U.S. patent application number 14/215581 was filed with the patent office on 2014-09-25 for ligands for the glp-1 receptor and methods for discovery thereof.
This patent application is currently assigned to TransTech Pharma, LLC. The applicant listed for this patent is TransTech Pharma, LLC. Invention is credited to Mohan Rao.
Application Number | 20140287499 14/215581 |
Document ID | / |
Family ID | 41162231 |
Filed Date | 2014-09-25 |
United States Patent
Application |
20140287499 |
Kind Code |
A1 |
Rao; Mohan |
September 25, 2014 |
Ligands for the GLP-1 Receptor and Methods for Discovery
Thereof
Abstract
Disclosed is the three-dimensional (3-D) structure of the GLP-1
receptor (GLP-1R) and methods by which the structure may be used to
develop compounds that bind to, and/or modulate the GLP-1R. The
technology described herein may be applied to the development of
compounds that target the GLP-1R, or may be used to develop target
compound that may bind to, and/or modulate the activity of the
GLP-1R.
Inventors: |
Rao; Mohan; (Greensboro,
NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TransTech Pharma, LLC |
High Point |
NC |
US |
|
|
Assignee: |
TransTech Pharma, LLC
High Point
NC
|
Family ID: |
41162231 |
Appl. No.: |
14/215581 |
Filed: |
May 1, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12936434 |
Oct 5, 2010 |
8718994 |
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PCT/US09/39905 |
Apr 8, 2009 |
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14215581 |
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61043574 |
Apr 9, 2008 |
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Current U.S.
Class: |
435/366 |
Current CPC
Class: |
G01N 2333/605 20130101;
C12N 5/0602 20130101; G01N 2333/72 20130101; G16C 20/50 20190201;
G16B 15/00 20190201; G16C 20/40 20190201 |
Class at
Publication: |
435/366 |
International
Class: |
C12N 5/071 20060101
C12N005/071 |
Claims
1. A method for agonizing the human GLP-1 receptor (GLP-1R), the
method comprising the steps of: contacting a cell expressing GLP-1R
with a compound, wherein the compound has a molecular weight of
less than 2000 daltons, wherein the compound does not interfere
with the binding of native GLP-1 to the GLP-1R; wherein there is at
least one atomic interaction between the compound and at least one
of the amino acid residues selected from the group consisting of
Ser31, Thr35, Trp39, Arg43, Thr65, Phe66, Glu68, Asp67, Tyr69,
Trp91, Leu89, Tyr88, Arg121, Arg102, Leu123, Glu127, Trp110 and
Glu128 of the GLP-1R; and wherein the atomic interaction between
the compound and the GLP-1R comprises at least one atomic
interaction between the potential modulator compound and at least
one of the amino acid residues selected from the group consisting
of Arg376 and Arg380 of the GLP-1R.
Description
FIELD OF INVENTION
[0001] The present invention relates to compounds that modulate the
human GLP-1 receptor (GLP-1R). More particularly, the present
invention comprises methods that use structural coordinates that
define the three-dimensional (3-D) structure of the GLP-1R to
develop compounds that modulate GLP-1R activity.
BACKGROUND
[0002] Glucagon-like peptide-1 (GLP-1) is a member of the incretin
family of neuroendocrine peptide hormones secreted from the L-cells
of the intestine in response to food ingestion. GLP-1 has multiple
metabolic effects that are attractive for an anti-diabetic agent. A
key function of GLP-1 is to activate its receptor, GLP-1R, on
pancreatic beta-cells to enhance glucose-dependent insulin
secretion. Positive metabolic benefits of GLP-1 may include, but
are not limited to, suppression of excessive glucagon production,
decreased food intake, delayed gastric emptying, and improvement of
b-cell mass and function. The positive effects of GLP-1 on
beta-cell mass and function offers the hope that GLP-1-based
therapies may delay early stage disease progression. In addition, a
GLP-1 agonist could be useful in combination therapies such as with
insulin in patients with type I diabetes. Unfortunately, the rapid
proteolysis of GLP-1 into an inactive metabolite limits its use as
a therapeutic agent.
[0003] Validation of GLP-1R agonists as a therapeutic modality was
achieved by Exendin-4 (Byetta.RTM. (Amylin Pharmaceuticals, Inc.)),
a peptide GLP-1 receptor agonist recently approved for the
treatment of Type II diabetes mellitus. Dosing of Exendin-4 by
subcutaneous administration lowers blood glucose and decreases
HbA1c levels, which are important biomarker measurements for
disease control. Therefore, an oral GLP-1 receptor agonist should
provide glycemic control while offering the convenience of oral
dosing.
[0004] GLP-1R belongs to the class B receptor sub-class of the G
protein-coupled receptor (GPCR) superfamily that regulates many
important physiological and pathophysiological processes. In
addition to the seven transmembrane domains characteristic of all
GPCR family members, class B GPCRs contain a relatively large
N-terminal domain. It is believed that the binding and activation
of these receptors by relatively large natural peptide ligands
require both the N-terminal domain and the transmembrane domain of
the receptor. As such, the identification of low molecular weight
non-peptide agonist molecules for class B GPCRs has proven
particularly difficult.
[0005] Further, because peptides, such as GLP-1, may lack
sufficient oral bioavailability for consideration as oral drug
agents, small molecule modulators of GLP-1R with oral
bioavailability are highly desired. The present invention describes
a class of tetrahydroisoquinoline derivatives that modulate
GLP-1R.
SUMMARY
[0006] Embodiments of the present invention provide modulators for
GLP-1R. The present invention may be embodied in a variety of
ways.
[0007] In one embodiment, the invention may comprise a method for
identifying a compound that has the ability to modulate the GLP-1R.
The method may comprise the step of generating a three-dimensional
model of the GLP-1R, or a portion thereof. The method may further
comprise generating a three-dimensional model of a potential
modulator compound of interest. Next, the method may comprise
determining the nature of at least one of the atomic interactions
between the potential modulator compound and the GLP-1R, or a
portion thereof, as defined by the three-dimensional models for the
potential modulator compound and the GLP-1R or a portion
thereof.
