U.S. patent application number 13/270051 was filed with the patent office on 2012-02-02 for crystal structure of aldehyde dehydrogenase and methods of use thereof.
Invention is credited to Che-Hong CHEN, Thomas D. HURLEY, Daria MOCHLY-ROSEN.
Application Number | 20120028287 13/270051 |
Document ID | / |
Family ID | 41265221 |
Filed Date | 2012-02-02 |
United States Patent
Application |
20120028287 |
Kind Code |
A1 |
CHEN; Che-Hong ; et
al. |
February 2, 2012 |
Crystal Structure of Aldehyde Dehydrogenase and Methods of Use
Thereof
Abstract
The present disclosure provides a crystal structure of aldehyde
dehydrogenase (ALDH) with a modulator of ALDH bound thereto. The
present disclosure provides a computer readable medium comprising
atomic coordinates for an ALDH polypeptide and a modulator bound to
a site within the polypeptide. A method is also provided. In
general terms, the method comprises computationally identifying a
compound that binds to an ALDH polypeptide, using the atomic
coordinates.
Inventors: |
CHEN; Che-Hong; (Fremont,
CA) ; MOCHLY-ROSEN; Daria; (Menlo Park, CA) ;
HURLEY; Thomas D.; (Indianapolis, IN) |
Family ID: |
41265221 |
Appl. No.: |
13/270051 |
Filed: |
October 10, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12435265 |
May 4, 2009 |
|
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13270051 |
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61126890 |
May 7, 2008 |
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Current U.S.
Class: |
435/26 ; 435/177;
703/11; 703/2 |
Current CPC
Class: |
C12Y 102/01004 20130101;
C07K 2299/00 20130101; C12Y 102/01003 20130101; G16C 20/50
20190201 |
Class at
Publication: |
435/26 ; 435/177;
703/2; 703/11 |
International
Class: |
C12Q 1/32 20060101
C12Q001/32; G06F 7/60 20060101 G06F007/60; G06G 7/58 20060101
G06G007/58; C12N 11/02 20060101 C12N011/02 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] The U.S. government may have certain rights in this
invention, pursuant to grant nos. AA11982, AA18123, and AA11417
awarded by the National Institutes of Health.
Claims
1. A crystal comprising an aldehyde dehydrogenase (ALDH)
polypeptide in crystalline form, wherein said crystal comprises a
chemical entity bound to the active site of the ALDH
polypeptide.
2. The crystal of claim 1, wherein the ALDH polypeptide is an ALDH2
polypeptide, wherein the crystal is characterized with space group
P2.sub.1, and has unit cell parameters of a=102 .ANG., b=177 .ANG.,
c=103 .ANG., bond angles of a=.gamma.=90.degree.,
b=94.5.degree..
3. The crystal of claim 1, wherein the ALDH polypeptide is an ALDH2
polypeptide that comprises a Glu at a position corresponding to
amino acid 504 of SEQ ID NO:1, wherein the crystal is characterized
with space group P2.sub.1, and has unit cell parameters of a=102
.ANG., b=177 .ANG., c=102 .ANG., bond angles of
a=.gamma.=90.degree., b=94.6.degree.
4. The crystal of claim 1, wherein the bound chemical entity is an
agonist.
5. The crystal of claim 4, wherein the agonist is
N-(1,3-benzodioxo1-5-ylmethyl)-2,6-dichlorobenzamide.
6. The crystal of claim 1, wherein the ALDH polypeptide has a
length of about 500 amino acids.
7. The crystal of claim 1, wherein the ALDH polypeptide comprises
an amino acid sequence having at least about 80% amino acid
sequence identity to amino acids 18-517 of the amino acid sequence
set forth in SEQ ID NO:1, wherein the ALDH polypeptide comprises a
Glu at a position corresponding to amino acid 504 of SEQ ID
NO:1.
8. The crystal of claim 1, wherein the ALDH polypeptide comprises
an amino acid sequence having at least about 80% amino acid
sequence identity to amino acids 18-517 of the amino acid sequence
set forth in SEQ ID NO:1, wherein the ALDH polypeptide comprises a
Lys at a position corresponding to amino acid 504 of SEQ ID
NO:1.
9. A composition comprising the crystal of claim 1.
10. The composition of claim 1, wherein the crystal diffracts
x-rays for a determination of structure coordinates to a resolution
of between 1.5 Angstroms and 2.0 Angstroms.
11. A method comprising: computationally identifying a compound
that binds to an aldehyde dehydrogenase (ALDH) polypeptide using
atomic coordinates for a complex comprising said ALDH polypeptide
and a ligand bound to a ligand-binding site within the ALDH
polypeptide.
12. The method of claim 11, wherein said atomic coordinates are set
forth in Table 1.
13. The method of claim 11, wherein said atomic coordinates are set
forth in Table 6.
14. The method of claim 11, further comprising: testing said
compound to determine if it modulates an enzymatic activity of said
ALDH polypeptide.
15. The method of claim 11, further comprising: testing said
compound to determine if it modulates a substrate specificity of
said ALDH polypeptide.
16. The method of claim 11, wherein said computationally
identifying employs a docking program that computationally tests
known compounds for binding to said ALDH polypeptide.
17. The method of claim 11, wherein said computationally
identifying includes designing a compound that binds to said ALDH
polypeptide.
18. The method of claim 17, wherein said compound is designed from
a known compound.
19. A method comprising: a) receiving a set of atomic coordinates
for a complex comprising an aldehyde dehydrogenase (ALDH)
polypeptide and a ligand bound to a ligand-binding site within said
ALDH polypeptide; b) identifying a compound that binds to said ALDH
polypeptide using said coordinates.
20. A method of identifying a drug candidate compound for the
treatment of a disorder, the method comprising: a) employing the
three-dimensional structural coordinates of an aldehyde
dehydrogenase (ALDH) polypeptide and determining the binding mode
of a test compound within the catalytic site of the polypeptide; b)
selecting a test compound having the best fit with the ALDH
catalytic site; and c) assaying the ability of the test compound to
modulate ALDH catalytic activity, wherein a test compound that
modulates ALDH catalytic activity is considered a candidate agent
for treating a disorder.
21. The method of claim 20, wherein the test agent blocks access of
a substrate to one or both of Cys 302 and Glu 268 of the active
site, wherein the test agent reduces catalytic activity of the ALDH
polypeptide, and wherein the test agent is considered a candidate
agent for treating a disorder that would benefit from reducing ALDH
activity.
22. The method of claim 21, wherein the disorder is cancer, and
wherein the test agent is considered a candidate agent for
sensitizing a cancer cell to a cancer chemotherapeutic agent.
23. The method of claim 20, wherein the test agent increases
binding of a substrate to one or both of Cys 302 and Glu 268 of the
active site, wherein the test agent increases catalytic activity of
the ALDH polypeptide, and wherein the test agent is considered a
candidate agent for treating a disorder that would benefit from
increasing ALDH activity.
24. The method of claim 23, wherein the disorder is a disorder
resulting from a toxic level of an aldehyde, cataract, oral cancer,
esophageal cancer, an upper digestive tract cancer, lung cancer,
atopic dermatitis, radiation dermatitis, an acute or chronic
ischemic or oxidative stress disease, nitroglycerin insensitivity,
seizure, or a neurodegenerative disease.
25. A computer-assisted method for identifying potential modulators
of aldehyde dehydrogenase (ALDH), using a programmed computer
comprising a processor, a data storage system, an input device, and
an output device, the method comprising: a) inputting into the
programmed computer through said input device data comprising the
three-dimensional coordinates of a subset of the atoms generated
from a complex of ALDH and an agonist or an antagonist bound at or
near the active site of the ALDH, thereby generating a criteria
data set; b) comparing, using said processor, said criteria data
set to a computer database of chemical structures stored in said
computer data storage system; c) selecting from said database,
using computer methods, chemical structures having a portion that
is structurally similar to said criteria data set; and d)
outputting to said output device the selected chemical structures
having a portion similar to said criteria data set.
26. A computer readable medium comprising: atomic coordinates for a
complex comprising: i) an aldehyde dehydrogenase (ALDH)
polypeptide; and ii) a ligand bound to a ligand-binding site in the
ALDH polypeptide.
27. The computer readable medium of claim 26, further comprising:
programming for displaying a molecular model of said ALDH
polypeptide.
28. The computer readable medium of claim 26, further comprising:
programming for identifying a compound that binds to said ALDH
polypeptide.
29. The computer readable medium of claim 28, further comprising: a
database of structures of known test compounds.
30. The computer readable medium of claim 26, wherein said atomic
coordinates are set forth in Table 1 or in Table 6.
31. A computer comprising the computer-readable medium of claim
26.
32. A computer system comprising: a memory comprising X-ray
crystallographic structure coordinates defining a ligand-binding
site of a complex comprising an aldehyde dehydrogenase (ALDH)
polypeptide with a ligand bound to a ligand-binding site within the
ALDH polypeptide; and a processor in electrical communication with
the memory; wherein the processor generates a molecular model
having a three dimensional structure representative of at least a
portion of said ALDH polypeptide-bound ligand complex.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 61/126,890, filed May 7, 2008, which
application is incorporated herein by reference in its
entirety.
TABLES PROVIDED IN ELECTRONIC FORM
[0003] This application includes Table 1 and Table 6. Table 1 is a
text file named "STAN-595_Table.sub.--1_atomic_coordinates" created
on May 4, 2009. The size of the
"STAN-595_Table.sub.--1_atomic_coordinates" text file is 11,001 KB.
Table 6 is a text file named
"STAN-595_Table.sub.--6_atomic_coordinates" created on May 4, 2009.
The size of "STAN-595_Table.sub.--6_atomic_coordinates" text file
is 2,677 KB. The information contained in Table 1 and in Table 6 is
hereby incorporated by reference in this application.
TABLE-US-LTS-CD-00001 LENGTHY TABLES The patent application
contains a lengthy table section. A copy of the table is available
in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120028287A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
BACKGROUND
[0004] Aldehyde dehydrogenase (ALDH) is a family of enzymes that
play a critical role in detoxification of many cytotoxic xenogenic
and biogenic aldehydes. The ALDH family includes at least 11
members with different substrate specificity and cellular
localization. Accumulation of cytotoxic aldehyde compounds, or
defects in ALDH genes, have been implicated in a variety of
diseases, including neurodegenerative diseases, cancer, myocardial
infarction, stroke, and diseases related to accumulation of
acetaldehyde from alcohol intake.
[0005] There is a need in the art for compounds that modulate the
activity of ALDH enzymes, and for methods of rational design of
such compounds.
[0006] Literature
[0007] Perez-Miller and Hurley (2003) Biochem. 42:7100; Larson et
al. (2005) J. Biol. Chem. 280:30550; and Li et al. (2006) J. Clin.
Invest. 116:506.
SUMMARY OF THE INVENTION
[0008] The present disclosure provides a crystal structure of an
aldehyde dehydrogenase (ALDH) polypeptide with a modulator of ALDH
bound thereto. The present disclosure provides a computer readable
medium comprising atomic coordinates for an ALDH polypeptide and a
modulator bound to a site within the polypeptide. A method is also
provided. In general terms, the method comprises computationally
identifying a compound that binds to an ALDH polypeptide, using the
atomic coordinates.
[0009] Features of the Disclosure
[0010] The present disclosure provides a crystal comprising an
aldehyde dehydrogenase (ALDH) polypeptide in crystalline form,
where the crystal comprises a chemical entity bound to the active
site of the ALDH polypeptide. In some aspects, the ALDH polypeptide
is an ALDH2 polypeptide, wherein the crystal is characterized with
space group P2.sub.1, and has unit cell parameters of a=102 .ANG.,
b=177 .ANG., c=103 .ANG., bond angles of a=.gamma.=90.degree.,
b=94.5.degree.. In other aspects, the ALDH polypeptide is an ALDH2
polypeptide that comprises a Glu at a position corresponding to
amino acid 504 of SEQ ID NO:1, wherein the crystal is characterized
with space group P2.sub.1, and has unit cell parameters of a=102
.ANG., b=177 .ANG., c=102 .ANG., bond angles of
a=.gamma.=90.degree., b=94.6.degree.. The bound chemical entity can
be an agonist or an antagonist. In some cases, the bound entity is
an agonist, where an exemplary agonist is
N-(1,3-benzodioxo1-5-ylmethyl)-2,6-dichlorobenzamide (also referred
to herein as "Alda-1").
[0011] The ALDH polypeptide present in a subject crystal will in
some cases have a length of about 500 amino acids. The ALDH
polypeptide present in a subject crystal will in some cases have a
length of about 500 amino acids and lack a leader peptide, e.g.,
amino acids 1-17 as shown in FIG. 1A. The ALDH polypeptide present
in a subject crystal can comprise an amino acid sequence having at
least about 80% amino acid sequence identity to amino acids 18-517
of the amino acid sequence set forth in SEQ ID NO:1, where the ALDH
polypeptide comprises a Glu at a position corresponding to amino
acid 504 of SEQ ID NO:1. The ALDH polypeptide present in a subject
crystal can comprise an amino acid sequence having at least about
80% amino acid sequence identity to amino acids 18-517 of the amino
acid sequence set forth in SEQ ID NO:1, where the ALDH polypeptide
comprises a Lys at a position corresponding to amino acid 504 of
SEQ ID NO:1. A subject crystal can in some embodiments diffract
x-rays for a determination of structure coordinates to a resolution
of between 1.5 Angstroms and 2.0 Angstroms. The present disclosure
further provides a composition comprising a subject crystal.
