U.S. patent application number 11/107658 was filed with the patent office on 2005-08-25 for extended tethering approach for rapid identification of ligands.
Invention is credited to Braisted, Andrew C., Erlanson, Daniel A., McDowell, Robert, Prescott, John.
Application Number | 20050186630 11/107658 |
Document ID | / |
Family ID | 46204328 |
Filed Date | 2005-08-25 |
United States Patent
Application |
20050186630 |
Kind Code |
A1 |
Erlanson, Daniel A. ; et
al. |
August 25, 2005 |
Extended tethering approach for rapid identification of ligands
Abstract
The invention concerns a method for rapid identification and
characterization of binding partners for a target molecule, and for
providing binding partners with improved binding affinity. More
specifically, the invention concerns an improved tethering method
for the rapid identification of at least two binding partners that
bind near one another to a target molecule.
Inventors: |
Erlanson, Daniel A.; (San
Francisco, CA) ; Braisted, Andrew C.; (San Francisco,
CA) ; McDowell, Robert; (San Francisco, CA) ;
Prescott, John; (San Francisco, CA) |
Correspondence
Address: |
HELLER EHRMAN LLP
275 MIDDLEFIELD ROAD
MENLO PARK
CA
94025-3506
US
|
Family ID: |
46204328 |
Appl. No.: |
11/107658 |
Filed: |
April 15, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11107658 |
Apr 15, 2005 |
|
|
|
09990421 |
Nov 21, 2001 |
|
|
|
60252294 |
Nov 21, 2000 |
|
|
|
60310725 |
Aug 7, 2001 |
|
|
|
Current U.S.
Class: |
435/6.11 ;
435/7.1 |
Current CPC
Class: |
G01N 2500/04 20130101;
C07D 333/70 20130101; C07D 333/38 20130101; G01N 33/6845 20130101;
C40B 30/04 20130101; C07B 2200/11 20130101; C07D 405/12 20130101;
C07D 401/04 20130101 |
Class at
Publication: |
435/006 ;
435/007.1 |
International
Class: |
C12Q 001/68; G01N
033/53 |
Claims
1-74. (canceled)
75. A method of synthesizing a non-peptide small molecule
comprising reacting a first ligand with a second ligand, wherein
(a) the first ligand has inherent binding affinity for a first site
of interest on a Target Biological Molecule (TBM) having a reactive
nucleophile; and (b) the second ligand has been identified from
among a library of ligand candidates to have inherent binding
affinity for a second site of interest on said TBM by screening
said library with a complex formed between a reactive derivative of
said first ligand and the nucleophile of said TBM resulting in the
formation of an irreversible covalent bond between said complex and
said second ligand:
76. The method of claim 75 wherein said first ligand has been
identified from among a library of ligand candidates by screening
said library with said TBM under conditions resulting in the
formation of a reversible covalent bond between said nuclephile and
said first ligand
77. The method of claim 75 wherein said nucleophile is selected
from the group consisting of thiol, protected thiol, reversible
disulfide, hydroxyl, protected hydroxyl, amino, protected amino,
carboxyl and protected carboxyl groups.
78. The method of claim 75 wherein said first and second ligands
are covalently linked to one another through a disulfide bond.
79. The method of claim 75 wherein said first and second ligands
are directly fused to one another.
80. The method of claim 75 wherein said small molecule consists
essentially of said first and second ligands, covalently linked
through a disulfide bond.
81. The method of claim 78 further comprising the step of
synthesizing derivatives of said small molecule.
82. The method of claim 81 wherein the disulfide bond covalently
linking said first and second ligands to one another is replaced by
a different covalent linkage.
83. The method of claim 82 wherein the different linkage is a
straight chain, branched chain or aromatic linker.
84. The method of claim 83 wherein the different linkage is a
straight chain linker of at least two atoms in length.
85. The method of claim 83 wherein the different linkage is a
straight chain linker of at least four atoms in length.
86. The method of claim 83 wherein the different linkage is a
straight chain linker of at least 12 atoms in length.
Description
[0001] This application is a continuation-in-part and claims
priority under 35 U.S.C. .sctn. 1.19(e) of U.S. Provisional
Application No. 60/252,294 filed on Nov. 21, 2000 and U.S.
Provisional Application No. 60/310,725 filed on Aug. 7, 2001.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to a method for
rapid identification and characterization of binding partners for a
target molecule, and for providing modified binding partners with
improved binding affinity. More specifically, the invention
concerns an improved tethering method for the rapid identification
of small molecule fragments that bind near one another on a target
molecule. The method is particularly suitable for rapid
identification of small molecule ligands that bind weakly near
sites of interest through a preformed linker on a target biological
molecule (TBM), such as a polypeptide or other macromolecule, to
produce higher affinity compounds.
[0004] 2. Description of the Related Art
[0005] The drug discovery process usually begins with massive
screening of compound libraries (typically hundreds of thousands of
members) to identify modest affinity leads (K.sub.d .about.1 to 10
.mu.M). Although some targets are well suited for this screening
process, most are problematic because moderate affinity leads are
difficult to obtain. Identifying and subsequently optimizing weaker
binding compounds would improve the success rate, but screening at
high concentrations is generally impractical because of compound
insolubility and assay artifacts. Moreover, the typical screening
process does not target specific sites for drug design, only those
sites for which a high-throughput assay is available. Finally, many
traditional screening methods rely on inhibition assays that are
often subject to artifacts caused by reactive chemical species or
denaturants.
[0006] Erlanson et al., Proc. Nat. Acad Sci. USA 97:9367-9372
(2000), have recently reported a new strategy, called "tethering",
to rapidly and reliably identify small (.about.250 Da) soluble drug
fragments that bind with low affinity to a specifically targeted
site on a protein or other macromolecule, using an intermediary
disulfide "tether." According to this approach, a library of
disulfide-containing molecules is allowed to react with a
cysteine-containing target protein under partially reducing
conditions that promote rapid thiol exchange. If a molecule has
even weak affinity for the target protein, the disulfide bond
("tether") linking the molecule to the target protein will be
entropically stabilized. The disulfide-tethered fragments can then
be identified by a variety of methods, including mass spectrometry
(MS), and their affinity improved by traditional approaches upon
removal of the disulfide tether. See also PCT Publication No. WO
00/00823, published on Jan. 6, 2000.
[0007] Although the tethering approach of Erlanson et al.
represents a significant advance in the rapid identification of
small low-affinity ligands, and is a powerful tool for generating
drug leads, there is a need for further improved methods to
facilitate the rational design of drug candidates.
SUMMARY OF THE INVENTION
[0008] The present invention describes a strategy to rapidly and
reliably identify ligands that have intrinsic binding affinity for
different sites on a target molecule by using an extended tethering
approach. This approach is based on the design of a Small Molecule
Extender (SME) that is tethered, via a reversible or irreversible
covalent bond, to a Target Molecule (TM) at or near a first site of
interest, and has a chemically reactive group reactive with small
organic molecules to be screened for affinity to a second site of
interest on the TM. Accordingly, the SME is used for screening a
plurality of ligand candidates to identify a ligand that has
intrinsic binding affinity for a second site of interest on the TM.
If desired, further SME's can be designed based on the
identification of the ligand with binding affinity for the second
site of interest, and the screening can be repeated to identify
further ligands having intrinsic binding affinity for the same or
other site(s) of interest on the same or related TM's.
[0009] One aspect of the invention concerns the design of a Small
Molecule Extender (SME). In this aspect, the invention concerns a
process comprising:
[0010] (i) contacting a Target Molecule (TM) having a first and a
second site of interest, and containing or modified to contain a
reactive nucleophile or electrophile at or near the first site of
interest with a plurality of first small organic ligand candidates,
the candidates having a functional group reactive with the
nucleophile or electrophile, under conditions such that a
reversible covalent bond is formed between the nucleophile or
electrophile and a candidate that has affinity for the first site
of interest, to form a TM-first ligand complex;
[0011] (ii) identifying the first ligand from the complex of (i);
and
[0012] (iii) designing a derivative of the first ligand identified
in (ii) to provide a SME having a first functional group reactive
with the nucleophile or electrophile on the TM and a second
functional group reactive with a second ligand having affinity for
the second site of interest.
[0013] In one embodiment of this aspect of the invention, the SME
of step (iii) above is designed such that it is capable of forming
an irreversible covalent bond with the nucleophile or electrophile
of the TM. In a preferred embodiment, the reactive group on the TM
is a nucleophile, preferably a thiol, protected thiol, reversible
disulfide, hydroxyl, protected hydroxyl, amino, protected amino,
carboxyl, or protected carboxyl group, and preferred first
functional groups on the SME are groups capable of undergoing
SN2-like additions or forming Michael-type adducts with the
nucleophile. SME's designed in this manner are then contacted with
the TM to form an irreversile TM-SME complex. This complex is then
contacted with a plurality of second small organic ligand
candidates, where such candidates have a functional group reactive
with the SME in the TM-SME complex. As a result, a candidate that
has affinity for the second site of interest on the TM forms a
reversible covalent bond with the TM-SME complex, whereby a ligand
having intrinsic binding affinity for the second site of interest
is identified.
[0014] In an alternative embodiment of the invention, the SME of
step (iii) above is designed to contain a first functional group
that forms a first reversible covalent bond with the nucleophile or
electrophile on the TM. The reactive group on the TM preferably is
a nucleophile. The reversible covalent bond preferably is a
disulfide bond which is formed with a thiol, protected thiol, or
reversible disulfide bond on the TM. SME's designed in this manner
are then contacted with the TM either prior to or simultaneously
with contacting the TM with a plurality of second small organic
ligand candidates, each small organic ligand candidate having a
free thiol, protected thiol, or a reversible disulfide group, under
conditions of thiol exchange, wherein a ligand candidate having
affinity for the second site of interest on the TM forms a
disulfide bond with the TM-SME complex, whereby a second ligand is
identified. The process may be performed in the presence of a
disulfide reducing agent, such as mercaptoethanol, dithiothreitol
(DTT), dithioerythreitol (DTE), mercaptopropanoic acid,
glutathione, cysteamine, cysteine, tri(carboxyethyl)phosphine
(TCEP), and tris(cyanoethyl)phosphine.
[0015] In a particular embodiment, the SME is designed based on
selection of a small organic molecule having a thiol or protected
thiol (disulfide monophore) from a library of such molecules by a
Target Biological Molecule (TBM) having a thiol at or near a site
of interest. In this case, the method of this invention is a
process comprising:
[0016] (i) contacting a TBM containing or modified to contain a
thiol, protected thiol or reversible disulfide group at or near a
first site of interest on the TBM with a library of small organic
molecules, each small organic molecule having a free thiol or a
reversible disulfide group (disulfide monophores), under conditions
of thiol exchange wherein a library member having affinity for a
first site of interest forms a disulfide bond with the TBM;
[0017] (ii) identifying the library member (selected disulfide
monophore) from (i); and
[0018] (iii) designing a derivative of the library member in (ii)
that is the SME having a first functional group reactive with the
thiol on the TBM and having a second functional group which is a
thiol, protected thiol or reversible disulfide group.
[0019] Just as before, the SME can be designed to contain a first
functional group that forms an irreversible or reversible covalent
bond with the TBM, and can be used to screen small molecule ligand
candidates, in particular libraries of small molecules, as
described above, to identify a second ligand.
[0020] Thus, in one embodiment, the, SME of step (iii) is designed
to contain a first functional group that forms an irreversible
covalent bond with the thiol on the TBM. Preferred first functional
groups of this embodiment are groups capable of undergoing SN2 like
additions or forming Michael-type adducts with the thiol. SME's
designed in this manner are then contacted with the TBM to form an
irreversible TBM-SME complex. This complex is then contacted with
second library of small organic molecules, each small organic
molecule having a free thiol or a reversible disulfide group, under
conditions of thiol exchange wherein the library member having
affinity, preferably the highest affinity, for a second site of
interest on the TBM (second ligand) forms a disulfide bond with the
TBM-SME complex.
[0021] In an alternative embodiment, the small molecule extender
(SME) of step (iii) is designed to contain a first functional group
that forms a first reversible disulfide bond with the thiol on the
TBM. SME's designed in this manner are then contacted with the TBM
either prior to or simultaneously with contacting the TBM with a
second library of small organic molecules, each small organic
molecule having a free thiol or a reversible disulfide group, under
conditions of thiol exchange under conditions wherein the member of
the second library having affinity, preferably the highest
affinity, for a second site of interest on the TBM (second ligand)
forms a disulfide bond with the TBM-SME complex.
[0022] The process may be performed in the presence of a disulfide
reducing agent, such as those listed above.
[0023] Determining the affinity of a ligand candidate (library
member) for a first or second site of interest on a TM or TBM can
be carried out by competition between different library members in
a pool, or by comparison (i.e. titration) with a reducing agent,
such as those listed above.
[0024] In a particular embodiment, the invention concerns a process
comprising:
[0025] (i) contacting a Target Biological Molecule (TBM) containing
or modified to contain a nucleophile at or near a site of interest
on the TBM with a small molecule extender having a first functional
group reactive with the nucleophile and having a second functional
group which is a thiol, protected thiol or reversible disulfide
group, thereby forming a TBM-Small Molecule Extender (TBM-SME)
complex;
[0026] (ii) contacting the TBM-SME complex with a library of small
organic molecules, each small organic molecule (ligand) having a
free thiol, protected thiol or a reversible disulfide group, under
conditions of thiol exchange wherein a library member having
affinity for the site of interest forms a disulfide bond with the
TBM-SME complex thereby forming a TBM-SME-ligand complex and
[0027] (iii) determining the ligand from (ii).
[0028] In another particular embodiment, the invention concerns a
process comprising:
[0029] (i) providing a Target Biological Molecule (TBM) containing
or modified to contain a reactive nucleophile near a first site of
interest on the TBM;
[0030] (ii) contacting the TBM from (i) with a small molecule
extender having a group reactive with the nucleophile on the TBM
and having a free thiol or protected thiol;
[0031] (iii) adjusting the conditions to cause a covalent bond to
be formed between the nucleophile on the TBM and the group on the
small molecule extender thereby forming a covalent complex
comprising the TBM and the small molecule extender, the complex
displaying a free thiol or protected thiol near a second site of
interest on the TBM;
[0032] (iv) contacting the complex from (iii) with a library of
small organic molecules, each molecule having a free thiol or
exchangeable disulfide linking group, under conditions of thiol
exchange wherein the library member having the highest affinity for
the second site of interest on the TBM forms a disulfide bond with
the complex; and
[0033] (v) identifying the library member from (iv).