[0008] The present invention also provides a method of generating a
three-dimensional model of a protein, or a portion thereof. The
method may comprise the steps of providing an amino acid sequence
of the protein of interest, and comparing the amino acid sequence
of the protein of interest to the amino acid sequence of other
proteins for which a three-dimensional structure has been defined
to identify a second protein having a predetermined level of
sequence identity to the protein of interest. Once a second protein
having a known 3-D structure has been identified, the method may
further include the step of aligning conserved residues from the
protein of interest with conserved residues from the second
protein. Next, the sequence for the protein of interest may be
threaded along the three-dimensional structure of the second
protein, such that the position of at least two conserved residues
from both proteins are aligned.
[0009] The present invention also comprises a computer model for
the GLP-1R or a portion thereof, comprising structural coordinates
for a three-dimensional model for the GLP-1R, or a portion thereof,
visualized on a computer screen.
[0010] The present invention also provides compounds that modulate
the GLP-1R. In one embodiment, the compounds may be useful in the
treatment of a disease state responsive to the modulation of the
GLP-1R. The compound may comprise a pharmacophore. For example, in
one embodiment, the present invention may comprise a pharmacophore
comprising at least one atom or molecular group that interacts with
at least one atom or molecular group of the GLP-1R, or a portion
thereof. In one embodiment, the compound interacts with the GLP-1R,
or a portion thereof, to modulate the activity of the GLP-1R. For
example, the compound may be a compound identified by docking a
computer representation of the compound, or a synthetic variant
thereof, with a computer representation of a three-dimensional
structure of the GLP-1R, or a portion thereof. In yet another
embodiment, the present invention may comprise a pharmaceutical
composition. For example, the present invention may comprise a
pharmaceutical composition comprising a compound identified by
docking a computer representation of the compound with a computer
representation of a structure of the GLP-1R, or a portion
thereof.
[0011] The present invention also comprises a method of conducting
a drug-discovery business. The method may comprise the step of
generating a three-dimensional structural model of a target
molecule of interest on a computer. Also, the method may comprise
generating a three-dimensional structural model of a potential
modulator compound of the target molecule on a computer, and
docking the model for the potential modulator compound with a 3-D
structural model of the target molecule so as to minimize the free
energy of the interaction between the target molecule and the
potential modulator. In this way, a modulator compound that may
interact with the target may be identified. The method may also
include the subsequent steps of providing a modified structure for
the modulator compound of interest, and assessing whether the
modified structure has a lower free energy of interaction with the
target than the original modulator compound.
[0012] The present invention comprises methods that use structural
coordinates that define the three-dimensional (3-D) structure of
the GLP-1R to develop compounds that modulate GLP-1R activity. In
one aspect of the present invention, structural models for human,
mouse, rat, pig, chicken, chimpanzee and dog GLP-1 receptor
sequences are disclosed. In another aspect of the present
invention, a structural model is disclosed for the ligand binding
domain of the GLP-1R, a synthetic mimic of the GLP-1R and
allosteric binding modulators of the GLP-1R are disclosed. In
another aspect of the present invention, a method is disclosed for
identifying a potential modulators (allosteric and orthosteric) for
human, mouse, rat, pig and dog GLP-1R by determining binding
interactions between a test compound and atomic contacts of ligand
binding domain residues of the GLP-1R is disclosed. This method may
comprise generating a atomic contacts using computational modeling
tools; and generating test compounds with a spatial structure as
shown in FIGS. 2A and 2B. In yet another aspect of the present
invention, a method is disclosed for identifying a potential
modulator of the GLP-1R function by docking a computer
representation of a test compound with a computer representation of
a structure of human, mouse, chicken, rat, pig and dog GLP-1R, or
ligand binding domain thereof, as shown in FIGS. 2A, 2B, 3A and 3B.
In still a further aspect of the present invention, a method is
disclosed for identifying modulators of the GLP-1R function by
binding a test compound with any of the residues of the GLP-1R
shown in FIGS. 2A and 2B. In another aspect of the present
invention, a method is disclosed for the design of ligands for
human, mouse, rat, pig, dog GLP-1R based on the three-dimensional
structure of ligand binding domains as shown in FIGS. 2A and 2B. In
a further aspect of the present invention, a method is disclosed
for the design of antagonist and agonists for Sites 1 and 2, as
shown in FIGS. 2A and 2b.
[0013] In another embodiment, the present invention comprises
treatment of a disease state responsive to the modulation of the
GLP-1R using compounds identified by the methods and systems of the
present invention. The disease state may include disorders wherein
activation of the GLP-1R is beneficial, including metabolic
disorders, wherein activation of the GLP-1R is beneficial; glucose
intolerance; hyperglycaemia; dyslipidemia; Type 1 diabetes; Type 2
diabetes; hypertriglyceridemia; syndrome X; insulin resistance;
IGT; obesity; diabetes as a consequence of obesity; diabetic
dyslipidemia; hyperlipidemia; cardiovascular diseases;
hypertension, and complications resulting from or associated with
diabetes including but not limited to neuropathy, retinopathy,
nephropathy, impaired wound healing, and the like, or any other
indications listed in US Patent Publication No. 2004/0127423A3 or
U.S. Pat. No. 6,927,214, the content of which is herein
incorporated by reference.
[0014] There may be advantages provided by certain embodiments of
the present invention. For example, the methods of the present
invention may provide a means to identify a plurality of putative
pharmacological agents based upon the known 3-D structure of a
target protein. Also, the present invention may provide a means to
modify the structure of a putative pharmacological agent in silico
to determine how such changes can effect the activity of the agent.
Making such determinations in silico provides the ability to
rapidly evaluate a large number of compounds. Also, making such
determinations in silico allows for a rational approach to drug
development, such that compounds may be systematically developed
and their activity evaluated.
[0015] There are, of course, additional features of the invention,
which will be described in more detail hereinafter. It is to be
understood that the invention is not limited in its application to
the specific details as set forth in the following description and
figures. The invention is capable of other embodiments and of being
practiced or carried out in various ways.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 shows a homology model for the GLP-1R sequence
(Primary accession number P43220) constructed using bovine
rhodopsin (PDB code: 1F88) and extracellular domain structure of
GIP-1R (PDB code: 2qkh) as a template. This model includes an
extracellular domain, transmembrane, and intracellular domains;
[0017] FIGS. 2A and 2B show three-dimensional homology threading
models of the GLP-1R generated using methods in accordance with the
present invention. The models depict two putative binding sites on
the extracellular portion of the GLP-1R: Site 1 and Site 2,
respectively;
[0018] FIGS. 3A and 3B show possible ligand binding modes within
the Sites 1 and 2 of FIGS. 2A and 2B; and
[0019] FIG. 4 shows a schematic of a method used to develop
modulators of a target receptor.
