U.S. patent application number 15/155643 was filed with the patent office on 2016-09-29 for methods for identifying inhibitors of amyloid protein aggregation.
This patent application is currently assigned to Treventis Corporation. The applicant listed for this patent is Treventis Corporation. Invention is credited to Christopher J. BARDEN, Michael D. Carter, Donald F. Weaver.
Application Number | 20160283652 15/155643 |
Document ID | / |
Family ID | 56975445 |
Filed Date | 2016-09-29 |
United States Patent
Application |
20160283652 |
Kind Code |
A1 |
BARDEN; Christopher J. ; et
al. |
September 29, 2016 |
METHODS FOR IDENTIFYING INHIBITORS OF AMYLOID PROTEIN
AGGREGATION
Abstract
Methods for identifying compounds that are inhibitors or are
likely to be inhibitors of amyloid protein aggregation, as well as
three-dimensional, non-crystallographic models (i.e.
"pseudo-crystal structures") of amyloid aggregation utilized in the
methods, are described. Means for creating the three-dimensional,
non-crystallographic models (i.e. "pseudo-crystal structures") of
amyloid aggregation are also described.
Inventors: |
BARDEN; Christopher J.;
(Toronto, CA) ; Carter; Michael D.; (Toronto,
CA) ; Weaver; Donald F.; (Halifax, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Treventis Corporation |
Bryn Mawr |
PA |
US |
|
|
Assignee: |
Treventis Corporation
Bryn Mawr
PA
|
Family ID: |
56975445 |
Appl. No.: |
15/155643 |
Filed: |
May 16, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13872234 |
Apr 29, 2013 |
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15155643 |
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12549851 |
Aug 28, 2009 |
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13872234 |
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61092845 |
Aug 29, 2008 |
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61092826 |
Aug 29, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G16B 35/00 20190201;
G16B 15/00 20190201; G16C 20/60 20190201 |
International
Class: |
G06F 19/16 20060101
G06F019/16; C40B 30/02 20060101 C40B030/02; G06F 19/12 20060101
G06F019/12 |
Claims
1. A method of creating non-crystallographic models of amyloid
protein aggregation comprising: applying molecular mechanics and/or
dynamics to a three-dimensional model comprising a model of
crystallized amyloid protein, a model of multiply anti-amyloid
compound bound in the pocket of the crystallized amyloid protein,
and a candidate model of an uncrystallized amyloid protein, such
that a pocket on the uncrystallized amyloid protein is formed
around the multiply anti-amyloid compound; extracting the model of
uncrystallized amyloid protein from said model; and validating said
model of uncrystallized amyloid protein with respect to
experimental data so as to confirm its utility as a
non-crystallographic model of amyloid protein aggregation.
2. A non-crystallographic model of amyloid protein aggregation
comprising: a three-dimensional model of a monomeric amyloid
peptide; and a three-dimensional model of an amyloid protein
consisting of one or more amyloid peptides, said amyloid protein
model to be positioned with respect to said amyloid peptide model
such that it forms a pocket in conjunction with said amyloid
peptide model, such that a candidate compound may be inserted into
said pocket thereby modulating amyloid aggregation.
3. The non-crystallographic model of amyloid protein aggregation of
claim 2, wherein said amyloid peptide model and said amyloid
protein model are both composed of beta-amyloid protein or an
amyloid-forming fragment thereof.
4. The non-crystallographic model of amyloid protein aggregation of
claim 2, wherein said amyloid peptide model is substantially SEQ
ID: 1.
5. The non-crystallographic model of amyloid protein aggregation of
claim 4, wherein said amyloid protein model is substantially SEQ
ID: 2.
6. The non-crystallographic model of amyloid protein aggregation of
claim 5, wherein said amyloid peptide model is further positioned
with respect to said amyloid protein model such that: the amino
acid Val at position 8 of SEQ ID: 1 interacts with the amino acid
Glu at position 6 of SEQ ID: 2; the amino acid Gly at position 9 of
SEQ ID: 1 interacts with the amino acid Glu at position 6 of SEQ
ID: 2; the amino acid Ser at position 10 of SEQ ID: 1 interacts
with the amino acid Gly at position 9 of SEQ ID: 2; the amino acid
Ser at position 10 of SEQ ID: 1 interacts with the amino acid Ser
at position 10 of SEQ ID:2; and the amino acid Lys at position 12
of SEQ ID: 1 interacts with the amino acid Glu at position 6 of SEQ
ID: 2.
7. The non-crystallographic model of amyloid protein aggregation of
claim 5, wherein said amyloid peptide model is further positioned
with respect to said amyloid protein model substantially in the
orientation shown in FIG. 2.
8. A method of identifying compounds that modulate amyloid
aggregation comprising the steps of: constructing the
non-crystallographic model of amyloid protein aggregation according
to claim 7 in a computer modeling program; selecting a list of
candidate compounds; constructing said candidate compounds in a
computer modeling program; performing an iterative docking and
scoring of all candidate compounds, by means of docking each
candidate compound into a pocket formed by said model and scoring
each candidate compound to reflect its degree of complementarity
with respect to said pocket; and identifying compounds that
modulate amyloid aggregation or better modulate amyloid aggregation
by reference to a score cutoff that substantially distinguishes
active compounds from inactive compounds, or more active compounds
from less active compounds, respectively.
9. A method of improving the potency of a compound known to
modulate amyloid aggregation according to claim 8, wherein the list
of candidate compounds includes both the compound known to be
active and analogs of said compound, and the score cutoff is that
score which belongs to the compound known to be active.
10. The non-crystallographic model of amyloid protein aggregation
of claim 1, wherein said amyloid peptide model and said amyloid
protein model are both composed of alpha-synuclein protein or an
amyloid-forming fragment thereof.
11. The non-crystallographic model of amyloid protein aggregation
of claim 1, wherein said amyloid peptide model and said amyloid
protein model are both composed of tau protein or an
amyloid-forming fragment thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 61/092,826, filed on Aug. 29, 2008, entitled
"Methods for identifying inhibitors of amyloid protein
aggregation," herein incorporated by reference in its entirety for
all purposes.
[0002] This application is related to U.S. Provisional Application
Ser. No. 61/092,845, entitled "Methods of treating amyloid disease
using analogs of 1-(4-nitrophenyl)piperazine," filed on Aug. 29,
2008, herein incorporated by reference in its entirety for all
purposes.
FIELD OF THE INVENTION
[0003] This invention relates to methods of identifying compounds
that are inhibitors or are likely to be inhibitors of amyloid
protein aggregation, and three-dimensional, non-crystallographic,
models of amyloid receptors and models of amyloid protein
aggregation utilized in the methods.
BACKGROUND OF THE INVENTION
[0004] The build-up of amyloid proteins in living tissue, a
condition known as amyloidosis, is either the cause or a major
factor in the pathology of many so-called "amyloid diseases," e.g.,
Alzheimer's, Parkinson's, Huntington's, and prion diseases.
[0005] Certain models of beta-amyloid aggregation that exist in
vitro or in vivo (rather than "virtual" (i.e. in silico)) are used
for high-throughput screening to discover new compounds that
modulate amyloid aggregation (Dolphin et al, 2007, ChemMedChem
2:1613-1623; Gazit, 2006, ACS Chem. Biol. 1:417-419). These
existing in vitro or in vivo models however require the synthesis
and/or procurement of every candidate compound, as well as the
synthesis or incubation of the model systems. In addition, these
existing in vitro and in vivo models do not describe a pocket in
which the candidate compounds may be inserted, and as such do not
furnish a means for de novo design of compounds or for improving
the potency of known modulating compounds "virtually" (i.e. in
silico).
[0006] Most prior work in the field of amyloid modeling focused on
the structure and dynamics of aggregation, without regard to the
discovery of modulating compounds (see for example Buchete, Tycho,
and Hummer, 2005, J. Molec. Biol. 353:804-821, which describes the
overall structure of beta-amyloid protofibrils; Luhrs et al, 2005,
Proc. Natl. Acad. Sci. USA. 102:17342-17347, which verifies the
singular axis of fibril addition). These works also do not describe
an amyloid protein pocket for non-peptidic compound insertion nor
suggest a means of modulating amyloid protein aggregation.
[0007] Mastrangelo et al. (2006, J. Molec. Biol. 358:106-119)
describes a model of beta-amyloid fibrillization, consisting of two
protofibrils of beta-amyloid and a pocket found between them that
can be used to insert modulating, peptidic compounds. These
compounds are specific modulators of beta-amyloid fibrillization
and not of aggregation, which is a fundamentally different process
from fibrillization.
[0008] One treatment of amyloid diseases utilizes peptides as
modulators of disease targets, being amyloid proteins in this case.
(Findeis et al, 1999, Biochemistry 38:6791-6800; Ghanta et al,
1996, J Biol Chem 271:29525-29528; Tjernberg, 1996, J Biol Chem
271:8545-8548; Kokkoni et al, 2006, Biochemistry 45:9906-9918).
However, utilities of the peptidic compounds described in these
references as drugs is doubtful, e.g., due to in vivo stability
issues.
[0009] While a number of non-peptidic compound classes have been
identified as inhibitors of beta-amyloid build-up, the discovery of
further compound classes, as well as the further optimization of
known compound classes to improve potency may be haphazard due to
the lack of known crystal structures for beta-amyloid protein.
[0010] The amyloid diseases remain invariably fatal using current
medical practice.
[0011] Accordingly, there exists a need for novel methods of
identifying compounds that are inhibitors or are likely to be
inhibitors of amyloid protein aggregation, and three-dimensional,
non-crystallographic, models of amyloid receptors and models of
amyloid protein aggregation which may be utilized in these
methods.
DEFINITIONS
[0012] The following terms should have the following meaning
whenever used in the present specification, regardless whether the
use is singular or plural.
[0013] "Crystallized amyloid protein" means an amyloid protein for
which a binding pocket is known.
[0014] "Uncrystallized amyloid protein" means an amyloid protein
for which a binding pocket is unknown.
[0015] "Multiply anti-amyloid compound" means a compound that has
activity against both a crystallized amyloid protein and an
uncrystallized amyloid protein.
[0016] "Modulating amyloid aggregation" means changing the
distribution of oligomers such that amyloid deposition is reduced
in a subject and encompasses a process of inhibition of
aggregation.
[0017] "Model" means a molecular model, which is a representation
of atoms and their positions with respect to each other,
substantially including such features as covalent bonds between
highly interacting atoms in a molecule and/or non-covalent
interactions between the atoms of two or more molecules.
[0018] "Super-model" means a model of models.
[0019] "Computer modeling program" means any program that can
visualize or otherwise manipulate molecular models on a computer,
preferably also featuring molecular mechanics or molecular dynamics
routines.
[0020] "Interacts" means chemical interactions, especially those
that result in covalent bonds being formed between atoms in a
molecule and/or non-covalent bonds between the atoms of two or more
molecules. If said molecules are composed of amino acid residues,
it is commonly said that two residues interact when at least one
atom from one residue interacts with at least one atom from the
other residue.
[0021] "Non-crystallographic" means that the model was not directly
obtained by the techniques of crystallography, especially X-ray
crystallography.
[0022] "Candidate model" means a model of an uncrystallized amyloid
protein that is to be evaluated for utility as a pseudo-crystal
structure of a crystallized or uncrystallized amyloid protein
and/or amyloid aggregation.
[0023] "Halogen" means fluorine, chlorine, bromine, or iodine.
[0024] "Extracting" means the step of operating the computer
modeling program such that the relevant subset is highlighted,
saved, and/or placed in a different workspace within the program
for the purposes of manipulating the subset without reference to
the rest of a model.
[0025] Such a subset may be considered a model in and of
itself.
[0026] "Validating" means the step of performing docking and/or
quantitative structure-activity relationship (QSAR) calculations in
a computer modeling program, in order to verify that a given model
can generally correctly classify and/or predict the anti-amyloid
activity of a set of known positive and negative controls for
anti-amyloid activity. In preferred embodiments of the invention,
the set is as described in the Examples below.
[0027] "Constructing" means operating a computer modeling program
to instantiate an in silico representation of a given model.
[0028] "Selecting a list of candidate compounds" means choosing a
set of compounds for evaluation in a model. Selection of said set
may be reference to commercially available screening libraries such
as Hit2Lead or Enamine, by reference to an internal list of
available and/or proprietary compounds for in vitro or in silico
screening, or by utilizing lead-hopping or de novo design
methods.
[0029] "Iterative docking" means docking each candidate compound in
turn into the model using a computer modeling program.
[0030] "Scoring" means applying formulas to obtain a score from
"scoring functions" pertaining to drug-target interactions, the
output of which furnishes a measurement of the degree of
complementarity of the compound with respect to the pocket.
[0031] "Score cutoff" means some value, usually an absolute energy,
that can be set higher or lower depending on the level of activity
that is deemed to separate active from inactive, or as a reference
between more active and less active.
[0032] "Improving potency" means providing a means to furnish a
compound analogous to an active compound that has minor structural
differences which produce a better modulating effect with respect
to the target.
OBJECTS AND SUMMARY OF THE INVENTION
[0033] The invention provides methods of utilizing
three-dimensional, non-crystallographic, models of amyloid
aggregation to identify compounds that are inhibitors or are likely
to be inhibitors of amyloid protein aggregation. It also provides
three-dimensional, non-crystallographic models, of amyloid
receptors and models of amyloid protein aggregation utilized in
these methods, as well as methods of producing such models for
these methods.
[0034] It is an object of the invention to provide
three-dimensional pseudo-crystal structures of uncrystallized
amyloid proteins.
[0035] It is a further object of the invention to provide virtual
three-dimensional models of amyloid aggregation and methods for
producing such models.
[0036] It is also an object of the invention to provide virtual
three-dimensional models of amyloid aggregation, indicating pockets
in which candidate anti-amyloid models may be placed.
[0037] It is another object of the invention to provide methods of
constructing three-dimensional pseudo-crystal structures or models
of uncrystallized amyloid proteins and of validating the models to
verify that the structures can classify and predict anti-amyloid
activity of candidate compounds.
[0038] It is a further object of the invention to provide methods
of utilizing the three-dimensional pseudo-crystal structures or
models to identify compounds that modulate or are likely to
modulate amyloid aggregation.
[0039] It is an additional object of the invention to provide
methods of improving the anti-amyloid potency of candidate
compounds.
[0040] It is a further object of the invention to provide methods
of predicting potential interactions between candidate compounds
and amyloid proteins.
[0041] It is also an object of the invention to provide methods for
identification and characterization of previously unknown binding
pockets or conformational regions of amyloid proteins.
[0042] It is yet another object of the invention to use compound
identified or improved using the methods of the invention as
therapies for diseases in which amyloidosis occurs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIG. 1 is a depiction of the super-model developed in
Example 1, in which the crystallized amyloid protein is a fragment
of transthyretin, the multiply anti-amyloid compound is
resveratrol, and the uncrystallized amyloid protein is a dimer of
A.beta..sub.17-42.
[0044] FIG. 2 is a stereographic view of the pseudo-crystal
structure, a model of amyloid protein aggregation, of Example 1
with resveratrol bound in its pocket.
DETAILED DESCRIPTION
[0045] Amyloidosis refers to a variety of conditions in which
amyloid proteins are abnormally deposited in organs and/or
tissues.
[0046] Certain types of amyloidosis occur principally in the
central nervous system, e.g., with aggregation of beta-amyloid
protein in Alzheimer's Disease, alpha-synuclein in Parkinson's
Disease, huntingtin protein in Huntington's Disease, and prion
protein in Creutzfeldt-Jacob and other prion diseases. Other types
of amyloidosis are systemic in nature, as, e.g., with aggregation
of transthyretin in senile systemic amyloidosis.
[0047] Historically, aggregations of protein were classified as
"amyloid" if they displayed apple-green birefringence under
polarized light when stained with the dyes Congo red or Thioflavin
T (ThT) (Sipe and Cohen, 2000, J. Struct. Biol. 130:88-98). That
definition of "amyloid protein" has been expanded in recent years
to apply to any fibrous structure which is ordered and insoluble,
and which is composed of fibrils of approximately 10 nm in width
and up to several microns in length (Zheng, Ma, and Nussinov, 2006,
Phys. Biol. 3:P1-P4).
[0048] An "amyloidogenic" polypeptide is one which, regardless of
sequence, can polymerize in a cross-.beta. sheet conformation in
vitro or in vivo to form amyloid protein (Xu, 2007, Amyloid
14:119-31). "Amyloidogenic" polypeptides include, e.g., amyloid
light chain, amyloid associated protein, .beta. amyloid,
transthyretin, .beta..sub.2 microglobulin, amylin, prion related
protein, and other amyloid proteins.
[0049] All amyloid proteins have commonalities. However, whether
certain amyloid proteins are related depends on the amyloid
proteins having commonality of both pathology and location. For
example, beta-amyloid protein and huntingtin protein are both
located in a human brain, but their divergence in pathology is
sufficient to consider them unrelated. Beta-amyloid protein, tau
protein, and alpha-synuclein protein on the other hand are related,
all being located in the human brain and being thought to have
overlapping roles in the pathology of neurodegenerative diseases,
e.g., Alzheimer's disease and Parkinson's disease. Nevertheless,
given the preponderance of non-peptidic anti-amyloid compounds that
inhibit aggregation of more than one amyloid protein, it is
reasonable to assume that models of amyloid protein aggregation
provided by the present invention will have significant utility
even with unrelated amyloid proteins, in addition to having utility
with the related amyloid proteins (i.e., beta-amyloid protein, tau
protein, and alpha-synuclein protein).
[0050] A lock and key analogy is commonly used to characterize
drug-target interactions where a specific "key" (compound/drug)
interacts only with its respective molecular "lock"
(target/receptor). An appropriate degree of shape and electronic
complimentarily between the drug and target must occur to produce
drug-target interactions which are necessary for a pharmacological
response. The specific location on the "lock" or target is often
referred to as the active or catalytic site, and the intuitive
shape of the active site is that of a pocket. Thus a "binding
pocket," or more simply "pocket," is used in the art to refer to
that space on the target in which a molecule can be inserted to
modulate activity of the target. The three-dimensional shape and
electronic properties of the pocket form the basis for rational
drug design and for virtual screening techniques (e.g., in silico)
utilized in the methods of the present invention, provides means
for identifying the pocket for non-peptidic compound insertion and
suggests means for modulating amyloid protein aggregation.
[0051] "Rational drug design" attempts to formulate drug design
hypotheses that specify and optimize the physical contacts between
a drug and its target, often by aid of a computer. Such
"computer-aided drug design" generally depends on high resolution,
three-dimensional models of the target, and such models are usually
constructed by X-ray crystallography of a crystal form of the
target. Crystallographic techniques are the primary means of
obtaining three-dimensional structure and binding information in
the art. In the absence of a solved, high-resolution (i.e. below 2
angstroms) crystal structure of a given amyloid protein indicating
the binding mode of an anti-amyloid compound and/or the chemical
interactions essential to aggregation, rational design of
anti-amyloid compounds for said protein was generally considered
haphazard and difficult prior to the methods and models of the
present invention.
[0052] The virtual screening techniques utilized in the methods of
the present invention and the virtual models of the present
invention allow, e.g., for the virtual identification of the
binding pockets of amyloid proteins, and construction of
three-dimensional non-crystallographic models of amyloid receptors
and models of amyloid aggregation. The methods of the present
invention generally do not require construction of crystal forms of
the target by X-ray crystallography, and instead provide or utilize
non-crystallographic models ("pseudo-crystal structures") that
could approximate or exceed the utility of traditional crystal
structures.
Candidate Models
[0053] The invention provides for candidate models of amyloid
receptors and of amyloid protein aggregation to be constructed,
evaluated and subsequently validated. The models of the present
invention are based, in part, both on the symmetry inherent in many
anti-amyloid compounds and on the multi-target activity of such
compounds.
[0054] The models of the present invention may be referred to as
"pseudo-crystal structures" or as surrogate models, as the models
are not obtained by the technique of crystallography. As stated
above and as demonstrated by the examples below, the utility of
"pseudo-crystal structures" of the present invention may approach
and exceed that of crystal structures.
[0055] Successful candidate models (e.g., pseudo-crystal
structures) will include elements directly related to the process
of aggregation and will be of sufficient detail to enable the
design of compounds for disrupting or otherwise altering the
interactions essential to amyloid protein aggregation. Indeed, if a
pseudo-crystal structure does not indicate the binding mode of an
anti-amyloid compound and/or the chemical interactions essential to
aggregation, then it is not a proper model of amyloid protein
aggregation and would not be expected to have utility to enable the
design of anti-amyloid compounds.
[0056] Successful candidate models in accordance with the present
invention may comprise pseudo-crystal structures of uncrystallized
amyloid proteins. Such pseudo-crystal structures generally comprise
two parts. The first part generally comprises an amyloid monomer,
and the second part generally comprises an amyloid monomer or an
amyloid oligomer. The first part is generally positioned with
respect to the second part such that it forms a pocket in
conjunction with the second part, such that a candidate compound
may be inserted into the pocket, thereby potentially modulating
amyloid aggregation.
[0057] The amyloid monomer comprises one peptide chain from an
amyloid oligomer or amyloid protein. The amyloid monomer in the
first part and the amyloid monomer (or each monomer in an amyloid
oligomer) in the second part may comprise the same sequence or
different sequences with respect to the first part.
[0058] The amyloid oligomer comprises a heterogeneous or
homogeneous link comprising an amyloid protein (or amyloidogenic
fragments thereof). For example, in certain embodiments, the
amyloid oligomer comprises an amyloid protein (e.g., either
beta-amyloid protein, tau protein, alpha-synuclein protein,
huntingtin protein, or prion protein) or amyloidogenic fragments
thereof; and in other embodiments the amyloid oligomer comprises
two or more amyloid proteins (e.g., a combination of beta-amyloid
protein and tau protein, a combination of beta-amyloid protein and
alpha-synuclein protein, etc.) or amyloidogenic fragments thereof.
The amyloid oligomer may also comprise a protein which has not yet
shown to be amyloidogenic or a portion of such protein.