[0034] In a particular embodiment, the processes of the present
invention may be performed with a library in which each member
forms a disulfide bond. An example of such a library is one in
which each member forms a cysteamine disulfide. When library
members form disulfides, a preferred molar ratio of reducing agent
to total disulfides is from about 1:100 to about 100:1 and more
preferably from about 1:1 to about 50:1.
[0035] The tethering process may be performed by contacting members
of the disulfide library one at a time with the TBM or in pools of
2 or more. When pools are used it is preferred to use from 5-15
library members per pool.
[0036] In all embodiments, the identity of the small molecules that
bind to the SME and/or a site of interest on a TM or TBM may be
determined, for example, by mass spectrometry (MS), or by means of
a detectable tag. When mass spectrometry is used to detect the
library member that binds to a TBM and pools are used, it is
preferred that each member of the pool differs in molecular weight,
preferably by about 10 Daltons. Identification can be performed by
measuring the mass of the TBM-library member complex, or by
releasing the library member form the complex first or by using a
functional assay, e.g. ELISA, enzyme assay etc.
[0037] In a different aspect, the invention concerns a molecule
comprising a first and/or second ligand identified by any of the
methods discussed above. In a particular embodiment, the molecule
comprises a first and a second ligand covalently linked to one
another. The covalent linkage may be provided by any covalent bond,
including but not limited to disulfide bonds.
[0038] In a further aspect, the invention concerns methods for
synthesizing such molecules. The molecules obtained can, of course,
be further modified, for example to impart improved properties,
such as solubility, bioavailability, affinity, and half-life. For
example, the disulfide bond can be replaced by a linker having
greater stability under standard biological conditions. Possible
linkers include, without limitation, alkanes, alkenes, aromatics,
heteroaromatics, ethers, and the like.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 is a schematic illustration of the basic tethering
approach for side-directed ligand discovery. A target molecule,
containing or modified to contain a free thiol group (such as a
cysteine-containing protein) is equilibrated with a
disulfide-containing library in the presence of a reducing agent,
such as 2-mercaptoethanol. Most of the library members will have
little or no intrinsic affinity for the target molecule, and thus
by mass action the equilibrium will lie toward the unmodified
target molecule. However, if a library member does show intrinsic
affinity for the target molecule, the equilibrium will shift toward
the modified target molecule, having attached to it the library
member with a disulfide tether.
[0040] FIG. 2 is a schematic illustration of the static extended
tethering approach. In the first step, a target molecule containing
or modified to contain a free thiol group (such as a
cysteine-containing protein) is modified by a thiol-containing
extender, comprising a reactive group capable of forming an
irreversible covalent bond with the thiol group on the target
molecule, a portion having intrinsic affinity for the target
molecule, and a thiol group. The complex formed between the target
molecule and the thiol-containing extender is then used to screen a
library of disulfide-containing monophores to identify a library
member that has the highest intrinsic binding affinity for a second
binding site on the target molecule. LG=ligand; PG=protecting
group; R=reactive group.
[0041] FIG. 3 illustrates the dynamic extended tethering strategy,
where the extender is bifunctional and contains two functional
groups (usually disulfide), each capable of forming reversible
covalent bonds. R=reactive group.
[0042] FIG. 4 illustrates the chemical synthesis of a specific
extender (2,6-dichloro-benzoic acid
3-(2-acetylsulfanyl-acetylamino)-4-carboxy-2-o- xo-butyl ester), as
described in Example 2.
[0043] FIG. 5 shows the structural comparison between a known
tetrapeptide inhibitor of Caspase-3 and a generic extender
synthesized based on the inhibitor.
[0044] FIG. 6 shows mass spectra of two representative extended
tethering experiments.
DETAILED DESCRIPTION OF THE INVENTION
[0045] 1. Definitions
[0046] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Singleton et al., Dictionary of Microbiology and Molecular Biology
2nd ed., J. Wiley & Sons (New York, N.Y. 1994), and March,
Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th
ed., John Wiley & Sons (New York, N.Y. 1992), provide one
skilled in the art with a general guide to many of the terms used
in the present application.
[0047] One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. Indeed, the
present invention is in no way limited to the methods and materials
described. For purposes of the present invention, the following
terms are defined below.
[0048] The terms "target," "Target Molecule," and "TM" are used
interchangeably and in the broadest sense, and refer to a chemical
or biological entity for which a ligand has intrinsic binding
affinity. The target can be a molecule, a portion of a molecule, or
an aggregate of molecules. The target is capable of reversible
attachment to a ligand via a reversible or irreversible covalent
bond (tether). Specific examples of target molecules include
polypeptides or proteins (e.g., enzymes, including proteases, e.g.
cysteine, serine, and aspartyl proteases), receptors, transcription
factors, ligands for receptors, growth factors, cytokines,
immunoglobulins, nuclear proteins, signal transduction components
(e.g., kinases, phosphatases), allosteric enzyme regulators, and
the like, polynucleotides, peptides, carbohydrates, glycoproteins,
glycolipids, and other macromolecules, such as nucleic acid-protein
complexes, chromatin or ribosomes, lipid bilayer-containing
structures, such as membranes, or structures derived from
membranes, such as vesicles. The definition specifically includes
Target Biological Molecules (TBMs) as defined below.
[0049] A "Target Biological Molecule" or "TBM" as used herein
refers to a single biological molecule or a plurality of biological
molecules capable of forming a biologically relevant complex with
one another for which a small molecule agonist or antagonist would
have therapeutic importance. In a preferred embodiment, the TBM is
a polypeptide that comprises two or more amino acids, and which
possesses or is capable of being modified to possess a reactive
group for binding to members of a library of small organic
molecules.
[0050] The term "polynucleotide", when used in singular or plural,
generally refers to any polyribonucleotide or
polydeoxribonucleotide, which may be unmodified RNA or DNA or
modified RNA or DNA. Thus, for instance, polynucleotides as defined
herein include, without limitation, single- and double-stranded
DNA, DNA including single- and double-stranded regions, single- and
double-stranded RNA, and RNA including single- and double-stranded
regions, hybrid molecules comprising DNA and RNA that may be
single-stranded or, more typically, double-stranded or include
single- and double-stranded regions. In addition, the term
"polynucleotide" as used herein refers to triple-stranded regions
comprising RNA or DNA or both RNA and DNA. The strands in such
regions may be from the same molecule or from different molecules.
The regions may include all of one or more of the molecules, but
more typically involve only a region of some of the molecules. One
of the molecules of a triple-helical region often is an
oligonucleotide. The term "polynucleotide" specifically includes
DNAs and RNAs that contain one or more modified bases. Thus, DNAs
or RNAs with backbones modified for stability or for other reasons
are "polynucleotides" as that term is intended herein. Moreover,
DNAs or RNAs comprising unusual bases, such as inosine, or modified
bases, such as tritylated bases, are included within the term
"polynucleotides" as defined herein. In general, the term
"polynucleotide" embraces all chemically, enzymatically and/or
metabolically modified forms of unmodified polynucleotides, as well
as the chemical forms of DNA and RNA characteristic of viruses and
cells, including simple and complex cells.
[0051] A "ligand" as defined herein is an entity which has an
intrinsic binding affinity for the target. The ligand can be a
molecule, or a portion of a molecule which binds the target. The
ligands are typically small organic molecules which have an
intrinsic binding affinity for the target molecule, but may also be
other sequence-specific binding molecules, such as peptides (D-, L-
or a mixture of D- and L-), peptidomimetics, complex carbohydrates
or other oligomers of individual units or monomers which bind
specifically to the target. The term "monophore" is used herein
interchangeably with the term "ligand" and refers to a monomeric
unit of a ligand. The term "diaphore" denotes two monophores
covalently linked to form a unit that has a higher affinity for the
target because of the two constituent monophore units or ligands
binding to two separate but nearby sites on the target. The binding
affinity of a diaphore that is higher than the product of the
affinities of the individual components is referred to as
"avidity." The term diaphore is used irrespective of whether the
unit is covalently bound to the target or existing separately after
its release from the target. The term also includes various
derivatives and modifications that are introduced in order to
enhance binding to the target.
[0052] A "site of interest" on a target as used herein is a site to
which a specific ligand binds, which may include a specific
sequence of monomeric subunits, e.g. amino acid residues, or
nucleotides, and may have a three-dimensional structure. Typically,
the molecular interactions between the ligand and the site of
interest on the target are non-covalent, and include hydrogen
bonds, van der Waals interactions and electrostatic interactions.
In the case of polypeptide, e.g. protein targets, the site of
interest broadly includes the amino acid residues involved in
binding of the target to a molecule with which it forms a natural
complex in vivo or in vitro.
[0053] "Small molecules" are usually less than 10 kDa molecular
weight, and include but are not limited to synthetic organic or
inorganic compounds, peptides, (poly)nucleotides,
(oligo)saccharides and the like. Small molecules specifically
include small non-polymeric (e.g. not peptide or polypeptide)
organic and inorganic molecules. Many pharmaceutical companies have
extensive libraries of such molecules, which can be conveniently
screened by using the extended tethering approach of the present
invention. Preferred small molecules have molecular weights of less
than about 300 DA and more preferably less than about 650 Da.
[0054] The term "tether" as used herein refers to a structure which
includes a moiety capable of forming a reversible or reversible
covalent bond with a target (including Target Biological Molecules
as hereinabove defined), near a site of interest.
[0055] The phrase "Small Molecule Extender" (SME) as used herein
refers to a small organic molecule having a molecular weight of
from about 75 to about 1,500 daltons and having a first functional
group reactive with a nucleophile or electrophile on a TM and a
second functional group reactive with a ligand candidate or members
of a library of ligand candidates. Preferably, the first functional
group is reactive with a nucleophile on a TBM (capable of forming
an irreversible or reversible covalent bond with such nucleophile),
and the reactive group at the other end of the SME is a free or
protected thiol or a group that is a precursor of a free of
protected thiol. In one embodiment, at least a portion of the small
molecule extender is capable of forming a noncovalent bond with a
first site of interest on the TBM (i.e. has an inherent affinity
for such first site of interest). Included within this definition
are small organic (including non-polymeric) molecules containing
metals such as Cd, Hg and As which may form a bond with the
nucleophile. e.g. SH of the TBM.
[0056] The phrase "reversible covalent bond" as used herein refers
to a covalent bond which can be broken, preferably under conditions
that do not denature the target. Examples include, without
limitation, disulfides, Schiff-bases, thioesters, and the like.
[0057] The term "reactive group" with reference to a ligand is used
to describe a chemical group or moiety providing a site at which
covalent bond with the ligand candidates (e.g. members of a library
or small organic compounds) may be formed. Thus, the reactive group
is chosen such that it is capable of forming a covalent bond with
members of the library against which it is screened.
[0058] The term "antagonist" is used in the broadest sense and
includes any ligand that partially or fully blocks, inhibits or
neutralizes a biological activity exhibited by a target, such as a
TBM. In a similar manner, the term "agonist" is used in the
broadest sense and includes any ligand that mimics a biological
activity exhibited by a target, such as a TBM, for example, by
specifically changing the function or expression of such TBM, or
the efficiency of signaling through such TBM, thereby altering
(increasing or inhibiting) an already existing biological activity
or triggering a new biological activity.
[0059] The phrases "modified to contain" and "modified to possess"
are used interchangeably, and refer to making a mutant, variant or
derivative of the target, or the reactive nucleophile or
electrophile, including but not limited to chemical modifications.
For example, in a protein one can substitute an amino acid residue
having a side chain containing a nucleophile or electrophile for a
wild-type residue. Another example is the conversion of the thiol
group of a cysteine residue to an amine group.
[0060] The term "reactive nucleophile" as used herein refers to a
nucleophile that is capable of forming a covalent bond with a
compatible functional group on another molecule under conditions
that do not denature or damage the target, e.g. TBM. The most
relevant nucleophiles are thiols, alcohols, activated carbonyls,
epoxides, aziridines, aromatic sulfonates, hemiacetals, and amines.
Similarly, the term "reactive electrophile" as used herein refers
to an electrophile that is capable of forming a covalent bond with
a compatible functional group on another molecule, preferably under
conditions that do not denature or otherwise damage the target,
e.g. TMB. The most relevant electrophiles are imines, carbonyls,
epoxides, aziridies, sulfonates, and hemiacetals.
[0061] A "first site of interest" on a target, e.g. TBM refers to a
site that can be contacted by at least a portion of the SME when it
is covalently bound to the reactive nucleophile or electrophile.
The first site of interest may, but does not have to possess the
ability to form a noncovalent bond with the SME.
[0062] The phrases "group reactive with the nucleophile,"
"nucleophile reactive group," "group reactive with an
electrophile," and "electrophile reactive group," as used herein,
refer to a functional group on the SME that can form a covalent
bond with the nucleophile/electrophile on the TM, e.g. TBM under
conditions that do not denature or otherwise damage the TM, e.g.
TBM.
[0063] The term "protected thiol" as used herein refers to a thiol
that has been reacted with a group or molecule to form a covalent
bond that renders it less reactive and which may be deprotected to
regenerate a free thiol.
[0064] The phrase "adjusting the conditions" as used herein refers
to subjecting a target, such as a TBM to any individual,
combination or series of reaction conditions or reagents necessary
to cause a covalent bond to form between the ligand and the target,
such as a nucleophile and the group reactive with the nucleophile
on the SME, or to break a covalent bond already formed.
[0065] The term "covalent complex" as used herein refers to the
combination of the SME and the TM, e.g. TBM which is both
covalently bonded through the nucleophile/electrophile on the TM,
e.g. TBM with the group reactive with the nucleophile/electrophile
on the SME, and non-covalently bonded through a portion of the
small molecule extender and the first site of interest on the TM,
e.g. TBM.
[0066] The phrase "exchangeable disulfide linking group" as used
herein refers to the library of molecules screened with the
covalent complex displaying the thiol-containing small molecule
extender, where each member of the library contains a disulfide
group that can react with the thiol or protected thiol displayed on
the covalent complex to form a new disulfide bond when the reaction
conditions are adjusted to favor such thiol exchange.
[0067] The phase "highest affinity for the second site of interest"
as used herein refers to the molecule having the greater
thermodynamic stability toward the second site of interest on the
TM, e.g. TBM that is preferentially selected from the library of
disulfide-containing library members.
[0068] "Functional variants" of a molecule herein are variants
having an activity in common with the reference molecule.