DETAILED DESCRIPTION
Definitions
[0020] The following definitions may be used to understand the
description herein. Unless defined otherwise, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art. Practitioners are
particularly directed to Current Protocols in Molecular Biology
(Ansubel) for definitions and terms of the art. Abbreviations for
amino acid residues are the standard 3-letter and/or 1-letter codes
used in the art to refer to one of the 20 common L-amino acids.
[0021] A GLP-1R or part thereof in the present invention may be a
wild type protein or part thereof, a mutant protein or part
thereof, or variant or homologue of such a protein.
[0022] As used herein, the term "wild type" refers to a polypeptide
having a primary amino acid sequence which is identical with the
native polypeptide. The term "mutant" refers to a polypeptide
having a primary amino acid sequence which differs from the wild
type sequence by one or more amino acid additions, substitutions or
deletions. A mutant may or may not be functional. As used herein,
the term "variant" refers to a naturally occurring polypeptide
which differs from a wild-type sequence. As used herein, when
referring to a protein, the terms "portion" or "part" indicate that
the polypeptide comprises a fraction (or fractions) of the amino
acid sequence referred to.
[0023] "Polypeptide" and "protein" are used interchangeably herein
to describe protein molecules that may comprise either partial or
full-length proteins.
[0024] As used herein, "small organic molecules" are molecules of
molecular weight less than 2,000 Daltons that contain at least one
carbon atom.
[0025] The term "vector" refers to a nucleic acid molecule that may
be used to transport a second nucleic acid molecule into a cell. In
one embodiment, the vector allows for replication of DNA sequences
inserted into the vector. The vector may comprise a promoter to
enhance expression of the nucleic acid molecule in at least some
host cells. Vectors may replicate autonomously (extra chromosomal)
or may be integrated into a host cell chromosome. In one
embodiment, the vector may comprise an expression vector capable of
producing a protein derived from at least part of a nucleic acid
sequence inserted into the vector.
[0026] As used herein, the term "interact" refers to a condition of
proximity between a ligand or compound, or portions or fragments
thereof, and a portion of a second molecule of interest. The
interaction may be non-covalent, for example, as a result of
hydrogen-bonding, van der Waals interactions, or electrostatic or
hydrophobic interactions, or it may be covalent.
[0027] As used herein, the term "atomic contacts" or "atomic
interaction" refers to the inter-atomic contact between atoms in a
test compound and atoms in a second molecule (e.g., the protein of
interest) for which a three-dimensional model is made. The atomic
interaction is governed by geometric and physiochemical
complementarity as well as steric fit between the two molecules for
which the atomic contacts/interaction is evaluated. For example,
potential atomic interactions between the GLP-1R ligand binding
domain and sample small molecules are described, at least in part,
by FIGS. 3a and 3b.
[0028] As used herein, the term "docking" refers to a process by
which a test compound is placed in close proximity with a second
molecule (e.g., the protein of interest). Docking is also used to
describe the process of finding low energy conformations of a test
compound and a second molecule (e.g., the protein or polypeptide of
interest, or portion thereof). Docking studies include molecular
modeling studies aimed at finding a proper fit between a ligand and
its binding site.
[0029] As used herein, the term "docked" refers to a favorable
configuration of a test compound positioned within a given site on
a molecule of interest.
[0030] As used herein, the term "hang point residues" refers to
residues on a first molecule of known structure that are then used
as anchors for the threading of a second molecule of unknown
structure along the structure of the first molecule so as to
determine a structure for the second molecule.
[0031] As used herein, the term "conserved residues" refers to
amino acids that are the same among a plurality of proteins having
the same structure and/or function. A region of conserved residues
may be important for protein structure or function. Thus,
contiguous conserved residues as identified in a three-dimensional
protein may be important for protein structure or function. To find
conserved residues, or conserved regions of 3-D structure, a
comparison amino acid sequence for the same or similar proteins
from different species, or of individuals of the same species, may
be made.
[0032] As used herein, the term "homologue" means a polypeptide
having a degree of homology with the wild-type amino acid sequence.
Homology comparisons can be conducted by eye, or more usually, with
the aid of readily available sequence comparison programs. These
commercially available computer programs can calculate percent
homology between two or more sequences (e.g. Wilbur, W. J. and
Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA, 80:726-730). For
example, homologous sequences may be taken to include an amino acid
sequences which in alternate embodiments are at least 75%
identical, 85% identical, 90% identical, 95% identical, or 98%
identical to each other.
[0033] The terms "identity" or "percent identical" refers to
sequence identity between two amino acid sequences or between two
nucleic acid sequences. Percent identity can be determined by
aligning two sequences and refers to the number of identical
residues (i.e., amino acid or nucleotide) at positions shared by
the compared sequences. Sequence alignment and comparison may be
conducted using the algorithms standard in the art (e.g. Smith and
Waterman, 1981, Adv. Appl. Math. 2:482; Needleman and Wunsch, 1970,
J. Mol. Biol. 48:443; Pearson and Lipman, 1988, Proc. Natl. Acad.
Sci., USA, 85:2444) or by computerized versions of these algorithms
(Wisconsin Genetics Software Package Release 7.0, Genetics Computer
Group, 575 Science Drive, Madison, Wis.) publicly available as
BLAST and FASTA. Also, ENTREZ, available through the National
Institutes of Health, Bethesda Md., may be used for sequence
comparison. In one embodiment, the percent identity of two
sequences may be determined using GCG with a gap weight of 1, such
that each amino acid gap is weighted as if it were a single amino
acid mismatch between the two sequences.
[0034] As used herein, a polypeptide or protein "domain" comprises
a region along a polypeptide or protein that comprises an
independent unit. Domains may be defined in terms of structure,
sequence and/or biological activity. In one embodiment, a
polypeptide domain may comprise a region of a protein that folds in
a manner that is substantially independent from the rest of the
protein. Domains may be identified using domain databases such as,
but not limited to, PFAM, PRODOM, PROSITE, BLOCKS, PRINTS, SBASE,
ISREC PROFILES, SAMRT, and PROCLASS.