[0059] In certain embodiments, the amyloid oligomer or
amyloidogenic fragment thereof comprises a region comprising
approximately residues 17 through 40 of beta-amyloid protein of
length 40; approximately residues 17 through 42 of beta-amyloid
protein of length 42; approximately residues 1 through 15 of
alpha-synuclein protein; approximately residues 25 through 40 or
275 through 280 of tau protein; approximately residues 90 through
110 of the major prion precursor protein; or approximately residues
5 through 20 of the huntingtin protein.
[0060] In certain embodiments, the model comprises the
pseudo-crystal structure formed by SEQ ID: 1 and SEQ ID: 2 and
interacting in the manner depicted stereographically in FIG. 2,
which is a model of amyloid protein aggregation of Example 1, with
resveratrol bound in its pocket. Three-dimensional features of the
model may be appreciated by a person of normal binocular vision by
superimposing the images visually.
[0061] In certain embodiments, the first part comprises
substantially SEQ ID: 1 and the second part comprise substantially
SEQ ID: 2, wherein SEQ ID: 1 is
TABLE-US-00001 Lys Val Phe Phe Ala Glu Asp Val Gly Ser Asn 1 6 11
Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val 16 21 Val Ile Ala
26
and,
SEQ ID: 2 is
TABLE-US-00002 [0062] Lys Val Phe Phe Ala Glu Asp Val Gly Ser Asn 1
6 11 Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val 16 21 Val Ile
Ala 26
[0063] In certain embodiments, the first part is further positioned
with respect to the second part such that the amino acid Val at
position 8 of SEQ ID: 1 interacts with the amino acid Glu at
position 6 of SEQ ID: 2; the amino acid Gly at position 9 of SEQ
ID: 1 interacts with the amino acid Glu at position 6 of SEQ ID: 2;
the amino acid Ser at position 10 of SEQ ID: 1 interacts with the
amino acid Gly at position 9 of SEQ ID: 2; the amino acid Ser at
position 10 of SEQ ID: 1 interacts with the amino acid Ser at
position 10 of SEQ ID:2; and the amino acid Lys at position 12 of
SEQ ID: 1 interacts with the amino acid Glu at position 6 of SEQ
ID: 2.
[0064] In certain embodiments, the first part is positioned with
respect to the second part in the orientation shown in FIG. 2. FIG.
2 is a stereographic view of the pseudo-crystal structure, a model
of amyloid protein aggregation, of Example 1 with resveratrol bound
in its pocket. The model of FIG. 2 features two A.beta..sub.17-42
monomers bound to each other near the N terminals in a "double
candycane" structure. Each monomer is folded in a loop from
residues 23 through 33, which is a conformation well supported by
electron microscopy and NMR spectroscopy. The loops are stabilized
by intra-loop cationic-anionic interactions between Asp.sub.23 and
Lys.sub.28, and they also show an inter-loop attraction through
these same residues. Short-run (up to 100 ns) molecular dynamics
calculations on this model indicate that the pocket is stable.
[0065] In certain embodiments, a virtual non-crystallographic model
of amyloid protein aggregation comprises a pseudo-crystal structure
comprising a three-dimensional model of an uncrystallized monomeric
amyloid protein and a three-dimensional model of an uncrystallized
amyloid protein comprising one or more amyloid peptides; said
three-dimensional model of an uncrystallized amyloid protein is
positioned with respect to said amyloid monomer model such that it
forms a pocket in conjunction with said amyloid monomer model, such
that a candidate compound may be inserted into said pocket thereby,
e.g., potentially modulating amyloid aggregation.
[0066] Aggregation is a fundamentally different process from
fibrillization. Aggregation occurs among monomers and oligomers of
incipient amyloid protein, while fibrillization is directed to the
further lengthening of pre-formed amyloid protofibrils and
subsequent assembly into fibrils. Modulators of aggregation are
preferred to modulators of fibrillization for treatment of amyloid
diseases, since soluble oligomers of beta-amyloid protein are
thought to be the pathogenic species in Alzheimer's Disease, rather
than the mature fibrils thereof (Klyubin et al, 2008, J. Neurosci.
28:4231-4237; Townsend et al, 2006, J. Physiol. 572:477-492).
[0067] For clarity, it should be understood that a model of amyloid
protein aggregation promulgated in the present application is
substantially a model of addition of monomeric amyloid peptide to
an amyloid oligomer or an amyloid protein. In certain embodiments,
the monomeric amyloid peptide and the amyloid oligomer (or the
amyloid protein) is composed of chains of different amyloid
peptides. In other embodiments, the monomeric amyloid peptide and
the amyloid oligomer or protein are composed of chains of the same
amyloid peptide.
[0068] In certain embodiments of the invention, modulating amyloid
aggregation may result in greatly increasing the proportion of
amyloid peptide assemblies with size n, where n is between 1 and
10. In other embodiments, modulating amyloid aggregation may result
in increasing the proportion of amyloid peptide assemblies with
size n, where n is between 1 and 5. In still other embodiments of
the invention, modulating amyloid aggregation may result in
increasing the proportion of amyloid peptide monomer (i.e. where n
is only 1).
[0069] In certain embodiments, a virtual non-crystallographic model
of amyloid protein aggregation comprises a pseudo-crystal structure
comprising an amyloid monomer model and a three-dimensional model
of an uncrystallized amyloid protein. The "amyloid protein" in the
minimal case may comprise only one amyloidogenic peptide having
substantially the same sequence as an amyloid protein or an
amyloidogenic fragment thereof. The "amyloid protein" may also
comprise a plurality of chains of one amyloid protein or
amyloidogenic portions thereof, and does not include other amyloid
protein or amyloidogenic portions thereof. In certain embodiments,
the plurality of chains are aggregated according, e.g., to a
building-up principle.
[0070] In certain embodiments, the amyloid monomer model and the
three-dimensional model of an uncrystallized amyloid protein are
both composed of beta-amyloid protein or an amyloidogenic fragment
thereof. For example, the amyloids may comprise substantially the
sequence of beta-amyloid 1-42 and/or the sequence of beta-amyloid
1-40 and/or the sequence of the beta-amyloid fragment 25-35 and/or
the sequences of beta-amyloid fragment 17-42, which are sequences
known to form aggregated beta-amyloid (Hughes et al, 2000, J Biol
Chem 275:25109-25115).
[0071] In certain embodiments, candidate models of amyloid protein
aggregation comprise a pseudo-crystal structure comprising a
three-dimensional model of an uncrystallized, monomeric amyloid
protein and a three-dimensional model of an uncrystalized amyloid
protein comprising one or more amyloid peptides positioned with
respect to the monomeric amyloid protein model such that it forms a
pocket in conjunction with the monomeric amyloid protein. In one
embodiment, the monomeric amyloid model is substantially SEQ ID: 1;
and the amyloid protein model comprises SEQ ID: 2.
[0072] In certain embodiments three-dimensional,
non-crystallographic models, of amyloid aggregation comprise
pseudo-crystal structures for amyloid proteins that lack extant
crystal structures (e.g., beta-amyloid protein, tau,
alpha-synuclein, huntingtin, and prion protein, etc.).
[0073] In certain embodiments of the invention, a model may be
recorded in a mathematical form using a combination of atomic
information and three-dimensional coordinates (e.g. in the PDB
format, or in the proprietary format of a computer modeling
program), which are then visualized or otherwise represented
virtually, e.g., in a computer modeling program. In certain
embodiments, the model reflects the likely protonation state at
physiological pH such that basic moieties of the model are
protonated and acidic moieties are deprotonated in a manner
consistent with valence considerations. In further embodiments,
force fields and charges are applied appropriate to the model such
that molecular mechanics and/or dynamics can be used; details on
these and other computational techniques characteristically found
in computer modeling programs are described, e.g., in A. Leach,
Molecular Modeling: Principles and Applications, 2.sup.nd edition,
Prentice Hall, 2001, herein incorporated by reference.
[0074] The models of the present invention may, e.g., enable the
identification of new chemical classes of modulators of their
respective amyloid protein and related amyloid proteins. For
example, if a candidate compound has a three-dimensional shape and
electronic properties that will allow it to "fit" into the
pseudo-crystal structure and interact with the pseudo-crystal
structure, it is likely to be a potential modulator of the amyloid
protein aggregation.
[0075] The models of the present invention may also provide a means
of improvement in potency for compounds known to have modulating
effects on a relevant amyloid protein, as they may suggest
structural modification to the compounds known to have modulating
effects on the amyloid protein, to allow the compounds to better
"fit" and interact with the three-dimensional pseudo-crystal
structure. They may also provide means to the de novo design of
compounds which may "fit" into the three-dimensional structure.
[0076] The models may further provide means for the identification
and discovery of the compounds which may be useful in modulation of
either or both aggregation and fibrilization.
Construction of Models
[0077] The present invention further provide means for constructing
surrogate models (i.e., non-crystallographic virtual models) of
amyloid protein aggregation which may be used for drug design much
as crystal structures are customarily used but at a lower cost and
without a need for synthesis of every candidate compound.
[0078] For example, the three-dimensional, non-crystallographic
models of amyloid aggregation models in accordance with the present
invention may be constructed, e.g., virtually (e.g., in a computer
modeling system), depicted pictorially, instantiated in a physical
form by way of a modeling kit, or visualized mentally. The virtual
modeling afforded by the present invention allows for greatly
reduced cost and labor as compared to in vitro or in vivo systems,
as it does not require the synthesis and/or procurement of every
candidate compound. In particular, it allows for the screening and
prediction of the anti-amyloid properties of a candidate compound
based on its three-dimensional shape and electronic configuration,
even before the compounds are synthesized. It disposes of a
potentially great number of candidate compounds which will not
"fit" into the three-dimensional, non-crystallographic, models of
the amyloid aggregation afforded by the present invention, and are
therefore are unlikely to have anti-amyloid activity.
[0079] Generally, construction of the non-crystallographic models
is performed manually, using a molecule or peptide/biomolecule
building routine as included in a computer modeling program. In
some embodiments of the invention, construction may proceed
automatically through the reading of a previously constructed file
or sequence.
[0080] In certain embodiments, the invention provides means for
constructing virtual non-crystallographic models of amyloid
aggregations for proteins for which the three-dimensional structure
and binding information (e.g., binding mode and chemical
interactions essential for aggregation) are known. In other
embodiments, the invention provides means for constructing virtual
non-crystallographic models of amyloid aggregations for proteins
for which the three-dimensional structure and binding information
(e.g., binding mode and chemical interactions essential for
aggregation) are unknown. In certain embodiments, the means for
constructing virtual non-crystallographic models of amyloid
aggregations for proteins for which the three-dimensional structure
and binding information are known and the means for constructing
virtual non-crystallographic models of amyloid aggregations for
proteins for which the three-dimensional structure and binding
information are unknown are the same.
[0081] In certain embodiments, a pseudo-crystal structure of an
uncrystallized amyloid protein can be virtually identified and
constructed by manipulating a computer program to orient an
uncrystallized amyloid protein and/or oligomer such that a pocket
is formed around a multiply anti-amyloid compound already bound in
the crystallized amyloid protein. The pocket has a particular
three-dimensional shape and electronic properties and represents,
e.g., a pseudo-crystal structure of the uncrystallized amyloid
protein. In certain embodiments, the multiply anti-amyloid compound
is removed and is not part of the pseudo-crystal structure. In
other embodiments, the multiply anti-amyloid compound is part of
the pseudo-crystal structure.
[0082] In certain embodiments, a binding pocket corresponds to a
common conformational region on a variety of amyloid proteins,
e.g., the Common Conformational Motif (CCM). For example, the CCM
may correspond to a conformational region found in both A.beta. and
tau; or a conformational region found in both A.beta. and
alpha-synuclein; or a conformational region found in both A.beta.
and transthyretin. The conformational region may also be common to
A.beta., tau and alpha-synuclein (which are all located in the
human brain and being thought to have overlapping roles in the
pathology of neurodegenerative diseases). The binding pocket may
therefore be useful for the identification of candidate compounds
which are likely to inhibit the aggregation of several amyloid
proteins simultaneously (e.g., A.beta. and tau; A.beta. and alpha
synuclein; A.beta., tau and alpha synuclein, etc.). It should be
understood for this reason that the CCM is not strictly
sequence-dependent.
[0083] In certain embodiments, a method of creating pseudo-crystal
structures of uncrystallized amyloid protein receptor comprises (1)
applying molecular mechanics and/or dynamics to a three-dimensional
super-model comprising a crystallized amyloid protein model, a
model of multiply anti-amyloid compound bound in the pocket of the
crystallized amyloid protein or oligomer, and a candidate model of
an uncrystallized amyloid protein, such that a pocket on the
uncrystallized amyloid protein or oligomer is formed around the
multiply anti-amyloid compound; (2) extracting said uncrystallized
amyloid protein model from said super-model; and (3) validating
said uncrystallized amyloid protein model with respect to
experimental data so as to confirm its utility as a pseudo-crystal
structure. In certain embodiments, the extracted model comprises
the multiply anti-amyloid compound. In other embodiments, the
extracted model is free of the multiply anti-amyloid compound.
[0084] Example 1 below describes an exemplary method for
construction of one pseudo-crystal structure of beta-amyloid
protein in accordance with one embodiment of the invention. In the
method of Example 1, the uncrystallized amyloid protein is an
amyloidogenic fragment of beta-amyloid protein, the crystallized
amyloid protein is transthyretin protein, and the multiply
anti-amyloid compound is resveratrol. In particular, the
pseudo-crystal structure of Example 1 has two parts: one monomer of
an amyloidogenic beta-amyloid fragment (i.e. the "amyloid
monomer"); and another monomer of an amyloidogenic beta-amyloid
fragment, which could have multiple additional peptides attached
thereon without disrupting its interaction with the amyloid monomer
(as such, this second part is sometimes referred to as the "amyloid
oligomer", as it is only contingently monomeric).
[0085] In certain embodiments, the invention provides a method of
constructing pseudo-crystal structures of uncrystallized amyloid
protein which are models of amyloid protein aggregation. This
method's first step is to apply molecular mechanics and/or dynamics
to a three-dimensional super-model comprising a crystallized
amyloid protein model, a model of multiply anti-amyloid compound
bound in the pocket of the crystallized amyloid protein, and a
candidate model of an uncrystallized amyloid protein or oligomer,
such that a pocket on the uncrystallized amyloid protein is formed
around the multiply anti-amyloid compound.
[0086] The method's second step is to extract said uncrystallized
amyloid protein or oligomer model from the super-model. In certain
embodiments, the extracted model comprises the multiply
anti-amyloid compound. In other embodiments, the extracted model is
free of the multiply anti-amyloid compound.
[0087] The method's third step is to validate said uncrystallized
amyloid protein model with respect to experimental data so as to
confirm its utility as a pseudo-crystal structure.
[0088] The crystallized amyloid protein may be transthyretin, islet
amyloid polypeptide, beta-2-microglobulin, and insulin protein; and
the uncrystallized amyloid protein may be either of beta-amyloid,
tau, alpha-synuclein, huntingtin, or prion protein; an
amyloidogenic fragment of any of the foregoing, or composed of a
heterogeneous combination of such proteins.
[0089] A multiply anti-amyloid compound may be resveratrol, or a
compound having low energy conformations that overlap geometrically
and electrostatically with resveratrol. In certain embodiments, a
multiply anti-amyloid compound is a compound selected from the
lists of compounds provided in paragraphs [00106] to [0117] of the
present application.
[0090] Once a model (i.e., a pseudo-crystal structure) is
constructed, it is then extracted and validated with respect to
experimental data to verify that it can classify and predict, e.g.,
the anti-amyloid activity of candidate compounds. In certain
embodiments, validation comprises performing docking and/or
quantitative structure-activity relationship (QSAR) calculations in
a computer modeling program, in order to verify that a given model
can generally correctly classify and/or predict the anti-amyloid
activity of a set of known positive and negative controls for
anti-amyloid activity. Examples of computer modeling programs
include but are not limited to MOE, Sybyl, Cerius.sup.2, CHARMm,
DOCK, AUTODOCK, GLIDE, and FlexX. Manuals of these computer
modeling programs are herein incorporated by reference in their
entirety.
[0091] Example 2 below describes an exemplary method for validating
an exemplary of amyloid protein aggregation.
[0092] Once validated, the resulting pseudo-crystal structure can
be used as a three-dimensional, non-crystallographic, model of
amyloid aggregation in the methods of the present invention.
The Methods of the Invention
[0093] The methods of the invention may be used to identify or
improve the potency of candidate compounds that are anti-amyloid
with respect to the amyloid protein or fragment thereof depicted in
a particular three-dimensional, non-crystallographic, model, or is
related to the amyloid protein or fragment thereof that is depicted
in the model. The methods of the invention may also be used to
identify, or improve the potency of, compounds that are
anti-amyloid with respect to an amyloid protein or fragment thereof
that is unrelated to the amyloid protein or fragment thereof that
is depicted in a particular three-dimensional non-crystallographic
model.
[0094] In certain embodiments, the methods of the present invention
utilize the "virtual" non-crystallographic models to identify new
compounds which may be inhibitors or likely to be inhibitors of
amyloid protein aggregation, as well as improve existing modulators
of amyloid proteins, all without the construction of crystal form
of the target by X-ray crystallography.
[0095] In certain embodiments, the models are used to predict the
potential interactions between a candidate compound and an amyloid
protein, e.g., by allowing visualization of the candidate compound
fitting into the binding pocket.
[0096] One empirical determinant for whether two atoms interact is
their proximity to each other in a given chemical system. For
example, a covalent bond between carbons is approximately 1.5
angstroms in interatomic distance, while a weak non-covalent bond
between atoms could be as long as 3 angstroms (although stronger
non-covalent bonds may approach 1.5 angstroms in length). The
hydrogens in the head group of a protonated Lys residue interacting
with the oxygens in the head group of a deprotonated Glu residue is
an example of a primarily ionic non-covalent bond. The hydrogen in
the head group of a Ser residue interacting with the carbonyl
oxygen in the backbone of a different Ser residue, or a hydrogen in
the backbone of a Gly residue interacting with the oxygen in the
head group of a Ser residue, are examples of primarily hydrogen
bonds. The virtual visualization provided by the methods of the
present invention supply a means of predicting the likelihood of
these interactions between a candidate model compound and a
particular binding pocket. If the visualization shows that the
modeling compound is unlikely to interact with a particular binding
pocket on an amyloid protein, it is unlikely that the modeling
compound will be useful as an inhibitor, e.g., of the amyloid
protein aggregation. To the contrary, if the compound is likely to
interact with the binding pocket of the amyloid aggregation, it is
likely that the compound may be capable of modulating activity and
potentially preventing or minimizing aggregation of this particular
amyloid protein.
[0097] In the context of a pseudo-crystal structures described in
the present application, one of the most important intermolecular
interactions are those which hold the amyloid oligomer together as
well as those which attract the amyloid monomer. The latter said
interactions are pivotal to the conformation and eventual
incorporation of the amyloid monomer into the amyloid oligomer, a
process which is believed to continue repeatedly until reaching the
critical size at which fibrillization occurs. By inserting into the
pocket where these intermolecular interactions occur, the candidate
compound may disrupt or otherwise alter the process of aggregation.
Whether the candidate compound can do so depends, e.g., on its
shape and electronic complementarity, i.e., the ability of moieties
on the compound to interact with important residues in the pocket.
The visualization of the fitting provided by the present invention
will allow the prediction of whether the candidate compound is
likely to interact with the important residues of the pocket. Other
intermolecular interactions, which may be less important to
describing aggregation, yet are potentially of importance in
achieving the desired accuracy with respect to a given model of
amyloid protein aggregation. The presently claimed methods may also
have utility in predicting these other intermolecular
interactions.
[0098] A common interaction within such systems involves ionic,
acid-base interactions such as Lys-Glu and Lys-Asp as well as
hydrogen-bonding interactions such as Ser-Ser. Therefore, regions
of amyloid proteins or amyloidogenic fragments thereof that should
make excellent models include but are not limited to the following:
in beta-amyloid protein of length 40, approximately residues 17
through 40; in beta-amyloid protein of length 42, approximately
residues 17 through 42; in alpha-synuclein, approximately residues
1 through 15; in tau protein, approximately residues 25 through 40
or approximately residues 275 through 280; in the major prion
precursor protein, approximately residues 90 through 110; and in
huntingtin, approximately residues 5 through 20.
[0099] Chemical interaction is a matter of degree; especially in
non-covalent bonds, the type of bonding involved can often be an
admixture of one or more types of bonding named above, possibly
including other effects such as hydrophobic or van der Waals
interactions.
[0100] Selected examples of chemical interactions which may be
predicted utilizing the methods of the present invention include
(but are not limited) to non-covalent bonds including, e.g., ionic,
dipole-dipole, dipole-induced dipole, and hydrogen bonding.
[0101] Covalent bonds may also be predicted by the methods of the
present invention. Covalent bonds are the most permanent of
chemical interactions and are generally not altered by
intermolecular interactions, which are governed primarily by
non-covalent bonds.
[0102] In certain embodiments, the present invention provides
method for identification and characterization of a previously
unknown binding pocket or a conformational region of an amyloid,
and subsequent utilization of the previously unknown
pocket/conformational region as a screening tool for identification
and optimization of anti-amyloid compounds. In these embodiments,
the binding pocket is identified in silico, e.g., by using
molecular modeling (especially optimization and dynamical
techniques) on a "super-model" comprising a model of amyloid
protein for which a binding pocket is known (a "crystallized
amyloid protein"); a model of a compound (a "multiply anti-amyloid
compound") that has activity against said crystallized amyloid
protein, as well as having activity against an amyloid protein for
which a binding pocket is unknown (an "uncrystallized amyloid
protein"); and a candidate model for said uncrystallized amyloid
protein. In certain embodiments, these methods further allow for
the construction of a pseudo-crystal structure of beta-amyloid
protein or a receptor having utility in identification and
optimization of anti-amyloid compounds.
[0103] A crystallized amyloid protein may be selected, e.g., from
the group consisting of transthyretin, islet amyloid polypeptide,
beta-2-microglobulin, and insulin protein. In certain embodiments,
the crystallized amyloid protein is transthyretin protein, or an
amyloidogenic fragments thereof.
[0104] An uncrystalized amyloid protein may be selected, e.g., from
the group consisting of beta-amyloid, tau, alpha-synuclein,
huntingtin, and prion protein. In certain embodiments, the
uncrystalized amyloid protein is beta-amyloid proteins, and
amyloidogenic fragments thereof.