[0069] "Active" or "activity" means a qualitative biological and/or
immunological property.
[0070] 2. Targets
[0071] Targets, such as target biological molecules (TBMs), that
find use in the present invention include, without limitation,
molecules, portions of molecules and aggregates of molecules to
which a ligand candidate may bind, such as polypeptides or proteins
(e.g., enzymes, receptors, transcription factors, ligands for
receptors, growth factors, immunoglobulins, nuclear proteins,
signal transduction components, allosteric enzyme regulators, and
the like), polynucleotides, peptides, carbohydrates, glycoproteins,
glycolipids, and other macromolecules, such as nucleic acid-protein
complexes, chromatin or ribosomes, lipid bilayer-containing
structures, such as membranes, or structures derived from
membranes, such as vesicles. The target can be obtained in a
variety of ways, including isolation and purification from natural
source, chemical synthesis, recombinant production and any
combination of these and similar methods.
[0072] Preferred enzyme target families are cysteine proteases,
aspartyl proteases, serine proteases, metalloproteases, kinases,
phosphatases, polymerases and integrases. Preferred protein:protein
targets are 4-helical cytokines, trimeric cytokines, signaling
modules, transcription factors and chemokines.
[0073] In a particularly preferred embodiment, the target is a TBM,
and even more preferably is a polypeptide, especially a protein.
Polypeptides, including proteins, that find use herein as targets
for binding ligands, preferably small organic molecule ligands,
include virtually any polypeptide (including short polypeptides
also referred to as peptides) or protein that comprises two or more
binding sites of interest, and which possesses or is capable of
being modified to possess a reactive group for binding to a small
organic molecule or other ligand (e.g. peptide). Polypeptides of
interest may be obtained commercially, recombinantly, by chemical
synthesis, by purification from natural source, or otherwise and,
for the most parts are proteins, particularly proteins associated
with a specific human disease or condition, such as cell surface
and soluble receptor proteins, such as lymphocyte cell surface
receptors, enzymes, such as proteases (e.g., serine, cysteine, and
aspartyl proteases) and thymidylate synthetase, steroid receptors,
nuclear proteins, allosteric enzymes, clotting factors, kinases
(both serine and threonine) and dephosphorylases (or phophatases,
either serine/threonine or protein tyrosine phosphatases, e.g.
PTP's, especially PTP1B), bacterial enzymes, fungal enzymes and
viral enzymes (especially those associate with HIV, influenza,
rhinovirus and RSV), signal transduction molecules, transcription
factors, proteins or enzymes associated with DNA and/or RNA
synthesis or degradation, immunoglobulins, hormones, receptors for
various cytokines including, for example, erythropoietin (EPO),
granulocyte colony stimulating (G-CSF) receptor, granulocyte
macrophage colony stimulating (GM-CSF) receptor, thrombopoietin
(TPO), interleukins, e.g. IL-2, IL-3, IL-4, IL-5, IL-6, IL-10,
IL-11, IL-12, growth hormone, prolactin, human placental lactogen
(LPL), CNTF, oncostatin, various chemokines and their receptors,
such as RANTES MIP.beta., 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.), nerve growth factor (NGF), various neurotrophins
and their ligands, other hormones and receptors such as, bone
morphogenic factors, follicle stimulating hormone (FSH), and
luteinizing hormone (LH), trimeric hormones including tissue
necrosis factor (TNF) and CD40 ligand, apoptosis factor-1 and -2
(AP-1 and AP-2), p53, bax/bcl2, mdm2, caspases, and proteins and
receptors that share 20% or more sequence identity to these.
[0074] An important group of human inflammation and immunology
targets includes: IgE/IgER, ZAP-70, lck, syk, ITK/BTK, TACE,
Cathepsin S and F, CD11a, LFA/ICAM, VLA-4, CD28/B7, CTLA4, TNF
alpha and beta, (and the p55 and p75 TNF receptors), CD40L, p38 map
kinase, IL-2, IL-4, Il-13, IL-15, Rac 2, PKC theta, IL-8, TAK-1,
jnk, IKK2 and IL-18.
[0075] Still other important specific targets include: caspases 1,
3, 8 and 9, IL-1/IL-1 receptor, BACE, HIV integrase, PDE IV,
Hepatitis C helicase, Hepatitis C protease, rhinovirus protease,
tryptase, cPLA (cytosolic Phospholipase A2), CDK4, c-jun kinase,
adaptors such as Grb2, GSK-3, AKT, MEKK-1, PAK-1, raf, TRAF's 1-6,
Tie2, ErbB 1 and 2, FGF, PDGF, PARP, CD2, C5a receptor, CD4, CD26,
CD3, TGF-alpha, NF-kB, IKK beta, STAT 6, Neurokinnin-1, PTP-1B,
CD45,Cdc25A, SHIP-2, TC-PTP, PTP-alpha, LAR and human p53, bax/bcl2
and mdm2.
[0076] The target, e.g. a TBM of interest will be chosen such that
it possesses or is modified to possess a reactive group which is
capable of forming a reversible or irreversible covalent bond with
a ligand having intrinsic affinity for a site of interest on the
target. For example, many targets naturally possess reactive groups
(for example, amine, thiol, aldehyde, ketone, hydroxyl groups, and
the like) to which ligands, such as members of an organic small
molecule library, may covalently bond. For example, 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, automates 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 target, e.g. a TBM,
during automated synthesis.
[0077] In one particular embodiment, the target 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, and preferably is associated with a cysteine residue
of the polypeptide, if the tether chosen is a free or protected
thiol group (see below). The target preferably contains, or is
modified to contain, only a limited number of free or protected
thiol groups, preferably not more than about 5 thiol groups, more
preferably not more than about 2 thiol groups, more preferably not
more than one free thiol group, although polypeptides having more
free thiol groups will also find use. The target, such as TBM, of
interest may be initially obtained or selected such that it already
possesses the desired number of thiol groups, or may be modified to
possess the desired number of thiol groups.
[0078] When the target is a polynucleotide, a tether can, for
example, be attached to the polynucleotide on a base at any
exocyclic amine or any vinyl carbon, such as the 5- or 6-position
of pyrimidines, 8- or 2-positions of purines, at the 5' or 3'
carbons, at the sugar phosphate backbone, or at internucleotide
phosphorus atoms. However, a tether can be introduced also at other
positions, such as the 5-position of thymidine or uracil. In the
case of a double-stranded DNA, for example, a tether can be located
in a major or minor groove, close to the site of interest, but not
so close as to result in steric hindrance, which might interfere
with binding of the ligand to the target at the site of
interest.
[0079] Those skilled in the art are well aware of various
recombinant, chemical, synthesis and/or other techniques that can
be routinely employed to modify a target, e.g. a polypeptide of
interest such that it possesses a desired number of free thiol
groups that are available for covalent binding to a ligand
candidate comprising a free thiol group. Such techniques include,
for example, site-directed mutagenesis of the nucleic acid sequence
encoding the target polypeptide such that it encodes a polypeptide
with a different number of cysteine residues. Particularly
preferred is site-directed mutagenesis using polymerase chain
reaction (PCR) amplification (see, for example, U.S. Pat. No.
4,683,195 issued 28 Jul. 1987; and Current Protocols In Molecular
Biology, Chapter 15 (Ausubel et al., ed., 1991). Other
site-directed mutagenesis techniques are also well known in the art
and are described, for example, in the following publications:
Ausubel et al, supra, Chapter 8; Molecular Cloning: A Laboratory
Manual., 2.sup.nd edition (Sambrook et al., 1989); Zoller et al.,
Methods Enzymol. 100:468-500 (1983); Zoller & Smith, DNA
3:479-488 (1984); Zoller et al., Nucl. Acids Res., 10:6487 (1987);
Brake et al., Proc. Natl. Acad. Sci. USA 81:4642-4646 (1984);
Botstein et al, Science 229:1193 (1985); Kunkel et al, Methods
Enzymol. 154:367-82 (1987), Adelman et al, DNA 2:183 (1983); and
Carter et al., Nucl. Acids Res., 13:4331 (1986). Cassette
mutagenesis (Wells et al., Gene, 34:315 [1985]), and restriction
selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London
SerA, 317:415 [1986]) may also be used.
[0080] Amino acid sequence variants with more than one amino acid
substitution may be generated in one of several ways. If the amino
acids are located close together in the polypeptide chain, they may
be mutated simultaneously, using one oligonucleotide that codes for
all of the desired amino acid substitutions. If, however, the amino
acids are located some distance from one another (e.g. separated by
more than ten amino acids), it is more difficult to generate a
single oligonucleotide that encodes all of the desired changes.
Instead, one of two alternative methods may be employed. In the
first method, a separate oligonucleotide is generated for each
amino acid to be substituted. The oligonucleotides are then
annealed to the single-stranded template DNA simultaneously, and
the second strand of DNA that is synthesized from the template will
encode all of the desired amino acid substitutions. The alternative
method involves two or more rounds of mutagenesis to produce the
desired mutant.
[0081] Sources of new reactive groups, e.g. cysteines can be placed
anywhere within the target. For example, if a cysteine is
introduced onto the surface of the protein in an area known to be
important for protein-protein interactions, small molecules can be
selected that bind to and block this surface.
[0082] The following tables exemplify target biological molecules
(TBM's) that can be used in accordance with the present
invention.
1 Immunology Indications IL-6 Inflammation B7/CD28 Graft rejection
CD4 Immunosuppression CD3 Immunosuppression CD2 Renal
Transplantation c-maf Inflammation/Immunosuppression CD11a/LFA1
(ICAM) Immunosuppression/Inflammation
[0083]
2 Enzymes Indications Phospholipase A2 Inflammation ZAP-70
Immunosuppression Phophodiesterase IV Asthma Interleukin converting
enzyme (ICE) Inflammation Inosine monophosphate dehydrogenase
Autoimmune diseases Tryptase Psoriasis/asthma CDK4 Cancer mTOR
Immunosuppression PARP (Cell death pathway) Stroke Phosphatases
Cancer Raf Cancer JNK3 Neurodegeneration MEK Cancer GSK-3 Diabetes
FABI (Fatty acid biosynthesis) Bacterial FABH (Fatty acid
biosynthesis) Bacterial BACE Alzheimer's IkB-ubiquitin Ligase
Inflammation/diabetes Lysophosphatidic acid acetlytransferase CD26
(dipeptidyl peptidase IV) Akt TNF converting enzyme
Inflammation
[0084]
3 Viral Targets Indications Rhinovirus protease Common cold
Parainfluenza neuraminidase Colds/Veterinary uses HIV fusion gp41
HIV infection/treatment Hepatitis C Helicase Hepatitis Hepatitis C
protease Hepatitis
[0085]
4 Protein-Protein Targets Indications ErbB Receptors Cancer
Neurokinin-1 Inflammation, Migraine IL-9 Asthma FGF Angiogenesis
PDGF Angiogenesis TIE2 Angiogenesis NF.kappa.B Dimerization
Inflammation Tissue Factor/Factor VII Cardiovascular Disease
Selectins Inflammation TGF-.alpha. Angiogenesis Angiopoietin I
Angiogenesis APAF-1/Caspase 9 CARD Stroke Bcl-2 Cancer
[0086]
5 7-Transmembrane Indications IL-8 Stroke, inflammation Rantes
Inflammation, Migraine CC Chemokine Receptors Asthma GPR
14/Urotensin Angiogenesis Orexin/Receptor Appetite C5a receptor
Sepsis/crohn's disease Histamine H3 receptor Allergy CCR5 HIV
attachment
[0087]
6 Target PDB Codes Accession No. Crystal Structure Ref. BACE 1FKN
GB AAF13715 Hong, L. et al., Science. 290(5489): 150-3 (2000).
Caspase 1 1BMQ SWS P29466 Okamoto, Y., et al., Chem Pharm Bull
(Tokyo), 47(1): 11-21 (1999). Caspase 4 none SWS P49662 NA Caspase
5 none SWS P51878 NA Caspase 3 1CP3 SWS P42574 Mittl, PR, et al., J
Biol Chem, 272(10): 6539-47(1997). Caspase 8 1I4E, 1QTN SWS P08160;
Xu, G., et al., Nature, 410(6827): 494-7 GB BAB32555 (2001).
Caspase 9 3YGS SWS P55211 Qin, H., et al., Nature, 399(6736):
549-57 (1999). RHV.multidot.Prot 1CQQ SWS P04936 Matthews, D., et
al., 96(20): 11000-7 (1999). Cathepsin K 1MEM SWS P43235 McGrath,
ME, et al., Nat Struct Biol, 4(2): 105-(1997). Cathepsin S 1BXF SWS
P25774 Fengler, A., et al., Protein (model) Eng, 11(11):
1007-13(1998). Tryptase 1A0L SWS P20231 Pereira, P. J. et al.,
Nature, 392(6673): 306-11 (1998). HCV Prot 1A1R, SWS Q81755 Di
Marco, et al., J Biol Chem. IDY9 275(10): 7152-7(2000). CD26 none
SWS P27487 NA TACE 1BKC GB U69612 Maskos, K., et al., PNAS, 95(7):
3408-12 (1998). ZAP-70 none SWS P43403 NA p38 MAP 1P38 SWS P47811
Wang, Z., et al., PNAS, 94(6): 2327-32 (1997). CDK-4 none SWS
Q9XTB6 NA c-jun kinase NA SWS P45983 (C- NA Jun Kinase-1) NA SWS
P45984 (C- NA Jun Kinase-2) 1JNK SWS P53779 (C- NA Jun Kinase-3)
GSK-3 NA SWS P49840 NA (GSK-3A) NA SWS P49841 NA (GSK-3B) AKT none
SWS P31749 NA MEK none SWS Q02750 NA Raf none SWS P04049 NA TIE-2
none SWS Q02763 NA ILK none SWS Q13418 NA IkB NA SWS O15111 NA
(IKappaBKinase) NA SWS O14920 NA (IKappBKinBeta) Jak1 none SWS
P23458 NA Jak2 none SWS O60674 NA Jak3 none SWS P52333 NA Tyk2 none
SWS P29597 NA EGF Kinase see Vasc. Endo. Growth Factor Receptor
(VEGFR) and EGFR both with tyrosine kinase activity(Below):
VEGFR2/KD NA SWS P35968 NA R Kinase EGFR NA SWS P00533 NA TC-PTP NA
SWS P17706 NA: T-cell Protein Tyrosine Phosphatase CDC25A NA SWS
P30304 NA CDC25A NA GB O14757 NA CDK (CHK1) CD45 NA SWS P08575 NA
PTP alpha NA SWS P18433 NA pol III NA SWS O14802 NA; DNA directed
RNA polymerase III (PolRIIIA) mur-D Ligase NA GB O14802 (E. Coli)
NA NA SWS P14900 (E. Coli) NA SHP NA SWS Q15466 NA PTP-1B 1PTP SWS
P00760 Finer-Moore, JS, et al., Proteins, 12(3): 203-22(1992).