[0035] As used herein, "ligand binding domain" (LBD) refers to a
domain of a protein responsible for binding a ligand. The term
"ligand binding domain" includes homologues of a ligand binding
domain or portions thereof. In this regard, deliberate amino acid
substitutions may be made in the LBD on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity,
and/or the amphipathic nature of the residues, as long as the
binding specificity of the ligand binding domain is retained.
[0036] As used herein, the "ligand binding site" comprises residues
in a protein that directly interact with a ligand, or residues
involved in positioning the ligand in close proximity to those
residues that directly interact with the ligand. The interaction of
residues in the ligand binding site may be defined by the spatial
proximity of the residues to a ligand in the model or structure.
The term "ligand binding site" includes homologues of a ligand
binding site or portions thereof. In this regard, deliberate amino
acid substitutions may be made in the ligand binding site on the
basis of similarity in polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues, as long as the binding specificity of the ligand
binding site is retained. For I7L, the ligand binding site may be
defined as comprising those residues described below for each of
Site 1 and Site 2. In another embodiment, the ligand binding site
may be defined as comprising those residues as described below for
each of Site 1 and Site 2 and any other residues that are within a
3 angstrom radius thereof.
[0037] As used herein, a "ligand" refers to a molecule or compound
or entity that associates with a ligand binding domain, including
substrates or analogues or parts thereof. As described herein, the
term "ligand" may refer to compounds that bind to the protein of
interest. A ligand may be a modulator. Or, a ligand may not have a
biological effect. Or, a ligand may block the binding of other
ligands thereby inhibiting a biological effect. Ligands may
include, but are not limited to, small molecule inhibitors of the
activity of protein. These small molecules may include peptides,
peptidomimetics, organic compounds and the like.
[0038] As used herein, a "modulator compound" refers to a molecule
which changes or alters the biological activity of a molecule of
interest. A modulator compound may increase or decrease activity,
or change the physical or chemical characteristics, or functional
or immunological properties, of the molecule of interest. A
modulator compound may include natural and/or chemically
synthesized or artificial peptides, modified peptides (e.g.,
phosphopeptides), antibodies, carbohydrates, monosaccharides,
oligosaccharides, polysaccharides, glycolipids, heterocyclic
compounds, nucleosides or nucleotides or parts thereof, and small
organic or inorganic molecules. A modulator compound may be an
endogenous physiological compound or it may be a natural or
synthetic compound. Or, the modulator compound may be a small
organic molecule. The term "modulator compound" also includes a
chemically modified ligand or compound, and includes isomers and
racemic forms.
[0039] The terms "structural coordinates" or "atomic coordinates"
as used herein refers to a set of values that define the position
of one or more amino acid residues or molecules with reference to a
system of axes. A data set of structural coordinates defines the
three dimensional structure of a molecule or molecules. Structural
coordinates can be slightly modified and still render nearly
identical three dimensional structures. A measure of a unique set
of structural coordinates is the root-mean-square deviation of the
resulting structure. In alternate embodiments, structural
coordinates that render three dimensional structures that deviate
from one another by a root-mean-square deviation of less than 3
angstroms, or less than 2.0 angstroms, or less than 0.5 angstroms,
or less than 0.3 angstroms, may be viewed by a person of ordinary
skill in the art as identical. Variations in structural coordinates
may be generated because of mathematical manipulations of the
structural coordinates of the GLP-1R. Variations in structure due
to mutations, additions, substitutions, and/or deletions of the
amino acids, or other changes in any of the components that make up
a structure of the invention may also account for modifications in
structural coordinates. If such modifications are within the
standard error as compared to the original structural coordinates,
the resulting structure may be considered to be the same or
equivalent. Therefore, a ligand that bound to a ligand binding
domain of a GLP-1R would also be expected to bind to another ligand
binding domain whose structural coordinates defined a shape that
fell within the margin of error defined by the first structure.
Such modified structures of a ligand binding domain are also within
the scope of the invention.
[0040] As used herein, a structural "model" of a protein of
interest, a polypeptide of interest, or any other compound of
interest, may be in two or three dimensions. For example, a
computer model may be in three dimensions despite the constraints
imposed by a computer screen, if it is possible to scroll along at
least a pair of axes, causing rotation of the image. Also, a model
of a protein or chemical compound of interest may be defined by the
structural coordinates for the protein or compound of interest.
[0041] As used herein, the terms "modeling" or "generating a model"
includes the quantitative and qualitative analysis of molecular
structure and/or function based on atomic structural information
and interaction models. The term may include conventional
numeric-based molecular dynamic and energy minimization models,
interactive computer graphic models, modified molecular mechanics
models, distance geometry, and other structure-based constraint
models.
[0042] The term "peptide mimetics" are structures which serve as
substitutes for peptides in interactions between molecules (Morgan
et al., 1989, Ann. Reports Med. Chem., 24:243-252). Peptide
mimetics may include synthetic structures that may or may not
contain amino acids and/or peptide bonds but that retain the
structural and functional features of a peptide, or agonist, or
antagonist. Peptide mimetics also include peptoids, oligopeptoids
(Simon et al., 1972, Proc. Natl. Acad, Sci., USA, 89:9367); and
peptide libraries containing peptides of a designed length
representing all possible sequences of amino acids corresponding to
a peptide, or agonist or antagonist of the invention.
[0043] As used herein, "pharmacophore" is a collection of steric
and electronic features that are necessary to ensure the optimal
supramolecular interactions with a specific biological target
structure. A pharmacophore may comprise a structural definition
that comprises a set of active molecules. A pharmacophore may
comprise a modulator compound.
[0044] As used herein, an "effective amount" as used herein means
the amount of an agent that is effective for producing a desired
effect in a subject. The term "therapeutically effective amount"
denotes that amount of a drug or pharmaceutical agent that will
elicit the therapeutic response of an animal or human that is being
sought. The actual dose which comprises the effective amount may
depend upon the route of administration, the size and health of the
subject, the disorder being treated, and the like.
[0045] The term "pharmaceutical composition" is used herein to
denote a composition that may be administered to a mammalian host,
e.g., orally, topically, parenterally, by inhalation spray, or
rectally, in unit dosage formulations containing conventional
non-toxic carriers, diluents, adjuvants, vehicles and the like. The
term "parenteral" as used herein, includes subcutaneous injections,
intravenous, intramuscular, intracisternal injection, or by
infusion techniques.