[0105] A multiply anti-amyloid compound may be any compound which
has activity against any crystallized amyloid protein. In a
preferred embodiment, the multiply anti-amyloid compound is
resveratrol or an analog thereof.
[0106] In certain embodiments, the crystallized amyloid protein is
selected from the group of transthyretin, islet amyloid
polypeptide, beta-2-microglobulin, and insulin protein; and the
uncrystallized amyloid protein is selected from the group of
beta-amyloid, tau, alpha-synuclein, huntingtin, and prion protein.
In certain embodiments, the crystallized amyloid protein is
transthyretin protein or an amyloidogenic fragment thereof, and the
uncrystallized amyloid protein is beta-amyloid protein or an
amyloidogenic fragment thereof, and the multiply anti-amyloid
compound is resveratrol.
[0107] The invention further encompasses and provides a method of
identifying compounds that modulate amyloid aggregation. The method
generally comprises the steps of constructing a pseudo-crystal
structure in a computer modeling program; selecting a list of
candidate compounds; constructing said candidate compounds in a
computer modeling program; performing an iterative docking and
scoring of all candidate compounds, by means of docking each
candidate compound into the pocket formed by said model and scoring
each candidate compound to reflect its degree of complementarity
with respect to said pocket; and identifying compounds that
modulate amyloid aggregation by reference to a score cutoff that
substantially distinguishes active compounds from inactive
compounds.
[0108] A candidate compound may, e.g., be a compounds of Formulas
Ia, Ib, Ic, pharmaceutically acceptable salts, stereo-isomers,
polymorphs, metabolites, prod-drugs, analogs and combinations of
any of the foregoing:
##STR00001##
wherein
[0109] R.sub.1 is selected from the group consisting of H, nitro,
carboxylic acid, alkylcarboxylic acid, acetamide connected in
either direction, N-(2-ethanol)amine, N-(2-morpholinethyl)amine,
amine optionally substituted with one or more alkyl groups, amide
optionally substituted with one or more alkyl groups, and
alkoxy;
[0110] R.sub.2 is selected from the group consisting of H,
carboxylic acid, alkyl, alkanoyl, alkanesulfonyl, benzenesulfonyl,
phenonyl optionally substituted with any one or more of alkoxy,
halogen, or alkyl groups, benzyl optionally substituted with any
one or more of alkoxy, halogen, or alkyl groups, and amide
optionally substituted with any one or more of alkyl or aryl
groups;
[0111] R.sub.3 is selected from the group consisting of H, alkyl,
furanylalkyl, thiophenealkyl, alkanoyl, phenyl optionally
substituted with any one or more halogen, alkyl, or alkoxy groups,
benzyl optionally substituted with any one or more halogen, alkyl,
or alkoxy groups, and phenonyl optionally substituted with any one
or more halogen, alkyl, or alkoxy groups; and
[0112] R.sub.4 is selected from the group consisting of H, alkyl,
or phenyl optionally substituted with any one or more halogen,
alkyl, or alkoxy groups.
[0113] In certain embodiments, R.sub.1 is selected from the group
consisting of nitro, acetamide connected in either direction,
N-(2-ethanol)amine, amino optionally substituted with any one or
more alkyl groups, and amide optionally substituted with any one or
more alkyl groups; R.sub.2 is selected from the group consisting of
carboxylic acid, amide optionally substituted with any one or more
of alkyl or aryl groups, and phenonyl optionally substituted with
any one or more of alkoxy, alkyl, or aryl groups; R.sub.3 is
selected from the group consisting of methyl, phenyl optionally
substituted with any one or more halogen, alkyl, or alkoxy groups,
benzyl optionally substituted with any one or more halogen, alkyl,
or alkoxy groups, and phenonyl optionally substituted with any one
or more halogen, alkyl, or alkoxy groups; and R.sub.4 is selected
from the group consisting of H, alkoxy, and aryl.
[0114] In other embodiments, R.sub.1 is selected from the group
consisting of nitro, acetamide connected in either direction,
N-(2-ethanol)amine, amino optionally substituted with methyl or
dimethyl, amide optionally substituted with methyl, ethyl,
dimethyl, or diethyl, and methoxy; R.sub.2 is selected from the
group consisting of phenonyl optionally substituted with any one or
more of methoxy, alkyl, or halogen, amide optionally substituted
with any one or more of methyl, phenyl, benzyl, or dimethyl, and
carboxylic acid; R.sub.3 is selected from the group consisting of
methyl, phenyl optionally substituted with any one or more of
halogen, alkyl, or methoxy, benzyl optionally substituted with any
one or more of halogen, alkyl, or methoxy, and phenonyl optionally
substituted with any one or more of halogen, alkyl, or methoxy; and
R.sub.4 is selected from the group consisting of H, methyl, and
phenyl optionally substituted with any one or more of halogen,
alkyl, or alkoxy.
[0115] The compounds disclosed in Formulas Ia, Ib and Ic should be
understood as also accommodating methyl, ethyl, methoxy, fluoro, or
chloro groups at any position otherwise occupied by a ring
hydrogen. Moreover, R.sub.3 and R.sub.4 may be used in combination
to produce a nitro moiety on the phenyl ring, or to create ring
systems such as morpholine, quinoline, or isoquinoline.
[0116] In certain embodiments, a candidate compound is a compound
of Formula Ia, pharmaceutically acceptable salts, stereo-isomers,
polymorphs, metabolites, prod-drugs and combinations thereof:
##STR00002##
wherein the NR.sub.3R.sub.4 moiety is connected ortho to the
R.sub.1 moiety on the phenyl ring;
[0117] R.sub.1 is selected from the group consisting of nitro,
amino optionally substituted with methyl or dimethyl, and amide
optionally substituted with methyl, dimethyl, ethyl, or
diethyl;
[0118] R.sub.2 is phenonyl optionally substituted with halogen or
methoxy;
[0119] R.sub.3 is selected from the group consisting of phenyl
optionally substituted with halogen or methoxy and benzyl
optionally substituted with halogen or methoxy; and
[0120] R.sub.4 is selected from the group consisting of H, methyl,
and phenyl; with the following exception: when R.sub.1 is nitro,
R.sub.4 is H, and R.sub.3 is benzyl optionally substituted with
fluoro or isopropyl.
[0121] In other embodiments, a candidate compound is a compound of
Formula Ic, pharmaceutically acceptable salts, stereo-isomers,
polymorphs, metabolites, prod-drugs and combinations thereof:
##STR00003##
wherein the NR.sub.3R.sub.4 moiety is connected ortho to the
R.sub.1 moiety on the phenyl ring;
[0122] R.sub.1 is selected from the group consisting of nitro,
amino optionally substituted with methyl or dimethyl, and amide
optionally substituted with methyl, dimethyl, ethyl, or diethyl;
the R.sub.2 moiety is connected meta with respect to the phenyl
ring;
[0123] R.sub.2 is carboxylic acid;
[0124] R.sub.3 is selected from the group consisting of phenyl
optionally substituted by any one or more of methoxy or halogen and
benzyl optionally substituted by any one or more of methoxy or
halogen; and
[0125] R.sub.4 is selected from the group consisting of H and
methyl.
[0126] In additional embodiments, a candidate compound is a
compound of Formula II:
##STR00004##
wherein
[0127] X is selected from the group consisting of hydrogen, methyl,
amine, methoxy, phenyl optionally substituted with up to a total of
three methyl and/or methoxy and/or halogen groups, cyclopentane,
morpholine, piperidine, N-methylpiperidine, N-ethylpiperidine,
(N,N-diethyl)formamide, pyridine, pyrazine, pyrrole, pyrrolidine,
furan, thiophene, tetrahydrofuran, pyran, tetrahydroisoquinoline,
isoquinoline, quinoline, N-phenylpiperazine optionally substituted
with up to a total of three methoxy and/or halogen groups, or
N-benzylpiperazine;
[0128] A is an optional spacer group, attachable in either
direction, selected from the group consisting of --NH--,
--NHCH.sub.2--, --NHCH.sub.2CH.sub.2--, --NHCH.sub.2CH.sub.2O--,
and --NHCH.sub.2(CH.sub.3);
[0129] D is selected from the group consisting of methyl,
isopropyl, tert-butyl, dimethylamine, morpholine, alcohol, phenyl
optionally substituted with up to a total of three methyl and/or
ethyl and/or methoxy and/or halogen and/or acetamide and/or ethoxy
and/or cyano groups, pyridine, pyrazine, pyrrole, pyrrolidine,
furan, thiophene, tetrahydrofuran, and pyran; and
[0130] Z is an optional spacer group, selected from the group
consisting of --CH.sub.2--, --SO.sub.2--, --SO.sub.2CH.sub.2--,
--CH.sub.2C(.dbd.O)--, --CH.sub.2CH.sub.2--, --C(.dbd.O)--, and
--C(.dbd.S)NHC(.dbd.O)--.
[0131] In other embodiments, A is absent (thus X is directly
connected to the phenyl ring at the position held by A); X is
tetrahydroisoquinoline, attached to the phenyl ring by its lone
nitrogen; Z is --C(.dbd.O)--; and D is methyl. In other
embodiments, A is absent; X is morpholine, attached to the phenyl
ring by its lone nitrogen; Z is --CH.sub.2--; and D is methyl.
[0132] In certain additional embodiments, a candidate compound is a
compound of Formula III:
##STR00005##
wherein
[0133] X is selected from the group consisting of methyl,
methylamine, halogen, and phenyl optionally substituted with up to
a total of three methyl and/or methoxy and/or halogen groups;
[0134] A is an optional spacer group, attachable in either
direction, selected from the group consisting of --NH--,
--N(CH.sub.3)H--, --O--, --OCH.sub.3--, --C(.dbd.O)NH--, and
--NHCH.sub.2--; and
[0135] Z is selected from the group consisting of phenyl optionally
substituted with up to a total of three methyl and/or ethyl and/or
methoxy and/or halogen and/or acetamide and/or ethoxy and/or cyano
groups; excepting those compounds that include X as phenyl and A as
--NHCH.sub.2--, the nitrogen in said A being connected to the
nitro-containing phenyl ring in said formula and the carbon in said
A being connected to said X in said formula.
[0136] In certain embodiments, a candidate compound is selected
from the group consisting of: [0137]
(4-(4-nitro-3-(phenylamino)phenyl)piperazin-1-yl)(phenyl)methanone;
[0138]
5-(4-dimethylcarbamylpiperazin-1-yl)-2-nitro-N-phenylbenzenamine;
[0139]
N-methyl-5-(4-benzoylpiperazin-1-yl)-2-nitro-N-phenylbenzenamine;
[0140] N,N-dimethyl-5-(4-ethylpiperazin-1-yl)-2-nitrobenzenamine;
[0141] N-(2-(4-benzoylpiperazin-1-yl)-5-nitrophenyl)acetamide;
[0142]
2-(benzylamino)-N,N-dimethyl-4-(4-benzoylpiperazin-1-yl)benzamide;
[0143]
2-(benzylamino)-N-ethyl-4-(4-benzoylpiperazin-1-yl)benzamide;
[0144] 3'-(benzylamino)-4'-nitrophenyl-3-carboxylic acid; [0145]
3'-(benzylamnino)-4'-nitro-N-phenylbiphenyl-3-carboxamide; [0146]
ethyl-1-(3-(benzylamino)-4-nitrophenyl) piperidine-4-carboxylate;
N-(2-(4-benzoylpiperazin-1-yl)-5-nitrophenyl)benzenamine; [0147]
(4-(4-amino-3-(phenylamino)phenyl)piperazin-1-yl)(phenyl)methanone;
[0148] 1-(3-(benzylamino)-4-nitrophenyl) piperidine-3-carboxylic
acid; [0149] 4'-nitro-3'-(phenylamino) biphenyl-3-carboxylic acid;
[0150] N,N-dimethyl-2-(4-benzoylpiperazin-1-yl)-5-nitrobenzenamine;
[0151] 4'-amino-3'-(phenylamino) biphenyl-3-carboxylic acid; [0152]
(4-(3-(N-benzyl-N-phenylamino)-4-aminophenyl)piperazin-1-yl)(phenyl)metha-
none; [0153]
(4-(3-(N-methyl-N-phenylamino)-4-(dimethylamino)phenyl)piperazin-1-yl)(ph-
enyl)methanone; [0154]
(4-(4-(dimethylamino)-3-(phenylamino)phenyl)piperazin-1-yl)(phenyl)methan-
one; [0155]
(4-(3-(N-methyl-N-phenylamino)-4-aminophenyl)piperazin-1-yl)(phenyl)metha-
none; [0156]
(4-(3-(N-methyl-N-phenylamino)-4-(methylamino)phenyl)piperazin-1-yl)(phen-
yl)methanone; [0157]
(4-(4-(methylamino)-3-(phenylamino)phenyl)piperazin-1-yl)(phenyl)methanon-
e; [0158]
2-(4-(4-benzoylpiperazin-1-yl)-2-(phenylamino)phenylamino)ethano-
l; [0159] N-benzyl-2-(4-benzoylpiperazin-1-yl)-5-nitrobenzenamine;
N-(4-(4-benzoylpiperazin-1-yl)-2-(phenylamino)phenyl)acetamide;
[0160]
4-(4-benzoylpiperazin-1-yl)-N1-(2-morpholinoethyl)-N2-phenylbenzene-1,2-d-
iamine, [0161] pharmaceutically acceptable salts, stereo-isomers,
polymorphs, metabolites, prod-drugs, analogs and combinations
thereof.
[0162] In other embodiments, a candidate compound is selected from
the group consisting of: [0163]
[4-[4-nitro-3-(tricyclo[3.3.1.13,7]dec-2-ylamino)phenyl]-1-piperazinyl]ph-
enylmethanone, [0164]
2-(4-benzoyl-1-piperazinyl)-5-nitrobenzonitrile, [0165]
[4-[3-[(4-methyl-4H-1,2,4-triazol-3-yl)thio]-4-nitrophenyl]-1-pipe-
razinyl]phenylmethanone, [0166]
[4-[4-nitro-3-(2-propen-1-ylamino)phenyl]-1-piperazinyl]phenylmethanone,
[0167]
[4-[3-[(2-methylpropyl)amino]-4-nitrophenyl]-1-piperazinyl]phenylm-
ethanone, [0168]
[4-[4-nitro-3-[[(tetrahydro-2-furanyl)methyl]amino]phenyl]-1-piperazinyl]-
phenylmethanone, [0169]
[4-[3-[(2,2-dimethylpropyl)amino]-4-nitrophenyl]-1-piperazinyl]phenylmeth-
anone, [0170]
[4-[3-(ethylamino)-4-nitrophenyl]-1-piperazinyl]phenylmethanone,
[0171] [4-(2-methyl-4-nitrophenyl)-1-piperazinyl]phenylmethanone,
[0172]
5-[[2-(4-benzoyl-1-piperazinyl)-5-nitrophenyl]methylene]-2,4,6(1H,3H,5H)--
pyrimidinetrione, [0173]
[4-[2-(2,5-dimethyl-1H-pyrrol-1-yl)-4-nitrophenyl]-1-piperazinyl]phenylme-
thanone, [0174]
[4-[4-nitro-2-(1H-pyrrol-1-yl)phenyl]-1-piperazinyl]phenylmethanone,
[0175]
2-[5-(4-benzoyl-1-piperazinyl)-2-nitrophenyl]-4-methyl-1(2H)phthal-
azinone, [0176]
[4-[3-(methylamino)-4-nitrophenyl]-1-piperazinyl]phenylmethanone,
[0177]
[4-[4-nitro-3-[(3-pyridinylmethyl)amino]phenyl]-1-piperazinyl]phenylmetha-
none, [0178]
[4-[3-(3,4-dihydro-2(1H)-isoquinolinyl)-4-nitrophenyl]-1-piperazinyl]phen-
ylmethanone, [0179]
[4-[4-nitro-3-(1-piperidinyl)phenyl]-1-piperazinyl]phenylmethanone,
[0180]
[4-[4-nitro-2-(trifluoromethyl)phenyl]-1-piperazinyl]phenylmethano-
ne, [0181]
[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-piperazinyl]pheny-
lmethanone, [0182]
[4-[3-(4-morpholinyl)-4-nitrophenyl]-1-piperazinyl]phenylmethanone,
[0183]
[4-[4-nitro-3-[(1-tricyclo[3.3.1.13,7]dec-1-ylethyl)amino]phenyl]--
1-piperazinyl]phenylmethanone, [0184]
[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl]phenylmethanone,
[0185]
[4-[4-nitro-3-[(2-phenylethyl)amino]phenyl]-1-piperazinyl]phenylme-
thanone, [0186]
[4-[3-[(2-furanylmethyl)amino]-4-nitrophenyl]-1-piperazinyl]phenylmethano-
ne, [0187]
[4-[3-(3,5-dimethyl-1H-pyrazol-1-yl)-4-nitrophenyl]-1-piperazin-
yl]phenylmethanone, [0188]
[4-[3-(cyclopropylamino)-4-nitrophenyl]-1-piperazinyl]phenylmethanone,
[0189] [4-(2-chloro-4-nitrophenyl)-1-piperazinyl]phenylmethanone,
[0190] [4-(2-fluoro-4-nitrophenyl)-1-piperazinyl]phenylmethanone,
[0191] 1-benzoyl-4-(3-formyl-4-nitrophenyl)piperazine, [0192]
1-benzoyl-4-[3-[(2,5-dioxo-4-imidazolidinylidene)methyl]-4-nitrophenyl]pi-
perazine, [0193] 1-(3-amino-4-nitrophenyl)-4-benzoylpiperazine,
1-benzoyl-4-(4-nitrophenyl)piperazine, [0194]
(2,4-dichlorophenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazi-
nyl]methanone, [0195]
(5-chloro-2-methoxyphenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-pi-
perazinyl]methanone, [0196]
[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl](4-propoxyphenyl-
)methanone, [0197]
(3,4-dimethoxyphenyl)[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-pipera-
zinyl]methanone, [0198]
(3,4-dimethoxyphenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperaz-
inyl]methanone, [0199]
(3-chloro-4-methylphenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-pip-
erazinyl]methanone, [0200] (2-methoxy-3-methylphenyl)
[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl]methanone,
[0201] (2-methoxy-3-methylphenyl)
[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-piperazinyl]methanone,
[0202]
[4-(1-methylethoxy)phenyl][4-[4-nitro-3-[(1-phenylethyl)amino]phen-
yl]-1-piperazinyl]methanone, [0203]
[4-(1-methylethoxy)phenyl][4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-pi-
perazinyl]methanone, [0204]
[3-(1-methylethoxy)phenyl][4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-p-
iperazinyl]methanone, [0205]
[3-(1-methylethoxy)phenyl][4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-pi-
perazinyl]methanone, [0206]
(4-ethylphenyl)[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-piperazinyl]-
methanone, [0207]
(4-ethylphenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl]m-
ethanone, [0208] (3-ethoxyphenyl)
[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-piperazinyl]methanone,
[0209]
(3-ethoxyphenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piper-
azinyl]methanone, [0210]
(3,4-dichlorophenyl)[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-piperaz-
inyl]methanone, [0211]
(3,4-dichlorophenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazi-
nyl]methanone, [0212]
[4-(1-methylethyl)phenyl][4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-pi-
perazinyl]methanone, [0213]
(2-iodophenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl]me-
thanone, [0214]
[4-(1,1-dimethylethyl)phenyl][4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-
-piperazinyl]methanone, [0215]
(3-bromophenyl)[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-piperazinyl]-
methanone, [0216]
[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-piperazinyl]phenylmethanone-
, [0217]
(2-bromophenyl)[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-pipe-
razinyl]methanone, [0218]
(4-butoxyphenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl]-
methanone, [0219]
[4-(1-methylethyl)phenyl][4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-pip-
erazinyl]methanone, [0220]
(4-methoxyphenyl)[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-piperaziny-
l]methanone, [0221]
(2-methylphenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl]-
methanone, [0222]
(2-methylphenyl)[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-piperazinyl-
]methanone, [0223]
(3-fluorophenyl)[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-piperazinyl-
]methanone, [0224]
(3-fluorophenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl]-
methanone, [0225]
(3-methoxyphenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl-
]methanone, [0226]
(3-bromophenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl]m-
ethanone, [0227]
[4-(1,1-dimethylethyl)phenyl][4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]--
1-piperazinyl]methanone, [0228]
(4-ethoxyphenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl]-
methanone, [0229]
[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl]phenylmethanone,
[0230]
(4-fluorophenyl)[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-pipe-
razinyl]methanone, [0231] (4-chlorophenyl)
[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-piperazinyl]methanone,
[0232]
(4-fluorophenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piper-
azinyl]methanone, [0233]
(2-chlorophenyl)[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-piperazinyl-
]methanone, [0234]
(4-methylphenyl)[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-piperazinyl-
]methanone, [0235]
[4-[3-[[(4-chlorophenyl)methyl]amino]-4-nitrophenyl]-1-piperazinyl](2-flu-
orophenyl)methanone, [0236]
(2-chlorophenyl)[4-[3-[[4-chlorophenyl)methyl]amino]-4-nitrophenyl]-1-pip-
erazinyl]methanone, [0237]
(4-bromophenyl)[4-[3-[[(4-chlorophenyl)methyl]amino]-4-nitrophenyl]-1-pip-
erazinyl]methanone, [0238]
(2-chloro-4,5-difluorophenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-
-piperazinyl]methanone, [0239]
(2-chlorophenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl]-
methanone, [0240]
(4-methylphenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl]-
methanone, [0241]
(2-bromophenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl]m-
ethanone, [0242]
[4-[3-[[(4-chlorophenyl)methyl]amino]-4-nitrophenyl]-1-piperazinyl](4-met-
hylphenyl)methanone, [0243]
[4-[3-[[[4-(1-methylethyl)phenyl]methyl]amino]-4-nitrophenyl]-1-piperazin-
yl](4-methylphenyl)methanone, [0244]
(3,5-dichloro-4-methoxyphenyl)[4-[3-[[[4-(1-methylethyl)phenyl]methyl]ami-
no]-4-nitrophenyl]-1-piperazinyl]methanone, [0245]
(2-fluorophenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl]-
methanone, [0246]
(2-fluorophenyl)[4-[3-[[[4-(1-methylethyl)phenyl]methyl]amino]-4-nitrophe-
nyl]-1-piperazinyl]methanone, [0247]
(4-chlorophenyl)[4-[3-[[[4-(1-methylethyl)phenyl]methyl]amino]-4-nitrophe-
nyl]-1-piperazinyl]methanone, [0248]
[4-[3-[[(4-chlorophenyl)methyl]amino]-4-nitrophenyl]-1-piperazinyl](3-met-
hylphenyl)methanone, [0249]
(3-methylphenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl]-
methanone, [0250]
(3-chlorophenyl)[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-piperazinyl-
]methanone, [0251]
(3-chlorophenyl)[4-[3-[[(4-chlorophenyl)methyl]amino]-4-nitrophenyl]-1-pi-
perazinyl]methanone, [0252]
(2-chloro-4,5-difluorophenyl)[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]--
1-piperazinyl]methanone, [0253]
(2-chloro-4,5-difluorophenyl)[4-[3-[[(4-chlorophenyl)methyl]amino]-4-nitr-
ophenyl]-1-piperazinyl]methanone, [0254]
(4-bromophenyl)[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-piperazinyl]-
methanone, [0255]
(3-chlorophenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl]-
methanone, [0256] (4-methoxyphenyl)
[4-[4-nitro-3-[(1-phenylethyl)amino]phenyl]-1-piperazinyl]methanone,
[0257]
[4-[3-[[(4-chlorophenyl)methyl]amino]-4-nitrophenyl]-1-piperazinyl-
](4-methoxyphenyl)methanone, [0258]
(4-methoxyphenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl-
]methanone, [0259]
[4-[3-[[(4-chlorophenyl)methyl]amino]-4-nitrophenyl]-1-piperazinyl](2-met-
hoxyphenyl)methanone, [0260]
(2-methoxyphenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl-
]methanone, [0261]
(4-bromophenyl)[4-[4-nitro-3-[(phenylmethyl)amino]phenyl]-1-piperazinyl]m-
ethanone, [0262]
(2-bromophenyl)[4-[3-[[(4-chlorophenyl)methyl]amino]-4-nitrophenyl]-1-pip-
erazinyl]methanone, [0263] pharmaceutically acceptable salts,
stereo-isomers, polymorphs, metabolites, prod-drugs, analogs and
combinations of any of the foregoing.