[0012] The present disclosure provides a method involving
computationally identifying a compound that binds to an ALDH
polypeptide using atomic coordinates for a complex comprising the
ALDH polypeptide and a ligand bound to a ligand-binding site within
the ALDH polypeptide. In some embodiments, the atomic coordinates
are those set forth in Table 1 or in Table 6. A subject method can
further involve testing the compound to determine if it modulates
an enzymatic activity of said ALDH polypeptide. A subject method
can further involve testing the compound to determine if it
modulates a substrate specificity of said ALDH polypeptide. In some
cases, computationally identifying a compound involves employing a
docking program that computationally tests known compounds for
binding to said ALDH polypeptide. In some cases, computationally
identifying a compound includes designing a compound that binds to
said ALDH polypeptide. The compound can be designed based on a
known compound.
[0013] The present disclosure provides a method that involves: a)
receiving a set of atomic coordinates for a complex comprising an
aldehyde dehydrogenase (ALDH) polypeptide and a ligand bound to a
ligand-binding site within the ALDH polypeptide; and b) identifying
a compound that binds to the ALDH polypeptide using said
coordinates.
[0014] The present disclosure provides a method of identifying a
drug candidate compound for the treatment of a disorder, the method
generally involving: a) employing the three-dimensional structural
coordinates of an ALDH polypeptide and determining the binding mode
of a test compound within the catalytic site of the ALDH
polypeptide; b) selecting a test compound having the best fit with
the ALDH catalytic site; and c) assaying the ability of the test
compound to modulate ALDH catalytic activity, wherein a test
compound that modulates ALDH catalytic activity is considered a
candidate agent for treating a disorder. In some embodiments, where
the test agent blocks access of a substrate to one or both of Cys
302 and Glu 268 of the active site, and the test agent reduces
catalytic activity of the ALDH polypeptide, the test agent is
considered a candidate agent for treating a disorder that would
benefit from reducing ALDH activity. For example, where the
disorder is cancer, and the test agent is considered a candidate
agent for sensitizing a cancer cell to a cancer chemotherapeutic
agent. In other embodiments, where the test agent increases binding
of a substrate to one or both of Cys 302 and Glu 268 of the active
site, and where the test agent increases catalytic activity of the
ALDH polypeptide, the test agent is considered a candidate agent
for treating a disorder that would benefit from increasing ALDH
activity. Examples of such disorders include a disorder resulting
from a toxic level of an aldehyde, cataract, oral cancer,
esophageal cancer, an upper digestive tract cancer, lung cancer,
atopic dermatitis, radiation dermatitis, an acute or chronic
ischemic or oxidative stress disease, nitroglycerin insensitivity,
seizure, and a neurodegenerative disease.
[0015] The present disclosure provides computer-assisted method for
identifying potential modulators of aldehyde dehydrogenase (ALDH),
using a programmed computer comprising a processor, a data storage
system, an input device, and an output device, the method
involving: a) inputting into the programmed computer through said
input device data comprising the three-dimensional coordinates of a
subset of the atoms generated from a complex of ALDH and an agonist
or an antagonist bound at or near the active site of the ALDH,
thereby generating a criteria data set; b) comparing, using the
processor, the criteria data set to a computer database of chemical
structures stored in the computer data storage system; c) selecting
from the database, using computer methods, chemical structures
having a portion that is structurally similar to the criteria data
set; and d) outputting to the output device the selected chemical
structures having a portion similar to the criteria data set.
[0016] The present disclosure provides a computer readable medium
comprising atomic coordinates for a complex comprising: i) an
aldehyde dehydrogenase (ALDH) polypeptide; and ii) a ligand bound
to a ligand-binding site in the ALDH polypeptide. A subject
computer readable medium can further include programming for
displaying a molecular model of said ALDH polypeptide. A subject
computer readable medium can further include programming for
identifying a compound that binds to said ALDH polypeptide. A
subject computer readable medium can further include a database of
structures of known test compounds. In some embodiments, the atomic
coordinates present in a subject computer-readable medium are those
set forth in Table 1. In some embodiments, the atomic coordinates
present in a subject computer-readable medium are those set forth
in Table 6.
[0017] The present disclosure provides a computer comprising a
subject computer-readable medium.
[0018] The present disclosure provides a computer system
comprising: a memory comprising X-ray crystallographic structure
coordinates defining a ligand-binding site of a complex comprising
an ALDH polypeptide with a ligand bound to a ligand-binding site
within the ALDH polypeptide; and a processor in electrical
communication with the memory; where the processor generates a
molecular model having a three dimensional structure representative
of at least a portion of the ALDH polypeptide-bound ligand
complex.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1A and FIG. 1B provide the amino acid sequence of an
E487K variant of human ALDH2 (SEQ ID NO:1) and the amino acid
sequence of "wild-type" human ALDH2 (SEQ ID NO:2),
respectively.
[0020] FIG. 2 depicts enzyme activation of homotetrameric wild type
ALDH2 (homo wild type), heterotetrameric ALDH2 (hetero wild
type/mutant; comprising a mixture of wild-type and mutant
monomers), and homotetrameric mutant ALDH2 by Alda-1 (100 .mu.M).
Enzymatic activity of recombinant ALDH2 proteins (20 .mu.g each) is
presented in percentage using homotetrameric wild type enzyme as a
100% control (n=3, **p<0.01 vs. control).
[0021] FIGS. 3A-C depict a structure of ALDH2 with Alda-1 bound.
(A) Ribbon diagram of the ALDH2 tetramer with different color
denoting the individual subunits and the bound Alda-1 molecules
indicated using the gray space-filling atom representation. (B)
Stereoview of the original Fo-Fc (top, contoured at 3 standard
deviations of the map). (C) Final refined 2Fo-Fc electron density
(contoured at 1.2 standard deviations of the map) for Alda-1 bound
to ALDH2*1. Produced using SPDBViewer and PovRay.
[0022] FIG. 4 depicts overlay of the aligned structures of ALDH2
with bound Alda-1 and with bound daidzin (pdb entry 1OF7).
[0023] FIGS. 5A and 5B depict Alda-1 competition with daidzin
inhibition. Dehydrogenase activity was measured at 0.1 mM
propionaldehyde, varying concentrations of daidzin. (A) Wild-type
ALDH2 at 0 .mu.M or 10 .mu.M Alda-1 and (B) ALDH2*2 at 0 .mu.M or
50 .mu.M Alda-1. NAD.sup.+ concentrations were 0.5 mM for wild-type
ALDH2 and 10 mM for ALDH2*2. Lines show fits to 4-parameter
logistic curve.
[0024] FIG. 6 shows M-M plot for the effects of Alda-1 on the
dehydrogenase activity of ALDH2*2 against varied NAD.sup.+.
[0025] FIG. 7 depicts a substrate-binding site surface of ALDH1A1
with the position of Alda-1 as found in ALDH2 overlayed onto this
surface.
[0026] FIG. 8 depicts a substrate-binding site surface of the model
of ALDH1B1 with the position of
[0027] Alda-1 as found in ALDH2 overlayed onto this surface.
[0028] FIG. 9 depicts a substrate-binding site surface of rat
ALDH3A1 with the position of Alda-1 as found in ALDH2 overlayed
onto this surface.
[0029] FIG. 10 depicts binding of Alda-1 to ALDH2*2.
[0030] FIG. 11 provides ribbon representations of the structure of
ALDH2*2 without (left) and with (right) Alda-1 bound. The helices
at the interface between the subunits are restored in the electron
density maps when Alda-1is bound to ALDH2*2 (grey space-filling
atoms).
DEFINITIONS
[0031] As used herein, the term "binding site" or "binding pocket"
refers to a region of a polypeptide (e.g., an ALDH polypeptide)
that binds or interacts with a particular compound.
[0032] As used herein, the term "interface" refers to the point or
surface at which two or more domains of one or more molecules
associate.
[0033] As used herein, the terms "associates with" or "interacts
with" refers to a condition of proximity between a chemical entity,
compound, or portions thereof, with another chemical entity,
compound or portion thereof. The association or interaction may be
non-covalent--wherein the juxtaposition is energetically favored by
hydrogen bonding or van der Waals or electrostatic interactions--or
it may be covalent.
[0034] As used herein, the term "pharmacophore" refers to an
ensemble of steric and electronic features that is necessary to
ensure the optimal supramolecular interactions with a specific
biological target structure and to trigger or block a biological
response. A pharmacophore may be used to design one or more
candidate compounds that comprise all or most of the ensemble of
steric and electronic features present in the pharmacophore and
that are expected to bind to a site and trigger or block a
biological response.
[0035] Structural similarity may be inferred from, e.g., sequence
similarity, which can be determined by one of ordinary skill
through visual inspection and comparison of the sequences, or
through the use of well-known alignment software programs such as
CLUSTAL (Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci.
USA, 80, 726-730 (1983)) or CLUSTALW (Thompson, J. D., Higgins, D.
G. and Gibson, T. J., CLUSTAL W: improving the sensitivity of
progressive multiple sequence alignment through sequence weighting,
positions-specific gap penalties and weight matrix choice, Nucleic
Acids Research, 22:4673-4680 (1994)) or BLAST (Altschul S F, Gish
W, et al., .J Mol. Biol., October 5;215(3):403-10 (1990)), a set of
similarity search programs designed to explore all of the available
sequence databases regardless of whether the query is protein or
DNA. CLUSTAL W is available on the internet at ebi.ac.uk/clustalw/;
BLAST is available on the internet at ncbi.nlm.nih.gov/BLAST/. A
residue within a first protein or nucleic acid sequence corresponds
to a residue within a second protein or nucleic acid sequence if
the two residues occupy the same position when the first and second
sequences are aligned.
[0036] The term "atomic coordinates" refers to the Cartesian
coordinates corresponding to an atom's spatial relationship to
other atoms in a molecule or molecular complex. Atomic coordinates
may be obtained using x-ray crystallography techniques or nuclear
magnetic resonance techniques, or may be derived using molecular
replacement analysis or homology modeling. Various software
programs allow for the graphical representation of a set of
structural coordinates to obtain a three dimensional representation
of a molecule or molecular complex. The atomic coordinates of the
present disclosure may be modified from the original set provided
in Table 1 or Table 6 by mathematical manipulation, such as by
inversion or integer additions or subtractions. As such, it is
recognized that the structural coordinates of the present invention
are relative, and are in no way specifically limited by the actual
x, y, z coordinates of Table 1 or Table 6.
[0037] "Root mean square deviation" is the square root of the
arithmetic mean of the squares of the deviations from the mean, and
is a way of expressing deviation or variation from the structural
coordinates described herein. The present disclosure includes all
embodiments comprising conservative substitutions of the noted
amino acid residues resulting in same structural coordinates within
the stated root mean square deviation. It will be apparent to the
skilled practitioner that the numbering of the amino acid residues
of ALDH may be different than that set forth herein, and may
contain certain conservative amino acid substitutions that yield
the same three dimensional structures as those defined by Table 1
or Table 6. Corresponding amino acids and conservative
substitutions in other isoforms or analogues are easily identified
by visual inspection of the relevant amino acid sequences or by
using commercially available homology software programs (e.g.,
MODELLER, Accelrys, San Diego, Calif.; Sali and Blundell (1993) J
Mol Biol 234:779-815; Sanchez and Sali (1997) Curr Opin Struct Biol
7: 206-214; and Sanchez and Sali (1998) Proc Natl Acad Sci USA 95:
13597-13602).
[0038] The terms "system" and "computer-based system" refer to the
hardware means, software means, and data storage means used to
analyze the information of the present disclosure. The minimum
hardware of the computer-based systems of the present invention
comprises a central processing unit (CPU), input means, output
means, and data storage means. As such, any convenient
computer-based system may be employed in the present disclosure.
The data storage means may comprise any manufacture comprising a
recording of the present information as described above, or a
memory access means that can access such a manufacture.
[0039] A "processor" references any hardware and/or software
combination which will perform the functions required of it. For
example, any processor herein may be a programmable digital
microprocessor such as available in the form of an electronic
controller, mainframe, server or personal computer (desktop or
portable). Where the processor is programmable, suitable
programming can be communicated from a remote location to the
processor, or previously saved in a computer program product (such
as a portable or fixed computer readable storage medium, whether
magnetic, optical or solid state device based). For example, a
magnetic medium or optical disk may carry the programming, and can
be read by a suitable reader communicating with each processor at
its corresponding station.
[0040] "Computer readable medium" as used herein refers to any
storage or transmission medium that participates in providing
instructions and/or data to a computer for execution and/or
processing. Examples of storage media include floppy disks,
magnetic tape, USB, CD-ROM, a hard disk drive, a ROM or integrated
circuit, a magneto-optical disk, or a computer readable card such
as a PCMCIA card and the like, whether or not such devices are
internal or external to the computer. A file containing information
may be "stored" on computer readable medium, where "storing" means
recording information such that it is accessible and retrievable at
a later date by a computer. A file may be stored in permanent
memory.