[0046] The term "a" or "an" as used herein may refer to more than
one object unless the context clearly indicates otherwise. The term
"or" is used interchangeably with the term "and/or" unless the
context clearly indicates otherwise.
[0047] Embodiments of the present invention provide a method for
identifying a compound having the ability to modulate the GLP-1R.
The method may comprise the steps of: (a) generating a
three-dimensional model of a the GLP-1R, or a portion thereof; (b)
generating a three-dimensional model of a potential modulator
compound of interest; and (c) determining at least one atomic
interaction between the potential modulator compound and the
GLP-1R, or a portion thereof, as defined by the three-dimensional
models of each.
[0048] The method may be performed using a computer. Thus, in one
embodiment, the method comprises the steps of: (a) generating a
three-dimensional computer model of the GLP-1R, or a portion
thereof; (b) generating a three-dimensional computer model of the
potential modulator compound of interest; (c1) using a computer to
dock the three-dimensional model of the potential modulator
compound within the model of the GLP-1R or a portion thereof; and
(c2) quantifying at least one atomic interaction between the
potential modulator compound and the GLP-1R, or a portion
thereof.
[0049] The method further allows for varying the structure of the
potential modulator compound to determine how changes to the
structure of the modulator may affect the fit of the compound with
the protein of interest, such as GLP-1R. Thus, the method may
further comprise the steps of modifying the computer model of the
potential modulator compound, and evaluating how modifying the
computer model of the potential modulator compound changes at least
one atomic interaction between of the model of the potential
modulator compound and the model of the GLP-1R, or portion thereof.
The potential modulator compound may be modified in silico. Thus,
in one embodiment, the step of modifying the computer model of the
potential modulator compound of interest comprises the step of
searching a library of molecular structures for molecular fragments
that can be linked to the potential modulator compound, wherein a
molecular fragment comprises at least one atom. The method may
further comprise linking a molecular fragment to the potential
modulator compound to generate a modified compound. The modified
compound may then be evaluated by docking the modified compound to
the GLP-1R and quantifying at least one atomic interaction between
the modified compound and the GLP-1R.
[0050] It may not be required to determine the entire structure of
the protein of interest to identify compounds that may act as
modulators of the protein. For example, the three-dimensional model
of the GLP-1R may comprise only a portion of the protein. Thus, the
model may comprise a ligand binding domain. Additionally or
alternatively, the model may comprise a ligand binding site.
[0051] It is also not necessarily required to determine how each
amino acid of the entire structure of the protein of interest
interacts with a potential modulator compound to identify compounds
that may act as modulators of the protein. For example, the amino
acid used to determine an atomic interaction between a potential
modulator compound and the GLP-1R may comprise a residue that is
conserved in the GLP-1R. Additionally, and/or alternatively, an
amino acid used to determine an atomic interaction between a
potential modulator compound and the GLP-1R may comprise a residue
that is present in, or affects the structure of, the ligand binding
domain and/or the ligand binding site.
[0052] The model of the GLP-1R may comprise a variety of formats.
In one embodiment, the model may comprise a three-dimensional
structural model. Or, the model of the GLP-1R may comprise
structural coordinates presented as the position of individual
atoms of the GLP-1R, or a portion thereof, in space. For example,
the model of the GLP-1R, or a portion thereof, may comprise the x,
y, and z atomic coordinates.
[0053] The method may be performed using a computer. Thus, in one
embodiment, the method comprises the steps of: (a) generating a
three-dimensional computer model of the GLP-1R, or a portion
thereof; (b) generating a three-dimensional computer model of the
potential modulator compound; (c1) using a computer to dock the
three-dimensional model of the potential modulator compound with
the model of the GLP-1R, or a portion thereof; and (c2) quantifying
at least one atomic interaction between the potential modulator
compound and the GLP-1R as defined by the docking of the model of
the potential modulator compound in the computer model of the
GLP-1R, or a portion thereof.
[0054] The method further allows for varying the structure of the
potential modulator compound to determine how changes in the
structure can affect the fit of the potential modulator compound
with the protein of interest. Thus, the method may further comprise
the steps of modifying the computer model of the potential
modulator compound, and evaluating how modifying the computer model
of the potential modulator compound affects the atomic interactions
between of the model of the potential modulator compound and the
model of the GLP-1R, or portion thereof. The potential modulator
compound may be modified in silico. Thus, in one embodiment, the
step of modifying the computer model of the potential modulator
compound of interest comprises the step of searching a library of
molecular structures for molecular fragments that can be linked to
the potential modulator compound, wherein a molecular fragment
comprises at least one atom. The method may further comprise
linking a molecular fragment to the potential modulator compound to
generate a modified compound. The modified compound may then be
evaluated by docking the modified compound to the GLP-1R, or a
portion thereof, and determining the atomic interactions between
the modified compound and the GLP-1R.
[0055] It is not necessarily required to determine the entire
structure of the protein of interest to identify compounds that may
act as modulators of the protein. For example, the
three-dimensional model of the protein of interest may comprise
only a portion of the protein.
[0056] It may not be required to determine how each amino acid of
the entire structure of the GLP-1R interacts with a potential
modulator compound to identify compounds that may act as modulators
of the GLP-1R. Additionally, or alternatively, an amino acid used
to determine an atomic interaction between a potential modulator
compound and the GLP-1R may comprise a residue that is present in,
or affects the structure of, the ligand binding domain and/or
ligand binding site.
[0057] Depending on the source of the protein used to generate a
three-dimensional structure of the protein of interest, such as
GLP-1R, there may be some variability in the absolute positioning
of each amino acid. Still, it is to be expected that the relative
positions of conserved amino acids may be maintained.
[0058] The analysis may further employ a modified protein. Thus,
the potential modulator compound may be evaluated for its
interaction with a modified GLP-1R, or portion thereof, wherein the
modified GLP-1R comprises at least one of an amino acid
substitution, an amino acid deletion, or an amino acid
insertion.
[0059] The nature of the interaction between the potential
modulator compound and the protein of interest may be defined in
terms of the atomic interaction between the compound and the
protein of interest.