SHIP-2 none SWS Q9R1V2 NA MEKK-1 NA SWS Q13233 NA PAK-1 NA SWS
Q13153 NA ICAM-1 NA SWS P05362 Bella, J., et al., Proc Natl Acad
Sci USA, 95(8): 4140-5 (1998). CD11A/LFA-1 NA SWS P20701 Qu, A., et
al., Proc Natl Acad Sci USA, 92(22: 10277-81 (1995) TAF1 UNSURE
UNSURE (see UNSURE below) NA SWS Q99142 (?? NA; tobacco Tumor
Activating Factor Tobacco Prot.) NA GB AAB30018 NA; Tumor-derived
Adhesion Factor NA GB D45198 NA; Template Activating Factor
HIV-Integrase 1BL3 (2.0) SWS P12497 Maignan, S., et al., J Mol
Biol, 282(2): 359-68 (1998). 1EXQ SWS P04585 Chen, J. C-H., et al.,
PNAS USA, 97(15): 8233-8 (2000) NA SWS O56380 NA 1HYZ SWS O56381;
Molteni, V., et al., Acta Crystallogr GB AAC37875 D Bio
Crystallog., 57: 536-44 (2001). 1HYV GB AAC37875 Molteni, V., et
al., Acta Crystallogr D Biol Crystallogr., 57(Pt 4): 536-44 (2001).
NA SWS O56382 NA NA SWS O56383 NA NA SWS O56384 NA NA SWS 056385 NA
HCV-Helicase 1N13, SWS Q81755 Di Marco, S., et al., J Biol Chem.,
1DY9, (1DY9) 275(10): 7152-7 (2000). others (Integrase) 1HEI SWS
P2664 Yao, N., et al., Nat Struct Biol, 4(6): 463-7 (Helicase)
(1997). Infl. 1A4G; many SWS P27907 Taylor, N., et al., J Med Chem,
Neuraminidase 41(6): 798-807 (1998). PDE-IV 1FOJ SWS Q07343 Xu, R.
X., et al., Science., (PDE4B2B) 288(5472): 1822-5 (2000). cPLA-2
1CJY SWS P47712 Dessen, A., et al., Cell., 97(3): 349-60 (1999).
IL-2 NA (in- SWS P01585 NA house IL-4 1HIK(apo) SWS P05112 Muller,
T., et al., J Mol Biol, 247(2): 360-72 (2.60) (1995). 1IAR SWS
P05112 Hage, T., et al., Cell., 97(2): 271-81 (complex) (2.30)**
(1999). IL-4R 1IAR SWS P24394 Hage, T., et al., Cell., 97(2):
271-81 (1999). IL-5 1HUL SWS P05113 Milburn, M. V., et al., Nature,
363(6425): 172-6 (1993). IL-6 1I1R(viral GB AAB62676 Chow, D., et
al., Science, IL6) (2.6) 291(5511): 2150-5 (2001). 1ALU SWS P05231
Somers, W., et al., EMBO J, 16(5): 989-97 (1.9) (1997). IL-7 1IL7
SWS P13232 Cosenza, L., et al., Protein Sci., 9(5): 916-26 (model)
(2000). IL-9 none SWS P15248 NA IL-13 1GA3 SWS P35225 NA (NMR) TNF
1TNF SWS P01375 Eck, MJ, et al., J Biol (TNF-alpha) Chem, 264(29)
17595-605(1989). CD-40 L 1ALY SWS P29965 Karpusas, M., et al.,
Structure, 3(12): 1426 (1995). OPGL none SWS O14788 NA BAFF none
SWS Q9Y275 NA TRAIL 1DG6 (1.30) GB AAC50332 Hymowitz, S. G., et
al., Biochemistry, 39(4): 633-40 (2000). 1DU3 (2.2) SWS P50591; Cha
SS, et al., J Biol Chem, GB AAC50332 275(40): 31171-7 (2000). 1D2Q
GB AAC50332 Cha and Oh, Immunity, 11(2): 253-61 (1999). IL-1 NA SWS
P01584 NA (IL-1 B Cytokine) IL-1R 1G0Y SWS P14778 Vigers, GPA, et
al., J Biol Chem., 275(47): 36927-33 (2000). IL-8 1QE6 SWS P10145
Gerber, N., et al., Proteins, 38(4): 361-7 (2000). RANTES-R NA SWS
P32246 NA RANTES NA GB XP_035842 NA NA SWS P13501 NA; (T-cell
specific RANTES protein) MCP-1 NA SWS Q14805 NA; (Metaphase
chromosomal protein) MCP-1 1D0K SWS P13500 Lubowski, J., et al.,
Nat Struct Biol., 4(1): 64-9 (1997). MCP-3 NA SWS P80098 Nat Struct
Biol, 4(1): 64-9 (1997). TRAF-A NA SWS Q13077 NA (TRAF-1?) (TRAF-1)
Target PDB Codes Accession No. Crystal Structure Ref. TRAF-B NA SWS
Q12933 NA (TRAF-2?) (TRAF-2) 1D00 GB S56163 Ye, H., et al., Mol
Cell, 4(3): 321-30 (TRAF-2) (TRAF-2) (2.0) (1999). TRAF-C NA SWS
Q13114 NA (TRAF-3?) (TRAF-3) TRAF-D NA GB XP_008483 NA (TRAF-4?)
(TRAF-4) TRAF-E NA GB XP_010656 NA (TRAF-5?) (TRAF-5) VEGF 1FLT SWS
P15692 Wiesmann, C., et al., Cell, 91(5): 695-704 (1997). Mineral
NA SWS P08235 NA Corticoid R. Estrogen 3ERD SWS P03372 Shiau, A.
K., Barstad, D., Loria, P. M., Receptor Cheng, L., Kushner, P. J.,
Agard, D. A., Greene, G. L., Cell, 95(7): 927-37 (1998).
Progesterone 1A28 SWS P06401 Williams, S. P., Sigler, P. B, Nature,
Rec. 393(6683): 392-6 (1998). NF-kappa-B-1 SWS P19838 P53 NA SWS
P04637 NA Y1CQ GB AAA59989 Kussie, P. H., et al., (2.3) Science,
74(5289): 948-53 (1996). MDM2 1YCR SWS Q00987 Kussie, P. H., et
al., Science, 74(5289): 948-53 (1996). STAT6 NA SWS P42226 NA
IL4R-alpha NA SWS P24394 NA IL6R-alpha NA SWS P08887 NA IL6R-beta
1BQU SWS P40189 Bravo, J., Staunton, D., Heath, J. K., chain Jones,
E. Y., EMBO J, 17(6): 1665-74 (1998). IL5R-alpha NA SWS Q01344 NA
IL7R NA SWS P16871 NA IL2R-alpha NA SWS P01589 NA IL2R-beta NA SWS
P14784 NA HIV GP41 1AIK SWS P19551 Chan, D. C., Fass, D., Berger,
J. M., Kim, P. S., Cell, 89(2): 263-73 (1997). HIV GP41 1AIK SWS
P04582 Chan, D. C., Fass, D., Berger, J. M., Kim, P. S., Cell,
89(2): 263-73 (1997). HIV GP41 SWS P03378 HIV GP41 SWS P03375 HIV
GP41 SWS P04582 HIV GP41 SWS P12488 HIV GP41 SWS P03377 HIV GP41
SWS P05879 HIV GP41 SWS P04581 HIV GP41 SWS P04578 HIV GP41 SWS
P04624 HIV GP41 SWS P12489 HIV GP41 SWS P20871 HIV GP41 SWS P31819
HIV GP41 SWS Q70626 HIV GP41 SWS P04583 HIV GP41 SWS P19551 HIV
GP41 SWS P05577 HIV GP41 SWS P18799 HIV GP41 SWS P20888 HIV GP41
SWS P03376 HIV GP41 SWS P04579 HIV GP41 SWS P19550 HIV GP41 SWS
P19549 HIV GP41 SWS P05878 HIV GP41 SWS P31872 HIV GP41 SWS P05880
HIV GP41 SWS P35961 HIV GP41 SWS P12487 HIV GP41 SWS P04580 HIV
GP41 SWS P05882 HIV GP41 SWS P05881 HIV GP41 SWS P18094 HIV GP41
SWS P24105 HIV GP41 SWS P17755 HIV GP41 SWS P15831 HIV GP41 SWS
P18040 HIV GP41 SWS Q74126 HIV GP41 SWS P05883 HIV GP41 SWS P04577
HIV GP41 SWS P32536 HIV GP41 SWS P12449 HIV GP41 SWS P20872 c-mal
NA GB NP_071884 NA; T-cell differentiation protein NA GB CAA54102
NA NA GB XP_017128 NA Mal NA SWS P21145 NA; T-LYMPHOCYTE
MATURATION-ASSOCIATED PROTEIN NA SWS P01732 NA; T-LYMPHOCYTE
DIFFERENTIATION ANTIGEN T8/CD8(?) Her-1 NA SWS P34704 NA: Cell
Signaling in C. elegans Sex Determination Her-2 NA SWS P04626 NA;
RECEPTOR PROTEIN-TYROSINE KINASE ERBB-2 E2F-1 NA SWS Q01094 NA
E2F-2 NA SWS Q14209 NA E2F-3 NA SWS O00716 NA E2F-4 NA SWS Q16254
NA E2F-5 NA SWS Q15329 NA E2F-6 NA SWS O75461 NA Cyclin A 1QMZ SWS
P20248 Brown, N. R., et al., Nat Cell Biol., 1(7): 438-43 (1999).
mTOR/FRAP 1NSG SWS P42345 Liang, J., et al., Acta Crystall D Biol
Crystall, 55 (Pt 4): 736-44 (1995). Survivin 1F3H SWS O15392
Verdecia, M. A., et al., Nat Struct Biol., 7(7): 602-8 (2000).
FGF-1 1EV2 SWS P05230 Plotnikov, A. N., et al., Cell., 101(4):
413-24 (2000).(Heparin Binding Growth Factor I) Basic FGF 1FGK SWS
P11362 Mohammadi, M., et al., Cell, 86(4): 577-87 Rec. I
(1996).(Basic FGF Rec. I) FGF-2 1CVS SWS P09038 Plotnikov, A. N.,
et al., Cell, 98(5): 641-50 (1999). FGF-3 NA SWS P11487 NA FGF-4 NA
SWS P08620 NA FGF-5 NA SWS P12034 NA FGF-6 NA SWS P10767 NA FGF-7
NA SWS P21781 NA FGF-8 NA SWS P55075 NA FGF-9 1IHK SWS P31371
Plotnikov, A. N., et al., J Biol Chem., 276(6): 4322-9 (2001). PARP
NA SWS P09874 NA PDGF-alpha NA SWS P04085 NA PDGF-beta NA SWS
P01127 NA C5a receptor NA SWS P21730 NA CCR5 NA SWS P51681(CC NA
Chemo R-V) GPR14/Urote NA SWS Q9UKP6 NA nsin IIR) Tissue Factor
2HFT SWS P13726 Muller, Y. A., et al., J Mol Biol, 256(1): 144-59
(1996). Factor VII 1JBU SWS P08709 Eigenbrot, C., et al.,
Structure, 9: 627 (2001). Histamine H3 NA GB CAC39434 NA rec.
Neurokinin-1 NA GB SPHUB NA orexin NA SWS O43613 NA receptor-1
orexin NA SWW O43614 NA receptor-2 CD-3 delta NA SWS P04234 NA
chain CD-3 epsilon NA SWS P07766 NA chain CD-3 gamma NA SWS P09693
NA chain CD-3 zeta NA SWS P20963 NA chain CD-4 1CDJ SWS P01730 Wu,
H., et al., Proc Natl Acad Sci USA, 93(26): 15030-5 (1996).
TGF-alpha NA SWS P01135 NA TGF-beta-1 NA SWS P01137 NA TGF-beta-2
NA SWS P08112 NA TGF-beta-3 NA SWS P10600 NA TGF-beta-4 NA SWS
O00292 NA GRB2 1GRI SWS P29354 Maignan S, et al., Science, (3.1)
268(5208): 291-3 (1995). 1ZFP SWS P29354 Rahuel, J., et al., J Mol
Biol, (1.8) 279(4): 1013-22 (1998). 1BMB SWS P29354 Ettmayer, P.,
et al., J Med Chem, (1.8) 42(6): 971-80 (1999). LCK 1LKK SWS Tong,
L., et al., J Mol Biol, 256(3): 601-10 P06239; (2nd = P07100)
(1996). SRC 2SRC SWS P12931 Xu, W., et al., Mol Cell., 3(5): 629-38
(1999). TRAFs? NA SWS Q13077 NA (TRAF-1) 1CZZ GB S56163 Ye, H., et
al., Mol Cell, 4(3): 321-30 (TRAF-2) (TRAF-2) (2.7) (1999). 1CZY GB
S56163 Ye, H., et al., Mol Cell, 4(3): 321-30 (TRAF-2) (TRAF-2)
(2.0) (1999). 1D00 GB S56163 Ye, H., et al., Mol Cell, 4(3): 321-30
(TRAF-2) (TRAF-2) (2.0) (1999). NA SWS Q12933 NA (TRAF-2) 1FLK GB
Q13114 Ni, C.-Z., et al., Proc Natl Acad Sci USA., (TRAF-3)
(TRAF-3) (2.8) 97(19): 10395-9 (2000). BAX/BCL-2 NA SWS Q07812 NA
(BAX alpha) NA SWS Q07814 NA (BAX beta) NA SWS Q07815 NA (BAX
gamma) NA SWS P55269 NA (BAX delta) NA SWS P10415 NA (BCL-2) IgE
1F6A (3.5) SWS P01854 Garman, S. C., et al., T. S., Nature., (IgE
chain C) 406(6793): 259-66 (2000). IgER NA SWS P06734 NA (IgE Fc
Receptor) 1F6A (3.5) SWS P12319 Garman, S. C., et al., T. S.,
Nature., (IgE Fc Rec. 406(6793): 259-66 (2000). alpha) 1F2Q (2.4)
SWS P12319 Garman, S. C., Kinet, J. P., Jardetzky, T. S., (IgE Fc
Rec. Cell, 95(7): 951-61 (1998). alpha) NA SWS Q01362 NA (IgE
FcRec. Beta) NA SWS P30273 NA (IgE FcRec. Gama) Rhinovirus NA SWS
P03303 NA Protease (HRV-14 polyprot.) NA SWS P12916 NA (HRV-1B)
1CQQ SWS P04936 Matthews, D., et al., Proc Natl Acad Sci (HRV-2)
(1.85) USA, 96(20): 11000-7 (1999). NA SWS P07210 NA (HRV-89) 1C8M
SWS Q82122 Chakravarty, S., et al., to be published (HRV-16)
B7/CD28LG/ 1DR9 SWS P33681 Ikemizu, S., et al., Immunity. 2000 CD80
Jan; 12(1): 51-60. CD28 NA SWS P10747 NA APAF1 NA SWS O14727 NA
[0088] 3. Site(s) of Interest
[0089] Broadly, the "site of interest" on a particular target, such
as a Target Biological Molecule (TBM), is defined by the residues
that are involved in binding of the target to a molecule with which
it forms a natural complex in vivo or in vitro. If the target is a
peptide, polypeptide, or protein, the site of interest is defined
by the amino acid residues that participate in binding to (usually
by non-covalent association) to a ligand of the target.