[0264] In certain embodiments, the candidate compound is
represented by one of one of the following structural formulas:
##STR00006## ##STR00007## ##STR00008## ##STR00009## ##STR00010##
##STR00011##
pharmaceutically acceptable salts, stereo-isomers, polymorphs,
metabolites, pro-drugs, analogs and mixtures of any of the
foregoing.
[0265] In certain embodiments, the candidate compound has activity
against both A.beta. and A-Syn, as determined by a ThT functional
aggregation assay.
[0266] In certain embodiments, the candidate compound has activity
against both A.beta. and tau, as determined by a ThT functional
aggregation assay.
[0267] In certain embodiments, the candidate compound only has
activity against A.beta., as determined by a ThT functional
aggregation assay.
[0268] In certain embodiments, the IC50/.mu.M for A.beta. for the
candidate compound is from about 0.5 to about 5734, as determined
by ThT functional aggregation assay. In certain embodiments, the
IC50/.mu.M for A.beta. is from about 0.8 to about 180. In certain
embodiments, the IC50/.mu.M for A.beta. is from about 0.8 to about
107. In certain embodiments, the IC50/.mu.M for A.beta. is 0.85,
0.88, 1.47, 1.9, 2.3, 2.29, 3.5, 7.5, 8.4, 9.64, 15.5, 18, 23, 25,
26.5, 54, 66, 84, 90, 92, and 100.
[0269] In certain embodiment, the IC50/.mu.M for A-syn for A-Syn,
as determined by A-Syn functional ThT aggregation assay, for the
candidate compound is from about 1 to about 70. In certain
embodiment, the IC50/.mu.M for A-syn is from about 5 to about 50.
In certain embodiment, the IC50/.mu.M for A-syn is 4, 5, 10, 15 or
45.
[0270] In certain embodiments, the IC50/.mu.M for A.beta. for the
candidate compound is 0.85, 9.64, 25, 1.47, 9.23, 27, 26.6, or 3.5;
and the IC50/.mu.M for A-syn is 6.34, 5, 4, 7, 8, 10, 15, or
45.
[0271] One example of a pseudo-crystal structure utilized in these
methods is the one formed by SEQ ID: 1 and SEQ ID: 2 when
interacting in the manner depicted in FIG. 2.
[0272] In certain embodiments, a method of identifying compounds
that modulate amyloid aggregation comprises the steps of
constructing a pseudo-crystal structure in a computer modeling
program; selecting a list of candidate compounds; constructing said
candidate compounds in a computer modeling program; performing an
iterative docking and scoring of all candidate compounds, by means
of docking each candidate compound into the pocket formed by said
model and scoring each candidate compound to reflect its degree of
complementarity with respect to said pocket; and identifying
compounds that modulate amyloid aggregation by reference to a score
cutoff that substantially distinguishes active compounds from
inactive compounds. A score cutoff is usually an absolute energy,
that can be set higher or lower depending on the level of activity
that is deemed to separate active from inactive, or as a reference
between more active and less active. In some embodiments, the score
cutoff is the value of a known active compound. In other
embodiments, the score cutoff is the value that distinguishes a
small part (i.e. 1% to 10%) of a set of compounds as being
substantially more active than other members of the set.
[0273] In certain embodiments, the pseudo-crystal structure
comprises a three-dimensional model of uncrystallized, monomeric
amyloid protein and a three-dimensional model of uncrystallized
amyloid protein or oligomer comprising one or more amyloid
peptides, said amyloid protein or oligomer model to be positioned
with respect to said amyloid monomer model such that it forms a
pocket in conjunction with said amyloid monomer model, such that a
compound may be inserted into said pocket thereby modulating
amyloid aggregation.
[0274] In certain embodiments, the amyloid monomer model and the
amyloid protein or oligomer model are both composed of beta-amyloid
protein or an amyloidogenic fragment thereof.
[0275] In other embodiments, said amyloid monomer model is
substantially SEQ ID: 1 and said amyloid oligomer model is
substantially SEQ ID: 2. In certain embodiments, said amyloid
monomer model is further positioned with respect to said amyloid
protein model such that the amino acid Val at position 8 of SEQ ID:
1 interacts with the amino acid Glu at position 6 of SEQ ID: 2; the
amino acid Gly at position 9 of SEQ ID: 1 interacts with the amino
acid Glu at position 6 of SEQ ID: 2; the amino acid Ser at position
10 of SEQ ID: 1 interacts with the amino acid Gly at position 9 of
SEQ ID: 2; the amino acid Ser at position 10 of SEQ ID: 1 interacts
with the amino acid Ser at position 10 of SEQ ID:2; and the amino
acid Lys at position 12 of SEQ ID: 1 interacts with the amino acid
Glu at position 6 of SEQ ID: 2. In certain embodiments, the amyloid
monomer model is positioned with respect to the amyloid protein
model substantially in the orientation shown in FIG. 2. In certain
embodiments, the candidate compounds are drawn from proprietary
compound collections and/or from commercially available screening
libraries.
[0276] The process of docking as implemented in the computer
modeling program or programs is described, e.g., in the manuals of
said program or programs. Docking may be performed, e.g.,
interactively or in batch mode. Computer modeling programs
especially suited to docking include, but are not restricted to,
DOCK, AUTODOCK, GLIDE, and FlexX. The manuals of these programs are
herein incorporated by reference in their entirety for all
purposes.
[0277] Scoring functions are generally applied after docking and
may be integral to a given docking method. Examples of scoring
functions include DrugScore, London, and Affinity dG. In some
embodiments, the scoring function may be a composite of two or more
scoring functions.
[0278] In certain embodiments, a method of improving potency of a
compound known to modulate amyloid aggregation comprising the steps
of constructing a pseudo-crystal structure in a computer modeling
program; selecting a list of candidate compounds; constructing said
candidate compounds in a computer modeling program; performing an
iterative docking and scoring of all candidate compounds, by means
of docking each candidate compound into the pocket formed by said
model and scoring each candidate compound to reflect its degree of
complementarity with respect to said pocket; and identifying
compounds that modulate amyloid aggregation by reference to a score
cutoff that substantially distinguishes more active compounds from
less active compounds. In certain embodiments of the invention, the
list of candidate compounds includes both the compound known to be
active and analogs of said compound, and the score cutoff is that
score which belongs to the compound known to be active. In certain
embodiments, the pocket comprises substantially SEQ ID: 1 and
amyloid oligomer model has substantially SEQ ID: 2, wherein SEQ ID:
1 is
TABLE-US-00003 Lys Val Phe Phe Ala Glu Asp Val Gly Ser Asn 1 6 11
Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val 16 21 Val Ile Ala
26
and,
SEQ ID: 2 is
TABLE-US-00004 [0279] Lys Val Phe Phe Ala Glu Asp Val Gly Ser Asn 1
6 11 Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val 16 21 Val Ile
Ala 26
In certain, embodiments the two sequences comprising the pocket are
not substantially the same.
[0280] In certain embodiments, the present invention uses known
non-peptidic compounds amyloid and/or amyloidogenic fragments to
identify new compounds which may be inhibitors or likely to be
inhibitors of amyloid protein aggregation. For example, a wide
variety of non-peptidic compounds have shown the ability to inhibit
the aggregation of amyloid proteins in vitro, and many such
compounds can inhibit the aggregation of beta-amyloid protein as
well as other kinds of amyloid (see for example Klabunde et al,
2000, Nat. Struct. Biol. 7:312-321; Green et al, 2003, J. Am. Chem.
Soc. 125:13404-13414; Masuda et al, 2006, Biochemistry
45:6085-6094; Ono et al, 2003, J. Neurochem 87:172-181; Tagliavini
et al, 2000, J. Mol Biol. 300:1309-1322). In addition, a number of
amyloid proteins or amyloidogenic fragments thereof have been
crystallized, including islet amyloid polypeptide or amylin
(Wilizius et al, 2008, Protein Sci. June 12 online);
beta-2-microglobulin or light chain (Iwata et al, 2007, J. Biochem.
142:413-419; Schormann et al, 1995, Proc. Natl. Acad. Sci. USA
92:9490-9494), insulin; and notably transthyretin, which has been
crystallized several times in co-crystal with known inhibitors
(Hamilton et al, 1993, J. Biol. Chem. 268:2416-2424; Peterson et
al, 1998, Proc. Natl. Acad. Sci. USA 95:12956-12960; Klabunde et
al, 2000, Nat. Struct. Biol. 7:312-321). Some known compounds have
also been shown to have beneficial in vivo effects, including
reducing the size of amyloid plaques and delaying mortality in
mouse models of amyloid disease (Chen et al, 2000, Nat. Med.
6:797-801; Imbimbo et al, 2007, Pharmacol. Res. 55:318-328). It is
postulated that compounds that modulate the aggregation of amyloid
protein in vitro (so-called "anti-amyloid compounds") and have in
vivo effects may be beneficial for the treatment of amyloid
diseases, e.g., Alzheimer's Disease. In certain embodiments, the
present invention uses these known compounds to identify new
compounds which may be inhibitors or likely to be inhibitors of
amyloid protein aggregation. One compound which may be used in the
methods of the present invention (e.g., as a multiply anti-amyloid
compound) is resveratrol, an antioxidant component of red wine and
an inhibitor of beta-amyloid aggregation at an effective
concentration of 5.6 .mu.M (Riviere et al, 2007, Bioorg. Med. Chem.
15:1160-1167).
[0281] In certain embodiments, the invention is directed to methods
of improving potency of a compound known to modulate amyloid
aggregation comprising the steps of constructing a pseudo-crystal
structure in a computer modeling program; selecting a list of
candidate compounds; constructing said candidate compounds in a
computer modeling program; performing an iterative docking and
scoring of all candidate compounds, by means of docking each
candidate compound into the pocket formed by said model and scoring
each candidate compound to reflect its degree of complementarity
with respect to said pocket; and identifying compounds that
modulate amyloid aggregation by reference to a score cutoff that
substantially distinguishes more active compounds from less active
compounds. In certain embodiments, the pseudo-crystal structure for
this method is the one formed by SEQ ID: 1 and SEQ ID: 2 when
interacting in the manner depicted in FIG. 2. The list of candidate
compounds may include, e.g., both the compound known to be active
anti-amyloid compounds and analogs of such compounds, and the score
cutoff is that score which belongs to the compound known to be
active. In certain embodiments, the known compound is a compound
selected from the lists recited in paragraphs [0106] to [0117] of
the application. In certain embodiments, the known compound is
resveratrol.
[0282] The invention also encompasses and provides for use of the
compounds identified or improved using the invention's methods as
therapies for diseases in which amyloidosis occurs. The disease
include (but are not limited to) Alzheimer's disease, Huntington's
disease, Parkinson's disease, Creutzfeldt-Jacob disease,
amyotrophic lateral sclerosis, and senile systemic amyloidosis. In
certain embodiments, the disease is Alzheimer's disease. In certain
embodiments, the therapeutic compound inhibits aggregation of one
or more of the following amyloidogenic proteins: beta-amyloid
protein, tau protein, alpha-synuclein protein, huntingtin protein,
prion precursor protein; other amyloidogenic protein not named
herein, and combinations of any of the foregoing.
[0283] In certain preferred embodiments, both the aggregation of
alpha-synuclein protein and of tau protein are inhibited. In
certain embodiments, both the aggregation of beta-amyloid protein
and of tau protein are inhibited. In certain embodiments, both the
aggregation of beta-amyloid protein and of alpha-synuclein protein
are inhibited. In certain embodiments, only the aggregation of tau
protein, beta-amyloid protein, or alpha-synuclein protein is
inhibited.
[0284] In certain embodiments, the aggregation of beta-amyloid
protein, tau protein, and alpha-synuclein protein are all
inhibited. In certain embodiments, huntingtin aggregation is
inhibited. In certain embodiments, prion protein aggregation is
inhibited.
[0285] In certain embodiments, the methods comprise administering
to a subject a therapeutic compound identified or improved using
the invention's methods (including analogs and derivatives of such
compounds) and/or pharmaceutically acceptable salts thereof, such
that the therapeutic compound inhibits the aggregation of an
amyloidogenic protein. The subject in these methods may be, e.g., a
vertebrate, a mammal, a human, or a non-human animal. The
administration may be, e.g., oral, parenteral, transdermal,
intrathecal, or intranasal.
[0286] In certain embodiments, the methods comprise using known
amyloid and/or amyloidogenic fragments to discover and/or optimize
compounds that modulate their respective amyloid protein, and other
amyloid proteins, e.g., due to the purported relative universality
of amyloid pathogenesis. (Kayed et al, 2003, Science 300:486-489;
Bucciantini et al, 2002, Nature 416:507-511). For example,
transthyretin is known to bind beta-amyloid protein in vivo,
probably as aggregated beta-amyloid (Tsuzuki et al, 2000, Neurosci.
Lett. 281:171-174; Liu and Murphy, 2006, Biochemistry
45:15702-15709), and several inhibitors of transthyretin are also
known to inhibit beta-amyloid protein. However, as such compounds
do not inhibit both transthyretin and beta-amyloid protein with
equal potency, the utility of transthyretin crystal structures per
se for discovering inhibitors of beta-amyloid protein does not
approach what might be expected if a similar crystal structure of
beta-amyloid were available. The methods of present invention in
certain embodiments provide for the utilization of these compounds
(despite the lack of known crystal structure of beta-amyloid) to
identify new compounds which may be inhibitors or likely to be
inhibitors of simultaneous inhibition of several amyloid
proteins.
[0287] In other embodiments, the invention relates to methods of
virtually screening of compound library, e.g., in silico screening.
More specifically, the invention relates to methods of virtually
screening a library or libraries to identify compounds that are
likely to inhibit amyloid protein aggregation. Such compounds would
be of use as therapies for neurodegenerative diseases such as
Alzheimer's disease, Parkinson's disease, Huntington's disease,
prion diseases, and systemic amyloidosis.
[0288] The utility and operation of the embodiments of the
invention can be further appreciated by reference to the following
non-limiting examples:
Example 1
Construction of a Common Conformational Motif Model and Subsequent
Extraction of a Model of Amyloid Protein Aggregation
[0289] A Common Conformational Motif model was constructed using a
computer modeling program in interactive session and validated.
[0290] First, resveratrol was drawn and minimized to a
root-mean-square gradient of 0.05 using the MMFF94 force field with
corresponding partial charges. The PDB structure 1BM7 (www.pdb.org)
of transthyretin bound to flufenamic acid was loaded, and
resveratrol was manually placed in a position coincident with one
of the two flufenamic acid binding sites. The binding site of the
other flufenamic acid (i.e. the other homodimer), the flufenamic
acid moiety in the resveratrol-occupied binding site, and any
solvent molecules were deleted. The 17-42 residue sequence of
beta-amyloid protein was then drawn and duplicated, and the two
strands of beta-amyloid protein were placed in close,
non-overlapping proximity to each other and to resveratrol. The
entire structure was then correctly protonated/deprotonated for
physiological pH and minimized to a root-mean-square gradient of
0.05 using the MMFF94 force field with corresponding partial
charges. The orientation of one strand of beta-amyloid protein with
respect to the other was a matter of routine experimentation, as
the method furnished both a means of adjusting the structure such
that a pocket is formed and a means of validating that the
candidate model was useful. Several dozen candidate models were
created and extracted. One model was validated as the Common
Conformational Motif model and is depicted in FIG. 1.
[0291] The model of amyloid protein aggregation was then extracted.
The extracted model of amyloid protein aggregation is depicted in
FIG. 2. FIG. 2 shows two A.beta..sub.17-42 monomers bound to each
other near the N terminals in a "double candycane" structure. Each
monomer is folded in a loop from residues 23 through 33. The loops
are stabilized by intra-loop cationic-anionic interactions between
Asp.sub.23 and Lys.sub.28, and they also show an inter-loop
attraction through these same residues. Short-run (up to 100 ns)
molecular dynamics calculations on this model indicate that the
pocket is stable.
[0292] When the model of amyloid protein aggregation depicted in
FIG. 2 was compared to the structure of transthyretin ("TTR")
depicted in FIG. 1, a number of similarities were observed. The
model of amyloid protein aggregation depicted in FIG. 2 provided a
similar expanse of hydrophobic contact by lining up along two
flattened sides of what would otherwise be a much smaller area of
.beta.-sheet, rather than forming a pocket across the top of
several .beta.-sheets. Anti-amyloid compounds in the amyloid
protein aggregation model of FIG. 2 were stabilized in the pocket
by Lys and Ser and by exterior Leu, just as in the structure of
TTR. Similarly, the positive charge of Lys was shared between the
substrate and a nearby Asp in both structures.
[0293] It was therefore postulated that a small negatively charged
compound might be able to fit between both Lys residues and benefit
from the salt bridge interaction, as was reported in TTR. It was
further postulated that model of amyloid protein aggregation
depicted in FIG. 2 may be a transient species on the way to a
stacked dimer using a "dock-lock" mechanism. It is known that many
anti-amyloid compounds are multiply anti-amyloid. As such, it was
postulated that the utility of the models of the present invention
extends beyond the prevention of beta-amyloid protein aggregation
and includes additional species of the amyloid proteins, therefore
we hypothesized that it can be considered a common conformational
motif, of utility for other amyloids beyond beta-amyloid
protein.
Example 2
Validation of the CCM Model of Amyloid Protein Aggregation of
Example 1
[0294] A burgeoning number of compounds have been shown to be
anti-amyloidogenic and/or anti-fibrillogenic. To validate the model
of amyloid protein aggregation constructed in Example 1, and in
view of its potential extension to other amyloid proteins like
.alpha.-synuclein, a set of these compounds was evaluated in an in
silico docking screen using a collection of 79 compounds for which
IC.sub.50s have been computed for AD, tau, and .alpha.-synuclein
inhibition. This diverse set comprised polyphenols, benzothiazoles,
phenothizaines, macrolides, prophyrins, steroids, derivatives of
Congo red, and other anti-amyloid molecules of note.
[0295] Among the 39 polyphenols evaluated in the screen, the best
docking scores were predicted for purpurogallin, epigallocatechin,
catechin gallate, hinokiflavone, myricetin, and gallocatechin
gallate--all of which have IC.sub.50s below 7 .mu.M. The worst
scores in the test were predicted for rutin (expt IC.sub.50 32
.mu.M) and chlorogenic acid (>40 .mu.M). Since each compound was
represented by several poses, computing a ranking for the remaining
compounds was somewhat ambiguous. Nevertheless, the preliminary
result was encouraging enough to consider a more stringent
criterion: the quantitative structure-activity relationship (QSAR).
The Masuda test set was subjected to a CoMFA (comparative molecular
field analysis) in order to demonstrate the validity of the model
of amyloid protein aggregation depicted in FIG. 2.
[0296] CoMFA is a 3-D QSAR technique generally used to posit a
"pseudoreceptor" when none is known. Because the algorithm is
highly sensitive to alignment of compounds with respect to each
other, it is often difficult to produce statistically significant
results, even with structurally similar compounds. However, when
statistically significant results are obtained, this can be a great
confirmation of a pharmacophore hypothesis.