[0060] The present invention also comprises a method of generating
a three-dimensional model of a protein of interest, or a portion
thereof. In one embodiment, method may comprise the steps of: (a)
providing an amino acid sequence of a protein of interest; (b)
comparing the amino acid sequence of the protein of interest to the
amino acid sequences of a plurality of other proteins; (c)
identifying a second protein for which a three-dimensional
structure has been defined, and that has a predetermined level of
sequence identity to the protein of interest; (d) aligning
conserved residues from the protein of interest with conserved
residues from the second protein; and (e) threading the protein of
interest along the three-dimensional structure of the second
protein such that the position of at least two conserved residues
from both proteins are aligned.
[0061] The protein aligned with the protein of interest may also
comprise a protein having a similar sequence to the protein of
interest. The level of sequence identity may range from at least 5%
sequence identity, to more than 10% sequence identity, to more than
20% sequence identity. Also, the protein aligned with the protein
of interest may comprise a protein having a similar function as the
protein of interest.
[0062] The present invention may also comprise a structural model
for a protein of interest, or a portion of a protein, that may be
manipulated using a computer. In one example embodiment, the
present invention may comprise a computer model for the GLP-1R, or
a portion thereof. The model may comprise atomic coordinates for a
three-dimensional model for the GLP-1R, or a portion thereof,
visualized on a computer screen.
[0063] In one embodiment, the computer model of the protein of
interest may comprise atomic coordinates presented as the position
of individual atoms of the GLP-1R, or a portion thereof, in
space.
[0064] Also, the model may comprise a three-dimensional computer
model of a potential modulator compound docked into the GLP-1R
structure such that the atomic interaction between the GLP-1R and
the potential modulator compound may be quantified. The atomic
interactions between the GLP-1R and the potential modulator
compound may be defined at least in part determining atomic
coordinates for the potential modulator compound as it interacts
with the GLP-1R.
[0065] The model allows for varying the structure of the potential
modulator compound to determine how changes in the structure of the
modified compound can effect the fit of the compound with the
protein of interest. Thus, the model may further comprise a
three-dimensional model of a modified compound docked with the
GLP-1R structure. The potential modulator compound may be modified
in silico. Thus, in one embodiment, the step of modifying the
computer model of the potential modulator compound of interest
comprises the step of searching a library of molecular structures
for molecular fragments that can be linked to the potential
modulator compound, wherein a molecular fragment comprises at least
one atom, and linking the fragments to the compound. The modified
compound may then be evaluated by docking the modified compound to
the GLP-1R, or a portion thereof, and determining the atomic
interactions between the modified compound and the GLP-1R.
[0066] The present invention also comprises a pharmacophore having
a structure required to modify the activity of the protein of
interest. For example, the pharmacophore may comprise at least one
atom or molecular group that interacts with at least one atom or
molecular group of the GLP-1R, or a portion thereof. Additionally,
the three dimensional structure of the pharmacophore may comprise a
plurality of atoms or molecular groups that interact with at least
one atom or molecular group of a three-dimensional structure of the
GLP-1R, or a portion thereof. To be active as a modulator of the
GLP-1R, the pharmacophore may interact with the ligand binding
domain of the GLP-1R, or a portion thereof, such as the ligand
binding site.
[0067] The nature of the interaction between the pharmacophore and
the protein of interest may be defined in terms of the atomic
interaction between the pharmacophore and the protein of
interest.
[0068] The pharmacophore may be defined by its ability to interact
with amino acids in the protein of interest that are important for
substrate binding.
[0069] The computer model may further employ a modified protein.
Thus, the pharmacophore may be evaluated for its interaction with a
modified GLP-1R, or portion thereof, wherein the modified GLP-1R
comprises at least one of an amino acid substitution, an amino acid
deletion, or an amino acid insertion.
[0070] In yet another embodiment, the present invention comprises
compounds that interact with at least one atom or molecular group
of the GLP-1R. In an embodiment, the compounds include molecules
that interact with residues known to be in the ligand binding
domain and/or ligand binding site. Examples of such compounds that
may modulate the activity of GLP-1R are disclosed in commonly owned
U.S. patent application Ser. No. 12/399,504, filed Mar. 6, 2009,
herein incorporated by reference.
[0071] The interaction between the compound and the GLP-1R may
comprise an in silico interaction defined by a computer model of
the structure of the compound and a computer model of the GLP-1R,
or a portion thereof. Thus, the present invention may also comprise
a compound identified by docking a computer representation of the
compound with a computer representation of a structure of the
GLP-1R, or a portion thereof.
[0072] The nature of the interaction between the compound and the
protein of interest may be defined in terms of the atomic
interaction between the compound and the protein of interest.
[0073] The present invention also comprises pharmaceutical
compositions comprising compounds able to modify the activity of a
protein of interest. In one embodiment, the protein of interest may
comprise the GLP-1R. In one embodiment, the present invention may
comprise a pharmaceutical composition comprising a compound
identified by docking a computer representation of the compound
with a computer representation of a three-dimensional structure of
the GLP-1R, or a portion thereof.
[0074] The nature of the interaction between the compound of the
pharmaceutical composition and the protein of interest may be
defined in terms of the atomic interaction between the compound and
the protein of interest.
[0075] The pharmaceutical composition may comprise the compound
present in a therapeutically effective amount. The dosage used for
the pharmaceutical compositions of the present invention may vary
depending on the specific compound being used, as well as the
methods of administration. In one embodiment, a therapeutically
effective amount may comprise a dose in a range from about 0.01 to
1,000 mg of active compound per kg body weight per day.
[0076] The present invention also comprises a method of conducting
a drug-discovery business. The method may comprise the step of
generating a three-dimensional structural model of a target
molecule of interest on a computer. Also, the method may comprise
generating a three-dimensional structural model of a potential
modulator compound of the target molecule on a computer, and
docking the model for the potential modulator compound with the
target molecule so as to minimize the free energy of the
interaction between the target molecule and the potential
modulator. In this way, a modulator compound that may interact with
the target may be identified. The method may also include the
subsequent steps of providing a modified structure for the
modulator compound of interest, and assessing whether the modified
structure has a lower free energy of interaction with the target
than the original structure for the modulator compound.
[0077] The method may further include evaluating at least some of
the potential modulator compounds identified by in silico screening
in a biological assay. Once compounds initially identified by the
in silico assay are corroborated by a biological assay, animal
studies may be used for detailed therapeutic profiling, and
pharmaceutical compositions may then be developed. Or, additional
in silico assays may be conducted on compounds that appear to be
promising based on the biological data.