[0090] When, for example, the target biological molecule is a
protein that exerts its biological effect through binding to
another protein, such as with hormones, cytokines or other proteins
involved in signaling, it may form a natural complex in vivo with
one or more other proteins. In this case, the site of interest is
defined as the critical contact residues involved in a particular
protein:protein binding interface. Critical contact residues are
defined as those amino acids on protein A that make direct contact
with amino acids on protein B, and when mutated to alanine decrease
the binding affinity by at least 10 fold and preferably at least 20
fold, as measured with a direct binding or competition assay (e.g.
ELISA or RIA). See (A Hot Spot of Binding Energy in a
Hormone-Receptor Interface by Clackston and Wells Science
267:383-386 (1995) and Cunningham and Wells J. Mol. Biol,
234:554-563 (1993)). Also included in the definition of a site of
interest are amino acid residues from protein B that are within
about 4 angstroms of the critical contact residues identified in
protein A.
[0091] Scanning amino acid analysis can be employed to identify one
or more amino acids along a contiguous sequence. Among the
preferred scanning amino acids are relatively small, neutral amino
acids. Such amino acids include alanine, glycine, serine, and
cysteine. Alanine is typically a preferred scanning amino acid
among this group because it eliminates the side-chain beyond the
beta-carbon and is less likely to alter the main-chain conformation
of the variant (Cunningham and Wells, Science, 244: 1081-1085
(1989)). Alanine is also typically preferred because it is the most
common amino acid. Further, it is frequently found in both buried
and exposed positions (Creighton, The Proteins, (W.H. Freeman &
Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)). If alanine
substitution does not yield adequate amounts of variant, an
isoteric amino acid can be used.
[0092] When the target biological molecule is an enzyme, the site
of interest can include amino acids that make contact with, or lie
within, about 4 angstroms of a bound substrate, inhibitor,
activator, cofactor or allosteric modulator of the enzyme. By way
of illustration, when the enzyme is a protease, the site of
interest would include the substrate binding channel from P4 to
P4', residues involved in catalytic function (e.g. the catalytic
triad) and any cofactor (e.g. Zn) binding site. For protein
kinases, the site of interest would include the substrate-binding
channel (as above) in addition to the ATP binding site. For
dehydrogenases, the site of interest would include the substrate
binding region as well as the site occupied by NAD/NADH. In
hydrolases such as PDE4, the site of interest would include all
residues contacting the cAMP substrate, as well as residues
involved in binding the catalytic divalent cations (Xu, R. X. et
al. Science 288:1822-1825 (2000)).
[0093] For an allosterically regulated enzyme, such as glycogen
phosphorylase B, the site of interest includes all residues in the
substrate binding region, residues in contact with the natural
allosteric inhibitor glucose-6-phosphate, and residues in novel
allosteric sites such as those identified in binding other
inhibitors such as CP320626 (Oikonomakos N G, et al. Structure Fold
Des 8:575-584 (2000)).
[0094] The TBM's either contain, or are modified to contain, a
reactive residue at or near a site of interest. Preferably, the
TBM's contain or are modified to contain a thiol-containing amino
acid residue at or near a site of interest. In this case, after a
TBM is selected, the site of interest is calculated. Once the site
of interest is known, a process of determining which amino acid
residue within, or near, the site of interest to modify is
undertaken. For example, one preferred modification results in
substituting a cysteine residue for another amino acid residue
located near the site of interest.
[0095] The choice of which residue within, or near, the site of
interest to modify is determined based on the following selection
criteria. First, a three dimensional description of the TBM is
obtained from one of several well-known sources. For example, the
tertiary structure of many TBMs has been determined through x-ray
crystallography experiments. These x-ray structures are available
from a wide variety of sources, such as the Protein Databank (PDB)
which can be found on the Internet at http://www.rcsb.org. Tertiary
structures can also be found in the Protein Structure Database
(PSdb) which is located at the Pittsburg Supercomputer Center at
http://www.psc.com.
[0096] In addition, the tertiary structure of many proteins, and
protein complexes, has been determined through computer-based
modeling approaches. Thus, models of protein three-dimensional
conformations are now widely available.
[0097] Once the three dimensional structure of the TBM is known, a
measurement is made based on a structural model of the wild type,
or a variant form, of the target biological molecule from any atom
of an amino acid within the site of interest across the surface of
the protein for a distance of approximately 10 angstroms. Variant,
which have been modified to contain the desired reactive groups
(e.g. thiol groups, or thiol-containing residues) are based on the
identification of one or more wild-type amino acid(s) on the
surface of the target biological molecule that fall within that
approximate 10-angstrom radius from the site of interest. For the
purposes of this measurement, any amino acid having at least one
atom falling within the about 10 angstrom radius from any atom of
an amino acid within the site of interest is a potential residue to
be modified to a thiol containing residue.
[0098] Preferred residues for modification are those that are
solvent-accessible. Solvent accessibility may be calculated from
structural models using standard numeric (Lee, B. & Richards,
F. M. J. Mol. Biol 55:379-400 (1971); Shrake, A. & Rupley, J.
A. J. Mol. Biol. 79:351-371 (1973)) or analytical (Connolly, M. L.
Science 221:709-713 (1983); Richmond, T. J. J. Mol. Biol. 178:63-89
(1984)) methods. For example, a potential cysteine variant is
considered solvent-accessible if the combined surface area of the
carbon-beta (CB), or sulfur-gamma (SG) is greater than 21
.ANG..sup.2 when calculated by the method of Lee and Richards (Lee,
B. & Richards, F. M. J. Mol. Biol 55:379-400 (1971)). This
value represents approximately 33% of the theoretical surface area
accessible to a cysteine side-chain as described by Creamer et al.
(Creamer, T. P. et al. Biochemistry 34:16245-16250 (1995)).
[0099] It is also preferred that the residue to be mutated to
cysteine, or another thiol-containing amino acid residue, not
participate in hydrogen-bonding with backbone atoms or, that at
most, it interacts with the backbone through only one hydrogen
bond. Wild-type residues where the side-chain participates in
multiple (>1) hydrogen bonds with other side-chains are also
less preferred. Variants for which all standard rotamers (chi1
angle of -60.degree., 60.degree., or 180.degree.) can introduce
unfavorable steric contacts with the N, CA, C, O, or CB atoms of
any other residue are also less preferred. Unfavorable contacts are
defined as interatomic distances that are less than 80% of the sum
of the van der Waals radii of the participating atoms.
[0100] Wild-type residues that fall within highly flexible regions
of the protein are less preferred. Within structures derived from
x-ray data, highly flexible regions can be defined as segments
where the backbone atoms possess weak electron density or high
temperature factors (>4 standard deviations above the mean
temperature factor for the structure). Within structures derived
from NMR data, highly flexible regions can be defined as segments
possessing <5 experimental restraints (derived from distance,
dihedral coupling, and H-bonding data) per residue, or regions
displaying a high variability (>2.0 A.sup.2 RMS deviation) among
the models in the ensemble. Additionally, residues found on convex
"ridge" regions adjacent to concave surfaces are more preferred
while those within concave regions are less preferred cysteine
residues to be modified. Convexity and concavity can be calculated
based on surface vectors (Duncan, B. S. & Olson, A. J.
Biopolymers 33:219-229 (1993)) or by determining the accessibility
of water probes placed along the molecular surface (Nicholls, A. et
al. Proteins 11:281-296 (1991); Brady, G. P., Jr. & Stouten, P.
F. J. Comput. Aided Mol. Des. 14:383-401 (2000)). Residues
possessing a backbone conformation that is nominally forbidden for
L-amino acids (Ramachandran, G. N. et al. J. Mol. Biol. 7:95-99
(1963); Ramachandran, G. N. & Sasisekharahn, V. Adv. Prot.
Chem. 23:283-437 (1968)) are less preferred targets for
modification to a cysteine. Forbidden conformations commonly
feature a positive value of the phi angle.
[0101] Other preferred variants are those which, when mutated to
cysteine and linked via a disulfide bond to an alkyl tether, would
possess a conformation that directs the atoms of that tether
towards the site of interest. Two general procedures can be used to
identify these preferred variants. In the first procedure, a search
is made of unique structures (Hobohm, U. et al. Protein Science
1:409-417 (1992)) in the Protein Databank (Berman, H. M. et al.
Nucleic Acids Research 28:235-242 (2000)) to identify structural
fragments containing a disulfide-bonded cysteine at position j in
which the backbone atoms of residues j-1, j, and j+1 of the
fragment can be superimposed on the backbone atoms of residues i-1,
i, and i+1 of the target molecule with an RMSD of less than 0.75
A.sup.2. If fragments are identified that place the CB atom of the
residue disulfide-bonded to the cysteine at position j closer to
any atom of the site of interest than the CB atom of residue i
(when mutated to cysteine), position i is considered preferred. In
an alternative procedure, the residue at position i is
computationally "mutated" to a cysteine and capped with an S-Methyl
group via a disulfide bond.
[0102] Further details of identifying site(s) of interest on the
targets of the invention are provided in co-pending application
Ser. No. 60/310,725, filed on Aug. 7, 2001, the entire disclosure
of which is hereby expressly incorporated by reference.
[0103] 4. Small Molecule Extender (SME)
[0104] (A) Static SME
[0105] In one embodiment of the invention the SME forms a "static"
or irreversible covalent bond through the nucleophile or
electrophile, preferably nucleophile, on the TBM, thereby forming
an irreversible TBM-SME complex. This method is illustrated in FIG.
2. Optionally the SME also forms a non-covalent bond with a first
site of interest on the TBM. Additionally the SME contains a second
functional group capable of forming a reversible bond with a
library member of a library of small organic molecules, each
molecule having a functional group capable of forming a reversible
bond with the second functional group of the SME. The TBM-SME
complex and library are subjected to conditions wherein the library
member having affinity, preferably the highest affinity, for the
second site of interest on the TBM forms a reversible bond with the
TBM-SME complex.
[0106] Preferred TBM's are proteins and the preferred nucleophiles
on the TBM's suitable for forming an irreversible TBM-SME complex
include --SH, --OH, --NH.sub.2 and --COOH usually arising from side
chains of cys, ser or thr, lys and asp or glu respectively. TBM's
may be modified (e.g. mutants or derivatives) to contain these
nucleophiles or may contain them naturally. For example, cysteine
proteases (e.g. Caspases, especially 1, 3, 8 and 9; Cathesepins,
especially S and K etc.) and phosphotases (e.g. PTP.alpha., PTP1B,
LAR, SHP1,2, PTP.beta. and CD45) are examples of suitable proteins
containing naturally occurring cystiene thiol nucleophiles.
Derivatizing such TBM's with a SME to produce a static TBM-SME
complex and its reaction with a library member is illustrated
below. 1
[0107] Here, the nucleophile on the TBM is the sulfur of a thiol,
usually a cysteine, which is reacted with 2, a SME containing a
substituent G capable of forming an irreversible (under conditions
that do not denature the target) covalent bond and a free thiol,
protected thiol or derivatized thiol SR'. Preferably G is a group
capable of undergoing SN2-like attack by the thiol or forming a
Michael-type adduct with the thiol to produce the irreversible
reaction product 3 of that attack having a new covalent linkage
--SG'-. The following are representative examples of G groups
capable of undergoing SN2-like or Michael-type addition.
[0108] 1) .alpha.-halo acids: F, Cl and Br substituted .alpha. to a
COOH, PO.sub.3H.sub.2 or P(OR)O.sub.2H acid that is part of the SME
can form a thioether with the thiol of the TBM. Simple examples of
such a G-SME-SR' are; 2
[0109] where X is the halogen and R' is H, SCH.sub.3,
S(CH.sub.2).sub.nA, where A is OH, COOH, SO.sub.3H, CONH.sub.2 or
NH.sub.2 and n is 2 or 3.
[0110] 2) Fluorophos(phon)ates: These can be Sarin-like compounds
which react readily with both SH and OH nucleophiles. For example,
cys 215 of PTP1B can be reacted with a simple G-SME-SR' represented
by the following: 3
[0111] Here the phenyl ring represents a simplified SME, R is a
substituted or unsubstituted loweralkyl and R' is as defined above.
These compounds form thiophos(phon)ate SME's with the thiol
nucleophile. These compounds also are capable of forming static
TBM-SME's with naturally occurring --OH from serine or threonine
phosphatases or .beta.-lactamases.
[0112] 3) Epoxides, aziridines and thiiranes: SME's containing
these reactive functional groups are capable of undergoing SN2 ring
opening reactions with --SH, --OH and --COOH nucleophiles.
Preferred examples of the latter are aspartyl proteases like
.beta.-secretase (BASE). Preferred generic examples of epoxides,
aziridines and thiiranes are shown below. 4
[0113] Here, R' is as defined above, R is usually H or lower alkyl
and R" is lower alkyl, lower alkoxy, OH, NH.sub.2 or SR'. In the
case of thiiranes the group SR' is optionally present because upon
nucleophilic attack and ring opening a free thiol is produced which
may be used in the subsequent extended tethering reaction.