[0041] With respect to computer readable media, "permanent memory"
refers to memory that is permanently stored on a data storage
medium. Permanent memory is not erased by termination of the
electrical supply to a computer or processor. Computer hard-drive
ROM (i.e. ROM not used as virtual memory), CD-ROM, floppy disk and
DVD are all examples of permanent memory. Random Access Memory
(RAM) is an example of non-permanent memory. A file in permanent
memory may be editable and re-writable.
[0042] To "record" data, programming or other information on a
computer readable medium refers to a process for storing
information, using any convenient method. Any convenient data
storage structure may be chosen, based on the means used to access
the stored information. A variety of data processor programs and
formats can be used for storage, e.g. word processing text file,
database format, etc.
[0043] A "memory" or "memory unit" refers to any device which can
store information for subsequent retrieval by a processor, and may
include magnetic or optical devices (such as a hard disk, floppy
disk, CD, or DVD), or solid state memory devices (such as volatile
or non-volatile RAM). A memory or memory unit may have more than
one physical memory device of the same or different types (for
example, a memory may have multiple memory devices such as multiple
hard drives or multiple solid state memory devices or some
combination of hard drives and solid state memory devices).
[0044] A system can include hardware components which take the form
of one or more platforms, e.g., in the form of servers, such that
any functional elements of the system, i.e., those elements of the
system that carry out specific tasks (such as managing input and
output of information, processing information, etc.) of the system
may be carried out by the execution of software applications on and
across the one or more computer platforms represented of the
system. The one or more platforms present in the subject systems
may be any convenient type of computer platform, e.g., such as a
server, main-frame computer, a work station, etc. Where more than
one platform is present, the platforms may be connected via any
convenient type of connection, e.g., cabling or other communication
system including wireless systems, either networked or otherwise.
Where more than one platform is present, the platforms may be
co-located or they may be physically separated. Various operating
systems may be employed on any of the computer platforms, where
representative operating systems include Windows, MacOS, Sun
Solaris, Linux, OS/400, Compaq Tru64 Unix, SGI IRIX, Siemens
Reliant Unix, and others. The functional elements of system may
also be implemented in accordance with a variety of software
facilitators, platforms, or other convenient method.
[0045] Items of data are "linked" to one another in a memory when
the same data input (for example, filename or directory name or
search term) retrieves the linked items (in a same file or not) or
an input of one or more of the linked items retrieves one or more
of the others.
[0046] Subject computer readable media may be at a "remote
location", where "remote location," means a location other than the
location at which the x-ray crystallographic or other analysis is
carried out. For example, a remote location could be another
location (e.g., office, lab, etc.) in the same city, another
location in a different city, another location in a different
state, another location in a different country, etc. As such, when
one item is indicated as being "remote" from another, what is meant
is that the two items may be in the same room but separated, or at
least in different rooms or different buildings, and may be at
least one mile, ten miles, or at least one hundred miles apart.
[0047] "Communicating" information references transmitting the data
representing that information as, e.g., electrical or optical
signals over a suitable communication channel (e.g., a private or
public network). "Forwarding" an item refers to any means of
getting that item from one location to the next, whether by
physically transporting that item or otherwise (where that is
possible) and includes, at least in the case of data, physically
transporting a medium carrying the data or communicating the data.
Examples of communicating media include radio or infra-red
transmission channels as well as a network connection to another
computer or networked device, and the Internet or Intranets
including email transmissions and information recorded on websites
and the like.
[0048] Before the present invention is further described, it is to
be understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0049] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0050] Unless defined otherwise, all 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. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0051] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "an aldehyde dehydrogenase polypeptide"
includes a plurality of such polypeptides and reference to "the
x-ray structure" includes reference to one or more x-ray structures
and equivalents thereof known to those skilled in the art, and so
forth. It is further noted that the claims may be drafted to
exclude any optional element. As such, this statement is intended
to serve as antecedent basis for use of such exclusive terminology
as "solely," "only" and the like in connection with the recitation
of claim elements, or use of a "negative" limitation.
[0052] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to, antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
DETAILED DESCRIPTION
[0053] The present disclosure provides a crystal of an aldehyde
dehydrogenase (ALDH) polypeptide with a modulator of ALDH bound
thereto. The present disclosure provides a crystal structure of
aldehyde dehydrogenase (ALDH) with a modulator of ALDH bound
thereto. The present disclosure also provides a computer readable
medium comprising atomic coordinates for an ALDH polypeptide and a
modulator bound to a site within the polypeptide. A subject crystal
structure allows for identification and design of additional
modulators of ALDH. Thus, the present disclosure provides
structures and methods for identifying and designing ALDH ligands,
as well as methods for studying the ALDH mechanism. Also provided
is a computer system comprising: a memory comprising x-ray
crystallographic structure coordinates defining a structure of an
ALDH polypeptide with a bound modulator.
[0054] Crystal Structures
[0055] The present disclosure provides a crystal structure of a
complex comprising an aldehyde dehydrogenase (ALDH) polypeptide and
a modulator (a "ligand") of ALDH bound to the ALDH polypeptide
(e.g., bound to a ligand-binding site of the ALDH polypeptide).
[0056] The terms "ALDH" and "ALDH polypeptide" are used
interchangeably herein to refer to an enzyme that exhibits at least
a dehydrogenase activity (e.g., dehydrogenase activity in oxidizing
an aldehyde to the corresponding acid. ALDH polypeptides are known
in the art, and include ALDH polypeptides from any of a variety of
biological sources, including, e.g., prokaryotic sources and
eukaryotic sources. Eukaryotic ALDH includes human ALDH, rodent
ALDH (e.g., murine ALDH, such as mouse ALDH, and rat ALDH),
ungulate ALDH (e.g., bovine, ovine, equine, etc. ALDH), and the
like. A variety of ALDH polypeptides are known, and are reviewed
in, e.g, Vasiliou et al. (1999) Pharmacogenetics 9:421; Sophos et
al. (2001) Chemico-Biological Interactions 130-132:323-337; Sophos
and Vasiliou (2003) Chem. Biol. Interact. 143-144:5-22; and
Vasiliou and Nebert (2005) Hum. Genomics 2:138-143. The term "ALDH"
includes and ALDH polypeptide of any ALDH family, including any
isoform of ALDH.
[0057] Amino acid sequences of various human ALDH family members
(e.g., "isozymes") are known in the art and are publicly available.
See, e.g., GenBank Accession No. NP.sub.--000680 (ALDH 1, member
A1); GenBank Accession No. NP.sub.--000684 (ALDH 1, member A3);
GenBank Accession Nos. AAH02967 and NP.sub.--000681 (ALDH 2);
GenBank Accession No. NP.sub.--001026976 (ALDH 3, member A2,
isoform 1); GenBank Accession No. CAI39494 (ALDH 4, member A1);
GenBank Accession No. CAA20248 (ALDH 5, member A1); GenBank
Accession No. EAW81160 (ALDH 6, member A1, isoform CRA_b); GenBank
Accession No. AAH02515 (ALDH 7, member A1); GenBank Accession No.
NP.sub.--072090 (ALDH 8, member A1, isoform 1); GenBank Accession
No. NP.sub.--000687 (ALDH 9, member A1); GenBank Accession No.
AAG42417 (ALDH 12); GenBank Accession No. AAG42417 (ALDH 12);
GenBank Accession No. NP.sub.--699160 (ALDH 16); and GenBank
Accession No. CAI16766 (ALDH 18, member A1).
[0058] In some embodiments, the ALDH polypeptide component of a
subject ALDH/bound ligand crystal is an ALDH2 polypeptide. The term
"ALDH2" encompasses ALDH2 from various species. Amino acid
sequences of ALDH2 from various species are publicly available. For
example, a human ALDH2 amino acid sequence is found under GenBank
Accession Nos. AAH02967 and NP.sub.--000681; a mouse ALDH2 amino
acid sequence is found under GenBank Accession No. NP.sub.--033786;
and a rat ALDH2 amino acid sequence is found under GenBank
Accession No. NP_115792.
[0059] The term "ALDH2" as used herein encompasses wild-type ALDH2,
e.g., a polypeptide comprising an amino acid sequence having at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95%, at least about 98%, at least about
99%, or 100%, amino acid sequence identity to amino acids 18-517 of
the amino acid sequence set forth in SEQ ID NO:2, and having a Lys
at position 487 of mature ALDH2 (e.g., ALDH2 lacking amino acids
1-17 as set forth in SEQ ID NO:2), and having at least about 75%,
at least about 80%, at least about 85%, at least about 90%, at
least about 95%, at least about 98%, at least about 99%, or 100%,
of the enzymatic activity of a polypeptide comprising amino acids
18-517 of SEQ ID NO:2. In some embodiments, a wild-type ALDH2
polypeptide lacks the MLRAAARFGPRLGRRLL (SEQ ID NO:3) peptide
depicted in FIG. 1B, and has a length of about 500 amino acids. In
some embodiments, a wild-type ALDH2 polypeptide comprises amino
acids 18-517 of. SEQ ID NO:2. Wild-type ALDH2 having the sequence
of amino acids 18-517 of SEQ ID NO:2 is sometimes referred to
herein as "wild-type ALDH2" or "ALDH2*1."
[0060] The term "ALDH2" as used herein also encompasses fragments,
fusion proteins, and variants (e.g., variants having one or more
amino acid substitutions, addition, deletions, and/or insertions)
that retain ALDH2 enzymatic activity. Specific enzymatically active
ALDH2 variants, fragments, fusion proteins, and the like can be
verified by adapting the methods described herein. An example of an
ALDH2 variant is an ALDH2 polypeptide that comprises a Glu-to-Lys
substitution at amino acid position 487 of mature human ALDH2, as
depicted in FIG. 1A (amino acid 504 of SEQ ID NO:1), at a position
corresponding to amino acid 487 of mature human ALDH2, or at a
position corresponding to amino acid 504 of SEQ ID NO:1). This
mutation is referred to as the "E487K mutation"; the "E487K
variant"; or as the "Glu504Lys polymorphism". See, e.g., Larson et
al. (2005) J. Biol. Chem. 280:30550; and Li et al. (2006) J. Clin.
Invest. 116:506. An ALDH2 variant retains at least about 1% of the
enzymatic activity of a corresponding wild-type ALDH2 enzyme. For
example, the E487K variant retains at least about 1% of the
activity of an enzyme comprising the amino acid sequence depicted
in FIG. 1B (SEQ ID NO:2). An ALDH2 polypeptide can have a length of
about 500 amino acids, and can lack the MLRAAARFGPRLGRRLL (SEQ ID
NO:3) peptide depicted in FIG. 1A and 1B. An E487K variant of ALDH2
can have the amino acid sequence of amino acids 18-517 of SEQ ID
NO:1, can have a length of about 500 amino acids, and can lack the
MLRAAARFGPRLGRRLL (SEQ ID NO:3) peptide depicted in FIG. 1A; such
an E487K variant of ALDH2 is sometimes referred to herein as
ALDH2*2. In some embodiments, an E487K variant of ALDH2 can have
the amino acid sequence of amino acids 18-517 of SEQ ID NO:1,
except for having an S302 mutation (e.g., a change from Cys to Ser
at amino acid 319 of the sequence depicted in FIG. 1A; see, e.g.,
Perez-Miller and Hurley (2003) Biochem. 42:7100), can have a length
of about 500 amino acids, and can lack the MLRAAARFGPRLGRRLL (SEQ
ID NO:3) peptide depicted in FIG. 1A.
[0061] The term "ALDH2" encompasses an enzymatically active
polypeptide having at least about 75%, at least about 80%, at least
about 85%, at least about 90%, at least about 95%, at least about
98%, at least about 99%, or 100%, amino acid sequence identity to
amino acids 18-517 of the amino acid sequence set forth in SEQ ID
NO:1 or SEQ ID NO:2. The term "ALDH2" encompasses an enzymatically
active polypeptide having at least about 75%, at least about 80%,
at least about 85%, at least about 90%, at least about 95%, at
least about 98%, at least about 99%, or 100%, amino acid sequence
identity to amino acids 18-517 of the amino acid sequence set forth
in SEQ ID NO:1, where the amino acid sequence at a position
corresponding to amino acid 504 of SEQ ID NO:1 is a Glu. The term
"ALDH2" encompasses an enzymatically active polypeptide having at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 95%, at least about 98%, at least about
99%, or 100%, amino acid sequence identity to amino acids 18-517 of
the amino acid sequence set forth in SEQ ID NO:1, where the amino
acid sequence at a position corresponding to amino acid 504 of SEQ
ID NO:1 is a Lys.
[0062] The term "ALDH" encompasses a polypeptide having a length of
from about 400 amino acids to about 600 amino acids (aa), e.g.,
from about 400 aa to about 450 aa, from about 450 aa to about 500
aa, from about 500 aa to about 550 aa, or from about 550 aa to
about 600 aa.
[0063] An ALDH polypeptide can exhibit one or more of the following
enzymatic activities: a) a dehydrogenase activity (e.g.,
dehydrogenase activity in oxidizing an aldehyde (e.g., a xenogenic
aldehyde, a biogenic aldehyde, or an aldehyde produced from a
compound that is ingested, inhaled, or absorbed) to the
corresponding acid); b) an esterase activity; and c) a reductase
activity.