[0078] The compound may comprise a small organic compound.
Structural Modeling of the GLP-1R
[0079] Embodiments of the present invention comprise computer
modeling methods and systems to identify and optimize specific
small molecules that bind to, and thus, are able to modulate the
activity of, a particular target protein. In one embodiment, the
protein is the GLP-1R. Also provided by the present invention are
compounds identified using the modeling methods described
herein.
[0080] Thus, in one embodiment, the present invention provides a
method of generating a three-dimensional model of a protein of
interest, such as GLP-1R, or a portion thereof. The method may
comprise the steps of providing an amino acid sequence of the
protein of interest and comparing the amino acid sequence of the
protein of interest to the amino acid sequences of other proteins
to identify a second protein for which a three-dimensional
structure has been defined, and that has a predetermined level of
sequence identity to the protein of interest. Once a second protein
having a known structure has been identified, the method may
include the step of aligning conserved residues from the protein of
interest with conserved residues from the second protein. Next, the
sequence for the protein of interest may be threaded along the
three-dimensional structure of the second protein such that the
position of at least two conserved residues from both proteins are
aligned. The conserved residues from the first protein and the
second protein may comprise residues that are essential for protein
function.
[0081] Thus, as a first step, a three-dimensional model of the
protein of interest may be generated. To generate a three
dimensional model of a protein of interest, a sequence comparison
to proteins with experimentally determined three-dimensional
structures may be performed. The protein aligned with the protein
of interest may comprise a protein having a similar sequence to the
protein of interest. The level of sequence identity may range from
at least 5% sequence identity, to more than 10% sequence identity,
to more than 20% sequence identity.
[0082] The protein aligned with the protein of interest may not
necessarily be functionally related to the protein of interest. Or,
the protein aligned with the protein of interest may comprise a
protein having a similar function to the protein of interest. In
this way, conserved residues that have similar functions in the two
proteins may be aligned.
[0083] In one embodiment, the protein of interest may comprise the
GLP-1R.
[0084] To develop a three dimensional structure for the GLP-1R,
TTPredict.RTM. site search algorithms may be used to identify the
ligand binding site of the GLP-1R based on the location of site
residues. Also, TTPredict.RTM. algorithms may be used to identify
known GLP-1R-homologous sequences using BLAST searches on protein
sequence databases. TTPredict.RTM. algorithms may also be used to
access a number of publicly available and vendor-supplied fold
recognition programs to analyze the GLP-1R sequence folds (e.g.,
MSI suite of programs, TTPGene).
[0085] A homology model for the GLP-1R receptor sequence (Primary
accession number P43220) was constructed using bovine rhodopsin
(PDB code: 1F88) and extracellular domain structure of the GIP-1R
(PDB code: 2qkh) as a template. This model may include a large
extracellular domain, a transmembrane domain, and an intracellular
domain as shown in FIG. 1. The transmembrane domain consists of
seven helices with a hydrophobic "exterior" in contact with the
membrane lipid bilayer. The intracellular domain consists of three
loops and the C-terminus. Similar models were constructed for mouse
(O35659), rat (P32301), chicken (NP.sub.--001094505), chimpanzee
(XP.sub.--527380), dog (XP.sub.--538899) GLP-1R sequences.
[0086] Two putative binding sites (Site 1 and Site 2) on the
extracellular portion of GLP-1R were identified as shown in FIGS.
2A and 2B. Comparing the experimentally determined binding
affinities of small molecules with their computed binding scores
for Sites 1 and 2, Site 1 was identified as the main ligand binding
site. However, Site 2 also has features for binding to small
molecule modulators. Site 2 may recognize allosteric-type
modulators. FIGS. 3A and 3B show possible ligand binding modes
within Site 1 and Site 2.
[0087] Site 1 may be defined by eighteen residues: Ser31, Thr35,
Trp39, Arg43, Thr65, Phe66, Glu68, Asp67, Tyr69, Trp91, Leu89,
Tyr88, Arg121, Arg102, Leu123, Glu127, Trp110 and Glu128. These
comprise several hydrophobic, polar, negatively, and positively
charged residues.
[0088] Site 2 may be defined by seventeen residues: Lys38, Glu34,
Trp87, Gln45, Ser49, Asp53, Pro55, Pro73, Val 83, Arg40, Tyr42,
Leu50, Ser93, Pro86, His99, Val81 and Ser84.
[0089] The identification of Site 1 as the main ligand binding site
is consistent with the results of site-directed mutagenesis
experiments that suggest that several of the binding site residues
listed in the previous paragraph may be critical for the binding of
GLP-1 and Exendin-4 to the GLP-1R.
[0090] Lead optimization was pursued by novel computational methods
to the parent leads (shown in mesh in FIGS. 3A and 3B) followed by
suggested structural alterations based on their observed binding
environments shown in FIGS. 2A, 2B, 3A and 3B.
[0091] The threading approach may reveal distantly homologous
proteins that share the same folding structure, but that do not
comprise a high amount of sequence similarity. Rather than relying
only on sequence alignment, the fold recognition method may blend
the sequence-to-structure fitness with other structural
characteristics, such as sequence similarity and predicted
secondary structures, to find conserved residues that appear in
both the template protein of interest (e.g., GLP-1R) as well as any
query sequences, and overlay both sequences, maintaining alignment
of the conserved residues. Next, the threading program may match
the query sequence on the three-dimensional structure of the
template using conserved residues of the query protein as the hang
points. The resulting model may then be cleaned-up using standard
energy minimization and molecular dynamics techniques.
[0092] The model may be further refined once the initial structural
coordinates are defined. Thus, specific aspects of the model, such
as the ligand binding site, may be refined to incorporate the
structures of ligands that may be bound at that site.
[0093] The structure of the GLP-1R, or a portion thereof, may be
defined by the atomic coordinates in three dimensional space. A
data set of structural coordinates may define the three dimensional
structure of a molecule or molecules. Structural coordinates can be
slightly modified and still render nearly identical three
dimensional structures. A measure of a unique set of structural
coordinates is the root-mean-square deviation of the resulting
structure. In alternate embodiments, structural coordinates that
render three dimensional structures that deviate from one another
by a root-mean-square deviation of less than 3.0 angstroms, or less
than 2.0 angstroms, or less than 0.5 angstroms, or less than 0.3
angstroms, may be viewed by a person of ordinary skill in the art
as identical or equivalent.