[0114] 4) Halo-methyl ketones/amides: These compounds have the form
--(C.dbd.O)--CH.sub.2--X. Where X may be a large number of good
leaving groups like halogens, N.sub.2, O--R (Where R may be
substituted or unsubstituted heteroaryl, Aryl, alkyl,
--(P.dbd.O)Ar.sub.2, --N--O--(C.dbd.O) aryl/alkyl, --(C.dbd.O)
aryl/alkyl/alkylaryl and the like), S-Aryl, S-heteroaryl and vinyl
sulfones. 5
[0115] Fluromethylketones are simple examples of this class of
activated ketones which result in the formation of a thioether when
reacted with a thiol containing protein. Other well known examples
include acyloxymethyl ketones like benzoyloxymethyl ketone,
aminomethyl ketones like phenylmethylaminomethyl ketone and
sulfonylaminomethyl ketones. These and other types of suitable
compounds are reviewed in J. Med. Chem. 43(18) p 3351-71, Sep. 7,
2000.
[0116] 5) Electrophilic aromatic systems: Examples of these include
7-halo-2,1,3-benzoxadiazoles and ortho/para nitro substituted
halobenzenes. 6
[0117] Compounds of this type form arylalkylthioethers with TBM's
containing a thiol.
[0118] 6) Other suitable SN2 like reactions suitable for formation
of static covalent bonds with TBM nucleophiles include formation of
a Schiff base between an aldehyde and the amine group of lysine an
enzymes like DNA repair proteins followed by reduction with for
example NaCNBH.sub.4. 7
[0119] 7) Michael-type additions: Compounds of the form
--RC.dbd.CR-Q, or --C.ident.C-Q where Q is C(.dbd.O)H, C(.dbd.O)R
(including quinines), COOR, C(.dbd.O)NH.sub.2, C(.dbd.O)NHR, CN,
NO.sub.2, SOR, SO.sub.2R, where each R is independently substituted
or unsubstituted alkyl, aryl, hydrogen, halogen or another Q can
form Michael adducts with SR (where R is H, glutathione or
S-loweralkyl substituted with NH.sub.2 or OH), OH and NH.sub.2 on
the TBM.
[0120] 8) Boronic acids: These compounds can be used to label ser
or thr hydroxyls to form TBM-SME complexes of the form shown below:
8
[0121] where R' is as defined above
[0122] In each of the foregoing cases a "static" or irreversible
covalent bond is formed through the nucleophile on the TBM
producing an irreversible TBM-SME complex containing a thiol or
protected thiol. These complexes are then exposed to a library of
thiol or disulfide containing organic compounds in the presence of
a reducing agent (e.g. mercaptoethanol) for selection of a small
molecule ligand capable of binding a second binding site on the
TBM.
[0123] As noted above, in this static approach, the SME may, but
does not have to, include a portion that has binding affinity (i.e.
is capable of bonding to) a first site of interest on the TBM. Even
if the SME does not include such portion, it must be of appropriate
length and flexibility to ensure that the ligand candidates have
free access to the second site of interest on the target.
[0124] (B) Dynamic SME
[0125] In another embodiment of the invention the SME is a double
reversible covalent bond SME ("double disulfide" extender), that
is, this SME is bifunctional and contains two functional groups
(usually disulfide) capable of forming reversible covalent bonds.
This SME forms a "dynamic" or first reversible covalent bond
through a first functional group on the SME with the nucleophile on
the TBM, thereby forming a reversible TBM-SME complex (7 below).
Optionally the SME also forms a non-covalent bond with a first site
of interest on the TBM (the portion of the SME that forms a
non-covalent bond with the TBM is referred to herein as SME').
Additionally the SME contains or is modified to contain a second
functional group capable of forming a second reversible bond with a
library member of a second library of small organic molecules, each
molecule having a functional group capable of forming a reversible
bond with the first or second functional group of the SME. The
TBM-SME complex and the second library are subjected to conditions
wherein the library member having the highest affinity for a second
site of interest on the TBM forms a reversible bond with the
TBM-SME complex (8 below). Preferably the covalent bonds are
disulfides, which may be reversible in the presence of a reducing
agent. 9
[0126] The dynamic extended tethering process is illustrated in
FIG. 3 where a TMB containing or modified to contain a thiol or
protected thiol is incubated with a first library of small organic
molecules containing a thiol or protected thiol (a
disulfide-containing monophore) under conditions, such as with a
reducing agent, wherein at least one member of the library forms a
disulfide bond linking the selected library member with the TBM.
Optionally this process is repeated with a library of TBM's
differing from one another by the location of the thiol or
protected thiol, i.e. different cysteine mutants of the same
protein. Preferably each member of the small molecule library
differs in molecular weight from each of the other library members.
Preferably the small molecule library contains from 1-100 members,
more preferable from 5-15 and most preferably about 10 members.
Optionally the selected small molecule library member (selected
monophore) also forms a noncovalent bond with a first site of
interest on the TBM. The selected monophore, or a derivative
thereof, is then modified to contain a second thiol or protected
thiol thereby forming a "double disulfide" extender. This synthetic
double disulfide extender is then incubated with the TBM in the
presence of a second library of small organic molecules containing
a thiol or protected thiol (the library may be the same or
different from the first library) under conditions, such as with a
reducing agent like mercaptoethanol, wherein at least one member of
the second library forms a disulfide bond linking the selected
library member with the TBM through the double disulfide extender
as shown in 8 above. Optionally thereafter a diaphore is
synthesized based on the two selected library members
(monophores).
[0127] Two basic strategies exist for synthesizing a "double
disulfide" extender. In the first, synthesis of the dynamic
extender proceeds generically, that is by modification of the
monophore linker without any modification of the portion of the
monophore that forms a non-covalent bond with the TBM. By way of
illustration, the extender usually arises from the screening of a
disulfide monophore library as shown in FIG. 3. A typical monophore
selected from the library or pool will contain a linker of 2 or 3
methylene units between the disulfide that links the monophore to
the TBM cysteine and the portion of the monophore that binds
non-covalently to the first site of interest on the TBM. This
monophore linker can be derivatized as shown below to produce a
double disulfide extender in which the "R" or variable group of the
monophore remains invariant and becomes the portion of the extender
(SME') that binds non-covalently with the first site of interest on
the TBM. 10
[0128] Here the monophore is derivatized either at the methylene
nearest the cysteamine nitrogen to produce dynamic double disulfide
extender 1 or at the cysteamine nitrogen itself to produce the
symmetrical dynamic double disulfide extender 2.
[0129] Alternatively, when the monophore is a 3-mercaptopropionic
acid derivative the alpha carbon can be derivatized to produce a
generic dynamic double disulfide extender of the form shown in 3
below. 11
[0130] Optionally the amide nitrogen may be derivatized with an
acyl or sulfonyl to produce an extender of the form shown in 4
above.
[0131] A second strategy involves derivatizing the portion of the
monophore that binds non-covalently to the first site of interest
on the TBM. The derivatization is preferably carried out at a site
that minimally alters the binding of the monophore to the first
site of interest as illustrated below. 12
[0132] Here the dynamic tether is shown bound to the TBM thiol
forming the TBM-SME complex, where R' is the cysteamine radical.
This complex can then be contacted with a disulfide monophore or
library of disulfide monophores to obtain a linked compound having
a higher affinity for the TBM than either the SME or selected
monophore alone.
[0133] A second example of a SME designed form a disulfide
monophore that binds to the TBM is shown below. This dynamic SME
can be contacted with the TBM in the presence of one or more
disulfide monophores to form a covalent TBM-SME-monophore complex
where the SME has an affinity for the first site of interest and
the monophore has an affinity for the second site of interest on
the TBM. 13
[0134] Detection and identification of the structure of the
TBM-SME-monophore complex can be carried out by mass spectrometry
or inhibition in a functional assay (e.g. ELISA, enzyme assay
etc.).
[0135] SME's are often customized for a particular TBM or family of
TBM's. For example quinazoline derivatives are capable of forming
static or dynamic extenders with the EGF receptor or an "RD"
kinase. In the case of the EGF receptor, cys 773 is a suitable
nucleophile for either a static or dynamic quinazoline extender as
shown below; 14
[0136] where R.sup.1 is linked to cys 773 through a Michael
acceptor or disulfide,
[0137] R'0 is selected from 15
[0138] R.sup.2 is --(CH.sub.2).sub.n--SR' and
.dbd.C(.dbd.O)--(CH.sub.2).s- ub.n--SR';
[0139] R.sup.3, R.sup.4 and R.sup.5 are --O--(CH.sub.2).sub.n--SR'
and --(CH.sub.2).sub.n--SR';
[0140] R.sup.6 are; --(CH.sub.2).sub.n--SR'; where n is 1, 2, or 3
and
[0141] R' is H, a disulfide or a thiol protecting group.
[0142] Phosphotyrosine (P-tyr), phosphoserine (P-ser) and
phoshpothreonine (P-thr) mimetics or surrogates may be used as
extenders in the present invention to identivy fragments that
interact with subsites nearby to improve specificity or affinity
for a target phosphotase. Thus extended tethering using known
substrates or inhibitors as "anchors" to find nearby fragments by
standard covalent tethering with the extender is one preferred
embodiment of the instant invention.
[0143] Phosphotyrosine (P-tyr) mimetics are examples of SME's that
may be customized for phosphotases like PTP-1B, LAR etc. Known
PTP-1B P-tyr mimetics derivitized with mercapto-propanoic acid
and/or cystaeamine or the protected forms thereof, shown below,
bind to the active site of a PTP-1B cys mutant. 16
[0144] Such a compound may be used as a dynamic extender to select
a second fragment by covalent tethering as described above. The
compound shown above when bound to the target and titrated against
.beta.-mercaptoethanol (BME) displays a BME.sub.50 (the
concentration of .beta.-mercaptoethanol that, at equalibrium, is
capable of displacing 50% of the bound compound from the target) of
about 2.5 mM. When using a dynamic extender it is preferred to
measure the BME.sub.50 for the dynamic extender and to screen for a
second fragment by covalent tethering at a total thiol
concentration (BME+library thiols) at or below the BME.sub.50 of
the dynamic extender. For example, with the dynamic extender shown
above having a BME.sub.50 of 2.5 mM, the total thiol concentration
in the second fragment screening step should be 2.5 mM or less and
more preferrabley about 2 fold less, e.g. about 1 mM or less.
Alternatively the dynamic extenter may be converted to a static
extender removing the second fragment screening total thiol
concentration issue. When converting a dynamic extender to a static
extender it is important to maintain the same atom count so that
non-covalent binding of the static extender to the target will not
be distorted. For similar reasons it is important to minimize
introduction of other other bulky atoms or groups. With these
factors in mind, the above dynamic extender may be converted into
the static extenders defined below. 17
[0145] where R.sup.1 is selected from 18
[0146] and where R.sup.2 is selected from 19
[0147] In still another embodiment of the invention, an extender
may be a peptide either reversibly or irreversibly bound to the
TBM. In this embodiment the peptide is from about 2-15 residues
long, preferable from 5 to 10 residues, and may be composed of
natural and/or artificial alpha amino acids. An example of such a
peptide extender is an alpha helical p53 fragment peptide (or
smaller known non-natural peptides) that are capable of binding to
the N-terminal domain of MDM2 in a deep hydrophobic cleft with nM
affinities. BCL-2 and BCL-xL are also known to contain deep
peptide-binding grooves analogous the MDM2. Peptides that bind to
these targets may also be useful peptide extenders according to the
present invention. For example, a fragment peptide of p53 may form
a reversible (e.g. disulfide) bond through an existing (e.g. cys)
thiol or an introduced thiol (introduced cys, cysteamine
derivatized with the carboxyl terminus or mercapto-propanoic acid
through the amino terminus) on the peptide with an existing or
introduced thiol on the TBM. In this case a TBM-peptide extender
complex will be formed which is capable of being used to select a
thiol or disulfide fragment from a subsequent covalent tether
screen. This dynamic peptide extender will have one other free or
protected thiol (e.g. one of the above not used to form the
TBM-peptide extender complex), which is contacted with a library of
thiol or protected thiol fragments under conditions suitable for
forming a covalent disulfide bond with a fragment having affinity
for the TBM. Optionally the peptide extender may be a static one
where an irreversible covalent bond is formed with a nucleophile or
electrophile on the TBM as described above. Optionally in this
embodiment, a photoaffinity label may be used to attach the peptide
extender to the TBM. As above, a free or protected thiol
pre-existing or introduced is used to form a disulfide in a
subsequent screen to find a small molecule fragment having affinity
for the TBM.
[0148] Such a peptide extender may also be a synthetic peptide such
as the "Z-WQPY" peptide where the TBM is the IL-1 receptor. Here,
the peptide FEWTPGYWQPYALPL or fragments, mutants or analogues
thereof can be used as a static or dynamic extender as described
above to discover fragments via covalent tethering, where the
disulfide tether is to or with the extender and the non-covalent
bond is between the selected fragment and the TBM.
[0149] Other chemistries available for forming a reversible or
irreversible covalent bond between reactive groups on a SME and a
target or ligand, respectively, or between two ligands, are well
known in the art, and are described in basic textbooks, such as,
e.g. March, Advanced Organic Chemistry, John Wiley & Sons, New
York, 4.sup.th edition, 1992. Reductive aminations between
aldehydes and ketones and amines are described, for example, in
March et al., supra, at pp. 898-900; alternative methods for
preparing amines at page 1276; reactions between aldehydes and
ketones and hydrazide derivatives to give hydrazones and hydrazone
derivatives such as semicarbazones at pp. 904-906; amide bond
formation at p. 1275; formation of ureas at p. 1299; formation of
thiocarbamates at p. 892; formation of carbamates at p. 1280;
formation of sulfonamides at p. 1296; formation of thioethers at p.
1297; formation of disulfides at p. 1284; formation of ethers at p.
1285; formation of esters at p. 1281; additions to epoxides at p.
368; additions to aziridines at p. 368; formation of acetals and
ketals at p. 1269; formation of carbonates at p. 392; formation of
denamines at p. 1264; metathesis of alkenes at pp. 1146-1148 (see
also Grubbs et al., Acc, Chem. Res. 28:446-453 [1995]); transition
metal-catalyzed couplings of aryl halides and sulfonates with
alkanes and acetylenes, e.g. Heck reactions, at p.p. 717-178; the
reaction of aryl halides and sulfonates with organometallic
reagents, such as organoboron, reagents, at p. 662 (see also
Miyaura et al., Chem. Rev. 95:2457 [1995]); organotin, and
organozinc reagents, formation of oxazolidines (Ede et al.,
Tetrahedron Letts. 28:7119-7122 [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.c50:416-422
[1972]), and the like. In particular, disulfide-containing small
molecule libraries may be made from commercially available
carboxylic acids and protected cysteamine (e.g.
mono-BOC-cysteamine) by adapting the method of Parlow et al., Mol.
