a target molecule/organic molecule conjugate as described
above and then "build off" of the first organic compound that
covalently bound to the chemically reactive group of the target
molecule. For example, the first organic compound that covalently
bound to the target biomolecule may itself provide a chemically
reactive group to which a second organic compound may covalently
bond. As such, a target biomolecule/organic compound conjugate may
be screened against a library of organic compound to identify a
second organic compound capable of covalently bonding to a
chemically reactive group on the first organic molecule. This
process may be repeated in an iterative process to obtain
progressively higher affinity organic molecules for binding to the
target molecule. As described above, the first organic compound may
itself possess a chemically reactive group that provides a site for
bonding to a second organic molecule or, in the alternative, the
first organic molecule may be modified (either chemically, by
binding a compound comprising a chemically reactive group thereto,
or otherwise) prior to screening against a second library of
organic compounds.
[0063] Further details of the invention are illustrated in the
following non-limiting examples.
EXPERIMENTAL
[0064] A plasmid containing the thymydilate synthase gene derived
from E. coli will be mutated such that the five normally occurring
cysteine residues are converted to serine residues using
site-directed mutagenesis. At the same time, a single cysteine
residue will be engineered into the enzyme active site. In one
case, this could be the normally occurring catalytic cysteine
(C146). In another case, this cysteine residue might take the place
of an arginine residue (such as R127) which has been shown not to
significantly affect the activity of the enzyme when it is mutated
(Carreras and Santi, Annu. Rev. Biochem. 64:721-762 (1995)). One
can make any number of different mutant proteins containing a
single cysteine residue in various locations in and around the
active site of the enzyme. These mutant proteins will be
overexpressed and purified as previously described (Maley and
Maley, J. Biol. Chem. 263:7620-7627 (1988)). Generally, the enzyme
will be tested for substrate binding and, in the case of the C146
mutant, activity, to ensure that the mutations do not significantly
perturb the structure of the protein. In all cases, the protein
could be subjected to one or more of the following three
treatments.
[0065] In the first case, the mutant protein will be reacted with
one molar equivalent of a cysteamine/thionitrobenzoic acid mixed
disulfide. This reagent would be prepared by reacting cysteamine
(otherwise known as 2-aminoethanethiol) with a thiol activating
agent such as 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) and
purifying the product using the standard techniques of organic
chemistry. The protein would react with the reagent to form a new
mixed disulfide in which the cysteine group on the protein is
attached to the cysteamine moiety through a disulfide bond. The
free primary amine group of the cysteamine would then be free to
react with aldehydes.
[0066] In a typical experiment, individual libraries each
consisting of a set of ten different aldehydes chosen to be of
similar reactivity and structure will be mixed with the
cysteamine-modified protein in aqueous buffered solution. Initial
experiments will dictate the concentration of aldehydes used; at
first, a wide range of different concentrations will be tested.
During this time the aldehyde functionality of individual library
members will react with the primary amine group of the
protein-bound-cysteamine to yield an imine. Because this reaction
is reversible, equilibrium will favor imine formation with the
library member that had the highest intrinsic affinity for the
active site of the protein. After allowing the libraries of
aldehydes to react with the protein for varying lengths of time,
the solution will be treated with sodium cyanoborohydride to reduce
the imines to secondary amines. The protein-cysteamine-compound
complex will then be purified away from the unreacted members of
the library by using dialysis, chromatography, precipitation, or
other methods. Next, the protein will be treated with a
disulfide-reducing agent such as dithiothreitol (DTT) or
tris-(2-carboxyethyl)-phosphine (TCEP), thereby cleaving the
disulfide bond and releasing the captured library member(s) from
the proteins These will then be analyzed directly using
mass-spectrometry (MS), or they will first be conjugated to a
fluorescent dye (such as fluorescein by reaction with
fluorescein-maleimide) through their thiol moieties and then
analyzed by a combination of chromatography (HPLC or CE) and MS.