[0064] The X-ray crystal structures described herein are useful as
models for rationally designing pharmacophores and/or candidate
compounds, either de novo or by modification of known compounds.
Pharmacophores and candidate compounds identified through the use
of the crystal structure coordinates are useful for altering the
enzymatic activity and/or substrate selectivity of an ALDH
polypeptide, and so have utility for treating a variety of
disorders related to ALDH activity. Pharmacophores and candidate
compounds may be determined according to any method known in the
art, including the methods described herein.
[0065] Crystals and Crystal Compositions
[0066] The present disclosure provides crystals that include an
ALDH polypeptide and a chemical entity (e.g., an agonist or an
antagonist) bound to a binding site of the ALDH polypeptide. In
some embodiments, the crystal is capable of diffracting x-rays at a
resolution of less than 5 Angstroms, less than 4 Angstroms, less
than 3 Angstroms, or less than 2 Angstroms. For example, in some
embodiments, a subject crystal is capable of diffracting x-rays at
a resolution of between 1.5 Angstroms and 2.0 Angstroms. For
example, in some embodiments, a subject crystal is capable of
diffracting x-rays at a resolution of 1.69 Angstroms. In some
embodiments, a subject crystal has a unit cell dimension of a=102
.ANG., b=177 .ANG., c=103 .ANG., with bond angles
a=.gamma.=90.degree. , b=94.5.degree., and belongs to space group
P2.sub.1. As another example, in some embodiments, a subject
crystal is capable of diffracting x-rays at a resolution of 1.9
Angstroms. In some embodiments, a subject crystal has a unit cell
dimension of a=102 .ANG., b=177 .ANG., c=102 .ANG., with bond
angles a=.gamma.=90.degree., b=94.6.degree. , and belongs to space
group P2.sub.1. A subject crystal can have atomic coordinates as
presented in Table 1 or Table 6, or similar coordinates.
[0067] The present disclosure also provides a composition
comprising a subject crystal.
[0068] In some embodiments, the chemical entity bound to the ALDH
polypeptide is an ALDH agonist.
[0069] In some embodiments, the chemical entity bound to the ALDH
polypeptide is an ALDH antagonist. In some embodiments, the
chemical entity is bound to the ALDH polypeptide Alda-1
(N-(1,3-benzodioxo1-5-ylmethyl)-2,6-dichlorobenzamide).
[0070] In some embodiments, the ALDH polypeptide comprises an amino
acid sequence having at least about 75%, at least about 80%, at
least about 85%, at least about 90%, at least about 95%, at least
about 98%, at least about 99%, or 100%, amino acid sequence
identity to amino acids 18-517 of the amino acid sequence set forth
in SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the ALDH
polypeptide comprises an amino acid sequence having at least about
75%, at least about 80%, at least about 85%, at least about 90%, at
least about 95%, at least about 98%, at least about 99%, or 100%,
amino acid sequence identity to amino acids 18-517 of the amino
acid sequence set forth in SEQ ID NO:1, where the amino acid
sequence at a position corresponding to amino acid 504 of SEQ ID
NO:1 is a Glu. In some embodiments, the ALDH polypeptide comprises
an amino acid sequence having at least about 75%, at least about
80%, at least about 85%, at least about 90%, at least about 95%, at
least about 98%, at least about 99%, or 100%, amino acid sequence
identity to amino acids 18-517 of the amino acid sequence set forth
in SEQ ID NO:1, where the amino acid sequence at a position
corresponding to amino acid 504 of SEQ ID NO:1 is a Lys. In some
embodiments, the ALDH polypeptide comprises amino acids 18-517 of
the amino acid sequence set forth in SEQ ID NO:1. In other
embodiments, the ALDH polypeptide comprises amino acids 18-517 of
the amino acid sequence set forth in SEQ ID NO:2.
[0071] The ALDH polypeptide can be produced using any of a variety
of well known methods, including, e.g., synthetic methods, such as
solid phase, liquid phase and combination solid phase/liquid phase
syntheses; recombinant DNA methods, including cDNA cloning,
optionally combined with site directed mutagenesis; and
purification of the polypeptide from a natural source.
[0072] The present disclosure further provides a method for
producing a crystal of an ALDH polypeptide and an agonist or
antagonist bound in the ligand-binding site of the ALDH
polypeptide. The method generally involves producing crystallizable
ALDH polypeptide; forming a complex between the ALDH polypeptide
and an agonist or antagonist; and obtaining a crystal from a
solution comprising the ALDH/agonist or ALDH/antagonist complex
using a precipitating agent. In some embodiments, the apo-enzyme
(ALDH without bound agonist or antagonist) is concentrated and
equilibrated against a crystallization solution comprising a
precipitating agent. A suitable crystallization solution comprising
a precipitating agent is 100 mM ACES
(N-(2-acetamido)-2-aminoethansulfonic acid), pH 6.4, 100 mM
guanidine-HCl, 10 mM MgCl.sub.2, and 16-17% (w/v) poly(ethylene
glycol) (PEG) 6000. A complex of agonist or antagonist and ALDH
polypeptide can be achieved by first equilibrating apo-enzyme (ALDH
without agonist or antagonist) against 1% dimethylsulfoxide (DMSO)
in a crystal stabilization solution (100 mM ACES, pH 6.4, 100 mM
guanidine-HCl, 10 mM MgCl.sub.2, and 19% (w/v) PEG 6000); allowing
equilibration to proceed for 4 to 24 hours; replacing the crystal
stabilization solution with crystal stabilization solution
comprising an agonist or antagonist at a suitable concentration
(e.g., at a concentration of from about 100 .mu.M to about 400
.mu.M, e.g., 200 .mu.M); and allowing the crystals to soak for a
sufficient time period in the crystal stabilization solution,
thereby forming crystal complexes comprising the ALDH polypeptide
and the agonist or antagonist. Crystal complexes can be soaked in a
solution comprising a cryoprotectant prior to freezing in liquid
N.sub.2. The person skilled in the art knows that additional
factors such as temperature may be crucial for crystal formation.
These and other conditions of crystallization as well as strategies
to optimize conditions of crystallization have been summarized in
"Crystallization of Biological Macromolecules" by Alexander
McPherson (Cold Spring Harbor Laboratory; 1st edition (Jan. 15,
1999).
[0073] Methods of Identifying and Designing ALDH Modulators
[0074] The present disclosure provides methods for identifying and
designing ALDH ligands, as well as methods for studying the ALDH
mechanism. A subject method generally involves computationally
identifying a compound that binds to an ALDH polypeptide (e.g., a
compound that binds to a target site (e.g., a ligand-binding site;
a catalytic site; an entrance to the active site) of an ALDH
polypeptide) using atomic coordinates for an ALDH polypeptide with
a bound ligand. For example, in some embodiments, the atomic
coordinates are those provided in Table 1. As another example, in
some embodiments, the atomic coordinates are those provided in
Table 6. A compound that binds to an ALDH polypeptide includes a
compound that modulates (increases or decreases) enzymatic activity
of the ALDH polypeptide; a compound that modulates substrate
specificity/selectivity of the ALDH polypeptide; and a compound
that both modulates enzymatic activity of the ALDH polypeptide and
modulates substrate specificity/selectivity of the ALDH
polypeptide.
[0075] The present disclosure provides a method of identifying a
compound that binds to an ALDH polypeptide (e.g., to a
ligand-binding site of an ALDH polypeptide; a catalytic site; an
entrance to the active site), the method generally involving:
designing a compound based upon a three-dimensional structure of a
complex comprising an ALDH polypeptide and a ligand bound to a
ligand-binding site within the ALDH polypeptide, where the
three-dimensional structure is defined by structure coordinates
within Table 1 or Table 6; contacting the compound with an ALDH
polypeptide; and determining whether the compound binds to a
ligand-binding site of the ALDH polypeptide. In some embodiments,
the compound is designed de novo. In other embodiments, the
compound is designed from a known compound. The compound can be an
inhibitor (e.g., an antagonist) or an activator (e.g., an agonist)
of an enzymatic activity of an ALDH polypeptide. In some
embodiments, the compound modulates dehydrogenase activity of an
ALDH polypeptide. In other embodiments, the compound modulates
esterase activity of an ALDH polypeptide. In other embodiments, the
compound modulates substrate specificity/selectivity of an ALDH
polypeptide. In other embodiments, the compound modulate both an
enzymatic activity and a substrate selectivity/specificity of an
ALDH polypeptide.
[0076] In certain cases, a subject method will further comprise a
testing a compound to determine if it binds and/or modulates an
ALDH polypeptide, using the atomic coordinates provided herein. In
some embodiments, a subject method will further comprise obtaining
the compound (e.g., purchasing or synthesizing the compound) and
testing the compound to determine if it modulates (e.g., activates
or inhibits) an enzymatic activity of an ALDH polypeptide (e.g.,
acts an agonist or an antagonist of an ALDH polypeptide). In some
embodiments, a subject method will further comprise obtaining the
compound (e.g., purchasing or synthesizing the compound) and
testing the compound to determine if it modulates substrate
specificity/selectivity of an ALDH polypeptide.
[0077] In other cases, a subject method involves designing a
compound that binds to an ALDH polypeptide, either de novo, or by
modifying an existing compound that is known to bind to the ALDH
polypeptide. In particular embodiments, a subject method involves
computationally identifying a compound that binds to an ALDH
polypeptide using the atomic coordinates set forth in Table 1 or
Table 6. In other embodiments, a subject method involves
computationally identifying a compound that binds to the ligand
binding site of an ALDH polypeptide, wherein the ligand binding
site includes the following amino acids: Met-124, Phe-170, Leu-173,
Phe-292, Phe-296, Cys-302, and Phe-459 of human ALDH2 (or
corresponding amino acids in another ALDH family member) as well as
those atoms that are close thereto, e.g., within 5 .ANG., within 10
.ANG., within 20 .ANG. or within 30 .ANG. of those amino acids.
[0078] A method that comprises receiving a set of atomic
coordinates for an ALDH polypeptide; and identifying a compound
that binds to the ALDH polypeptide using the coordinates is also
provided, as is a method comprising: forwarding to a remote
location a set of atomic coordinates for the ALDH polypeptide; and
receiving the identity of a compound that binds to the ALDH
polypeptide.
[0079] In some embodiments, a subject method of identifying a
compound that binds to an ALDH polypeptide (e.g., a ligand-binding
site of an ALDH polypeptide), comprises the steps of: (a) providing
a molecular model comprising one or more ligand-binding regions of
an ALDH polypeptide, wherein the molecular model is made: (i) from
the atomic co-ordinates depicted in Table 1 or Table 6; or (ii)
from atomic co-ordinates derived by molecular modeling using the
atomic coordinates depicted in Table 1 or Table 6; (b) using the
molecular model to identify a candidate molecule that can bind to
the molecular model; and (c) producing the candidate molecule
identified in step (b).
[0080] A subject method can provide for one or more of: 1)
improving the potency of a "lead" compound or a known compound; 2)
designing new compound structures that exhibit improved
structure/function relationships for ALDH modulation; 3) designing
activator (agonist) compounds that are isozyme-selective activators
(e.g., compounds that are selective agonists for a particular ALDH
isozyme); 4) designing activator compounds that activate two or
more ALDH isozymes; 5) designing inhibitor (antagonist) compounds
that are isozyme-selective inhibitors (e.g., compounds that are
selective inhibitors for a particular ALDH isozyme); 6) designing
inhibitor compounds that inhibit two or more ALDH isozymes; 7)
designing compounds that exhibit both ALDH agonist and ALDH
antagonist activity; 8) designing compounds that can be directed,
controlled, or switched to function as either an ALDH agonist or an
ALDH antagonist; and 9) designing or selecting compounds that
modulate substrate specificity of an ALDH polypeptide. Compounds
that modulate substrate specificity of an ALDH polypeptide include
compounds that narrow the substrate specificity of an ALDH
polypeptide, e.g., such that the ALDH polypeptide demonstrates a
preference, or selectivity, for short-chain, long-chain, aliphatic,
or aromatic aldehyde or ester substrates; and compounds that
broaden the substrate specificity of an ALDH polypeptide.
[0081] In certain embodiments, a computer system comprising a
memory comprising the atomic coordinates of an ALDH polypeptide
having a bound ligand (ALDH/bound ligand) is provided. The atomic
coordinates are useful as models for rationally identifying
compounds that a ligand binding site of an ALDH polypeptide. Such
compounds may be designed either de novo, or by modification of a
known compound, for example. In other cases, binding compounds may
be identified by testing known compounds to determine if the "dock"
with a molecular model of an ALDH polypeptide. Such docking methods
are generally well known in the art.