Diversity 1:266-269 (1995), and can be screened for binding to
polypeptides that contain, or have been modified to contain,
reactive cysteines. All of the references cited in this section are
hereby expressly incorporated by reference.
[0150] While it is usually preferred that the attachment of the SME
does not denature the target, the TBM-SME complex may also be
formed under denaturing conditions, followed by refolding the
complex by methods known in the art. Moreover, the SME and the
covalent bond should not substantially alter the three-dimensional
structure of the target, so that the ligands will recognize and
bind to a site of interest on the target with useful site
specificity. Finally, the SME should be substantially unreactive
with other sites on the target under the reaction and assay
conditions.
[0151] 5. Detection and Identification of Ligands Bound to a
Target
[0152] The ligands bound to a target can be readily detected and
identified by mass spectroscopy (MS). MS detects molecules based on
mass-to-charge ratio (m/z) and thus can resolve molecules based on
their sizes (reviewed in Yates, Trends Genet. 16: 5-8 [2000]). A
mass spectrometer first converts molecules into gas-phase ions,
then individual ions are separated on the basis of m/z ratios and
are finally detected. A mass analyzer, which is an integral part of
a mass spectrometer, uses a physical property (e.g. electric or
magnetic fields, or time-of-flight [TOF]) to separate ions of a
particular m/z value that then strikes the ion detector. Mass
spectrometers are capable of generating data quickly and thus have
a great potential for high-throughput analysis. MS offers a very
versatile tool that can be used for drug discovery. Mass
spectroscopy may be employed either alone or in combination with
other means for detection or identifying the organic compound
ligand bound to the target. Techniques employing mass spectroscopy
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]; Siudzak, 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]).
[0153] However, the scope of the instant invention is not limited
to the use of MS. In fact, any other suitable technique for the
detection of the adduct formed between the biological target
molecule and the library member can be used. For example, one may
employ various chromatographic techniques such as liquid
chromatography, thin layer chromatography and likes 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
spectroscopy or separate from mass spectroscopy. 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. In yet another
embodiment, the formation of the new bonds liberates a labeled
probe, which can then be monitored. A simple functional assay, such
as an ELISA or enzymatic assay may also be used to detect binding
when binding of the extender or second fragment to the target
occurs in an area essential for what the assay measures (e.g.
binding to a "Hot Spot" in a protein:protein ELISA or binding in
the substrate binding pocket for an enzyme assay). Other techniques
that may find use for identifying the organic compound bound to the
target molecule include, for example, nuclear magnetic resonance
(NMR), capillary electrophoresis, X-ray crystallography, and the
like, all of which will be well known to those skilled in the
art.
[0154] 6. Preparation of Conjugate Molecules (e.g. Diaphores)
[0155] 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 uhsubstituted
or substituted, preferably with alkyl, alkoxyl, hydroxyalkyl or
hydroxyalkyl 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. The first organic compound that covalently bound to the
target biomolecule may itself possess a chemically reactive group
that provides a site for bonding to a second organic compound.
Alternatively, 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.
[0156] 7. Compounds of the Invention
[0157] The compounds of the present invention are characterized by
encompassing at least one, preferably at least two, ligands at
least one of which has been identified by the extended tethering
approach disclosed herein, and analogs of such compounds.
Accordingly, the compounds of the present invention encompass
numerous chemical classes, including but not limited to small
organic molecules, peptides, (poly)nucleotides, (oligo)saccharides,
etc. The ligands identified by the present methods typically serve
as lead compounds for the development of further variants and
derivatives designed by following well known techniques. In
particular, the ligands identified (including monophores,
diaphores, and more complex structures) are amenable to medicinal
chemistry and affinity maturation, and can be rapidly optimized
using structure-aided design. The present extended tethering
approach is superior over other known techniques, including
combinatorial chemistry, in that it allows further chemical
modifications focused on ligands which have already been shown to
to bind to different sites on a target, e.g. a TBM.
[0158] 8. Uses of Compounds Identified
[0159] The method of the present invention is a powerful technique
for generating drug leads, allows the identification of two or more
fragments that bind weakly or with moderate binding affinity to a
target at sites near one another, and the synthesis of diaphores or
larger molecules comprising the identified fragments (monophores)
covalently linked to each other to produce higher affinity
compounds. The diaphores or similar multimeric, compounds including
further ligand compounds, are valuable tools in rational drug
design, which can be further modified and optimized using medicinal
chemistry approaches and structure-aided design.
[0160] The diaphores identified in accordance with the present
invention and the modified drug leads and drugs designed therefrom
can be used, for example, to regulate a variety of in vitro and in
vivo biological processes which require or depend on the
site-specific interaction of two molecules. Molecules which bind to
a polynucleotide can be used, for example, to inhibit or prevent
gene activation by blocking the access of a factor needed for
activation to the target gene, or repress transcription by
stabilizing duplex DNA or interfering with the transcriptional
machinery.
[0161] 9. Pharmaceutical Compositions
[0162] The ligands identified in accordance with the present
invention, and compounds comprising such ligands, as well as
analogues of such compounds, can be used in pharmaceutical
compositions to prevent and/or treat a targeted disease or
condition. The target disease or condition depends on the
biological/physiological function of the target, e.g. TBM to which
the ligand or the compounds designed based on such ligand(s) binds.
Examples of such diseases and conditions are listed in the table of
TBM's above.
[0163] Suitable forms of pharmaceutical compositions, in part,
depend upon the use or route of entry, for example oral,
transdermal, inhalation, or by injections. Such forms should allow
the agent or composition to reach a target cell whether the target
cell is present in a multicellular host or in culture. For example,
pharmacological agents or compositions injected into the blood
stream should be soluble. Other factors are known in the art, and
include considerations such as toxicity and forms that prevent the
agent or composition from exerting its effect.
[0164] The active ingredient, when appropriate, can also be
formulated as pharmaceutically acceptable salts (e.g., acid
addition salts) and/or complexes. Pharmaceutically acceptable salts
are non-toxic at the concentration at which they are administered.
Pharmaceutically acceptable salts include acid addition salts such
as those containing sulfate, hydrochloride, phosphate, sulfonate,
sulfamate, sulfate, acetate, citrate, lactate, tartarate,
methanesulfonate, ethanesulfonate, benzenesulfonate,
p-toluenesulfonate, cyclohexylsulfonate, cyclohexylsulfamate an
quinate.
[0165] Pharmaceutically acceptable salts can be obtained from acids
such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfonic
acid, sulfamic acid, acetic acid, citric acid, lactic acid,
tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic
acid, benzenesulfonic acid, p-toluenesulfonic acid,
cyclobexylsulfonic acid, cyclohexylsulfamic acid, and quinic acid.
Such salts may be prepared by, for example, reacting the free acid
or base forms of the product with one or more equivalents of the
appropriate base or acid in a solvent or medium in which the salt
is insoluble, or in a solvent such as water, which is then removed
in vacuo or by freeze-drying or by exchanging the ions of an
existing salt for another ion on a suitable ion exchange resin.
[0166] Carriers or excipients can also be used to facilitate
administration of the compound. Examples of carriers and excipients
include calcium carbonate, calcium phosphate, various sugars such
as lactose, glucose, or sucrose, or types of starch, cellulose
derivatives, gelatin, vegetable oils, polyethylene glycols and
physiologically compatible solvents. The compositions or
pharmaceutical compositions can be administered by different routes
including, but not limited to, intravenous, intra-arterial,
intraperitoneal, intrapericardial, intracoronary, subcutaneous,
intramuscular, oral topical, or transmucosal.
[0167] The desired isotonicity of the compositions can be
accomplished using sodium chloride or other pharmaceutically
acceptable agents such as dextrose, boric acid, sodium tartarate,
propylene glycol, polyols (such as mannitol and sorbitol), or other
inorganic or organic solutes.
[0168] Techniques and ingredients for making, pharmaceutical
formulations generally may be found, for example, in Remington's
Pharmaceutical Sciences, 18.sup.th Edition, Mack Publishing Co.,
Easton, Pa. 1990. See also, Wang and Hanson "Parental Formulations
of Proteins and Peptides: Stability and Stabilizers," Journal of
Parental Science and technology, Technical Report No. 10, Supp.
42-2S (1988). A suitable administration format can be best
determined by a medical practitioner for each disease or condition
individually, and also in view of the patient's condition.
[0169] Pharmaceutical compositions are prepared by mixing the
ingredients following generally accepted procedures. For example,
the selected components can be mixed simply in a blender or other
standard device to produce a concentrated mixture which can then be
adjusted to the final concentration and viscosity by the addition
of water or thickening agent and possibly a buffer to control pH or
an additional solute to control tonicity.
[0170] The amounts of various compounds for use in the compositions
of the invention to be administered can be determined by standard
procedures. Generally, a therapeutically effective amount is
between about 100 mg/kg and 10.sup.-12 mg/kg depending on the age
and size of the patient, and the disease or disorder associated
with the patient. Generally, it is an amount between about 0.05 and
50 mg/kg of the individual to be treated. The determination of the
actual dose is well within the skill of an ordinary physician.
[0171] 10. Description of Preferred Embodiments
[0172] In a preferred embodiment, the methods of the present
invention are used to identify low molecular weight ligands that
bind to at least two different sites of interest on target proteins
through intermediary disulfide tethers formed between a first
ligand and the protein, and a reactive group on the first ligand
and a second ligand, respectively.
[0173] The low molecular weight ligands screened in preferred
embodiments of the invention will be, for the most part, small
chemical molecules that will 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. In one preferred embodiment, such small chemical molecules
are small organic molecules, other than polypeptides or
polynucleotides. In another preferred embodiment, the small organic
molecules are non-polymeric, i.e. are not peptide, polypeptides,
polynucleotides, etc.
[0174] Organic molecules may be 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., Sr. Louis, Mo., or may be obtained by chemical
synthesis. The methods of the present invention are preferably used
to screen libraries of small organic compounds carrying appropriate
reactive group, preferably thiol or protected thiol groups.
[0175] In recent years, combinatorial libraries, typically having
from dozens to hundreds of thousands of members, have become a
major tool for ligand discovery and drug development. In general,
libraries of organic compounds which find use herein will 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,
preferably at 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. in
separate microtiter well) or two or more members of the population
may be combined if methods for deconvolution are readily available.
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.
[0176] In a preferred embodiment, the target biological molecule
(TBM) is a polypeptide that contains or has been modified to
contain a thiol group, protected thiol group or reversible
disulfide bond. The TBM is then reacted with a Small Molecule
extender (SME), which includes a portion having affinity for a
first site of interest on the TBM and a group reactive with the
thiol, protected thiol or reversible disulfide bond on the TBM. As
discussed above, the linkage between the TBM and the SME may be
either an irreversible covalent bond ("static" extended tethering),
or a reversible covalent bond ("dynamic" extended tethering) to
form a TBM-SME complex. Whether the static or dynamic approach is
used, the TBM-SME complex is then used to screen a library of
disulfide-containing monophores to identify a library member that
has intrinsic affinity, most preferably the highest intrinsic
affinity, for a second binding site (site of interest) on the
target molecule. In a preferred embodiment, the reactive group on
the modified TBM is a free-thiol group contributed by the extender,
and the library is made up of small molecular weight compounds
containing reactive thiol group. For disulfide tethering to capture
the most stable ligand, the reaction must be under rapid exchange
to allow for equilibration. In a preferred embodiment, the reaction
is carried out in the presence of catalytic amount of a reducing
agent such as 2-mercaptoethanol. Thermodynamic equilibrium reached
in the presence of a reducing agent will favor the formation of
disulfide bond between thiol group of the extender on the modified
TBM and thiol group of a member of the library having intrinsic
affinity for the TBM. Thus, two different ligands with intrinsic
affinity for two different sites on the same TBM will be covalently
linked to form a diaphore. The diaphore will bind to the TBM with a
higher affinity than any of the constituent monophore units. The
monophore units in a diaphore may be from the same or different
chemical classes. By "same chemical class" is meant that each
monophore component is of the same chemical type, i.e., both are
aldehyde or amines etc.
[0177] In a particular embodiment, the target can be present on a
chip contacted with the ligand candidates. In this case, the
covalent bond linking the first ligand to the target may be formed
with the chip, in which case, the chip will become part of the
covalent bond, representing a special class of "Small Molecule
Extenders."
[0178] The library of the ligand candidates, e.g. small organic
molecule ligands, can be attached to a solid surface, e.g.
displayed on beads, for example as described in PCT publication WO
98/11436 published on Mar. 19, 1998. In a particular embodiment,
beads are modified to introduce reactive groups, e.g. a low level
of sulfhyrdyl groups. A library of ligand candidates is then
synthesized on the modified beads. Subsequently, the library is
incubated, under oxidizing conditions, with the target containing
or modified to contain a reactive group, e.g. a sulfhydryl group
such that s disulfide bond can be formed between the target and the
sulfhydryl on the bead. The beads are then washed in the presence
of a reducing agent, followed by incubation in the presence of a
sulfhydryl quenching agent, such as iodoacetate. The beads may then
be washed under denaturing conditions to remove any non-covalently
bound target.
EXAMPLES
[0179] The invention is further illustrated by the following,
non-limiting examples. Unless otherwise noted, all the standard
molecular biology procedures are performed according to protocols
described in (Molecular Cloning: A Laboratory Manual, vols. 1-3,
edited by Sambrook, J., Fritsch, E. F., and Maniatis, T., Cold
Spring Harbor Laboratory Press, 1989; Current Protocols in
Molecular Biology, vols. 1-2, edited by Ausbubel, F., Brent, R.,
Kingston, R., Moore, D., Seidman, J. G., Smith, J., and Struhl, K.,
Wiley Interscience, 1987).
[0180] The concept of basic tethering approach has been described
by Erlanson et al., supra, and in PCT Publication No. WO 00/00823.
The "extended tethering" approach is illustrated in this
application using caspase-3 as a target biological molecule (TBM).