[0297] Using the three best poses from the docking calculation as
the initial test set (3.times.79=237 total poses), a partial least
squares analysis of the fit indicated a cross-validated r.sup.2
(i.e. q.sup.2) of 0.248. Removing 25 outlying poses improved the
leave-one-out q.sup.2 to 0.616. A more laborious bootstrapping
calculation produced a q.sup.2 of 0.619. This result indicated that
the CoMFA pseudoreceptor had a good agreement with experiment when
the molecules are aligned in accordance with the model of amyloid
protein aggregation depicted in FIG. 2. Only eight of the 79
compounds were predicted as false positives:
TABLE-US-00005 Predicted Expt Polyphenols Apigenin 1.4 >40
Baicalein 7.8 4.5 Catechin 10.6 >40 Catechin gallate 6.2 5.0
chlorogenic acid >40 >40 curcumin 2.0 1.7 cyanidin 4.8 4.0
daidzein 2.8 >40 delphinidin 4.4 3.0 2,2'-dihydroxybenzophenone
>40 >40 4,4'-dihydroxybenzophenone 1.6 >40 dopamine
chloride 6.7 28.6 epicatechin 16.1 >40 epicatechin 3-gallate 2.3
3.0 epigallocatechin 8.7 7.0 epigallocatechin gallate 2.5 2.0
exifone 2.2 0.7 gallocatechin 12.4 7.0 gallocatechin gallate 2.1
1.5 gingerol 19.8 25.0 gossypetin 1.2 1.3 hinokiflavone 5.8 5.0
hypericin 1.5 0.9 kaempferol 8.3 8.0 luteolin 3.6 3.0 myricetin 0.9
0.9 naringenin 3.2 25.0 2,3,4,2',4'-pentahydroxybenzophenone 2.2
2.8 procyanidin B1 21.6 14.0 procyanidin B2 >40 >40
purpurogallin 3.2 0.5 quercetin 4.3 5.0 rosmarinic acid 8.7 12.0
rutin >40 32.0 taxifolin 1.0 >40
2,2',4,4'-tetrahydroxybenzophenone 3.2 >40 theaflavone 2.8 2.0
tocopherol >40 >40 2,3,4-trihydroxybenzophenone 6.4 3.1
anthracycline daunorubicin hydrochloride 1.3 1.4 benzothiazoles
2-(4-aminophenyl)-6-methylbenzothiazole 2.4 2.0 basic blue 41 1.2
1.4 2-[4-(dimethylamino)phenyl]-6- 2.0 2.0 methylbenzothiazole
3,3'-dipropyl thiodicarbocyanine iodine 0.3 0.3 lignans magnelol
>40 >40 sesamin 19.2 >40 phenothiazines acetopromazine
maleate salt >40 >40 azure A 0.5 0.4 azure C 0.5 0.2
chlorpromazine hydrochloride >40 >40 lacmoid 3.8 1.4
methylene blue 20.0 2.3 perphenazine >40 >40 promazine
hydrochloride >40 >40 propionylpromazine >40 >40
hydrochloride quinacrine 8.9 8.4 quinacrine mustard 1.8 1.2 polyene
macrolides amphotericin B 2.0 2.2 filipin III 14.7 14.6 porphyrins
ferric dehydroporphyrin IX 0.2 0.2 hematin 0.2 0.2 phthalocyanine
tetrasulfonate 3.9 3.2 rifamycin rifampicin 4.0 4.9 steroids
taurochenodeoxycholic acid >40 >40 taurohydroxycholic acid
>40 >40 taurolithocholic acid >40 >40 taurolithocholic
acid 3-sulfate >40 >40 tauroursodeoxycholic acid >40
>40 Congo red and derivatives Congo red 1.0 0.9 chlorazol black
E 0.2 0.3 BSB 5.5 6.4 FSB 3.0 1.9 Ponceau SS 1.2 1.2 terpenoids
asiatic acid >40 >40 ginkgolide A >40 >40 ginkgolide B
14.4 11.0 ginkgolide C >40 >40 others
4,5-dianilinophthalimide 5.4 2.9 methyl yellow 2.2 1.5
[0298] It was therefore concluded that the model of amyloid protein
aggregation of FIG. 2 has utility in predicting the anti-amyloid
effects of candidate compounds tested in the example, and that
these compounds have activity against aggregation of .beta. amyloid
protein. It was also postulated that, given conformational
similarities between certain amyloid proteins, the candidate
compounds may inhibit aggregation of other amyloid proteins, and
that dimers of beta-amyloid protein in general may have significant
utility in predicting the anti-amyloid effects of candidate
compounds on other amyloids such as tau, alpha-synuclein,
huntingtin, and/or prion protein.
Example 3
Identification of Compounds that Modulate Amyloid Protein
Aggregation and Improvement of Potency Using the Common
Conformational Motif Model
[0299] The Common Conformational Motif model created in Example 1
was used to demonstrate its utility for rapid lead discovery.
[0300] A simple geometric query against a diverse collection of
around 750,000 drug-like compounds was constructed and yielded
1,260 compounds with non-hydrogenic atoms lying within 0.1 .ANG. of
each point in the 3-point pharmacophore. Searching in this manner
allowed for a quick (<90 min) pre-screening prior to the more
computationally laborious docking procedure. Using the parameters
for docking as described above, each of the 1,260 compounds were
fit into the CCM model (i.e., depicted in FIG. 2).
[0301] The top 125 compounds were examined for commercial
availability, and six of these compounds, evenly distributed with
respect to docking score, were acquired from Hit2Lead for in vitro
testing. Three of these compounds were inactive in the ThT assay,
one was weakly active, and two were highly active (see U.S.
Provisional Application Ser. No. 61/092,845 for initial compound
disclosures and subsequent use of the model for improving.
[0302] One-half of compounds (50%) initially tested had some
activity, and one-third (33.3%) had significant activity, as
compared to the typical hit rate of a high-throughput screen
(generally no more than 0.5%) with traditional screening
techniques. These results therefore confirmed that the screening
technique utilized in this example is highly hit-enriched screening
technique and may be more effective, as compared to traditional
screening techniques.
[0303] The present example therefore confirms, e.g., that the model
created in Example 1 has utility for rapid lead discovery.
[0304] It was then postulated that, e.g., because a number of the
compounds described in the application were shown to be multiply
anti-amyloid compounds, that pseudo-crystal structures described in
the application may be useful for identification and optimization
of anti-tau, anti-alpha-synuclein, and other anti-amyloid
applications, in addition to being useful in rapid lead discovery
of compounds that may prevent aggregation of beta-amyloid
protein.
Example 4
Identification of Known Compounds that Modulate Amyloid Protein
Aggregation Using the Model of FIG. 2
[0305] Further validation of the CCM model of protein aggregation
(i.e., the model of FIG. 2) was performed using a screen of the
PubChem collection (http://www.ncbi.nlm.nih.gov) using a blank
structure query and a limit query of "Pharmacological Action." The
query returned 14,172 compounds, consisting entirely of molecules
known to have a pharmacological effect.
[0306] This set of 14,172 compounds was then downloaded in MDL
SDfile format, imported into a molecular modeling program, stripped
of counterions and assigned physiologically appropriate protonation
states, and minimized using a molecular mechanics force field.
[0307] Next, an unguided docking study was performed, using docking
parameters and binding site identification identical to that above,
on the PubChem set. The results for the top 25, in decreasing order
of binding score and repeated hits in parenthesis, were as follows:
chlorophyllin (2), epigallocatechin gallate (3), coumermycin A1,
chlorotetracycline (2), doxycycline (2), hygromycin B,
rolitetracycline, desfarrioxamine, epicatechin gallate,
transcycline, oligoadenylade, 8-azido-ADP, citrinin,
tetrakis(4-N-methylpyridyl)porphyrin, tetracycline, acteoside,
actinonin, lactitol, minocycline, and rocephalin.
[0308] What was striking in these results was that they included a
number of known anti-amyloid compounds and compound families.
Specifically, porphyrins, polyphenols, and tetracyclines
constituted fully 60% of the top scores, which is significantly
better than most high-throughput screening campaigns. The list also
included known anti-amyloid compounds not considered part of any
family, including desferrioxamine and the neuroprotectant
acteoside.
[0309] It was therefore postulated that most of the remaining top
twenty five compounds were also likely to have effects against
A.beta. aggregation, especially given their highly symmetrical
structures (coumermycin, hygromycin, oligoadenylate), structural
affinity with NSAIDs (citrinin), and/or reported utility in related
protein misfolding/aggregation diseases such as Huntington's
disease (actinonin) and ALS (rocephilin).
[0310] Moreover, some compounds which were known for uses other
than anti-amyloid have been identified as being anti-amyloid by the
screen. It was therefore postulated that these compounds may have
utility in treating Alzheimer's Disease and related disorders
(e.g., Parkinson's Disease). For example, exploration of hits in
the top 1% included those mentioned above as well as the following,
in no particular order of activity: Pradimicin, Oleuropein,
Tunicamycin, Hyaluronic acid, Coumermycin, Ouabain, Amikacin,
Rifaximin, delavirdine, almitrine, and fluvastatin, lumiracoxib,
and fenoterol. It was therefore postulated that these compounds may
have utility in treating amyloid diseases.
[0311] In this regard, it is notable that even compounds that do
not ordinarily cross the blood-brain barrier are often capable of
crossing it in patients with neurologic disorders, and that
neurologic disorders of the peripheral nervous system also can
benefit from anti-amyloid compounds. As such, it was postulated
that the physiochemical parameters of an identified anti-amyloid
molecule may not need to fit into Log P, molecular weight, and
other ranges associated with blood-brain barrier penetration in
order to have utility in treatment of neurologic disorders (e.g.,
Alzheimer's Disease and/or related disorders).
Data Listing
[0312] The model of FIG. 2 in PDB format.
[0313] This listing can be transcribed into a text file (Unicode,
ASCII, or other standard encoding), saved to disc as with extension
.PDB, and loaded into a PDB-aware visualization or modeling program
in order to reconstruct the model.
TABLE-US-00006 HEADER PROTEIN FIBRIL REMARK 99 MOE v2007.09
(Chemical Computing Group Inc) Mon Jun 16 15:27:17 2008 ATOM 1 N
LEU E 17 -4.245 -9.375 10.249 1.00 0.00 N1+ ATOM 2 H1 LEU E 17
-4.274 -8.787 9.368 0.00 0.00 H ATOM 3 H2 LEU E 17 -4.396 -8.764
11.070 0.00 0.00 H ATOM 4 CA LEU E 17 -5.267 -10.479 10.168 1.00
0.00 C ATOM 5 C LEU E 17 -4.618 -11.861 10.006 1.00 0.00 C ATOM 6 O
LEU E 17 -3.393 -11.958 9.924 1.00 0.00 O ATOM 7 CB LEU E 17 -6.281
-10.169 9.008 1.00 0.00 C ATOM 8 CG LEU E 17 -7.425 -9.144 9.299
1.00 0.00 C ATOM 9 CD1 LEU E 17 -6.943 -7.714 9.627 1.00 0.00 C
ATOM 10 CD2 LEU E 17 -8.432 -9.109 8.124 1.00 0.00 C ATOM 11 H3 LEU
E 17 -3.272 -9.775 10.284 1.00 0.00 H ATOM 12 HA LEU E 17 -5.791
-10.502 11.115 1.00 0.00 H ATOM 13 HB2 LEU E 17 -5.722 -9.849 8.101
1.00 0.00 H ATOM 14 HB3 LEU E 17 -6.795 -11.117 8.729 1.00 0.00 H
ATOM 15 HG LEU E 17 -7.992 -9.514 10.187 1.00 0.00 H ATOM 16 HD11
LEU E 17 -7.811 -7.028 9.744 1.00 0.00 H ATOM 17 HD12 LEU E 17
-6.372 -7.687 10.576 1.00 0.00 H ATOM 18 HD13 LEU E 17 -6.304
-7.337 8.802 1.00 0.00 H ATOM 19 HD21 LEU E 17 -9.283 -8.433 8.356
1.00 0.00 H ATOM 20 HD22 LEU E 17 -7.942 -8.747 7.195 1.00 0.00 H
ATOM 21 HD23 LEU E 17 -8.844 -10.122 7.930 1.00 0.00 H ATOM 22 N
VAL E 18 -5.424 -12.964 9.972 1.00 0.00 N ATOM 23 CA VAL E 18
-4.953 -14.332 9.721 1.00 0.00 C ATOM 24 C VAL E 18 -4.942 -14.537
8.195 1.00 0.00 C ATOM 25 O VAL E 18 -5.993 -14.639 7.554 1.00 0.00
O ATOM 26 CB VAL E 18 -5.740 -15.453 10.461 1.00 0.00 C ATOM 27 CG1
VAL E 18 -5.106 -16.852 10.206 1.00 0.00 C ATOM 28 CG2 VAL E 18
-5.825 -15.184 11.990 1.00 0.00 C ATOM 29 HN VAL E 18 -6.412
-12.896 10.080 1.00 0.00 H ATOM 30 HA VAL E 18 -3.933 -14.389
10.082 1.00 0.00 H ATOM 31 HB VAL E 18 -6.781 -15.475 10.063 1.00
0.00 H ATOM 32 HG11 VAL E 18 -5.679 -17.636 10.745 1.00 0.00 H ATOM
33 HG12 VAL E 18 -5.126 -17.113 9.127 1.00 0.00 H ATOM 34 HG13 VAL
E 18 -4.054 -16.887 10.561 1.00 0.00 H ATOM 35 HG21 VAL E 18 -6.395
-15.996 12.491 1.00 0.00 H ATOM 36 HG22 VAL E 18 -4.811 -15.143
12.443 1.00 0.00 H ATOM 37 HG23 VAL E 18 -6.346 -14.228 12.203 1.00
0.00 H ATOM 38 N PHE E 19 -3.722 -14.584 7.596 1.00 0.00 N ATOM 39
CA PHE E 19 -3.476 -14.796 6.177 1.00 0.00 C ATOM 40 C PHE E 19
-3.187 -16.283 5.942 1.00 0.00 C ATOM 41 O PHE E 19 -2.095 -16.776
6.243 1.00 0.00 O ATOM 42 CB PHE E 19 -2.318 -13.880 5.682 1.00
0.00 C ATOM 43 CG PHE E 19 -2.186 -13.850 4.177 1.00 0.00 C ATOM 44
CD1 PHE E 19 -2.922 -12.924 3.413 1.00 0.00 C ATOM 45 CD2 PHE E 19
-1.293 -14.712 3.515 1.00 0.00 C ATOM 46 CE1 PHE E 19 -2.769
-12.862 2.022 1.00 0.00 C ATOM 47 CE2 PHE E 19 -1.130 -14.644 2.126
1.00 0.00 C ATOM 48 CZ PHE E 19 -1.871 -13.721 1.379 1.00 0.00 C
ATOM 49 HN PHE E 19 -2.899 -14.472 8.145 1.00 0.00 H ATOM 50 HA PHE
E 19 -4.374 -14.519 5.636 1.00 0.00 H ATOM 51 HB2 PHE E 19 -2.509
-12.835 6.008 1.00 0.00 H ATOM 52 HB3 PHE E 19 -1.350 -14.202 6.121
1.00 0.00 H ATOM 53 HD1 PHE E 19 -3.604 -12.243 3.901 1.00 0.00 H
ATOM 54 HD2 PHE E 19 -0.719 -15.434 4.080 1.00 0.00 H ATOM 55 HE1
PHE E 19 -3.337 -12.145 1.447 1.00 0.00 H ATOM 56 HE2 PHE E 19
-0.430 -15.302 1.632 1.00 0.00 H ATOM 57 HZ PHE E 19 -1.746 -13.671
0.307 1.00 0.00 H ATOM 58 N PHE E 20 -4.188 -17.030 5.405 1.00 0.00
N ATOM 59 CA PHE E 20 -4.165 -18.482 5.235 1.00 0.00 C ATOM 60 C
PHE E 20 -3.313 -18.915 4.027 1.00 0.00 C ATOM 61 O PHE E 20 -3.832
-19.325 2.985 1.00 0.00 O ATOM 62 CB PHE E 20 -5.608 -19.088 5.165
1.00 0.00 C ATOM 63 CG PHE E 20 -6.384 -18.903 6.453 1.00 0.00 C
ATOM 64 CD1 PHE E 20 -7.240 -17.798 6.628 1.00 0.00 C ATOM 65 CD2
PHE E 20 -6.277 -19.845 7.494 1.00 0.00 C ATOM 66 CE1 PHE E 20
-7.970 -17.638 7.813 1.00 0.00 C ATOM 67 CE2 PHE E 20 -7.010
-19.689 8.678 1.00 0.00 C ATOM 68 CZ PHE E 20 -7.858 -18.586 8.836
1.00 0.00 C ATOM 69 HN PHE E 20 -5.048 -16.597 5.154 1.00 0.00 H
ATOM 70 HA PHE E 20 -3.690 -18.900 6.114 1.00 0.00 H ATOM 71 HB2
PHE E 20 -6.180 -18.626 4.331 1.00 0.00 H ATOM 72 HB3 PHE E 20
-5.547 -20.180 4.967 1.00 0.00 H ATOM 73 HD1 PHE E 20 -7.341
-17.060 5.846 1.00 0.00 H ATOM 74 HD2 PHE E 20 -5.629 -20.702 7.382
1.00 0.00 H ATOM 75 HE1 PHE E 20 -8.617 -16.782 7.935 1.00 0.00 H
ATOM 76 HE2 PHE E 20 -6.922 -20.421 9.468 1.00 0.00 H ATOM 77 HZ
PHE E 20 -8.422 -18.464 9.750 1.00 0.00 H ATOM 78 N ALA E 21 -1.962
-18.826 4.174 1.00 0.00 N ATOM 79 CA ALA E 21 -0.961 -19.299 3.227
1.00 0.00 C ATOM 80 C ALA E 21 0.265 -19.892 3.935 1.00 0.00 C ATOM
81 O ALA E 21 0.658 -19.456 5.022 1.00 0.00 O ATOM 82 CB ALA E 21