[0082] The structure data provided herein can be used in
conjunction with computer-modeling techniques to develop models of
binding of various ALDH-binding compounds by analysis of the
crystal structure data. The structure data provided herein can be
used in conjunction with computer-modeling techniques to design
compounds that modulate ALDH enzymatic activity. The site models
characterize the three-dimensional topography of site surface, as
well as factors including van der Waals contacts, electrostatic
interactions, and hydrogen-bonding opportunities. Computer
simulation techniques are then used to map interaction positions
for functional groups including but not limited to protons,
hydroxyl groups, amine groups, divalent cations, aromatic and
aliphatic functional groups, amide groups, alcohol groups, etc.
that are designed to interact with the model site. These groups may
be designed into a pharmacophore or candidate compound with the
expectation that the candidate compound will specifically bind to
the site. Pharmacophore design thus involves a consideration of the
ability of the candidate compounds falling within the pharmacophore
to interact with a site through any or all of the available types
of chemical interactions, including hydrogen bonding, van der
Waals, electrostatic, and covalent interactions, although in
general, pharmacophores interact with a site through non-covalent
mechanisms.
[0083] The ability of a pharmacophore or candidate compound to bind
to an ALDH polypeptide can be analyzed prior to actual synthesis
using computer modeling techniques. Only those candidates that are
indicated by computer modeling to bind the target (e.g., an ALDH
polypeptide binding site) with sufficient binding energy (i.e.,
binding energy corresponding to a dissociation constant with the
target on the order of 10.sup.-2 M or tighter) may be synthesized
and tested for their ability to bind to an ALDH polypeptide and to
modulate ALDH enzymatic function using enzyme assays known to those
of skill in the art and/or as described herein. The computational
evaluation step thus avoids the unnecessary synthesis of compounds
that are unlikely to bind an ALDH polypeptide with adequate
affinity.
[0084] An ALDH pharmacophore or candidate compound may be
computationally evaluated and designed by means of a series of
steps in which chemical entities or fragments are screened and
selected for their ability to associate with individual binding
target sites on an ALDH polypeptide. One skilled in the art may use
one of several methods to screen chemical entities or fragments for
their ability to associate with an ALDH polypeptide, and more
particularly with target sites on an ALDH polypeptide. The process
may begin by visual inspection of, for example a target site on a
computer screen, based on the ALDH polypeptide coordinates, or a
subset of those coordinates, as set forth in Table 1 or Table
6.
[0085] Selected fragments or chemical entities may then be
positioned in a variety of orientations or "docked" within a target
site of an ALDH polypeptide as defined from analysis of the crystal
structure data. Manual docking may be accomplished using software
such as Insight II (Accelrys, San Diego, Calif.) MOE (Chemical
Computing Group, Inc., Montreal, Quebec, Canada); and SYBYL
(Tripos, Inc., St. Louis, Mo., 1992), followed by energy
minimization and/or molecular dynamics with standard molecular
mechanics force fields, such as CHARMM (Brooks, et al., J. Comp.
Chem. 4:187-217, 1983), AMBER (Weiner, et al., J. Am. Chem. Soc.
106: 765-84, 1984) and C.sup.2 MMFF (Merck Molecular Force Field;
Accelrys, San Diego, Calif.). More automated docking may be
accomplished by using programs such as DOCK (Kuntz et al., J. Mol.
Biol., 161:269-88, 1982; DOCK is available from University of
California, San Francisco, Calif.); AUTODOCK (Goodsell & Olsen,
Proteins: Structure, Function, and Genetics 8:195-202, 1990;
AUTODOCK is available from Scripps Research Institute, La Jolla,
Calif.); GOLD (Cambridge Crystallographic Data Centre (CCDC); Jones
et al., J. Mol. Biol. 245:43-53, 1995); and FLEXX (Tripos, St.
Louis, Mo.; Rarey, M., et al., J. Mol. Biol. 261:470-89, 1996).
[0086] Specialized computer programs may also assist in the process
of selecting fragments or chemical entities. These include but are
not limited to: GRID (Goodford, P. J., "A Computational Procedure
for Determining Energetically Favorable Binding Sites on
Biologically Important Macromolecules," J. Med. Chem., 28, pp.
849-857 (1985)); GRID is available from Oxford University, Oxford,
UK; MCSS (Miranker, A. and M. Karplus, "Functionality Maps of
Binding Sites: A Multiple Copy Simultaneous Search Method,"
Proteins: Structure, Function and Genetics, 11, pp. 29-34 (1991));
MCSS is available from Molecular Simulations, Inc., San Diego,
Calif.; AUTODOCK (Goodsell, D. S. and A. J. Olsen, "Automated
Docking of Substrates to Proteins by Simulated Annealing,"
Proteins: Structure, Function, and Genetics, 8, pp. 195-202
(1990)); AUTODOCK is available from Scripps Research Institute, La
Jolla, Calif.; DOCK (Kunts, I. D., et al. "A Geometric Approach to
Macromolecule-Ligand Interactions," J. Mol. Biol., 161, pp. 269-288
(1982)); DOCK is available from University of California, San
Francisco, Calif.; CERIUS II (available from Accelrys, Inc., San
Diego, Calif.); and Flexx (Raret, et al. J. Mol. Biol. 261, pp.
470-489 (1996)).
[0087] After selecting suitable chemical entities or fragments,
they can be assembled into a single compound. Assembly may proceed
by visual inspection of the relationship of the fragments to each
other on a three-dimensional image of the fragments in relation to
the ALDH/modulator structure or portion thereof displayed on a
computer screen. Visual inspection may be followed by manual model
building using software such as the Quanta or Sybyl programs
described above.
[0088] Software programs also may be used to aid one skilled in the
art in connecting the individual chemical entities or fragments.
These include, but are not limited to CAVEAT (Bartlett, P. A., et
al. "CAVEAT: A Program to Facilitate the Structure-Derived Design
of Biologically Active Molecules" In "Molecular Recognition in
Chemical and Biological Problems," Special Publ, Royal Chem. Soc.,
78, pp. 182-196 (1989)); CAVEAT is available from the University of
California, Berkeley, Calif.; 3D Database systems such as MACCS-3D
(MDL Information Systems, San Leandro, Calif.); this area is
reviewed in Martin, Y. C., "3D Database Searching in Drug Design,"
J. Med. Chem., 35:2145-2154 (1992)); and HOOK (available from
Molecular Simulations Inc., San Diego, Calif.).
[0089] As an alternative to building candidate pharmacophores or
candidate compounds up from individual fragments or chemical
entities, they may be designed de novo using the structure of an
ALDH target site, optionally, including information from
co-factor(s) or known activators or inhibitor(s) that bind to the
target site. De novo design may be included by programs including,
but not limited to LUDI (Bohm, H. J., "The Computer Program LUDI: A
New Method for the De Novo Design of Enzyme Inhibitors, J. Comp.
Aid. Molec. Design, 6, pp. 61-78 (1992)); LUDI is available from
Molecular Simulations, Inc., San Diego, Calif.; LEGEND (Nishibata,
Y., and Itai, A., Tetrahedron 47, p. 8985 (1991); LEGEND is
available from Molecular Simulations, San Diego, Calif.; and
LeapFrog (available from Tripos Associates, St. Louis, Mo.).
[0090] The functional effects of known ALDH ligands also may be
altered through the use of the molecular modeling and design
techniques described herein. This may be carried out by docking the
structure of the known ALDH ligand into an ALDH model structure and
modifying the structure and charge distribution of the ligand to
optimize the binding interactions with the ALDH enzyme. The
modified structure may be synthesized or obtained from a library of
compounds and tested for its binding affinity and/or effect on ALDH
enzymatic activity. This information can be used in design of
optimized ligands. The crystals and structures provided in the
present disclosure are especially well suited for the docking,
co-crystallization, structure-based drug design and optimization of
ligands that modulate one or more enzymatic activities of an ALDH.
The present disclosure permits the use of molecular, biochemical
and computer modeling techniques to design and select novel ligands
that interact with an ALDH and affect one or more enzymatic
activities of an ALDH.
[0091] Additional molecular modeling techniques also may be
employed in accordance with the invention. See, e.g., Cohen, N. C.,
et al. "Molecular Modeling Software and Methods for Medicinal
Chemistry," J. Med. Chem., 33, pp. 883-894 (1990); Navia, M. A. and
Murcko, M. A., "The Use of Structural Information in Drug Design,"
Curr. Opin. Biotechnol. 8, pp. 696-700 (1997); and Afshar, et al.
"Structure-Based and Combinatorial Search for New RNA-Binding
Drugs," Curr. Opin. Biotechnol. 10, pp. 59-63 (1999).
[0092] Following pharmacophore or candidate compound design or
selection according to any of the above methods or other methods
known to one skilled in the art, the efficiency with which a
candidate compound falling within the pharmacophore definition
binds to an ALDH polypeptide may be tested and optimized using
computational evaluation. A candidate compound may be optimized,
e.g., so that in its bound state it would lack repulsive
electrostatic interaction with the target site. These repulsive
electrostatic interactions include repulsive charge-charge,
dipole-dipole, and charge-dipole interactions. In some embodiments,
the sum of all electrostatic interactions between the candidate
compound and an ALDH when the candidate compound is bound to the
ALDH make a neutral or favorable contribution to the binding
enthalpy.
[0093] Specific computer software is available in the art to
evaluate compound deformation energy and electrostatic interaction.
Examples of programs designed for such uses include: Gaussian 94,
revision C (Frisch, Gaussian, Inc., Pittsburgh, Pa. (1995); AMBER,
version 7. (Kollman, University of California at San Francisco,
(2002); QUANTA/CHARMM (Accelrys, Inc., San Diego, Calif., (1995);
Insight II/Discover (Accelrys, Inc., San Diego, Calif., (1995);
DelPhi (Accelrys, Inc., San Diego, Calif., (1995); and AMSOL
(University of Minnesota) (Quantum Chemistry Program Exchange,
Indiana University). These programs may be implemented, for
instance, using a computer workstation, as are well known in the
art, for example, a LINUX, SGI or Sun workstation. Other hardware
systems and software packages will be known to those skilled in the
art.
[0094] Once a pharmacophore or candidate compound has been
optimally selected or designed, as described above, substitutions
may then be made in some of its atoms or side groups to improve or
modify its binding properties. Generally, initial substitutions are
conservative in that the replacement group will have either
approximately same size, or overall structure, or hydrophobicity,
or charge as the original group. Components known in the art to
alter conformation should be avoided in making substitutions.
Substituted candidates may be analyzed for efficiency of fit to an
ALDH using the same methods described above.
[0095] Once a candidate compound has been identified using any of
the methods described above, it can be screened for biological
activity. Any one of a number of assays of for ALDH enzymatic known
to those of skill in the art may be used.
[0096] Assays for dehydrogenase activity of an ALDH polypeptide are
known in the art, and any known assay can be used. Examples of
dehydrogenase assays are found in various publications, including,
e.g., Sheikh et al. ((1997) J. Biol. Chem. 272:18817-18822);
Vallari and Pietruszko (1984) J. Biol. Chem. 259:4922; and Farres
et al. ((1994) J. Biol. Chem. 269:13854-13860).
[0097] As an example of an assay for dehydrogenase activity, ALDH2
is assayed at 25.degree. C. in 50 mM sodium pyrophosphate HCl
buffer, pH 9.0, 100 mM sodium phosphate buffer, pH 7.4, or 50 mM
sodium phosphate buffer, pH 7.4, where the buffer includes
NAD.sup.+ (e.g., 0.8 mM NAD.sup.+, or higher, e.g., 1 mM, 2 mM, or
5 mM NAD.sup.+) and an aldehyde substrate such as 14 .mu.M
propionaldehyde. Reduction of NAD.sup.+ is monitored at 340 nm
using a spectrophotometer, or by fluorescence increase using a
fluoromicrophotometer. Enzymatic activity can be assayed using a
standard spectrophotometric method, e.g., by measuring a reductive
reaction of the oxidized form of nicotinamide adenine dinucleotide
(NAD.sup.+) to its reduced form, NADH, at 340 nm, as described in
US 2005/0171043; and WO 2005/057213. In an exemplary assay, the
reaction is carried out at 25.degree. C. in 0.1 NaPP.sub.i buffer,
pH 9.5, 2.4 mM NAD.sup.+ and 10 mM acetaldehyde as the substrate.
Enzymatic activity is measured by a reductive reaction of NAD.sup.+
to NADH at 340 nm, as described in US 2005/0171043; and WO
2005/057213. Alternatively, the production of NADH can be coupled
with another enzymatic reaction that consumes NADH and that
provides for a detectable signal. An example of such an enzymatic
reaction is a diaphorase-based reaction, which reduces resazurin to
its oxidized fluorescent compound resorufin, as described in US
2005/0171043; and WO 2005/057213. Detection of fluorescent
resorufin at 590 nm provides amplified and more sensitive signals
for any change in ALDH2 enzymatic activity.
[0098] Esterase activity of ALDH2 can be determined by monitoring
the rate of p-nitrophenol formation at 400 nm in 25 mM N,N-Bis
(2-hydroxyethyl)-2-amino ethanesulfonic acid (BES) (pH 7.5) with
800 .mu.M p-nitrophenyl acetate as the substrate at room
temperature in the absence or presence of added NAD.sup.+. A
pH-dependent molar extinction coefficient of 16 mM.sup.-1cm.sup.-1
at 400 nm for nitrophenol can be used. See, e.g., Larson et al.
(2007) J. Biol. Chem. 282:12940). Esterase activity of ALDH2 can be
determined by measuring the rate of p-nitrophenol formation at 400
nm in 50 mM Pipes (pH 7.4) with 1 mM p-nitrophenylacetate as the
substrate. A molar extinction coefficient of 18.3.times.10.sup.3
M.sup.-1cm.sup.-1 at 400 nm for p-nitrophenolate can be used for
calculating its rate of formation. See, e.g., Ho et al. (2005)
Biochemistry 44:8022).