The later method will allow quantitation of the released library
members, and will facilitate analysis if more than a single library
member bound to the cysteamine-portion of the protein. It should be
noted that the initial library can contain more or fewer than ten
compounds; the ideal number being determined empirically, and will
probably vary with different combinations of mutants and
libraries.
[0067] A second methodology will involve reacting the
single-cysteine-containing mutant protein with a
thioglycerol/thionitrobenzoic acid mixed disulfide, which will be
synthesized analogously to the cysteamine/thionitrobenzoic acid
mixed disulfide described above. Once the thioglycerol is attached
to the protein through a disulfide bond, the modified protein will
be treated with a 15 mM sodium periodate solution for 15 minutes at
room temperature (Acharya and Manjula, Biochemistry 26:3524-3530
(1987)) so as to oxidize the glycol portion to an aldehyde. This
aldehyde-containing protein will then be reacted with libraries
consisting of pools of primary or secondary amines, and the rest of
the procedure would be as described above.
[0068] A variation on this second methodology will involve using
specially constructed libraries of amines that also contained the
glycol functionality. After reacting these libraries with the
protein and reducing the resulting imines to secondary amines, the
proteins will be treated a second time with sodium periodate to
oxidize the newly introduced glycol to an aldehyde. The
protein-compound-aldehyde will then be reacted with a second
amine-containing library and subsequently reduced with sodium
cyanoborohydride. In principle, this process could be repeated
several times so as to, actually build an organic molecule within
the active site of the protein. This is similar to the method of
Huc and Lehn (Huc and Lehn, Proc. Natl. Acad. Sci. USA 94:2106-2110
(1997)), but with the significant advantage that the molecule is
built selectively into a specified site of interest. Another
advantage is that it is a linear, stepwise process, where we have
control over each individual step.
[0069] Another variation on this second methodology is made
possible by the fact that after reduction of the imine a secondary
amine is formed, and this can in principle be reacted with a
library of aldehydes. In practice, primary amine libraries will be
screened against the original aldehyde-containing protein target,
and the amine that binds most tightly will be identified. This
amine alone will then be conjugated to the aldehyde-containing
protein and reduced to form a secondary amine. In other words, a
new target protein will be prepared, consisting of the original
target protein coupled to the amine selected from the first library
and containing a secondary amine. This new target protein will then
be reacted with a library of aldehydes, and the aldehyde that binds
most tightly will be identified. There are several advantages to
this methodology. First, as described in the preceding paragraph,
it is a stepwise approach, where each step can be optimized for
speed and accuracy. Second, two separate libraries are screened so
as to maximize the diversity with a minimum degree of effort. For
example, if the amine library and the aldehyde library each contain
a mere 1000 members, then although there are one million possible
combinations, in practice only 1000 of these need to be sampled in
order to identify the tightest-binder (i.e., the single
tightest-binding amine pre-bound to the protein and screened
against the library of 1000 aldehydes). Finally, this variation
requires only simple primary amines and aldehydes or ketones, of
which a large number are readily available. It should be noted that
a similar approach can be used for the first (cysteamine-based)
methodology, as that method also has the potential to generate a
secondary amine.
[0070] A third methodology will involve reacting the
single-cysteine-containing mutant proteins with libraries of
disulfides. Because disulfide formation, like imine formation, is
reversible, the process should be equilibrium-driven, such that
library members that have the highest inherent affinity for the
active site will tend to form disulfide bonds with the protein most
often. The thiol-disulfide exchange will be further promoted by
adding various concentrations of reduced and oxidized
2-mercaptoethanol so as to fine tune the reactivity. The protein
will be purified away from the unbound library members and analyzed
as described in the first method.
[0071] The foregoing description details specific methods which can
be employed to practice the present invention. Having detailed such
specific methods, those skilled in the art will well enough know
how to devise alternative reliable methods at arriving at the same
information in using the fruits of the present invention. Thus,
however, detailed the foregoing may appear in text, it should not
be construed as limiting the overall scope thereof; rather, the
ambit of the present invention is to be determined only by the
lawful construction of the appended claims. All documents cited
herein are expressly incorporated by reference.
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