-0.506 -18.178 2.263 1.00 0.00 C ATOM 83 HN ALA E 21 -1.593 -18.392
4.999 1.00 0.00 H ATOM 84 HA ALA E 21 -1.420 -20.086 2.641 1.00
0.00 H ATOM 85 HB1 ALA E 21 0.004 -17.364 2.821 1.00 0.00 H ATOM 86
HB2 ALA E 21 0.203 -18.574 1.505 1.00 0.00 H ATOM 87 HB3 ALA E 21
-1.381 -17.749 1.731 1.00 0.00 H ATOM 88 N GLU E 22 0.889 -20.915
3.294 1.00 0.00 N ATOM 89 CA GLU E 22 2.127 -21.571 3.695 1.00 0.00
C ATOM 90 C GLU E 22 2.991 -21.853 2.456 1.00 0.00 C ATOM 91 O GLU
E 22 2.485 -22.046 1.343 1.00 0.00 O ATOM 92 CB GLU E 22 1.869
-22.866 4.533 1.00 0.00 C ATOM 93 CG GLU E 22 3.110 -23.651 5.058
1.00 0.00 C ATOM 94 CD GLU E 22 4.098 -22.807 5.870 1.00 0.00 C
ATOM 95 OE1 GLU E 22 4.107 -22.938 7.119 1.00 0.00 O1- ATOM 96 OE2
GLU E 22 4.857 -22.025 5.232 1.00 0.00 O ATOM 97 HN GLU E 22 0.553
-21.237 2.412 1.00 0.00 H ATOM 98 HA GLU E 22 2.673 -20.873 4.318
1.00 0.00 H ATOM 99 HB2 GLU E 22 1.263 -22.574 5.421 1.00 0.00 H
ATOM 100 HB3 GLU E 22 1.253 -23.567 3.929 1.00 0.00 H ATOM 101 HG2
GLU E 22 2.758 -24.477 5.711 1.00 0.00 H ATOM 102 HG3 GLU E 22
3.668 -24.105 4.214 1.00 0.00 H ATOM 103 N ASP E 23 4.330 -21.871
2.667 1.00 0.00 N ATOM 104 CA ASP E 23 5.370 -22.061 1.567 1.00
0.00 C ATOM 105 C ASP E 23 6.435 -22.990 2.332 1.00 0.00 C ATOM 106
O ASP E 23 6.210 -23.825 1.448 1.00 0.00 O ATOM 107 CB ASP E 23
6.040 -20.683 1.370 1.00 0.00 C ATOM 108 CG ASP E 23 6.797 -20.649
0.046 1.00 0.00 C ATOM 109 OD1 ASP E 23 7.643 -21.546 -0.204 1.00
0.00 O1- ATOM 110 OD2 ASP E 23 6.537 -19.695 -0.736 1.00 0.00 O
ATOM 111 HN ASP E 23 4.638 -21.817 3.645 1.00 0.00 H ATOM 112 HA
ASP E 23 4.879 -22.397 0.748 1.00 0.00 H ATOM 113 HB2 ASP E 23
5.239 -19.918 1.301 1.00 0.00 H ATOM 114 HB3 ASP E 23 6.728 -20.384
2.185 1.00 0.00 H ATOM 115 N VAL E 24 7.231 -24.037 2.574 1.00 0.00
N ATOM 116 CA VAL E 24 8.168 -24.125 3.703 1.00 0.00 C ATOM 117 C
VAL E 24 9.537 -24.715 3.298 1.00 0.00 C ATOM 118 O VAL E 24 10.575
-24.135 3.628 1.00 0.00 O ATOM 119 CB VAL E 24 7.544 -24.791 4.968
1.00 0.00 C ATOM 120 CG1 VAL E 24 8.427 -24.572 6.227 1.00 0.00 C
ATOM 121 CG2 VAL E 24 7.165 -26.285 4.774 1.00 0.00 C ATOM 122 HN
VAL E 24 7.136 -24.802 1.922 1.00 0.00 H ATOM 123 HA VAL E 24 8.382
-23.101 3.985 1.00 0.00 H ATOM 124 HB VAL E 24 6.587 -24.245 5.166
1.00 0.00 H ATOM 125 HG11 VAL E 24 7.907 -24.960 7.128 1.00 0.00 H
ATOM 126 HG12 VAL E 24 8.621 -23.490 6.387 1.00 0.00 H ATOM 127
HG13 VAL E 24 9.400 -25.099 6.131 1.00 0.00 H ATOM 128 HG21 VAL E
24 6.624 -26.658 5.670 1.00 0.00 H ATOM 129 HG22 VAL E 24 6.494
-26.404 3.898 1.00 0.00 H ATOM 130 HG23 VAL E 24 8.068 -26.914
4.628 1.00 0.00 H ATOM 131 N GLY E 25 9.571 -25.782 2.570 1.00 0.00
N ATOM 132 CA GLY E 25 10.728 -26.711 2.245 1.00 0.00 C ATOM 133 C
GLY E 25 11.732 -26.887 3.369 1.00 0.00 C ATOM 134 O GLY E 25
11.461 -27.541 4.381 1.00 0.00 O ATOM 135 HN GLY E 25 8.703 -26.152
2.117 1.00 0.00 H ATOM 136 HA2 GLY E 25 10.344 -27.695 2.018 1.00
0.00 H ATOM 137 HA3 GLY E 25 11.215 -26.256 1.394 1.00 0.00 H ATOM
138 N SER E 26 12.939 -26.294 3.198 1.00 0.00 N ATOM 139 CA SER E
26 13.972 -26.116 4.226 1.00 0.00 C ATOM 140 C SER E 26 14.983
-25.119 3.655 1.00 0.00 C ATOM 141 O SER E 26 16.115 -25.473 3.305
1.00 0.00 O ATOM 142 CB SER E 26 14.553 -27.454 4.655 1.00 0.00 C
ATOM 143 OG SER E 26 13.746 -28.270 5.393 1.00 0.00 O ATOM 144 HN
SER E 26 13.143 -25.862 2.316 1.00 0.00 H ATOM 145 HA SER E 26
13.512 -25.665 5.095 1.00 0.00 H ATOM 146 HB2 SER E 26 14.987
-28.007 3.751 1.00 0.00 H ATOM 147 HB3 SER E 26 15.537 -27.247
5.297 1.00 0.00 H ATOM 148 HG SER E 26 12.849 -28.037 5.051 1.00
0.00 H ATOM 149 N ASN E 27 14.542 -23.833 3.507 1.00 0.00 N ATOM
150 CA ASN E 27 15.010 -22.894 2.483 1.00 0.00 C ATOM 151 C ASN E
27 14.612 -23.372 1.059 1.00 0.00 C ATOM 152 O ASN E 27 14.216
-24.523 0.833 1.00 0.00 O ATOM 153 CB ASN E 27 16.529 -22.500 2.633
1.00 0.00 C ATOM 154 CH ASN E 27 16.990 -21.234 1.884 1.00 0.00 C
ATOM 155 OD1 ASN E 27 16.265 -20.531 1.173 1.00 0.00 O ATOM 156 ND2
ASN E 27 18.302 -20.924 2.051 1.00 0.00 N ATOM 157 HN ASN E 27
13.700 -23.565 3.970 1.00 0.00 H ATOM 158 HA ASN E 27 14.436
-21.994 2.667 1.00 0.00 H ATOM 159 HB2 ASN E 27 16.730 -22.330
3.713 1.00 0.00 H ATOM 160 HB3 ASN E 27 17.156 -23.355 2.302 1.00
0.00 H ATOM 161 HD21 ASN E 27 18.883 -21.499 2.621 1.00 0.00 H ATOM
162 HD22 ASN E 27 18.654 -20.116 1.585 1.00 0.00 H ATOM 163 N LYS E
28 14.695 -22.445 0.075 1.00 0.00 N ATOM 164 CA LYS E 28 14.381
-22.629 -1.341 1.00 0.00 C ATOM 165 C LYS E 28 15.351 -21.770
-2.154 1.00 0.00 C ATOM 166 O LYS E 28 16.222 -22.307 -2.839 1.00
0.00 O ATOM 167 CB LYS E 28 12.877 -22.355 -1.687 1.00 0.00 C ATOM
168 CG LYS E 28 11.905 -23.328 -0.984 1.00 0.00 C ATOM 169 CD LYS E
28 10.407 -23.125 -1.274 1.00 0.00 C ATOM 170 CE LYS E 28 9.565
-24.127 -0.470 1.00 0.00 C ATOM 171 N2 LYS E 28 8.121 -23.991
-0.712 1.00 0.00 N1+ ATOM 172 HN LYS E 28 15.075 -21.548 0.336 1.00
0.00 H ATOM 173 HA LYS E 28 14.600 -23.660 -1.591 1.00 0.00 H ATOM
174 HB2 LYS E 28 12.608 -21.320 -1.392 1.00 0.00 H ATOM 175 HB3 LYS
E 28 12.733 -22.451 -2.786 1.00 0.00 H ATOM 176 HG2 LYS E 28 12.188
-24.371 -1.257 1.00 0.00 H ATOM 177 HG3 LYS E 28 12.040 -23.226
0.119 1.00 0.00 H ATOM 178 HD2 LYS E 28 10.112 -22.090 -0.985 1.00
0.00 H ATOM 179 HD3 LYS E 28 10.218 -23.245 -2.362 1.00 0.00 H ATOM
180 HE2 LYS E 28 9.855 -25.173 -0.714 1.00 0.00 H ATOM 181 HE3 LYS
E 28 9.735 -23.955 0.615 1.00 0.00 H ATOM 182 HZ1 LYS E 28 7.642
-24.701 -0.075 1.00 0.00 H ATOM 183 HZ2 LYS E 28 7.824 -22.985
-0.460 1.00 0.00 H ATOM 184 HZ3 LYS E 28 7.862 -24.209 -1.698 1.00
0.00 H ATOM 185 N GLY E 29 15.238 -20.414 -2.069 1.00 0.00 N ATOM
186 C GLY E 29 16.221 -19.472 -2.602 1.00 0.00 C ATOM 187 C GLY E
29 16.329 -18.178 -1.820 1.00 0.00 C ATOM 188 O GLY E 29 16.342
-17.105 -2.425 1.00 0.00 O ATOM 189 HN GLY E 29 14.466 -20.019
-1.577 1.00 0.00 H ATOM 190 HA2 GLY E 29 17.197 -19.939 -2.578 1.00
0.00 H ATOM 191 HA3 GLY E 29 15.907 -19.224 -3.607 1.00 0.00 H ATOM
192 N ALA E 30 16.422 -18.252 -0.458 1.00 0.00 N ATOM 193 CA ALA E
30 16.545 -17.145 0.489 1.00 0.00 C ATOM 194 C ALA E 30 15.354
-16.174 0.492 1.00 0.00 C ATOM 195 O ALA E 30 14.532 -16.217 1.409
1.00 0.00 O ATOM 196 CB ALA E 30 17.924 -16.432 0.416 1.00 0.00 C
ATOM 197 HN ALA E 30 16.394 -19.157 -0.010 1.00 0.00 H ATOM 198 HA
ALA E 30 16.518 -17.620 1.461 1.00 0.00 H ATOM 199 HB1 ALA E 30
18.057 -15.934 -0.568 1.00 0.00 H ATOM 200 HB2 ALA E 30 18.013
-15.667 1.216 1.00 0.00 H ATOM 201 HB3 ALA E 30 18.745 -17.169
0.545 1.00 0.00 H ATOM 202 N ILE E 31 15.230 -15.285 -0.535 1.00
0.00 N ATOM 203 CA ILE E 31 14.218 -14.224 -0.629 1.00 0.00 C ATOM
204 C ILE E 31 12.989 -14.791 -1.375 1.00 0.00 C ATOM 205 O ILE E
31 12.672 -14.414 -2.507 1.00 0.00 O ATOM 206 CB ILE E 31 14.773
-12.883 -1.203 1.00 0.00 C ATOM 207 CG1 ILE E 31 16.143 -12.433
-0.582 1.00 0.00 C ATOM 208 CG2 ILE E 31 13.711 -11.745 -1.112 1.00
0.00 C ATOM 209 CD1 ILE E 31 16.172 -12.182 0.938 1.00 0.00 C ATOM
210 HN ILE E 31 15.848 -15.388 -1.317 1.00 0.00 H ATOM 211 HA ILE E
31 13.897 -14.006 0.382 1.00 0.00 H ATOM 212 HB ILE E 31 14.972
-13.051 -2.289 1.00 0.00 H ATOM 213 HG12 ILE E 31 16.911 -13.200
-0.825 1.00 0.00 H ATOM 214 HG13 ILE E 31 16.462 -11.498 -1.097
1.00 0.00 H ATOM 215 HG21 ILE E 31 13.430 -11.548 -0.056 1.00 0.00
H ATOM 216 HG22 ILE E 31 14.122 -10.808 -1.543 1.00 0.00 H ATOM 217
HG23 ILE E 31 12.794 -12.002 -1.681 1.00 0.00 H ATOM 218 HD11 ILE E
31 15.913 -13.106 1.499 1.00 0.00 H ATOM 219 HD12 ILE E 31 17.189
-11.863 1.253 1.00 0.00 H ATOM 220 HD13 ILE E 31 15.456 -11.383
1.222 1.00 0.00 H ATOM 221 N ILE E 32 12.276 -15.746 -0.715 1.00
0.00 N ATOM 222 CA ILE E 32 11.097 -16.464 -1.206 1.00 0.00 C ATOM
223 C ILE E 32 10.373 -17.088 -0.006 1.00 0.00 C ATOM 224 O ILE E
32 10.993 -17.721 0.856 1.00 0.00 O ATOM 225 CB ILE E 32 11.393
-17.479 -2.356 1.00 0.00 C ATOM 226 CG1 ILE E 32 10.127 -18.060
-3.076 1.00 0.00 C ATOM 227 CG2 ILE E 32 12.450 -18.553 -1.962 1.00
0.00 C ATOM 228 CD1 ILE E 32 9.433 -19.286 -2.452 1.00 0.00 C ATOM
229 HN ILE E 32 12.602 -16.048 -0.185 1.00 0.00 H ATOM 230 HA ILE E
32 10.449 -15.702 -1.620 1.00 0.00 H ATOM 231 HB ILE E 32 11.890
-15.860 -3.146 1.00 0.00 H ATOM 232 HG12 ILE E 32 9.378 -17.249
-3.211 1.00 0.00 H ATOM 233 HG13 ILE E 32 10.447 -18.361 -4.101
1.00 0.00 H ATOM 234 HG21 ILE E 32 13.389 -18.065 -1.627 1.00 0.00
H ATOM 235 HG22 ILE E 32 12.064 -19.187 -1.136 1.00 0.00 H ATOM 236
HG23 ILE E 32 12.676 -19.197 -2.837 1.00 0.00 H ATOM 237 HD11 ILE E
32 10.124 -20.154 -2.407 1.00 0.00 H ATOM 238 HD12 ILE E 32 9.064
-19.088 -1.426 1.00 0.00 H ATOM 239 HD13 ILE E 32 8.554 -19.585
-3.062 1.00 0.00 H ATOM 240 N GLY E 33 9.029 -16.901 0.075 1.00
0.00 N ATOM 241 CA GLY E 33 8.210 -17.456 1.144 1.00 0.00 C ATOM
242 C GLY E 33 6.829 -16.852 1.184 1.00 0.00 C ATOM 243 O GLY E 33
6.600 -15.880 1.908 1.00 0.00 O ATOM 244 HN GLY E 33 8.548 -16.391
0.632 1.00 0.00 H ATOM 245 HA2 GLY E 33 8.131 -18.520 0.977 1.00
0.00 H
ATOM 246 HA3 GLY E 33 8.691 -17.222 2.083 1.00 0.00 H ATOM 247 N
LEU E 34 5.864 -17.435 0.423 1.00 0.00 N ATOM 248 CA LEU E 34 4.456
-17.032 0.382 1.00 0.00 C ATOM 249 C LEU E 34 3.710 -17.618 1.602
1.00 0.00 C ATOM 250 O LEU E 34 2.849 -18.495 1.492 1.00 0.00 O
ATOM 251 CB LEU E 34 3.807 -17.395 -0.999 1.00 0.00 C ATOM 252 CG
LEU E 34 2.606 -16.518 -1.483 1.00 0.00 C ATOM 253 CD1 LEU E 34
2.248 -16.841 -2.954 1.00 0.00 C ATOM 254 CD2 LEU E 34 1.347
-16.601 -0.591 1.00 0.00 C ATOM 255 HN LEU E 34 6.101 -18.272
-0.128 1.00 0.00 H ATOM 256 HA LEU E 34 4.439 -15.953 0.474 1.00
0.00 H ATOM 257 HB2 LEU E 34 4.604 -17.288 -1.771 1.00 0.00 H ATOM
258 HB3 LEU E 34 3.517 -18.468 -1.000 1.00 0.00 H ATOM 259 HG LEU E
34 2.950 -15.455 -1.466 1.00 0.00 H ATOM 260 HD11 LEU E 34 1.898
-17.891 -3.047 1.00 0.00 H ATOM 261 HD12 LEU E 34 1.446 -16.167
-3.320 1.00 0.00 H ATOM 262 HD13 LEU E 34 3.136 -16.709 -3.503 1.00
0.00 H ATOM 263 HD21 LEU E 34 1.569 -16.255 0.440 1.00 0.00 H ATOM
264 HD22 LEU E 34 0.532 -15.965 -0.996 1.00 0.00 H ATOM 265 HD23
LEU E 34 0.984 -17.650 -0.534 1.00 0.00 H ATOM 266 N MEI E 35 4.075
-17.119 2.814 1.00 0.00 N ATOM 267 CA MEI E 35 3.624 -17.583 4.123
1.00 0.00 C ATOM 268 C MEI E 35 2.523 -16.670 4.714 1.00 0.00 C
ATOM 269 O MEI E 35 1.822 -15.947 3.995 1.00 0.00 O ATOM 270 CB MEI
E 35 4.861 -17.768 5.073 1.00 0.00 C ATOM 271 CG MEI E 35 5.942
-18.759 4.568 1.00 0.00 C ATOM 272 SD MEI E 35 7.383 -18.952 5.668
1.00 0.00 S ATOM 273 CE MEI E 35 8.170 -17.332 5.411 1.00 0.00 C
ATOM 274 HN MEI E 35 4.792 -16.420 2.823 1.00 0.00 H ATOM 275 HA
MEI E 35 3.166 -18.555 3.990 1.00 0.00 H ATOM 276 HB2 MEI E 35
5.330 -16.775 5.244 1.00 0.00 H ATOM 277 HB3 MEI E 35 4.519 -18.161
6.055 1.00 0.00 H ATOM 278 HG2 MEI E 35 5.456 -19.752 4.443 1.00
0.00 H ATOM 279 HG3 MEI E 35 6.290 -18.447 3.561 1.00 0.00 H ATOM
280 HE1 MEI E 35 8.373 -17.150 4.334 1.00 0.00 H ATOM 281 HE2 MEI E
35 7.526 -16.507 5.783 1.00 0.00 H ATOM 282 HE3 MEI E 35 9.138
-17.276 5.953 1.00 0.00 H ATOM 283 N VAL E 36 2.330 -16.695 6.067
1.00 0.00 N ATOM 284 CA VAL E 36 1.301 -15.972 6.822 1.00 0.00 C
ATOM 285 C VAL E 36 1.640 -14.462 6.866 1.00 0.00 C ATOM 286 O VAL
E 36 2.086 -13.905 7.875 1.00 0.00 O ATOM 287 C VAL E 36 1.004
-16.594 8.226 1.00 0.00 C ATOM 288 CG1 VAL E 36 -0.158 -15.864
8.962 1.00 0.00 C ATOM 289 CG2 VAL E 36 0.676 -18.113 8.129 1.00
0.00 C ATOM 290 HN VAL E 36 2.894 -17.297 6.623 1.00 0.00 H ATOM
291 HA VAL E 36 0.386 -16.0.74 6.251 1.00 0.00 H ATOM 292 HB VAL E
36 1.921 -16.486 8.850 1.00 0.00 H ATOM 293 HG11 VAL E 36 0.075
-14.792 9.128 1.00 0.00 H ATOM 294 HG12 VAL E 36 -1.101 -15.941
8.383 1.00 0.00 H ATOM 295 HG13 VAL E 36 -0.326 -16.326 9.958 1.00
0.00 H ATOM 296 HG21 VAL E 36 0.481 -18.535 9.137 1.00 0.00 H ATOM
297 HG22 VAL E 36 -0.224 -18.282 7.499 1.00 0.00 H ATOM 298 HG23
VAL E 36 1.517 -18.684 7.683 1.00 0.00 H ATOM 299 N GLY E 37 1.403
-13.749 5.730 1.00 0.00 N ATOM 300 CA GLY E 37 1.476 -12.292 5.611
1.00 0.00 C ATOM 301 C GLY E 37 0.283 -11.611 6.248 1.00 0.00 C
ATOM 302 O GLY E 37 -0.559 -11.032 5.560 1.00 0.00 O ATOM 303 HN
GLY E 37 1.196 -14.271 4.095 1.00 0.00 H ATOM 304 HA2 GLY E 37
2.377 -11.951 6.105 1.00 0.00 H ATOM 305 HA3 GLY E 37 1.465 -12.057
4.556 1.00 0.00 H ATOM 306 N GLY E 37 0.186 -11.673 7.601 1.00 0.00
H ATOM 307 CA GLY E 38 -0.986 -11.294 8.387 1.00 0.00 C ATOM 308 C
GLY E 38 -1.293 -19.816 8.541 1.00 0.00 C ATOM 309 O GLY E 38
-1.509 -9.350 9.663 1.00 0.00 O ATOM 310 HN GLY E 38 0.930 -12.143
8.086 1.00 0.00 H ATOM 311 HA2 GLY E 38 -1.847 -11.747 7.917 1.00
0.00 H ATOM 312 HA3 GLY E 38 -0.823 -11.687 9.380 1.00 0.00 H ATOM
313 N VAL E 39 -1.365 -9.070 7.407 1.00 0.00 N ATOM 314 CA VAL E 39
-1.581 -7.621 7.265 1.00 0.00 C ATOM 315 C VAL E 39 -2.828 -7.129
8.043 1.00 0.00 C ATOM 316 O VAL E 39 -3.865 -7.803 8.123 1.00 0.00
O ATOM 317 CB VAL E 39 -1.591 -7.158 5.770 1.00 0.00 C ATOM 318 CG1
VAL E 39 -1.813 -5.628 5.590 1.00 0.00 C ATOM 319 CG2 VAL E 39
-0.276 -7.557 5.037 1.00 0.00 C ATOM 320 HN VAL E 39 -0.196 -9.555
6.546 1.00 0.00 H ATOM 321 HA VAL E 39 -0.718 -7.166 7.733 1.00
0.00 H ATOM 322 HB VAL E 39 -2.433 -7.680 5.258 1.00 0.00 H ATOM
323 HG11 VAL E 39 -2.801 -5.311 5.981 1.00 0.00 H ATOM 324 HD12 VAL
E 39 -1.021 -5.044 6.104 1.00 0.00 H ATOM 325 HD13 VAL E 39 -1.791
-5.361 4.511 1.00 0.00 H ATOM 326 HG21 VAL E 39 -0.312 -7.229 3.976
1.00 0.00 H ATOM 327 HG22 VAL E 39 0.607 -7.082 5.516 1.00 0.00 H
ATOM 328 HG23 VAL E 39 -0.125 -8.655 5.035 1.00 0.00 H ATOM 329 N
VAL E 40 -2.722 -5.916 8.658 1.00 0.00 N ATOM 330 CA VAL E 40
-3.784 -5.198 9.369 1.00 0.00 C ATOM 331 C VAL E 40 -4.604 -4.416
8.325 1.00 0.00 C ATOM 332 O VAL E 40 -4.082 -3.559 7.606 1.00 0.00
O ATOM 333 CB VAL E 40 -3.280 -4.281 10.522 1.00 0.00 C ATOM 334
CG1 VAL E 40 -4.454 -3.576 11.262 1.00 0.00 C ATOM 335 CG2 VAL E 40
-2.414 -5.071 11.545 1.00 0.00 C ATOM 336 HN VAL E 40 -1.866 -5.410
8.597 1.00 0.00 H ATOM 337 HA VAL E 40 -4.422 -5.947 9.817 1.00
0.00 H ATOM 338 HB VAL E 40 -2.635 -3.486 10.078 1.00 0.00 H ATOM
339 HG11 VAL E 40 -4.060 -2.931 12.077 1.00 0.00 H ATOM 340 HG12
VAL E 40 -5.031 -2.922 10.575 1.00 0.00 H ATOM 341 HG13 VAL E 40