[0099] A reductase activity of an ALDH polypeptide (e.g., ALDH2)
can be determined by measuring the rate of 1,2-glyceryl dinitrate
and 1,3-glyceryl dinitrate formation using a thin layer
chromatography (TLC) or liquid scintillation spectrometry method,
using a radioactively labeled substrate. For example, 0.1 mM or 1
mM GTN (glyceryl trinitrate) is incubated with the assay mixture (1
ml) containing 100 mM KPi (pH 7.5), 0.5 mM EDTA, 1 mM NADH, 1 mM
NADPH in the presence an ALDH polypeptide. After incubation at
37.degree. C. for about 10 minutes to about 30 minutes, the
reaction is stopped and GTN and its metabolites are extracted with
3.times.4 ml ether and pooled, and the solvent is evaporated by a
stream of nitrogen. The final volume is kept to less than 100
microliter in ethanol for subsequent thin layer chromatographic
(TLC) separation and scintillation counting. See, e.g., Zhang and
Stamler (2002) Proc. Natl. Acad. Sci. USA 99:8306.
[0100] Computer Models, Computer-Readable Media, and Computer
Systems
[0101] One embodiment of the present disclosure includes
representations, or models, of a three dimensional structure of an
ALDH with a bound ligand, such as a computer model. A computer
model of the present disclosure can be produced using any suitable
software program, including, but not limited to, PYMOL, GRASP, or O
software. Suitable computer hardware useful for producing an image
of the present invention are known to those of skill in the art
(e.g., a Silicon Graphics Workstation, Linux PC, or MacIntosh
PC).
[0102] The representations, or models, of a three dimensional
structure of an ALDH with a bound ligand can also be determined
based on the crystals provided in the present disclosure, with use
of techniques which include molecular replacement or SIR/MIR
(single/multiple isomorphous replacement). Methods of molecular
replacement are generally known by those of skill in the art
(generally described in Brunger, Meth Enzym 1997, 276:558-80;
Navaza and Saludjian, Meth Enzym 1997, 276, 581-94; Tong and
Rossmann, Meth Enzym 1997, 276:594-611; and Bentley, Meth Enzym
1997, 276:611-19, 1997, each of which is incorporated by this
reference herein in its entirety) and are performed by a software
program including, for example, the Phaser program (McCoy et al.,
Acta Crystallogr D Biol Crystallogr 2005, 61:458-64; Stroni et al.,
Acta Crystallogr D Biol Crystallogr 2004, 60:432-38).
[0103] Briefly, X-ray diffraction data are collected from the
crystal of an ALDH having a bound ligand. The X-ray diffraction
data are transformed to calculate a Patterson function. The
Patterson function of the crystallized target structure is compared
with a Patterson function calculated from a known structure
(referred to herein as a search structure). The Patterson function
of the crystallized target structure is rotated on the search
structure Patterson function to determine the correct orientation
of the crystallized target structure in the crystal. The
translation function is then calculated to determine the location
of the target structure with respect to the crystal axes. Once the
crystallized target structure has been correctly positioned in the
unit cell, initial phases for the experimental data can be
calculated. These phases are necessary for calculation of an
electron density map from which structural differences can be
observed, and for refinement of the structure. Alternatively, the
phases for the diffraction data can be deduced without an initial
structural model through the introduction of a heavy element, such
as selenium, mercury or the like. Location of the heavy atoms
within the structure using their intrinsic anomalous scattering
properties permits calculation of the phases for the complete
structure. These methods are known to those skilled in the art. The
structural features (e.g., amino acid sequence, conserved
di-sulfide bonds, and .beta.-strands or .beta.-sheets) of the
search molecule can be related to the crystallized target
structure.
[0104] As used herein, the term "model" refers to a representation
in a tangible medium of the three dimensional structure of an ALDH
enzyme in a complex with a bound ligand. For example, a model can
be a representation of the three dimensional structure in an
electronic file, on a computer screen, on a piece of paper (i.e.,
on a two dimensional medium), and/or as a ball-and-stick figure.
Physical three-dimensional models are tangible and include, but are
not limited to, stick models and space-filling models. The phrase
"imaging the model on a computer screen" refers to the ability to
express (or represent) and manipulate the model on a computer
screen using appropriate computer hardware and software technology
known to those skilled in the art. Such technology is available
from a variety of sources including, for example, Accelrys Inc.,
San Diego, Calif.. The phrase "providing a picture of the model"
refers to the ability to generate a "hard copy" of the model. Hard
copies include both motion and still pictures. Computer screen
images and pictures of the model can be visualized in a number of
formats including space-filling representations, backbone traces,
ribbon diagrams, and electron density maps.
[0105] One embodiment of the present disclosure relates to a
computer readable medium with ALDH/bound ligand structural data
and/or information stored thereon. As used herein, the phrase
"computer readable medium" refers to storage media readable by a
computer, which media may be used to store and retrieve data and
software programs incorporating computer code. Exemplary computer
readable media include floppy disk, CD-ROM, tape, memory (such as
flash memory or system memory), hard drive, and the like.
[0106] Thus, the present invention provides a computer readable
medium comprising atomic coordinates of an ALDH polypeptide with a
ligand bound to a ligand-binding site within the polypeptide. In
some embodiments, the atomic coordinates ate those set forth in
Table 1. In some embodiments, the atomic coordinates are those set
forth in Table 6. In some embodiments, a subject computer-readable
medium further comprises programming for displaying a molecular
model of the ALDH polypeptide with a ligand bound to a
ligand-binding site within the polypeptide. In some embodiments, a
subject computer-readable medium further comprises programming for
identifying a compound that binds to an ALDH polypeptide. For
example, the programming for identifying a compound that binds to
an ALDH polypeptide can comprise a database of structures of known
test compounds.
[0107] In another embodiment, the invention provides a computer
system having a memory comprising: X-ray crystallographic structure
coordinates defining a structure of an ALDH with a bound ligand;
and a processor in electrical communication with the memory,
wherein the processor generates a molecular model having a three
dimensional structure representative of an ALDH with a bound
ligand. The processor can be adapted for identifying a candidate
compound having a structure that is capable of binding to the ALDH
polypeptide.
[0108] As used herein, the term "computer system" is understood to
mean any general or special purpose system which includes a
processor in electrical communication with both a memory and at
least one input/output device, such as a terminal. Such a system
may include, but is not limited to, personal computers,
workstations, and mainframes. The processor may be a general
purpose processor or microprocessor or a specialized processor
executing programs located in RAM memory. The programs may be
placed in RAM from a storage device, such as a disk or
preprogrammed ROM memory. The RAM memory in one embodiment is used
both for data storage and program execution. The term computer
system also embraces systems where the processor and memory reside
in different physical entities but which are in electrical
communication by means of a network.
[0109] The processor executes a modeling program which accesses
data representative of an ALDH with a bound ligand. In addition,
the processor also can execute another program, a compound modeling
program, which uses the three-dimensional model of the ALDH with a
bound ligand to identify compounds having a chemical structure that
binds to the ALDH. In one embodiment the compound modeling program
and the ALDH/bound ligand structure modeling program are the same
program. In another embodiment, the compound modeling program and
the ALDH/bound ligand structure modeling program are different
programs, which programs may be stored on the same or different
storage medium. For example, the ALDH/bound ligand structure
modeling program may either store the three-dimensional model of
ALDH with a bound ligand in a region of memory accessible both to
it and to the compound modeling program, or the ALDH/bound ligand
model may be written to external storage, such as a disk, CD ROM,
or magnetic tape for later access by the compound modeling
program.
[0110] Compound Libraries for Screening
[0111] Inhibitors and/or activators identified according to the
methods of the invention can be provided from libraries of
compounds available from a number of sources or may be derived by
combinatorial chemistry approaches known in the art. Such libraries
include but are not limited to the available Chemical Director,
Maybridge, and natural product collections. In an exemplary
embodiment, libraries of compounds with known or predicted
structures may be docked to a subject ALDH/bound ligand
structure.
[0112] Utility
[0113] Compounds identified using a method as described above are
useful, for example, in the treatment of a condition or disorder
that is amenable to treatment by modulating ALDH activity. Such
conditions and disorders include, e.g., conditions involving
ischemic stress; chronic free-radical associated diseases; acute
free-radical associated diseases; insensitivity to nitroglycerin
(e.g., in angina and heart failure); hypertension; diabetes;
osteoporosis; cancer; alcohol (e.g., ethanol; ethyl alcohol)
addiction; narcotic addiction; aldehyde toxicity; and the like.
EXAMPLES
[0114] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Celsius, and pressure
is at or near atmospheric. Standard abbreviations may be used,
e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or
sec, second(s); min, minute(s); h or hr, hour(s); aa, amino
acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s);
i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c.,
subcutaneous(ly); and the like.
Example 1
Crystal Structure of ALDH2 with Alda-1
[0115] Methods
[0116] ALDH Expression, Purification, and Kinetic Studies
[0117] ALDH2 and ALDH2*2 were produced and purified using an E.
coli expression system as previously described (Larson et al.,
2005, J. Biol. Chem. 280, 30550-30556 and Larson et al., 2007, J.
Biol. Chem., 282, 12940-12950). All enzyme assays were performed in
25 mM BES, pH 7.5 and included a final concentration of 2% (v/v)
DMSO as a cosolvent in all cases whether or not Alda-1 was present.
Enzyme concentrations in the dehydrogenase assays were between 0.03
and 0.06 .mu.M for ALDH2 and between 0.3 and 0.5 .mu.M for ALDH2*2.
Esterase assays utilized 0.97 mM para-nitrophenylacetate (pNPA) as
a standard substrate concentration and enzyme concentrations for
0.06 .mu.M for ALDH2 and 0.7 .mu.M for ALDH2*2. All kinetic data
were analyzed with SigmaPlot (v10.0, StatSys). All activation data
for the dehydrogenase and esterase reactions were fit to the
expression v=V.sub.o+{(V.sub.max[S])/(K.sub.Act+[S])}, where
V.sub.o is the initial velocity for the reaction in the absence of
activator and K.sub.Act is the concentration of activator required
for half-maximal activation. All single-vary experiments for Alda-1
activation utilized a minimum of 10 different concentrations across
a range from 0-200 .mu.M. The covariation data between NAD.sup.+
and Alda-1 for ALDH2*2 utilized Alda-1 concentrations between 0-30
.mu.M and NAD.sup.+ concentrations between 0.5-10 mM and were fit
to the nonessential activator equation
v=(V.sub.max[S])/{K.sub.M[(1+[A]/K.sub.A)/(1+.beta.[A]/.alpha.K.sub.A)]+-
[S][(1+[A]/.alpha.K.sub.A)/(1+.beta.[A]/.alpha.K.sub.A)]},
where [S] is the varied concentration of coenzyme, [A] the varied
concentration of activator, a is the modifier on K.sub.M, .beta. is
the modifier on V.sub.max and K.sub.A is the half-maximal
concentration of activator. The daidzin inhibition data were fit to
the four parameter EC.sub.50 equation,
v=V.sub.min+{(V.sup.max-V.sub.min)/(1+[S]/EC.sub.50).sup.Hillslope}.
All data represent the average of a minimum of three independent
experiments with at least two different enzyme preparations.
[0118] Crystallization and Structure Determination
[0119] Crystals of the wild-type ALDH2 polypeptide (ALDH2*1) or
E487K mutant ALDH2 polypeptide (ALDH2*2) were grown under
conditions similar to that previously reported (Perez-Miller and
Hurley (2003) Biochem. 42:7100). Briefly, the apo-enzyme was
concentrated to 8 mg/ml and equilibrated against a crystallization
solution that contained 100 mM ACES
(N-(2-acetamido)-2-aminoethansulfonic acid), pH 6.4, 100 mM
guanidine-HCl, 10 mM MgCl.sub.2, and 16-17% (w/v) poly(ethylene
glycol) (PEG) 6000. The complex with Alda-1 was prepared through a
direct soaking experiment in which the apo-enzyme crystals were
first equilibrated against 1% dimethylsulfoxide (DMSO) in crystal
stabilization solution (100 mM ACES, pH 6.4, 100 mM guanidine-HCl,
10 mM MgCl.sub.2, and 19% (w/v) PEG 6000). Following an overnight
incubation, the stabilization solution was replaced with an
identical solution to which 200 .mu.M Alda-1 was added. The
crystals were allowed to soak overnight and prepared for cryogenic
freezing, a two-step protocol to introduce 18% (v/v) ethylene
glycol into the soaking solution. Diffraction data were collected
at beamline 19-ID operated by the Structural Biology Consortium
located within the Advanced Photon Source at Argonne National
Laboratory. All diffraction data were indexed, integrated, and
scaled using the HKL2000/HKL3000 program suite (Otwinowski and
Minor (1997) Meth. Enzymol. 276:307). X-ray diffraction data and
refinement statistics is shown in Table 2.