Caspases are a family of cysteine proteases, that are known to
participate in the initiation and execution of programmed cell
death (apoptosis). The first caspase (now referred to as Caspase-1)
was originally designated as interleukin-1.beta.-converting enzyme
(ICE) (Thomburry et al., Nature 356:768-774 [1992]; Cerretti et
al., Science 356:97-100 [1992]). Subsequently a large number of
caspases have been identified and characterized forming a caspase
family. Presently there are at least 10 members in the family
(Caspase-1 to Caspase-10). Caspases are expressed in cells in an
enzymatically inactive form and become activated by proteolytic
cleavage in response to an apoptotic stimulus. The inactive
proenzyme form consists of a large and a small domain (subunit), in
addition to an inhibitory N-terminal domain. Caspase activation
involves the processing of the proenzyme into the large and small
subunits, which occurs internally within the molecule. Caspases are
activated either by self-aggregation and autoprocessing (as in the
initiation of apoptosis), or via cleavage by an activated upstream
caspase (as in the execution phase of apoptosis). For review, see,
for example, Cohen G. M. Biochem. J. 326: 1-16 (1997).
[0181] Based on a known tetrapeptide inhibitor of caspase (Ator and
Dolle, Current Pharmaceutical Design 1:191-210 (1995)), an extender
was synthesized: 2,6-Dichloro-benzoic acid
3-(2-acetylsulfanyl-acetylamino)-4- -carboxy-2-oxo-butyl ester
(shown as compound 5 in FIG. 4), the synthesis of which is
described in Example 2 below. A generic structure of extender is
shown in FIG. 4. Caspase was modified by reacting with the extender
(Example 3) and subsequently used as a biological target molecule
for screening of disulfide library prepared as described in Example
1, by using the extended tethering approach.
[0182] All commercially available materials were used as received.
All synthesized compounds were characterized by .sup.1H NMR [Bruker
(Billerica, Mass.) DMX400 MHz Spectrometer] and HPLC-MS
(Hewlett-Packard Series 1100 MSD).
Example 1
Disulfide Libraries
[0183] Disulfide libraries were synthesized using standard
chemistry from the following classes of compounds: aldehydes,
ketones, carboxylic acids, amines, sulfonyl chlorides, isocyanates,
and isothiocyanates. For example, the disulfide-containing library
members were made from commercially available carboxylic acids and
mono-N-(tert-butoxycarbonyl)-- protected cystamine
(mono-BOC-cystamine) by adapting the method of Parlow and coworkers
(Parlow and Normansell, Mol. Diversity 1: 266-269 [1995]). Briefly,
260 .mu.mol of each carboxylic acid was immobilized onto 130
.mu.mol equivalents of 4-hydroxy-3-nitrobenzophenone on polystyrene
resin using 1,3-diisopropylcarbodiimide (DIC) in
N,N-dimethylformamide (DMF). After 4 h at room temperature, the
resin was rinsed with DMF (.times.2), dichloromethane (DCM,
.times.3), and tetrahydrofuran (THF, .times.1) to remove uncoupled
acid and DIC. The acids were cleaved from the resin via amide
formation with 66 .mu.mol of mono-BOC protected cystamine in THF.
After reaction for 12 h at ambient temperature, the solvent was
evaporated, and the BOC group was removed from the uncoupled half
of each disulfide by using 80% trifluoroacetic acid (TFA) in DCM.
The products were characterized by HPLC-MS, and those products that
were substantially pure were used without further purification. A
total of 530 compounds were made by using this methodology.
[0184] Libraries were also constructed from mono-BOC-protected
cystamine and a variety of sulfonyl chlorides, isocyanates, and
isothiocyanates. In the case of the sulfonyl chlorides, 10 .mu.mol
of each sulfonyl chloride was coupled with 10.5 .mu.mol of
mono-BOC-protected cystamine in THF (with 2% diisopropyl ethyl
amine) in the presence of 15 mg of poly(4-vinyl pyridine). After 48
h, the poly(4-vinylpyridine) was removed via filtration, and the
solvent was evaporated. The BOC group was removed by using 50% TFA
in DCM. In the case of the isothiocyanates, 10 .mu.mol of each
isocyanate or isothiocyanate was coupled with 10.5 .mu.mol of
mono-BOC-protected cystamine in THF. After reaction for 12 h at
ambient temperature, the solvent was evaporated, and the BOC group
was removed by using 50% TFA in DCM. A total of 212 compounds were
made by using this methodology.
[0185] Finally, oxime-based libraries were constructed by reacting
10 .mu.mol of specific aldehydes or ketones with 10.5 .mu.mol of
HO(CH.sub.2).sub.2S--S(CH.sub.2).sub.2ONH.sub.2 in 1:1
methanol/chloroform (with 2% acetic acid added) for 12 h at ambient
temperature to yield the oxime product. A total of 448 compounds
were made by using this methodology.
[0186] Individual library members were redissolved in either
acetonitrile or dimethyl sulfoxide to a final concentration of 50
or 100 mM. Aliquots of each of these were then pooled into groups
of 8-15 discrete compounds, with each member of the pool having a
unique molecular weight.
Example 2
Extender (SME) Synthesis
[0187] For extended tethering approach, extender
(2,6-Dichloro-benzoic acid
3-(2-acetylsulfanyl-acetylamino)-4-carboxy-2-oxo-butyl ester, shown
as compound 5 in FIG. 4) was synthesized using a series of chemical
reactions as shown in FIG. 4, and described below.
Synthesis of 2-(2-Acetylsulfanyl-acetylamino)-succinic acid
4-tert-butyl ester (compound 2, FIG. 4)
[0188] Acetylsulfanyl-acetic acid pentafluorophenyl ester (1.6 g,
5.3 mmol) and H-Asp(OtBu)-OH (1 g, 5.3 mmol) were mixed in 20 ml of
dry dichloromethane (DCM). Then 1.6 ml of triethylamine (11.5 mmol)
was added, and the reaction was allowed to proceed at ambient
temperature for 3.5 hours. The organic layer was then extracted
with 3.times.15 ml of 1 M sodium carbonate, the combined aqueous
fractions were acidified with 100 ml of 1 M sodium hydrogensulfate
and extracted with 3.times.30 ml ethyl acetate. The combined
organic fractions were then rinsed with 30 ml of 1 M sodium
hydrogensulfate, 30 ml of 5 M NaCl, dried over sodium sulfate,
filtered, and evaporated under reduced pressure to yield 1.97 g of
a nearly colorless syrup which was used without further
purification. MW=305 (found 306, M+1).
Synthesis of
3-(2-Acetylsulfanyl-acetylamino)-5-chloro-4-oxo-pentanoic acid
tert-butyl ester (compound 3, FIG. 4)
[0189] The free acid (compound 2) was dissolved in 10 ml of dry
tetrahydrofuran (THF), cooled to 0.degree. C., and treated with
0.58 ml N-methyl-morpholine (5.3 mmol) and 0.69
isobutylchloroformate. Dense white precipitate immediately formed,
and after 30 minutes the reaction was filtered through a glass frit
and transferred to a new flask with an additional 10 ml of THF.
Meanwhile, diazomethane was prepared by reacting
1-methyl-3-nitro-1-nitrosoguanidine (2.3 g, 15.6 mmol) with 7.4 ml
of 40% aqueous KOH and 25 ml diethyl ether for 45 minutes at
0.degree. C. The yellow ether layer was then decanted into the
reaction containing the mixed anhydride, and the reaction allowed
to proceed while slowly warming to ambient temperature over a
period of 165 minutes. The reaction was cooled to 8.degree. C., and
1.5 ml of 4 N HCl in dioxane (6 mmol total) was added dropwise.
This resulted in much bubbling, and the yellow solution became
colorless. The reaction was allowed to proceed for two hours while
gradually warming to ambient temperature and then quenched with 1
ml of glacial acetic acid. The solvent was removed under reduced
pressure and the residue redissolved in 75 ml ethyl acetate, rinsed
with 2.times.50 ml saturated sodium bicarbonate, 50 ml 5 M NaCl,
dried over sodium sulfate, filtered, and evaporated to dryness
before purification by flash chromatography using 90:10 chloroform
: ethyl acetate to yield 0.747 g of light yellow oil (2.2 mmol, 42%
from (1)). Expected MW=337.7, found 338 (M+1).
Synthesis of 2,6-Dichloro-benzoic acid
3-(2-acetylsulfanyl-acetylamino)-4--
tert-butoxycarbonyl-2-oxo-butyl ester (compound 4, FIG. 4)
[0190] The chloromethylketone (compound 3) (0.25 g, 0.74 mmol) was
dissolved in 5 ml of dry N,N-dimethylformamide (DMF), to which was
added 0.17 g 2,6-dichlorobenzoic acid (0.89 mmol) and 0.107 g KF
(1.84 mmol). The reaction was allowed to proceed at ambient
temperature for 19 hours, at which point it was diluted with 75 ml
ethyl acetate, rinsed with 2.times.50 ml saturated sodium
bicarbonated, 50 ml 1 M sodium hydrogen sulfate, 50 ml 5 M NaCl,
dried over sodium sulfate, filtered, and dried under reduced
pressure to yield a yellow syrup which HPLC-MS revealed to be about
75% product and 25% unreacted (3). This was used without further
purification. Expected MW=492.37, found 493 (M+1).
Synthesis of 2,6-Dichloro-benzoic acid
3-(2-acetylsulfanyl-acetylamino)-4-- carboxy-2-oxo-butyl ester
(compound 5, FIG. 4)
[0191] The product of the previous step (compound 4) was dissolved
in 10 ml of dry DCM, cooled to 0.degree. C., and treated with 9 ml
trifluoroacetic acid (TFA). The reaction was then removed from the
ice bath and allowed to warm to ambient temperature over a period
of one hour. Solvent was removed under reduced pressure, and the
residue redissoved twice in DCM and evaporated to remove residual
TFA. The crude product was purified by reverse-phase high-pressure
liquid chromatography to yield 101.9 mg (0.234 mmol, 32% from (3))
of white hygroscopic powder. Expected MW=436.37, found 437 (M+1).
This was dissolved in dimethylsulfoxide (DMSO) to yield a 50 mM
stock solution.
Example 3
Modification of Caspase 3 with Extender
[0192] Caspase 3 was cloned, overexpressed, and purified using
standard techniques (Rotonda et al., Nature Structural Biology
3(7):619-625 (1996)). To 2 ml of a 0.2 mg/ml Caspase 3 solution was
added 10 ml of 50 mM 2,6-Dichloro-benzoic acid
3-(2-acetylsulfanyl-acetylamino)-4-carboxy-2- -oxo-butyl ester
(compound 5, FIG. 3) synthesized as described in Example 2, and the
reaction was allowed to proceed at ambient temperature for 3.5
hours, at which point mass spectroscopy revealed complete
modification of the caspase 3 large subunit (MW 16861Da, calculated
MW 16860Da). The thioester was deprotected by adding 0.2 ml of 0.5
M hydroxylamine buffered in PBS buffer, and allowing the reaction
to proceed for 18 hours, at which point the large subunit had a
mass of 16819Da (calculated 16818Da). The protein was concentrated
in a Ultrafree 5 MWCO unit (Millipore) and the buffer exchanged to
0.1 M TES pH 7.5 using a Nap-5 column (Amersham Pharmacia Biotech).
The structure of the resulting "extended" caspase-3 is shown in
FIG. 6.
[0193] The protein was then screened against a disulfide library
prepared as described above, in Example 1, and using the
methodology described in Example 4 below.
Example 4
Screening of Disulfide Library
[0194] In a typical experiment, 1 .mu.l of a DMSO solution
containing a library of 8-15 disulfide-containing compounds was
added to 49 .mu.l of buffer containing extender-modified protein.
When mass spectroscopy was used for the identification of the bound
ligand, the compounds were chosen so that each has a unique
molecular weight. For example, these molecular weights differ by at
least 10 atomic mass units so that deconvolution is unambiguous.
Although pools of 8-15 disulfide-containing compounds were
typically chosen for screening because of the ease of
deconvolution, larger pools can also be used. The protein was
present at a concentration of .about.15 .mu.M, each of the
disulfide library members was present at .about.0.2 mM, and thus
the total concentration of all disulfide library members was
.about.2 mM. The reaction was done in a buffer containing 25 mM
potassium phosphate (pH 7.5) and 1 mM 2-mercaptoethanol, although
other buffers and reducing agents can be used. The reactions were
allowed to equilibrate at ambient temperature for at least 30 min.
These conditions can be varied considerably depending on the ease
with which the protein ionizes in the mass spectrometer, the
reactivity of the specific cysteine(s), etc.
[0195] After equilibration of aspartyl-conjugated caspase-3
(Example 3) and library (Example 1), the reaction was injected onto
an HP1100 HPLC and chromatographed on a C.sub.18 column attached to
a mass spectrometer (Finnigan-MAT LCQ, San Jose, Calif.). The
multiply charged ions arising from the protein were deconvoluted
with available software (XCALIBUR) to arrive at the mass of the
protein. The identity of any library member bonded through a
disulfide bond to the protein was then easily determined by
subtracting the known mass of the unmodified protein from the
observed mass. This process assumes that the attachment of a
library member does not dramatically change the ionization
characteristics of the protein itself, a conservative assumption
because in most cases the protein will be at least 20-fold larger
than any given library member. This assumption was confirmed by
demonstrating that small molecules selected by one protein are not
selected by other proteins.
[0196] The results of a representative experiment are shown in FIG.
6. The spectrum on the right side of FIG. 6 shows the result of
reacting "extended" Casase-3 (synthesized as described in Example
3), with a disulfide-containing molecule identified from a pool as
modifying extended Caspase-3. The predominant peak obtained (mass
of 17,094) corresponds to Caspase-3 covalently linked to the small
molecule ligand which has an intrinsic affinity for a second site
of interest on Caspase-3, resulting in the diaphore compound shown
above the peak.
[0197] The mass spectrum shown on the left side is a deconvoluter
mass spectrum of unmodified Caspase-3 (a cysteine-containing
polypeptide target), and the same disulfide-containing small
molecule ligand used above. The spectrum reveals a predominant peak
corresponding to the mass of unmodified Caspase-3 (16,614 DA). A
significantly smaller peak represents Caspase-3 disulfide-bonded to
2-aminoethanethiol (combined mass: 16,691 Da). Note that here the
small molecule ligand is not selected because its binding site is
too far from the reactive cysteine and no extender has
introduced.
[0198] The initial lead compound, identified as describe above, was
then modified in order to evaluate the relative importance of
various substituents in specific binding to Caspase-3.
[0199] All references cited throughout the specification are hereby
expressly incorporated by reference.
[0200] While the present invention has been described with
reference to the specific embodiment thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the object, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
* * * * *
References