-5.148 -4.316 11.712 1.00 0.00 H ATOM 342 HG21 VAL E 40 -2.043
-4.389 12.340 1.00 0.00 H ATOM 343 HG22 VAL E 40 -3.003 -5.878
12.029 1.00 0.00 H ATOM 344 HG23 VAL E 40 -1.527 -5.526 11.056 1.00
0.00 H ATOM 345 N ILE E 41 -5.924 -4.729 8.236 1.00 0.00 N ATOM 346
CA ILE E 41 -6.893 -4.168 7.297 1.00 0.00 C ATOM 347 C ILE E 41
-7.889 -3.330 8.114 1.00 0.00 C ATOM 348 O ILE E 41 -8.504 -3.828
9.064 1.00 0.00 O ATOM 349 CB ILE E 41 -7.608 -5.248 6.430 1.00
0.00 C ATOM 350 CG1 ILE E 41 -6.621 -6.237 5.713 1.00 0.00 C ATOM
351 CG2 ILE E 41 -8.613 -4.614 5.420 1.00 0.00 C ATOM 352 CD1 ILE E
41 -5.635 -5.625 4.699 1.00 0.00 C ATOM 353 HN ILE E 41 -6.322
-5.394 8.860 1.00 0.00 H ATOM 354 HA ILE E 41 -6.357 -3.510 6.624
1.00 0.00 H ATOM 355 HB ILE E 41 -8.215 -5.878 7.125 1.00 0.00 H
ATOM 356 HG12 ILE E 41 -6.032 -6.778 6.487 1.00 0.00 H ATOM 357
HG13 ILE E 41 -7.228 -7.008 5.187 1.00 0.00 H ATOM 358 HG21 ILE E
41 -9.411 -4.048 5.944 1.00 0.00 H ATOM 359 HG22 ILE E 41 -8.092
-3.930 4.717 1.00 0.00 H ATOM 360 HG23 ILE E 41 -9.106 -5.412 4.824
1.00 0.00 H ATOM 361 HD11 ILE E 41 -4.985 -6.419 4.272 1.00 0.00 H
ATOM 362 HD12 ILE E 41 -6.178 -5.137 3.863 1.00 0.00 H ATOM 363
HD13 ILE E 41 -4.982 -4.870 5.185 1.00 0.00 H ATOM 364 N ALA E 42
-8.047 -2.029 7.739 1.00 0.00 N ATOM 365 CA ALA E 42 -8.966 -1.076
8.334 1.00 0.00 C ATOM 366 C ALA E 42 -10.273 -1.087 7.568 1.00
0.00 C ATOM 367 G ALA E 42 -10.303 -0.735 6.660 0.00 0.00 H ATOM
368 O ALA E 42 -11.305 -1.536 8.069 1.00 0.00 O ATOM 369 CB ALA E
42 -8.369 0.348 8.322 1.00 0.00 C ATOM 370 HN ALA E 42 -7.500
-1.661 6.994 1.00 0.00 H ATOM 371 HA ALA E 42 -9.170 -1.376 9.356
1.00 0.00 H ATOM 372 HB1 ALA E 42 -7.417 0.368 8.894 1.00 0.00 H
ATOM 373 HB2 ALA E 42 -8.162 0.694 7.287 1.00 0.00 H ATOM 374 HB3
ALA E 42 -9.068 1.058 8.801 1.00 0.00 H TER 375 ALA E 42 1.00 0.00
ATOM 376 N LEU E 17 -4.694 -36.140 7.746 1.00 0.00 N1+ ATOM 377 H1
LEU E 17 -4.458 -36.755 8.545 0.00 0.00 H ATOM 378 H2 LEU E 17
-5.616 -36.439 7.314 0.00 0.00 H ATOM 379 CA LEU E 17 -4.787
-34.713 8.198 1.00 0.00 C ATOM 380 C LEU E 17 -4.985 -33.740 7.028
1.00 0.00 C ATOM 381 O LEU E 17 -4.889 -34.132 5.863 1.00 0.00 O
ATOM 382 CB LEU E 17 -3.484 -34.357 8.997 1.00 0.00 C ATOM 383 CG
LEU E 17 -3.348 -34.891 10.461 1.00 0.00 C ATOM 384 CD1 LEU E 17
-3.351 -36.431 10.610 1.00 0.00 C ATOM 385 CD2 LEU E 17 -2.088
-34.291 11.132 1.00 0.00 C ATOM 386 H3 LEU E 17 -3.931 -36.221
7.013 1.00 0.00 H ATOM 387 HA LEU E 17 -5.656 -34.629 8.839 1.00
0.00 H ATOM 388 HB2 LEU E 17 -2.595 -34.674 8.409 1.00 0.00 H ATOM
389 HB3 LEU E 17 -3.420 -33.246 9.076 1.00 0.00 H ATOM 390 HG LEU E
17 -4.222 -34.505 11.040 1.00 0.00 H ATOM 391 HD11 LEU E 17 -3.145
-36.712 11.667 1.00 0.00 H ATOM 392 HD12 LEU E 17 -4.339 -36.863
10.355 1.00 0.00 H ATOM 393 HD13 LEU E 17 -2.563 -36.894 9.978 1.00
0.00 H ATOM 394 HD21 LEU E 17 -2.027 -34.598 12.198 1.00 0.00 H
ATOM 395 HD22 LEU E 17 -1.165 -34.631 10.616 1.00 0.00 H ATOM 396
HD23 LEU E 17 -2.117 -33.181 11.098 1.00 0.00 H ATOM 397 N VAL E 18
-5.250 -32.431 7.323 1.00 0.00 N ATOM 398 CA VAL E 18 -5.140
-31.322 6.362 1.00 0.00 C ATOM 399 C VAL E 18 -3.628 -31.029 6.265
1.00 0.00 C ATOM 400 O VAL E 18 -3.062 -30.266 7.053 1.00 0.00 O
ATOM 401 CB VAL E 18 -6.000 -30.072 6.707 1.00 0.00 C ATOM 402 CG1
VAL E 18 -5.834 -28.954 5.638 1.00 0.00 C ATOM 403 CG2 VAL E 18
-7.504 -30.432 6.853 1.00 0.00 C ATOM 404 HN VAL E 18 -5.442
-32.132 8.254 1.00 0.00 H ATOM 405 HA VAL E 18 -5.481 -31.687 5.400
1.00 0.00 H ATOM 406 HB VAL E 18 -5.647 -29.662 7.682 1.00 0.00 H
ATOM 407 HG11 VAL E 18 -6.453 -28.072 5.907 1.00 0.00 H ATOM 408
HG12 VAL E 18 -4.779 -28.612 5.576 1.00 0.00 H ATOM 409 HG13 VAL E
18 -6.153 -29.308 4.635 1.00 0.00 H ATOM 410 HG21 VAL E 18 -8.091
-29.534 7.135 1.00 0.00 H ATOM 411 HG22 VAL E 18 -7.914 -30.840
5.913 1.00 0.00 H ATOM 412 HG23 VAL E 18 -7.656 -31.191 7.662 1.00
0.00 H ATOM 413 N PHE E 19 -2.950 -31.706 5.300 1.00 0.00 N ATOM
414 CA PHE E 19 -1.507 -31.863 5.235 1.00 0.00 C ATOM 415 C PHE E
19 -0.908 -31.009 4.116 1.00 0.00 C ATOM 416 O PHE E 19 -1.194
-31.223 2.934 1.00 0.00 O ATOM 417 CB PHE E 19 -1.144 -33.374 5.046
1.00 0.00 C ATOM 418 CG PHE E 19 0.246 -33.730 5.530 1.00 0.00 C
ATOM 419 CD1 PHE E 19 0.416 -34.446 6.732 1.00 0.00 C ATOM 420 CD2
PHE E 19 1.389 -33.412 4.774 1.00 0.00 C ATOM 421 CE1 PHE E 19
1.690 -34.842 7.157 1.00 0.00 C ATOM 422 CE2 PHE E 19 2.665 -33.796
5.201 1.00 0.00 C ATOM 423 C2 PHE E 19 2.815 -34.517 6.390 1.00
0.00 C ATOM 424 HN PHE E 19 -3.455 -32.243 4.629 1.00 0.00 H ATOM
425 HA PHE E 19 -1.099 -31.539 6.186 1.00 0.00 H ATOM 426 HB2 PHE E
19 -1.860 -33.993 5.630 1.00 0.00 H ATOM 427 HB3 PHE E 19 -1.236
-33.677 3.981 1.00 0.00 H ATOM 428 HD1 PHE E 19 -0.444 -34.712
7.329 1.00 0.00 H ATOM 429 HD2 PHE E 19 1.294 -32.865 3.849 1.00
0.00 H ATOM 430 HE1 PHE E 19 1.808 -35.396 8.078 1.00 0.00 H ATOM
431 HE2 PHE E 19 3.534 -33.535 4.610 1.00 0.00 H ATOM 432 H2 PHE E
19 3.799 -34.822 6.714 1.00 0.00 H ATOM 433 N PHE E 20 -0.039
-30.030 4.488 1.00 0.00 N ATOM 434 CA PHE E 20 0.810 -29.269 3.578
1.00 0.00 C ATOM 435 C PHE E 20 2.023 -30.138 3.216 1.00 0.00 C
ATOM 436 O PHE E 20 2.811 -30.498 4.094 1.00 0.00 O ATOM 437 CB PHE
E 20 1.247 -27.904 4.195 1.00 0.00 C ATOM 438 CG PHE E 20 1.908
-27.003 3.173 1.00 0.00 C ATOM 439 CD1 PHE E 20 1.129 -26.172 2.345
1.00 0.00 C ATOM 440 CD2 PHE E 20 3.310 -26.966 3.043 1.00 0.00 C
ATOM 441 CE1 PHE E 20 1.735 -25.324 1.410 1.00 0.00 C ATOM 442 CE2
PHE E 20 3.916 -26.119 2.106 1.00 0.00 C ATOM 443 C2 PHE E 20 3.129
-25.300 1.291 1.00 0.00 C ATOM 444 HN PHE E 20 0.114 -29.837 5.453
1.00 0.00 H ATOM 445 HA PHE E 20 0.227 -29.072 2.686 1.00 0.00 H
ATOM 446 HB2 PHE E 20 0.355 -27.366 4.583 1.00 0.00 H ATOM 447 HB3
PHE E 20 1.948 -28.061 5.043 1.00 0.00 H ATOM 448 HD1 PHE E 20
0.052 -26.176 2.434 1.00 0.00 H ATOM 449 HD2 PHE E 20 3.929 -27.588
3.674 1.00 0.00 H ATOM 450 HE1 PHE E 20 1.134 -24.674 0.791 1.00
0.00 H ATOM 451 HE2 PHE E 20 4.991 -26.076 2.016 1.00 0.00 H ATOM
452 H2 PHE E 20 3.601 -24.631 0.585 1.00 0.00 H ATOM 453 N ALA E 21
2.164 -30.498 1.906 1.00 0.00 N ATOM 454 CA ALA E 21 3.079 -31.491
1.341 1.00 0.00 C ATOM 455 C ALA E 21 4.497 -31.514 1.936 1.00 0.00
C ATOM 456 O ALA E 21 4.883 -32.512 2.545 1.00 0.00 O ATOM 457 CB
ALA E 21 3.099 -31.403 -0.206 1.00 0.00 C ATOM 458 HN ALA E 21
1.535 -30.112 1.238 1.00 0.00 H ATOM 459 HA ALA E 21 2.642 -32.445
1.597 1.00 0.00 H ATOM 460 HB1 ALA E 21 3.737 -32.206 -0.634 1.00
0.00 H ATOM 461 HB2 ALA E 21 2.074 -31.531 -0.614 1.00 0.00 H ATOM
462 HB3 ALA E 21 3.492 -30.422 -0.548 1.00 0.00 H ATOM 463 N GLU E
22 5.263 -30.400 1.771 1.00 0.00 N ATOM 464 CA GLU E 22 6.475
-30.018 2.485 1.00 0.00 C ATOM 465 C GLU E 22 7.570 -31.077 2.664
1.00 0.00 C ATOM 466 O GLU E 22 7.654 -31.678 3.738 1.00 0.00 O
ATOM 467 CB GLU E 22 7.018 -28.644 1.978 1.00 0.00 C ATOM 468 CG
GLU E 22 7.380 -28.531 0.468 1.00 0.00 C ATOM 469 CD GLU E 22 7.607
-27.099 -0.012 1.00 0.00 C ATOM 470 OE1 GLU E 22 7.580 -26.133
0.797 1.00 0.00 O1- ATOM 471 OE2 GLU E 22 7.823 -26.935 -1.242 1.00
0.00 O ATOM 472 HN GLU E 22 4.917 -29.682 1.178 1.00 0.00 H ATOM
473 HA GLU E 22 6.131 -29.822 3.493 1.00 0.00 H ATOM 474 HB2 GLU E
22 6.226 -27.892 2.186 1.00 0.00 H ATOM 475 HB3 GLU E 22 7.902
-28.351 2.586 1.00 0.00 H ATOM 476 HG2 GLU E 22 8.310 -29.089 0.241
1.00 0.00 H ATOM 477 HG3 GLU E 22 6.560 -28.945 -0.157 1.00 0.00 H
ATOM 478 N ASP E 23 8.453 -31.311 1.649 1.00 0.00 N ATOM 479 CA ASP
E 23 9.659 -32.116 1.788 1.00 0.00 C ATOM 480 C ASP E 23 10.698
-31.346 2.631 1.00 0.00 C ATOM 481 O ASP E 23 11.428 -30.480 2.139
1.00 0.00 O ATOM 482 CB ASP E 23 10.222 -32.625 0.425 1.00 0.00 C
ATOM 483 CG ASP E 23 11.244 -33.725 0.658 1.00 0.00 C ATOM 484 OD1
ASP E 23 10.844 -34.910 0.826 1.00 0.00 O1- ATOM 485 OD2 ASP E 23
12.465 -33.425 0.700 1.00 0.00 O ATOM 486 HN ASP E 23 8.323 -30.871
0.766 1.00 0.00 H ATOM 487 HA ASP E 23 9.361 -32.997 2.342 1.00
0.00 H ATOM 488 HB2 ASP E 23 9.402 -33.049 -0.193 1.00 0.00 H ATOM
489 HB3 ASP E 23 10.701 -31.795 -0.134 1.00 0.00 H ATOM 490 N VAL E
24 10.738 -31.665 3.954 1.00 0.00 N ATOM 491 CA VAL E 24 11.639
-31.109 4.967 1.00 0.00 C ATOM 492 C VAL E 24 13.004 -31.808 4.869
1.00 0.00 C ATOM 493 O VAL E 24 13.086 -33.050 4.779 1.00 0.00 O
ATOM 494 CB VAL E 24 11.046 -31.152 6.410 1.00 0.00 C ATOM 495 CG1
VAL E 24 12.015 -30.571 7.480 1.00 0.00 C ATOM 496 CG2 VAL E 24
9.688 -30.395 6.481 1.00 0.00 C
ATOM 497 HN VAL E 24 10.056 -32.300 4.304 1.00 0.00 H ATOM 498 HA
VAL E 24 11.782 -30.069 4.705 1.00 0.00 H ATOM 499 HB VAL E 24
10.854 -32.218 6.675 1.00 0.00 H ATOM 500 HG11 VAL E 24 11.547
-30.618 8.486 1.00 0.00 H ATOM 501 HG12 VAL E 24 12.961 -31.149
7.527 1.00 0.00 H ATOM 502 HG13 VAL E 24 12.257 -29.509 7.262 1.00
0.00 H ATOM 503 HG21 VAL E 24 9.816 -29.327 6.201 1.00 0.00 H ATOM
504 HG22 VAL E 24 8.938 -30.846 5.799 1.00 0.00 H ATOM 505 HG23 VAL
E 24 9.272 -3.439 7.510 1.00 0.00 H ATOM 506 N GLY E 25 14.111
-31.024 4.883 1.00 0.00 H ATOM 507 CA GLY E 25 15.491 -31.477 4.729
1.00 0.00 C ATOM 508 C GLY E 25 16.017 -31.220 3.332 1.00 0.00 C
ATOM 509 O GLY E 25 15.815 -32.033 2.430 1.00 0.00 O ATOM 510 HN
GLY E 25 13.991 -30.028 5.022 1.00 0.00 H ATOM 511 HA2 GLY E 25
16.089 -30.927 5.442 1.00 0.00 H ATOM 512 HA3 GLY E 25 15.532
-32.543 4.902 1.00 0.00 H ATOM 513 N SER E 26 16.721 -30.075 3.137
1.00 0.00 N ATOM 514 CA SER E 26 17.327 -29.655 1.868 1.00 0.00 C
ATOM 515 C SER E 26 18.644 -30.379 1.577 1.00 0.00 C ATOM 516 O SER
E 26 19.451 -30.630 2.474 1.00 0.00 O ATOM 517 CB SER E 26 17.571
-28.122 1.768 1.00 0.00 C ATOM 518 OG SER E 26 16.328 -27.439 1.628
1.00 0.00 O ATOM 519 HN SER E 26 16.861 -29.447 3.893 1.00 0.00 H
ATOM 520 HA SER E 26 16.623 -29.917 1.088 1.00 0.00 H ATOM 521 HB2
SER E 26 18.115 -27.758 2.668 1.00 0.00 H ATOM 522 HB3 SER E 26
18.180 -27.881 0.869 1.00 0.00 H ATOM 523 HG SER E 26 16.363
-26.617 2.166 1.00 0.00 H ATOM 524 N ASN E 27 18.871 -30.711 0.274
1.00 0.00 N ATOM 525 CA ASN E 27 20.009 -31.447 -0.279 1.00 0.00 C
ATOM 526 C ASN E 27 20.023 -32.925 0.159 1.00 0.00 C ATOM 527 O ASN
E 27 20.841 -33.343 0.984 1.00 0.00 O ATOM 528 CB ASN E 27 21.374
-30.702 -0.060 1.00 0.00 C ATOM 529 CG ASN E 27 22.516 -31.239
-0.934 1.00 0.00 C ATOM 530 OD1 ASN E 27 22.885 -30.637 -1.944 1.00
0.00 O ATOM 531 ND2 ASN E 27 23.095 -32.400 -0.539 1.00 0.00 N ATOM
532 HN ASN E 27 18.204 -30.396 -0.412 1.00 0.00 H ATOM 533 HA ASN E
27 19.842 -31.445 -1.348 1.00 0.00 H ATOM 534 HB2 ASN E 27 21.243
-29.629 -0.315 1.00 0.00 H ATOM 535 HB3 ASN E 27 21.668 -30.750
1.008 1.00 0.00 H ATOM 536 HD21 ASN E 27 22.675 -32.914 0.214 1.00
0.00 H ATOM 537 HD22 ASN E 27 23.857 -32.7500 -1.077 1.00 0.00 H
ATOM 538 N LYS E 28 19.102 -33.739 -0.428 1.00 0.00 N ATOM 539 CA
LYS E 28 18.955 -35.175 -0.209 1.00 0.00 C ATOM 540 C LYS E 28
19.695 -35.968 -1.294 1.00 0.00 C ATOM 541 O LYS E 28 20.870
-36.290 -1.118 1.00 0.00 O ATOM 542 CB LYS E 28 17.450 -35.571
-0.050 1.00 0.00 C ATOM 543 CG LYS E 28 16.846 -35.172 1.314 1.00
0.00 C ATOM 544 CD LYS E 28 15.304 -35.182 1.346 1.00 0.00 C ATOM
545 CE LYS E 28 14.738 -35.040 2.763 1.00 0.00 C ATOM 546 N2 LYS E
28 13.306 -34.710 2.730 1.00 0.00 N1+ ATOM 547 HN LYS E 28 18.436
-33.340 -1.050 1.00 0.00 H ATOM 548 HA LYS E 28 19.452 -35.425
0.721 1.00 0.00 H ATOM 549 HB2 LYS E 28 16.859 -35.104 -0.868 1.00
0.00 H ATOM 550 HB3 LYS E 28 17.340 -36.675 -0.145 1.00 0.00 H ATOM
551 HG2 LYS E 28 17.245 -35.869 2.086 1.00 0.00 H ATOM 552 HG3 LYS
E 28 17.179 -34.143 1.587 1.00 0.00 H ATOM 553 HD2 LYS E 28 14.952
-34.334 0.713 1.00 0.00 H ATOM 554 HD3 LYS E 28 14.918 -36.122
0.900 1.00 0.00 H ATOM 555 HE2 LYS E 28 14.783 -35.978 3.343 1.00
0.00 H ATOM 556 HE3 LYS E 28 15.255 -34.220 3.305 1.00 0.00 H ATOM
557 H21 LYS E 28 12.695 -35.544 2.088 1.00 0.00 H ATOM 558 H22 LYS
E 28 13.082 -34.111 3.574 1.00 0.00 H ATOM 559 H23 LYS E 28 13.075
-34.124 1.855 1.00 0.00 H ATOM 560 N GLY E 29 19.027 -36.300 -2.437
1.00 0.00 N ATOM 561 CA GLY E 29 19.565 -37.146 -3.501 1.00 0.00 C
ATOM 562 C GLY E 29 18.480 -38.474 -3.542 1.00 0.00 C ATOM 563 O
GLY E 29 18.975 -39.289 -2.625 1.00 0.00 O ATOM 564 HN GLY E 29
18.087 -35.992 -2.549 1.00 0.00 H ATOM 565 HA2 GLY E 29 19.426
-36.618 -4.434 1.00 0.00 H ATOM 566 HA3 GLY E 29 20.606 -37.358
-3.299 1.00 0.00 H ATOM 567 N ALA E 30 18.044 -38.713 -4.623 1.00
0.00 N ATOM 568 CA ALA E 30 17.195 -39.887 -4.853 1.00 0.00 C ATOM
569 C ALA E 30 16.033 -40.028 -3.855 1.00 0.00 C ATOM 570 O ALA E
30 14.870 -10.004 -4.264 1.00 0.00 O ATOM 571 CB ALA E 30 17.996
-41.209 -5.016 1.00 0.00 C ATOM 572 HN ALA E 30 18.002 -38.032
-5.347 1.00 0.00 H ATOM 573 HA ALA E 30 15.728 -39.708 -5.812 1.00
0.00 H ATOM 574 HB1 ALA E 30 18.515 -41.475 -4.071 1.00 0.00 H ATOM
575 HB2 ALA E 30 17.320 -42.045 -5.296 1.00 0.00 H ATOM 576 HB3 ALA
E 30 18.761 -41.098 -5.813 1.00 0.00 H ATOM 577 N ILE E 31 16.329
-40.162 -2.532 1.00 0.00 N ATOM 578 CA ILE E 31 15.363 -40.317
-1.441 1.00 0.00 C ATOM 579 C ILE E 31 14.825 -38.916 -1.073 1.00
0.00 C ATOM 580 O ILE E 31 15.252 -38.282 -0.104 1.00 0.00 O ATOM
581 CB ILE E 31 15.912 -41.130 -0.230 1.00 0.00 C ATOM 582 CG1 ILE
E 31 16.630 -42.468 -0.626 1.00 0.00 C ATOM 583 CG2 ILE E 31 14.803
-41.375 0.839 1.00 0.00 C ATOM 584 CD1 ILE E 31 15.789 -43.516
-1.380 1.00 0.00 C ATOM 585 HN ILE E 31 17.299 -40.107 -2.271 1.00
0.00 H ATOM 586 HA ILE E 31 14.533 -40.888 -1.840 1.00 0.00 H ATOM
587 HB ILE E 31 16.694 -40.500 0.259 1.00 0.00 H ATOM 588 HG12 ILE
E 31 17.520 -42.222 -1.248 1.00 0.00 H ATOM 589 HG13 ILE E 31
17.021 -42.937 0.304 1.00 0.00 H ATOM 590 HG21 ILE E 31 13.963
-41.963 0.413 1.00 0.00 H ATOM 591 HG22 ILE E 31 15.222 -41.940
1.698 1.00 0.00 H ATOM 592 HG23 ILE E 31 14.402 -40.417 1.230 1.00
0.00 H ATOM 593 HD11 ILE E 31 15.426 -43.112 -2.349 1.00 0.00 H
ATOM 594 HD12 ILE E 31 16.402 -44.418 -1.591 1.00 0.00 H ATOM 595
HD13 ILE E 31 14.911 -43.829 -0.776 1.00 0.00 H ATOM 596 N ILE E 32
13.866 -38.413 -1.894 1.00 0.00 N ATOM 597 CA ILE E 32 13.175
-37.125 -1.794 1.00 0.00 C ATOM 598 C ILE E 32 11.673 -37.376
-2.006 1.00 0.00 C ATOM 599 O ILE E 32 11.276 -38.069 -2.950 1.00