TABLE-US-00001 TABLE 2 ALDH2 ALDH2*2 Data Collection.sup..dagger.:
Space Group P2.sub.1 P2.sub.1 Cell Dimensions a = 102 .ANG., b =
177 .ANG., c = 103 .ANG. a = 102 .ANG., b = 177 .ANG., c = 102
.ANG. .alpha. = .gamma. = 90.degree., .beta. = 94.5.degree. .alpha.
= .gamma. = 90.degree., .beta. = 94.6.degree. Resolution 46.0-1.69
.ANG. 50.0-1.9 .ANG. Total observations 1,025,775 884,944 Unique
Reflections 375,531 281,043 Completeness 93.1% (90.9%)* 99.3%
(100%).sup.# <I>/.sigma..sub.<I> 11.4 (2.7)* 9.6
(2.8).sup.# R.sub.merge 0.077 (0.27)* 0.107 (0.40).sup.#
Refinement: R.sub.free/R.sub.work 0.21/0.25 (0.24/0.32)* 0.14/0.18
(0.20/0.27).sup.# R.m.s.d. ideal bonds 0.011 .ANG. 0.007 .ANG.
R.m.s.d. ideal angles 1.36 .ANG. 1.09 .ANG. Bound activator
molecules 8 8 Bound solvent atoms 3,135 2,731 .sup..dagger.Data
collected at SBC beamline 19-ID, Argonne National Laboratory
*Values for the highest resolution shell (1.74-1.69 .ANG.)
.sup.#Values for the highest resolution shell (1.93-1.90 .ANG.)
[0120] The structure of Alda-1 is as follows:
##STR00001##
[0121] Alda-1 is an ALDH2 agonist. Alda-1 (at 100 .mu.M) increased
the activity of the homotetrameric mutant,ALDH2*2 11 fold, the
heterotetrameric ALDH2 2.2 fold (similar to the base levels of wild
type ALDH2) and the homotetrameric wild type ALDH2*1/*1 2.1 fold
(FIG. 2). Chen et al. (2008) Science 321:1493-1495; PMID:
18787169.
[0122] Crystals of ALDH2 tend to form two different lattice groups:
primitive orthorhombic P2.sub.12.sub.12.sub.1 and a pseudo-centered
monoclinic lattice that indexes in the C222.sub.1 space group with
the same cell dimensions as the primitive orthorhombic lattice.
However, the intensities of the diffraction pattern lack the strict
orthorhombic symmetry and generally require integration in the
primitive monoclinic lattice. As the structure is essentially
isomorphous with the wild-type ALDH2 monoclinic data set (Zhou et
al. (1999) supra), the structure was solved by direct refinement
using the wild-type human ALDH2 structure (with ligands and solvent
removed) as the starting model (PDB code 1cw3). Confirmation of the
binding of Alda-1
(N-(1,3-benzodioxo1-5-ylmethyl)-2,6-dichlorobenzamide) was
evaluated through inspection of the initial Fo-Fc electron density
maps. Refinement of the structures utilized the program Refmac
(Murshudov et al. (1997) Acta Crystallogr. D. Biol. Crystallogr.
53:240) or Phenix (P. D. Adams et al. (2002) Acta Cryst. D58,
1948-1954), and was visually inspected and adjusted using the
visualization program Coot (Emsley and Cowtan (2004) Acta
Crystallogr. D. Biol. Crystallogr. 60:2126).
[0123] Results
[0124] Structure of Alda-1 Bound to ALDH2
[0125] The atomic coordinates of the crystal structure of ALDH2
with Alda-1 are provided in Table 1. The atomic coordinates of the
crystal structure of an E487K mutant of ALDH2 with Alda-1 are
provided in Table 6.
[0126] The structure of wild-type ALDH2 in a binary complex with
Alda-1 was determined to 1.69 .ANG. and that of the binary complex
between Alda-1 and an S302 mutant of ALDH2*2 was determined to 1.9
.ANG. (Table 2). Alda-1 binds to both forms of ALDH2 at the exit of
the substrate tunnel and extending in toward the active site (FIGS.
3 and 11), leaving the catalytic Cys302 unimpeded, though it adopts
two distinct rotamer positions. The benzodioxol group of Alda-1 is
bound within an aromatic and hydrophobic collar comprised of amino
acids Val 120, Met124, Phe170, Leu173, Phe292, Phe296, and Phe459
solely through hydrophobic interactions. A single highly critical
hydrogen bond is formed between the amide nitrogen that links the
two ring structures in Alda-1 and the mainchain carbonyl oxygen
atom of Asp457. The dichlorobenzamide ring is bound also primarily
through hydrophobic interactions between the benzamide ring with
Va1458, Phe292 and Met124. It is interesting to note that diadzin,
an ALDH2 inhibitor, occupies a site that overlaps with Alda-1 (12,
PDB code 2VLE). However, the additional phenolic arm of daidzin
reaches further into the catalytic site and contacts Cys302 and
Glu268, thus blocking catalytic function (FIG. 4). FIG. 4 was
generated using PYMOL and atom type coloring is utilized for both
structures. The available molecular surface in this region for
ALDH2 is displayed using the daidzin structure and the molecule is
sliced above the plane of the bound ligands. Critical active site
residues are labeled. The cleft through which the nicotinamide
moiety accesses the active site lies to the left of Glu268 and
Cys302 in this view and is labeled. For the complex between Alda-1
and ALDH2*2, it is important to note that the active site loop
comprised of residues 465-477 and the alpha-helix comprised of
residues 245-262 are visible in this crystal structure, where these
sections of protein structure were disordered in the ALDH2*2
crystal structure reported in the absence of ligands (Larson et al.
2005, supra) (FIG. 10).
[0127] FIGS. 10 and 11. The structure of Alda-1 bound to ALDH2*2 at
1.9 Angstroms resolution shows that Alda-1 binds in the same
location as to the wild-type ALDH2 structure (FIG. 10). The binding
of Alda-1 to ALDH2*2 restores the coenzyme-binding sites of ALDH2*2
to that more similar to wild-type ALDH2 than to that of ALDH2*2 in
the absence of Alda-1 (FIG. 11). Consequently, both the kinetic
data and structural data support a mechanism for Alda-1 activation
that is based on the partial restoration of ALDH2*2 to a state that
is more similar to the wild-type enzyme.
[0128] Kinetic Characterization of Alda-1 Activation for the
Dehydrogenase Activity of Wild-type ALDH2 and ALDH2*2
[0129] The location of Alda-1 within the substrate-binding tunnel
of ALDH2 raises two possibilities: a) the activation effect of
Alda-1 is substrate length dependent and b) Alda-1 activation and
daidzin inhibition are mutually exclusive. At pH 7.5 a strong
dependence of activation on the length and nature of the substrate
aldehydes was found, with linear aliphatic aldehydes up to
butyraldehyde activated by Alda-1 with .mu.M K.sub.Act values
(Table 3) and aromatic aldehydes such as benzaldehyde,
phenylacetaldehyde and 4-trans-(N,N-dimethylamino)-cinnamaldehyde
(DACA) exhibiting minimal effects at 20 .mu.M Alda-1 and saturating
concentrations of substrate. Thus, the space between Cys302 and the
benzodioxal ring of Alda-1 can accommodate up to 4 carbons in
length. As shown in Table 3, smaller linear aliphatic aldehydes
were activated by Alda-1 and the extent of activation decreased
with length.
TABLE-US-00002 TABLE 3 Substrate Dependence for Alda-1 Activation
(25 mM BES, pH 7.5, 0.5 mM NAD.sup.+) Substrate V.sub.max.sup.(app)
(min.sup.-1) K.sub.Act.sup.(app) (.mu.M) V.sub.m/V.sub.o 100 .mu.M
Acetaldehyde 107 +/- 12 0.98 +/- 0.20 1.8 +/- 0.1 100 .mu.M 78.5
+/- 9.6 5.1 +/- 1.2 1.7 +/- 0.1 Propionaldehyde 100 .mu.M
Butyraldehyde 86.6 +/- 3.1 1.8 +/- 0.5 1.3 +/- 0.1
[0130] Alda-1 had little effect on activity with benzaldehyde, even
at the maximum concentration used; 200 .mu.M. Alda-1 also had
little effect on ALDH2 activity with phenylaceteldehyde or DACA,
although high concentrations of Alda-1 (>100 .mu.M) were weakly
inhibitory.
[0131] Alda-1 antagonized daidzin inhibition of both ALDH2 and
ALDH2*2 in a manner consistent with their overlapping binding sites
in ALDH2 (Table 4 and FIG. 5).
TABLE-US-00003 TABLE 4 Alda-1 induced antagonism of Daidzin
Inhibition [25 mM BES, pH 7.5, 100 .mu.M propionaldehyde and 0.5 mM
NAD.sup.+ (ALDH2) or 10 mM NAD.sup.+ (ALDH2*2)] ALDH2 ALDH2 ALDH2*2
ALDH2*2 (no Alda-1) (10 .mu.M Alda-1) (no Alda-1) (50 .mu.M Alda-1)
V.sub.max.sup.(app) (min.sup.-1) 61.5 +/- 6.1 74.9 +/- 12.1 11.6
+/- 1.3 23.0 +/- 1.7 V.sub.min.sup.(app) (min.sup.-1) 5.1 +/- 4.7
2.5 +/- 5.2 0.2 +/- 0.6 0.2 +/- 0.2 Daidzin IC.sub.50 (.mu.M) 8.0
+/- 0.6 72.0 +/- 16.9 44.8 +/- 10.4 113.0 +/- 15.6 Hill Slope 0.9
+/- 0.3 1.0 +/- 0.1 1.1 +/- 0.1 1.1 +/- 0.1
[0132] The nature of the activation of ALDH2*2 was analyzed in
detail through a covariation experiment between NAD.sup.+ and
Alda-1 and the data was fitted to the non-essential activator
equation. This analysis showed that Alda-1 increases the V.sub.max
of ALDH2*2 by 2-fold and decreases the apparent Km for NAD.sup.+ by
6.7-fold (FIG. 6). The plot in FIG. 6 shows the average values from
3 experiments: K.sub.act=16+/-3 .mu.M; K.sub.M.sup.NAD7.4+/-0.7 mM;
m .alpha.-factor=0.15+/-0.03; .beta.-factor=2.0+/-0.2. The
concentrations of Alda-1 were varied from 0 to 30 .mu.M. The
.alpha.- and .beta.-factors describe the manner in which Alda-1
impacts the observed K.sub.M.sup.NAD and V.sub.max, respectively.
Thus, Alda-1 restores the K.sub.M for NAD.sup.+from 7.4 mM to 1.1
mM and increases the V.sub.max2-fold.
[0133] Kinetic Characterization of Alda-1 Activation for the
Esterase Activity of Wild-type ALDH2 and ALDH2*2
[0134] In addition to the dehydrogenation reaction, members of the
aldehyde dehydrogenase family also exhibit the ability to hydrolyze
esters, such as p-nitrophenylacetate, and coenzyme is known to
stimulate the hydrolytic activity (Feldman and Weiner, 1972, J.
Biol. Chem. 247, 267-272 and Takahashi and Weiner, 1981,
Biochemistry 20, 2720-2726). The ability of Alda-1 to stimulate the
hydrolysis of p-nitrophenylacetate both in the presence and absence
of coenzyme was examined. Alda-1 alone was found to activate the
esterase activity of both ALDH2 and ALDH2*2 between 6- and 7-fold
and the combined activating effects of both Alda-1 and NAD.sup.+
increase ester hydrolysis 10-fold for ALDH2 and over 100-fold for
ALDH2*2 (Table 5).
TABLE-US-00004 TABLE 5 Esterase Activity Activation Constants for
ALDH2 and ALDH2*2 (25 mM BES, pH 7.5, 0.97 mM p-Nitrophenylacetate)
ALDH2 ALDH2*2 ALDH2*2 Constant ALDH2 (0.5 mM NAD.sup.+) ALDH2*2
(1.0 mM NAD.sup.+) (50 .mu.M Alda-1) V.sub.o (min-1) 24.9 +/- 2.0
96.3 +/- 2.2 0.40 +/- 0.03 1.36 +/- 0.28 0.64 +/- 0.19 V.sub.max
(min.sup.-1) 181 +/- 6.8 248 +/- 22 2.3 +/- 0.2 14.7 +/- 1.3 49.5
+/- 4.3 K.sub.Act.sup.(app) (.mu.M) 3.4 +/- 0.5 2.6 +/- 0.1 16.1
+/- 5.8 11.2 +/- 1.3 2,820 +/- 330 V.sub.max (min.sup.-1) -- 242
+/- 14 -- -- -- K.sub.i.sup.(app) (.mu.M) -- 328 +/- 24 -- --
--
[0135] At higher concentrations Alda-1 and NAD.sup.+ become
antagonistic for ALDH2, a behavior not noted for ALDH2*2 at the
concentrations examined herein. Similar to that observed for
coenzyme-binding kinetics in the dehydrogenation reaction catalyzed
by ALDH2*2, Alda-1 lowered the half-maximal activating
concentration of NAD.sup.+ for the esterase reaction from 7.5 mM
(8) to 2.8 mM for ALDH2*2.
Example 2
Use of Model to Predict Ligand Binding
[0136] A homology model of ALDH5 (now called ALDH1B1) was built.