0.00 O ATOM 600 CB ILE E 32 13.798 -36.045 -2.733 1.00 0.00 C ATOM
601 CG1 ILE E 32 13.397 -34.568 -2.405 1.00 0.00 C ATOM 602 CG2 ILE
E 32 13.703 -36.399 -4.249 1.00 0.00 C ATOM 603 CD1 ILE E 32 12.021
-34.061 -2.875 1.00 0.00 C ATOM 604 HN ILE E 32 13.600 -38.950
-2.697 1.00 0.00 H ATOM 605 HA ILE E 32 13.305 -36.780 -0.776 1.00
0.00 H ATOM 606 HB ILE E 32 14.892 -36.078 -2.497 1.00 0.00 H ATOM
607 HG12 ILE E 32 13.485 -34.405 -1.308 1.00 0.00 H ATOM 608 HG13
ILE E 32 14.163 -33.909 -2.875 1.00 0.00 H ATOM 609 HG21 ILE E 32
14.149 -37.394 -4.455 1.00 0.00 H ATOM 610 HG22 ILE E 32 12.648
-36.415 -4.583 1.00 0.00 H ATOM 611 HG23 ILE E 32 14.247 -35.643
-4.853 1.00 0.00 H ATOM 612 HD11 ILE E 32 11.908 -34.171 -3.974
1.00 0.00 H ATOM 613 HD12 ILE E 32 11.192 -34.601 -2.378 1.00 0.00
H ATOM 614 HD13 ILE E 32 11.913 -32.983 -2.623 1.00 0.00 H ATOM 615
N GLY E 33 10.798 -36.830 -1.114 1.00 0.00 N ATOM 616 CA GLY E 33
9.354 -37.053 -1.143 1.00 0.00 C ATOM 617 C GLY E 33 8.801 -37.518
0.184 1.00 0.00 C ATOM 618 O GLY E 33 8.339 -38.654 0.302 1.00 0.00
O ATOM 619 HN GLY E 33 11.092 -36.185 -0.384 1.00 0.00 H ATOM 620
HA2 GLY E 33 8.892 -36.106 -1.383 1.00 0.00 H ATOM 621 HA3 GLY E 33
9.122 -37.819 -1.871 1.00 0.00 H ATOM 622 N LEU E 34 8.823 -36.622
1.207 1.00 0.00 N ATOM 623 CA LEU E 34 8.145 -36.731 2.499 1.00
0.00 C ATOM 624 C LEU E 34 6.660 -37.126 2.362 1.00 0.00 C ATOM 625
O LEU E 34 5.918 -36.579 1.541 1.00 0.00 O ATOM 626 CB LEU E 34
8.344 -34.415 3.327 1.00 0.00 C ATOM 627 CG LEU E 34 7.941 -35.418
4.838 1.00 0.00 C ATOM 628 CD1 LEU E 34 8.825 -34.451 5.664 1.00
0.00 C ATOM 629 CD2 LEU E 34 6.448 -35.085 5.072 1.00 0.00 C ATOM
630 HN LEU E 34 9.416 -35.803 1.082 1.00 0.00 H ATOM 631 HA LEU E
34 8.659 -37.529 3.017 1.00 0.00 H ATOM 632 HB2 LEU E 34 9.434
-35.189 3.272 1.00 0.00 H ATOM 633 HB3 LEU E 34 7.822 -34.577 2.813
1.00 0.00 H ATOM 634 HG LEU E 34 8.133 -36.439 5.243 1.00 0.00 H
ATOM 635 HD11 LEU E 34 8.647 -33.401 5.351 1.00 0.00 H ATOM 636
HD12 LEU E 34 8.585 -34.534 6.746 1.00 0.00 H ATOM 637 HD13 LEU E
34 9.901 -34.588 5.530 1.00 0.00 H ATOM 638 HD21 LEU E 34 5.779
-35.811 4.569 1.00 0.00 H ATOM 639 HD22 LEU E 34 6.209 -35.100
6.156 1.00 0.00 H ATOM 640 HD23 LEU E 34 6.211 -34.073 4.675 1.00
0.00 H ATOM 641 N MET E 35 6.217 -38.123 3.183 1.00 0.00 N ATOM 642
CA MET E 35 4.910 -38.770 3.124 1.00 0.00 C ATOM 643 C MET E 35
3.765 -37.812 3.481 1.00 0.00 C ATOM 644 O MET E 35 3.585 -37.420
4.638 1.00 0.00 O ATOM 645 C MET E 35 4.833 -40.051 4.011 1.00 0.00
C ATOM 646 CG MET E 35 5.798 -41.192 3.604 1.00 0.00 C ATOM 647 SD
MET E 35 5.642 -42.707 4.610 1.00 0.00 S ATOM 648 CE MET E 35 6.391
-42.115 6.158 1.00 0.00 C ATOM 649 HN MET E 35 6.827 -38.499 3.871
1.00 0.00 H ATOM 650 HA MET E 35 4.779 -39.088 2.097 1.00 0.00 H
ATOM 651 HB2 MET E 35 5.024 -39.765 5.068 1.00 0.00 H ATOM 652 HB3
MET E 35 3.798 -40.460 3.961 1.00 0.00 H ATOM 653 HG2 MET E 35
5.593 -41.452 2.541 1.00 0.00 H ATOM 654 HG3 MET E 35 6.847 -40.826
3.644 1.00 0.00 H ATOM 655 HE1 MET E 35 7.428 -41.752 5.989 1.00
0.00 H ATOM 656 HE2 MET E 35 5.802 -41.284 6.601 1.00 0.00 H ATOM
657 HE3 MET E 35 6.437 -42.932 6.909 1.00 0.00 H ATOM 658 N MET E
35 2.966 -37.423 2.450 1.00 0.00 N ATOM 659 CA MET E 35 1.879
-36.446 2.512 1.00 0.00 C ATOM 660 C VAL E 36 0.537 -37.124 3.120
1.00 0.00 C ATOM 661 O VAL E 36 -0.166 -37.757 2.424 1.00 0.00 O
ATOM 662 CB VAL E 36 1.582 -35.759 1.142 1.00 0.00 C ATOM 663 CG1
VAL E 36 0.490 -34.657 1.256 1.00 0.00 C ATOM 664 CG2 VAL E 36
2.873 -35.169 0.501 1.00 0.00 C ATOM 665 HN VAL E 36 3.162 -37.755
1.533 1.00 0.00 H ATOM 666 HA VAL E 36 2.204 -35.668 3.190 1.00
0.00 H ATOM 667 HB VAL E 36 1.193 -36.535 0.443 1.00 0.00 H ATOM
668 HG11 VAL E 36 -0.456 -35.076 1.643 1.00 0.00 H ATOM 669 HG12
VAL E 36 0.814 -33.845 1.946 1.00 0.00 H ATOM 670 HG13 VAL E 36
0.286 -34.209 0.270 1.00 0.00 H ATOM 671 HG21 VAL E 36 2.633
-34.629 -0.439 1.00 0.00 H ATOM 672 HG22 VAL E 36 3.379 -34.468
1.197 1.00 0.00 H ATOM 673 HG23 VAL E 36 3.597 -35.972 0.250 1.00
0.00 H ATOM 674 N GLY E 37 0.443 -36.995 4.463 1.00 0.00 N ATOM 675
CA GLY E 37 -0.684 -37.578 5.203 1.00 0.00 C ATOM 676 C GLY E 37
-2.018 -36.872 5.009 1.00 0.00 C ATOM 677 O GLY E 37 -2.704 -36.541
5.983 1.00 0.00 O ATOM 678 HN GLY E 37 1.129 -36.501 5.000 1.00
0.00 H ATOM 679 HA2 GLY E 37 -0.802 -38.602 4.875 1.00 0.00 H ATOM
680 HA3 GLY E 37 -0.432 -37.512 6.252 1.00 0.00 H ATOM 681 N GLY E
38 -2.423 -36.650 3.731 1.00 0.00 N ATOM 682 CA GLY E 38 -3.565
-35.839 3.306 1.00 0.00 C ATOM 683 C GLY E 38 -4.940 -36477 3.363
1.00 0.00 C ATOM 684 O GLY E 38 -5.800 -36.135 2.551 1.00 0.00 O
ATOM 685 HN GLY E 38 -1.826 -37.022 3.007 1.00 0.00 H ATOM 686 HA2
GLY E 38 -3.596 -34.961 3.934 1.00 0.00 H ATOM 687 HA3 GLY E 38
-3.385 -35.582 2.272 1.00 0.00 H ATOM 688 N VAL E 39 -5.190 -37.396
4.335 1.00 0.00 H ATOM 689 CA VAL E 39 -6.488 -38.012 4.618 1.00
0.00 C ATOM 690 C VAL E 39 -7.270 -37.008 5.483 1.00 0.00 C ATOM
691 O VAL E 39 -7.001 -36.850 6.682 1.00 0.00 O ATOM 692 CB VAL E
39 -6.406 -39.429 5.261 1.00 0.00 C ATOM 693 CG1 VAL E 39 -7.819
-40.027 5.516 1.00 0.00 C ATOM 694 CG2 VAL E 39 -5.568 -40.403
4.385 1.00 0.00 C ATOM 695 HN VAL E 39 -4.429 -37.670 4.917 1.00
0.00 H ATOM 696 HA VAL E 39 -7.000 -38.124 3.670 1.00 0.00 H ATOM
697 HB VAL E 39 -5.895 -39.340 6.247 1.00 0.00 H ATOM 698 HG11 VAL
E 39 -8.406 -39.393 6.213 1.00 0.00 H ATOM 699 HG12 VAL E 39 -8.388
-40.127 4.566 1.00 0.00 H ATOM 700 HG13 VAL E 39 -7.733 -41.035
5.974 1.00 0.00 H ATOM 701 HG21 VAL E 39 -5.510 -41.402 4.867 1.00
0.00 H ATOM 702 HG22 VAL E 39 -6.027 -40.529 3.382 1.00 0.00 H ATOM
703 HG23 VAL E 39 -4.530 -40.034 4.251 1.00 0.00 H ATOM 704 N VAL E
40 -8.241 -36.292 4.858 1.00 0.00 N ATOM 705 CA VAL E 40 -9.103
-35.281 5.475 1.00 0.00 C ATOM 706 C VAL E 40 -10.416 -35.968 5.885
1.00 0.00 C ATOM 707 O VAL E 40 -11.125 -36.545 5.054 1.00 0.00 O
ATOM 708 CB VAL E 40 -9.351 -34.024 4.589 1.00 0.00 C ATOM 709 CG1
VAL E 40 -10.227 -32.964 5.318 1.00 0.00 C ATOM 710 CG2 VAL E 40
-8.011 -33.379 4.132 1.00 0.00 C ATOM 711 HN VAL E 40 -8.399
-36.424 3.882 1.00 0.00 H ATOM 712 HA VAL E 40 -8.501 -34.939
-6.371 1.00 0.00 H ATOM 713 HB VAL E 40 -9.899 -34.345 3.673 1.00
0.00 H ATOM 714 HG11 VAL E 40 -10.381 -32.079 4.663 1.00 0.00 H
ATOM 715 HG12 VAL E 40 -11.230 -33.369 5.567 1.00 0.00 H ATOM 716
HG13 VAL E 40 -9.743 -32.619 6.256 1.00 0.00 H ATOM 717 HG21 VAL E
40 -8.207 -32.485 3.502 1.00 0.00 H ATOM 718 HG22 VAL E 40 -7.403
-33.066 5.006 1.00 0.00 H ATOM 719 HG23 VAL E 40 -7.410 -34.091
3.527 1.00 0.00 H ATOM 720 N ILE E 41 -10.751 -35.908 7.203 1.00
0.00 N ATOM 721 CA ILE E 41 -11.966 -36.466 7.800 1.00 0.00 C ATOM
722 C ILE E 41 -13.051 -35.375 7.786 1.00 0.00 C ATOM 723 O ILE E
41 -12.874 -34.293 8.357 1.00 0.00 O ATOM 724 CB ILE E 41 -11.750
-37.057 9.220 1.00 0.00 C ATOM 725 CG1 ILE E 41 -10.532 -38.054
9.311 1.00 0.00 C ATOM 726 CG2 ILE E 41 -13.054 -37.722 9.772 1.00
0.00 C ATOM 727 CD1 ILE E 41 -10.581 -39.309 8.417 1.00 0.00 C ATOM
728 HN ILE E 41 -10.162 -35.435 7.484 1.00 0.00 H ATOM 729 HA ILE E
41 -12.287 -37.278 7.159 1.00 0.00 H ATOM 730 HB ILE E 41 -11.507
-36.219 9.904 1.00 0.00 H ATOM 731 HG12 ILE E 41 -9.601 -37.494
9.073 1.00 0.00 H ATOM 732 HG13 ILE E 41 -10.437 -38.384 10.370
1.00 0.00 H ATOM 733 HG21 ILE E 41 -13.880 -36.983 9.847 1.00 0.00
H ATOM 734 HG22 ILE E 41 -13.387 -38.557 9.121 1.00 0.00 H ATOM 735
HG23 ILE E 41 -12.874 -38.127 10.790 1.00 0.00 H ATOM 736 HD11 ILE
E 41 -9.674 -39.929 8.578 1.00 0.00 H ATOM 737 HD12 ILE E 41
-11.470 -39.931 8.656 1.00 0.00 H ATOM 738 HD13 ILE E 41 -10.623
-39.030 7.343 1.00 0.00 H ATOM 739 N ALA E 42 -14.200 -35.665 7.117
1.00 0.00 N ATOM 740 CA ALA E 42 -15.376 -34.818 7.035 1.00 0.00 C
ATOM 741 C ALA E 42 -16.315 -35.048 8.212 1.00 0.00 C ATOM 742 H
ALA E 42 -16.713 -34.272 8.647 1.00 0.00 H ATOM 743 O ALA E 42
-16.591 -35.175 8.635 1.00 0.00 O ATOM 744 CB ALA E 42 -16.139
-35.030 5.708 1.00 0.00 C ATOM 745 HN ALA E 42 -14.299 -36.539
6.652 1.00 0.00 H ATOM 746 HA ALA E 42 -15.047 -33.788 7.079 1.00
0.00 H ATOM 747 HB1 ALA E 42 -15.467 -34.837 4.844 1.00 0.00 H
ATOM 748 HB2 ALA E 42 -16.519 -36.071 5.631 1.00 0.00 H ATOM 749
HB3 ALA E 42 -17.002 -34.335 5.635 1.00 0.00 H TER 750 ALA 42 1.00
0.00 HETATM 751 C * 53 9.744 -25.831 -3.543 1.00 0.00 C HETATM 752
H * 53 9.721 -26.606 -2.793 1.00 0.00 H HETATM 753 C * 53 10.953
-25.485 -4.191 1.00 0.00 C HETATM 754 C * 53 12.240 -26.077 -3.862
1.00 0.00 C HETATM 755 H * 53 13.086 -25.808 -4.512 1.00 0.00 H
HETATM 756 C * 53 12.487 -26.907 -2.815 1.00 0.00 C HETATM 757 H *
53 11.648 -27.207 -2.169 1.00 0.00 H HETATM 758 C * 53 13.793
-27.472 -2.513 1.00 0.00 C HETATM 759 C * 53 10.919 -24.274 -5.175
1.00 0.00 C HETATM 760 H * 53 11.823 -24.190 -5.694 1.00 0.00 H
HETATM 761 C * 53 9.724 -23.811 -5.478 1.00 0.00 C HETATM 762 O *
53 9.702 -22.789 -6.448 1.00 0.00 O HETATM 763 H * 53 10.584
-22.705 -5.812 1.00 0.00 H HETATM 764 C * 53 8.547 -24.143 -4.805
1.00 0.00 C HETATM 765 H * 53 7.632 -23.614 -5.026 1.00 0.00 H
HETATM 766 C * 53 8.557 -25.151 -3.839 1.00 0.00 C HETATM 767 O *
53 7.372 -25.430 -3.127 1.00 0.00 O HETATM 768 H * 53 7.560 -26.138
-2.437 1.00 0.00 H HETATM 769 C * 53 15.002 -26.920 -3.000 1.00
0.00 C HETATM 770 H * 53 15.006 -26.007 -3.577 1.00 0.00 H HETATM
771 C * 53 10.227 -27.545 -2.744 1.00 0.00 C HETATM 772 H * 53
17.133 -27.111 -3.140 1.00 0.00 H HETATM 773 C * 53 16.268 -28.721
-1.992 1.00 0.00 C HETATM 774 O * 53 17.505 -29.359 -1.788 1.00
0.00 O HETATM 775 H * 53 18.150 -28.868 -2.305 1.00 0.00 H HETATM
776 C * 53 15.089 -29.261 -1.468 1.00 0.00 C HETATM 777 H * 53
15.123 -30.135 -0.878 1.00 0.00 H HETATM 778 C * 53 13.859 -28.637
-1.721 1.00 0.00 C HETATM 779 H * 53 12.960 -29.082 -1.315 1.00
0.00 H CONECT 5 22 CONECT 24 38 CONECT 40 58 CONECT 60 78 CONECT 80
88 CONECT 90 103 CONECT 105 115 CONECT 117 131 CONECT 133 138
CONECT 140 149 CONECT 151 163 CONECT 165 185 CONECT 187 192 CONECT
194 202 CONECT 204 221 CONECT 223 240 CONECT 242 247 CONECT 249 266
CONECT 268 283 CONECT 285 299 CONECT 301 306 CONECT 308 313 CONECT
315 329 CONECT 331 345 CONECT 347 364 CONECT 380 397 CONECT 399 413
CONECT 415 433 CONECT 435 453 CONECT 455 463 CONECT 465 478 CONECT
480 490 CONECT 492 506 CONECT 508 513 CONECT 516 524 CONECT 526 538
CONECT 540 560 CONECT 562 567 CONECT 269 577 CONECT 579 596 CONECT
298 615 CONECT 617 622 CONECT 624 641 CONECT 643 658 CONECT 660 674
CONECT 676 681 CONECT 683 688 CONECT 690 704 CONECT 706 720 CONECT
722 739 CONECT 751 752 753 766 CONECT 753 754 759 CONECT 754 755
756 CONECT 756 757 758 CONECT 758 769 778 CONECT 759 760 761 CONECT
761 762 764 CONECT 762 763 CONECT 764 765 765 CONECT 766 767 CONECT
767 768 CONECT 769 770 771 CONECT 771 772 773 CONECT 773 774 776
CONECT 774 775 CONECT 776 777 778 CONECT 778 779 END
[0314] The present example is further evidence that pseudo-crystal
structures described in the application have utility and may be
used for virtual (in silico) screening in Alzheimer Disease, and,
possibly, related disorders, e.g., Parkinson's Disease.
Example 5
[0315] Using the methods described in the applications, many
compounds in the following Table were identified and predicted to
have an anti-amyloid activity at A.beta. and/or A-Syn amyloid
proteins. Some of these compounds were synthesized and some of the
compounds were obtained from commercial sources.
[0316] The anti-amyloid activity of these compounds was evaluated
by ThT aggregation assays of A.beta. 1-40 and alpha synuclein, as
set forth below. The column "A-Syn" below is a functional ThT
aggregation assay of alpha synuclein.
TABLE-US-00007 A.beta. - ThT Structure (IC.sub.50/.mu.M) A-syn
##STR00012## >20 ##STR00013## >20 ##STR00014## >20
##STR00015## >20 ##STR00016## 4.03 ##STR00017## 2.7 ##STR00018##
>20 ##STR00019## >20 ##STR00020## >20 ##STR00021## 2.1
##STR00022## 1.4 ##STR00023## >20 ##STR00024## -- ##STR00025##
>20 ##STR00026## >20 ##STR00027## >20 ##STR00028## >20
##STR00029## >100 ##STR00030## >20 ##STR00031## >20
##STR00032## >100 ##STR00033## >100 ##STR00034## 20-50
##STR00035## -- ##STR00036## 10-20 51 ##STR00037## 9.0 11.61
##STR00038## 0.85 6.34 ##STR00039## 2.5 142 ##STR00040## 25.8
##STR00041## >50 ##STR00042## >50 ##STR00043## >50
##STR00044## >50 ##STR00045## 8.82 ##STR00046## >50
##STR00047## 23.2 ##STR00048## >50 ##STR00049## >50
##STR00050## INACTIVE ##STR00051## 23 ##STR00052## >50
##STR00053## 8.4 ##STR00054## 39 ##STR00055## 177.5 ##STR00056##
9.64 5 ##STR00057## 44 ##STR00058## 25 4 ##STR00059## 100
##STR00060## 41.9 ##STR00061## 184 ##STR00062## 152 ##STR00063## 42
##STR00064## 217 ##STR00065## 1035 ##STR00066## 631 ##STR00067##
189 ##STR00068## ND ##STR00069## 84 ##STR00070## 196 ##STR00071##
265 ##STR00072## 66.2 ##STR00073## 63 ##STR00074## 174 ##STR00075##
18 ##STR00076## 105 ##STR00077## 1.47 8 ##STR00078## 5734
##STR00079## 14935 ##STR00080## 23 ##STR00081## 544 ##STR00082##
975 ##STR00083## 92 ##STR00084## 1.9 ##STR00085## 361 ##STR00086##
21.7 ##STR00087## 9.23 10 ##STR00088## 27 15 ##STR00089## 360
##STR00090## 428 ##STR00091## 539 ##STR00092## 26.6 45 ##STR00093##
106.6 ##STR00094## 148 ##STR00095## 3.5 ~7 ##STR00096## 15.5
##STR00097## 7.8 ##STR00098## 2.29 ##STR00099## 90 ##STR00100##
54.4 ##STR00101## 238.4
[0317] In the preceding specification, the invention has been
described with reference to specific exemplary embodiments and
examples thereof. It will, however, be evident that various
modifications and changes may be made thereto without departing
from the broader spirit and scope of the invention as set forth in
the claims that follow. The specification and drawings are
accordingly to be regarded in an illustrative manner rather than a
restrictive sense.
Sequence CWU 1
1
2126PRTUnknownpeptide 1Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn
Lys Gly Ala Ile Ile 1 5 10 15 Gly Leu Met Val Gly Gly Val Val Ile
Ala 20 25 226PRTunknownpeptide 2Leu Val Phe Phe Ala Glu Asp Val Gly
Ser Asn Lys Gly Ala Ile Ile 1 5 10 15 Gly Leu Met Val Gly Gly Val
Val Ile Ala 20 25
* * * * *
References