The structures of ALDH3A1 and ALDH1A1 were aligned to that of ALDH2
with Alda-1 bound for significant changes with the residues that
contribute to Alda-1 binding.
[0137] ALDH1A1b
[0138] Within the substrate-binding site of ALDH1A1 (FIG. 7)
substitutions of Gly for Met124 and Val for Leu173 enlarges area
"A" which will result in the loss of many van der Waals
interactions. The substitution of His for Phe292 enlarges site "B"
and makes the area more hydrophilic. Lastly the substitution of Val
for Phe459, "C", removes a major aromatic stacking interaction with
the 1,3-benzodioxol ring and forms a pocket below the ring.
[0139] Alda-1 is not expected to bind to ALDH1A1 strongly, as the
substitutions at positions 124, 173, 292 and 459 greatly increase
the available space surrounding Alda-1. The available van der Waals
contacts are likely too distantly spaced to support a similar mode
of binding.
[0140] ALDH1B1
[0141] Within the substrate-binding site of ALDH1B1 (FIG. 8)
substitutions of Glu for Met124 and Glu for Phe292 create
unfavorable electrostatic interactions with the dichlorobenzamide
ring. The substitution of Val for Leu173 and Val for Phe459
enlarges the area around the 1,3-benzodioxol ring ("A") and removes
a major aromatic stacking interaction and, like ALDH1A1, creates a
pocket below the 1,3-benzodioxol ring.
[0142] Alda-1 is not expected to bind to ALDH1B1 strongly, as the
Glutamates at positions 124 and 292 create a negatively charge area
on both sides of the Alda-1 binding site and the loss of Phe459
removes a major contact surface area under the bicyclic ring of
Alda-1.
[0143] ALDH3A1
[0144] Within the substrate-binding site of ALDH3A1 (FIG. 9)
substitutions of Tyr for Met124, the substitution of Trp for Phe292
and a two amino acid insertion at the position equivalent to Phe459
greatly narrows the entry to the substrate-binding pocket (A). In
addition, the substitution of Asn for Leu173 and Gln for Trp178
enlarges the area around the 1,3-benzodioxol ring (B). A C-terminal
extension present in ALDH3A1 adds additional basic residues,
including Arg501 near the exit of the substrate-binding pocket.
[0145] The substrate binding site region of ALDH3A1 is very
different from ALDH2; it is predicted that Alda-1 would not bind to
ALDH3A1.
[0146] The structures of the binary complexes between Alda-1 and
ALDH2 and of Alda-1 and ALDH2*2 were solved to 1.69 .ANG. and 1.9
.ANG. resolution. The location of Alda-1 binding within the
substrate entrance tunnel of ALDH2 is reminiscent of the binding of
daidzin, a known potent inhibitor of ALDH2. If the positions of
Alda-1 and daidzin in their respective crystal structures are
correct, it was reasoned that Alda-1 should antagonize daidzin
inhibition. This was found to be true for both the wild-type ALDH2
and for ALDH2*2, confirming that Alda-1 and daidzin share
overlapping binding sites. The very different effects of daidzin
and Alda-1 on ALDH2 activity can be explained, in part, from their
crystal structures. In the daidzin bound structure, the phenolic
moiety interacts directly with two essential active site residues,
Cys302 and Glu268, inhibiting the enzyme by restricting substrate
binding and catalysis. In contrast, the structure shown herein with
ALDH2 and Alda-1 shows that Alda-1 binds at the entrance to the
active site, but does not interact with the catalytic residues,
leaving Cys302 and Glu268 free to function. Because Alda-1 does
block part of the substrate site, it was predicted that ALDH2
activation would depend on substrate size. Modeling of the complex
suggests that the space between Cys302 and the benzodioxol ring of
Alda-1 could accommodate acyl-enzyme intermediates up to 4 carbons
in length. The concentration dependence of Alda-1 activation at
saturating concentrations of acetaldehyde, propionaldehyde,
butyraldehyde, benzaldehyde, phenylacetaldehyde, and
4-trans-(N,N-dimethylamino)-cinnamaldehyde (DACA) was examined. It
was found that only the smaller linear aliphatic aldehydes were
activated by Alda-1 and the extent of activation decreased with
length. Alda-1 had little effect on activity with benzaldehyde,
even at the maximum concentration used; 200 .mu.M. Alda-1 also had
little effect on ALDH2 activity with phenylacetaldehyde or DACA,
although high concentrations of Alda-1 (>100 .mu.M) were weakly
inhibitory.
[0147] While the present invention has been described with
reference to the specific embodiments 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 objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
Sequence CWU 1
1
31517PRTHomo sapiens 1Met Leu Arg Ala Ala Ala Arg Phe Gly Pro Arg
Leu Gly Arg Arg Leu1 5 10 15Leu Ser Ala Ala Ala Thr Gln Ala Val Pro
Ala Pro Asn Gln Gln Pro 20 25 30Glu Val Phe Cys Asn Gln Ile Phe Ile
Asn Asn Glu Trp His Asp Ala 35 40 45Val Ser Arg Lys Thr Phe Pro Thr
Val Asn Pro Ser Thr Gly Glu Val 50 55 60Ile Cys Gln Val Ala Glu Gly
Asp Lys Glu Asp Val Asp Lys Ala Val65 70 75 80Lys Ala Ala Arg Ala
Ala Phe Gln Leu Gly Ser Pro Trp Arg Arg Met 85 90 95Asp Ala Ser His
Arg Gly Arg Leu Leu Asn Arg Leu Ala Asp Leu Ile 100 105 110Glu Arg
Asp Arg Thr Tyr Leu Ala Ala Leu Glu Thr Leu Asp Asn Gly 115 120
125Lys Pro Tyr Val Ile Ser Tyr Leu Val Asp Leu Asp Met Val Leu Lys
130 135 140Cys Leu Arg Tyr Tyr Ala Gly Trp Ala Asp Lys Tyr His Gly
Lys Thr145 150 155 160Ile Pro Ile Asp Gly Asp Phe Phe Ser Tyr Thr
Arg His Glu Pro Val 165 170 175Gly Val Cys Gly Gln Ile Ile Pro Trp
Asn Phe Pro Leu Leu Met Gln 180 185 190Ala Trp Lys Leu Gly Pro Ala
Leu Ala Thr Gly Asn Val Val Val Met 195 200 205Lys Val Ala Glu Gln
Thr Pro Leu Thr Ala Leu Tyr Val Ala Asn Leu 210 215 220Ile Lys Glu
Ala Gly Phe Pro Pro Gly Val Val Asn Ile Val Pro Gly225 230 235
240Phe Gly Pro Thr Ala Gly Ala Ala Ile Ala Ser His Glu Asp Val Asp
245 250 255Lys Val Ala Phe Thr Gly Ser Thr Glu Ile Gly Arg Val Ile
Gln Val 260 265 270Ala Ala Gly Ser Ser Asn Leu Lys Arg Val Thr Leu
Glu Leu Gly Gly 275 280 285Lys Ser Pro Asn Ile Ile Met Ser Asp Ala
Asp Met Asp Trp Ala Val 290 295 300Glu Gln Ala His Phe Ala Leu Phe
Phe Asn Gln Gly Gln Cys Cys Cys305 310 315 320Ala Gly Ser Arg Thr
Phe Val Gln Glu Asp Ile Tyr Asp Glu Phe Val 325 330 335Glu Arg Ser
Val Ala Arg Ala Lys Ser Arg Val Val Gly Asn Pro Phe 340 345 350Asp
Ser Lys Thr Glu Gln Gly Pro Gln Val Asp Glu Thr Gln Phe Lys 355 360
365Lys Ile Leu Gly Tyr Ile Asn Thr Gly Lys Gln Glu Gly Ala Lys Leu
370 375 380Leu Cys Gly Gly Gly Ile Ala Ala Asp Arg Gly Tyr Phe Ile
Gln Pro385 390 395 400Thr Val Phe Gly Asp Val Gln Asp Gly Met Thr
Ile Ala Lys Glu Glu 405 410 415Ile Phe Gly Pro Val Met Gln Ile Leu
Lys Phe Lys Thr Ile Glu Glu 420 425 430Val Val Gly Arg Ala Asn Asn
Ser Thr Tyr Gly Leu Ala Ala Ala Val 435 440 445Phe Thr Lys Asp Leu
Asp Lys Ala Asn Tyr Leu Ser Gln Ala Leu Gln 450 455 460Ala Gly Thr
Val Trp Val Asn Cys Tyr Asp Val Phe Gly Ala Gln Ser465 470 475
480Pro Phe Gly Gly Tyr Lys Met Ser Gly Ser Gly Arg Glu Leu Gly Glu
485 490 495Tyr Gly Leu Gln Ala Tyr Thr Glu Val Lys Thr Val Thr Val
Lys Val 500 505 510Pro Gln Lys Asn Ser 5152517PRTHomo sapiens 2Met
Leu Arg Ala Ala Ala Arg Phe Gly Pro Arg Leu Gly Arg Arg Leu1 5 10
15Leu Ser Ala Ala Ala Thr Gln Ala Val Pro Ala Pro Asn Gln Gln Pro
20 25 30Glu Val Phe Cys Asn Gln Ile Phe Ile Asn Asn Glu Trp His Asp
Ala 35 40 45Val Ser Arg Lys Thr Phe Pro Thr Val Asn Pro Ser Thr Gly
Glu Val 50 55 60Ile Cys Gln Val Ala Glu Gly Asp Lys Glu Asp Val Asp
Lys Ala Val65 70 75 80Lys Ala Ala Arg Ala Ala Phe Gln Leu Gly Ser
Pro Trp Arg Arg Met 85 90 95Asp Ala Ser His Arg Gly Arg Leu Leu Asn
Arg Leu Ala Asp Leu Ile 100 105 110Glu Arg Asp Arg Thr Tyr Leu Ala
Ala Leu Glu Thr Leu Asp Asn Gly 115 120 125Lys Pro Tyr Val Ile Ser
Tyr Leu Val Asp Leu Asp Met Val Leu Lys 130 135 140Cys Leu Arg Tyr
Tyr Ala Gly Trp Ala Asp Lys Tyr His Gly Lys Thr145 150 155 160Ile
Pro Ile Asp Gly Asp Phe Phe Ser Tyr Thr Arg His Glu Pro Val 165 170
175Gly Val Cys Gly Gln Ile Ile Pro Trp Asn Phe Pro Leu Leu Met Gln
180 185 190Ala Trp Lys Leu Gly Pro Ala Leu Ala Thr Gly Asn Val Val
Val Met 195 200 205Lys Val Ala Glu Gln Thr Pro Leu Thr Ala Leu Tyr
Val Ala Asn Leu 210 215 220Ile Lys Glu Ala Gly Phe Pro Pro Gly Val
Val Asn Ile Val Pro Gly225 230 235 240Phe Gly Pro Thr Ala Gly Ala
Ala Ile Ala Ser His Glu Asp Val Asp 245 250 255Lys Val Ala Phe Thr
Gly Ser Thr Glu Ile Gly Arg Val Ile Gln Val 260 265 270Ala Ala Gly
Ser Ser Asn Leu Lys Arg Val Thr Leu Glu Leu Gly Gly 275 280 285Lys
Ser Pro Asn Ile Ile Met Ser Asp Ala Asp Met Asp Trp Ala Val 290 295
300Glu Gln Ala His Phe Ala Leu Phe Phe Asn Gln Gly Gln Cys Cys
Cys305 310 315 320Ala Gly Ser Arg Thr Phe Val Gln Glu Asp Ile Tyr
Asp Glu Phe Val 325 330 335Glu Arg Ser Val Ala Arg Ala Lys Ser Arg
Val Val Gly Asn Pro Phe 340 345 350Asp Ser Lys Thr Glu Gln Gly Pro
Gln Val Asp Glu Thr Gln Phe Lys 355 360 365Lys Ile Leu Gly Tyr Ile
Asn Thr Gly Lys Gln Glu Gly Ala Lys Leu 370 375 380Leu Cys Gly Gly
Gly Ile Ala Ala Asp Arg Gly Tyr Phe Ile Gln Pro385 390 395 400Thr
Val Phe Gly Asp Val Gln Asp Gly Met Thr Ile Ala Lys Glu Glu 405 410
415Ile Phe Gly Pro Val Met Gln Ile Leu Lys Phe Lys Thr Ile Glu Glu
420 425 430Val Val Gly Arg Ala Asn Asn Ser Thr Tyr Gly Leu Ala Ala
Ala Val 435 440 445Phe Thr Lys Asp Leu Asp Lys Ala Asn Tyr Leu Ser
Gln Ala Leu Gln 450 455 460Ala Gly Thr Val Trp Val Asn Cys Tyr Asp
Val Phe Gly Ala Gln Ser465 470 475 480Pro Phe Gly Gly Tyr Lys Met
Ser Gly Ser Gly Arg Glu Leu Gly Glu 485 490 495Tyr Gly Leu Gln Ala
Tyr Thr Lys Val Lys Thr Val Thr Val Lys Val 500 505 510Pro Gln Lys
Asn Ser 515317PRTHomo sapiens 3Met Leu Arg Ala Ala Ala Arg Phe Gly
Pro Arg Leu Gly Arg Arg Leu1 5 10 15Leu
* * * * *
References