In Silico Screening of Putative GLP-1R Modulators
[0094] The present invention further provides methods to dock
compounds of interest, such as putative therapeutic agents, into
the structure of the modeled protein to determine whether such
putative therapeutic agents may interact with the protein. In one
embodiment, the protein of interest is the GLP-1R, and the putative
therapeutic agents are putative modulator compounds. Thus, the
putative therapeutic agents may bind to the ligand binding site to
modify activity of the GLP-1R.
[0095] To generate a three dimensional model of a potential
modulator compound of interest, or a plurality of potential
modulator compounds, a database of in silico structures for
potential modulator compounds of interest, such as provided by
TTProbes.RTM., may be used. Once the three-dimensional structures
of the modulator compounds of interest have been generated, the
compounds may be docked into the ligand binding site of the protein
of interest.
[0096] The putative therapeutic agents (i.e., potential modulator
compounds) may comprise a variety of compounds. In one embodiment,
the putative therapeutic agent may comprise a peptide or a
peptidomimetic. Or, the putative therapeutic agent may comprise an
antibody. Alternatively, the putative therapeutic agent may
comprise a small organic compound.
[0097] The structure of a putative ligand may be provided as a
three-dimensional space-filling model, as a rotatable model on a
computer screen, or as atomic coordinates in three-dimensional
space. In one embodiment, the compounds that dock into the ligand
binding site with a negative free energy are considered to be
favorable. In alternative embodiments, a compound having an free
energy of interaction with the GLP-1R (or another molecule of
interest) of less than -2 kcal/mol, or less than -5 kcal/mol, or
less than -10 kcal/mole, are considered to provide favorable
binding to the protein of interest.
[0098] The duration of GLP-1R activation by a putative GLP-1R
modulator (hereinafter, "the modulator compound") was studied in
HEK293 cells expressing human GLP-1R to assess the effect of the
modulator compound on GLP-1R desensitization. In this particular
experiment, the maximum amount of cAMP production induced by the
modulator compound was about .about.80% relative to the maximum
amount of cAMP production induced by GLP-1. To assess receptor
desensitization, cells were treated with the modulator compound or
GLP-1 for 30 minutes. The cells were then washed and incubated for
an additional 30 minutes in the absence of ligand and/or modulator
compound. The amount of cAMP produced was then quantified.
Interestingly, cAMP production is sustained (90%) following removal
of the modulator compound or GLP-1. However, unlike cells treated
with GLP-1 initially, cells treated with the modulator compound
followed by further addition of the modulator compound or GLP-1
were further stimulated suggesting that the GLP-1R was not
desensitized by the modulator compound to the same extent that it
was by the native ligand. In conclusion, activation of the GLP-1R
by the modulator compound is prolonged and the GLP-1R can be
re-stimulated with GLP-1 peptide, illustrating that the modulator
compound does not down-regulate the human GLP-1R function in HEK293
cells expressing the human GLP-1R. This may allow for a prolonged
efficacy of in vivo with the modulator compound when compared to
native ligand.
[0099] In order to confirm that the apparent GLP-1R modulation seen
in the cell-based assay was specific, a modulator compound was
evaluated in an in vitro receptor-binding assay. This assay was
designed as a competition assay where the modulator compound was
evaluated for its ability to inhibit .sup.125I labeled human GLP-1
(7-36) from binding to membrane preparations derived from cells
expressing the human or mouse GLP-1R. The modulator compound
effectively inhibited binding of .sup.125I GLP-1 (7-36) to the
human or mouse GLP-1R with EC.sub.50 values of 236 nM and 188 nM,
respectively. These data suggest that the modulator compound binds
to the GLP-1R derived from human or mouse cells with comparable
efficiency; however, functionally it stimulates the human receptor
to a greater degree than the mouse and rat receptors.
[0100] To determine whether the binding of the modulator compound
to GLP-1R is competitive with respect to the binding of the GLP-1
peptide, the modulator compound's binding was studied in the
presence of varying concentrations of .sup.125I-labeled human GLP-1
(7-36). Increasing the concentrations of .sup.125I-labeled human
GLP-1 (7-36) did not change the modulator compound's potency for
inhibition of .sup.125I-labeled human GLP-1 (7-36) binding to
membranes. This data suggests the binding of the modulator compound
to GLP-1R is non-competitive with respect to the binding of GLP-1
(7-36). Finally, the selectivity of the GLP-1R for the modulator
compound was further tested against the GLP-1, GIP and the Glucagon
Receptors in a cAMP functional cell-based assay. The modulator
compound did not stimulate activation of the GIP and Glucagon
Receptor's cAMP production at concentrations up to 100 .mu.M. In
addition, the modulator compound did not block glucagon receptor
function in the presence of glucagon peptide (data not shown).
[0101] The modulator compound may be any compound known to modulate
the activity of the GLP-1R. In one embodiment, the modulator
compound may be a compound known to be an agonist of the GLP-1R. In
another embodiment, the modulator compound may be a compound as
identified in U.S. Provisional Patent applications 61/034,599 and
61/034,606, both disclosures of which are herein incorporated by
reference.
[0102] A schematic of a method used to develop modulators of the
GLP-1R is shown in FIG. 4. Thus, the method may include a first
stage 100 of developing a three-dimensional model of a protein or
polypeptide of interest (e.g., the GLP-1R). As described herein,
the method may comprise providing the amino acid sequence for the
protein or polypeptide of interest 110. The sequence of the protein
or polypeptide of interest may then be compared to amino acid
sequences available in protein sequence databases 120 to identify
proteins or polypeptides that have a known structure, and that may
be homologous in structure to the protein or polypeptide of
interest 130. If a second polypeptide or protein of known structure
that has a sequence that includes regions of identity to the
protein or polypeptide of interest is identified, the second
protein may be used to align conserved residues from the second
protein or polypeptide with the first protein or polypeptide of
interest 140. The aligned residues (hang-points) may then be used
as anchors as the first polypeptide or protein of interest is
threaded along the structure of the second protein or polypeptide
of interest to construct a three-dimensional model of the first
polypeptide or protein of interest 150.
[0103] Once a three-dimensional model of the protein or polypeptide
of interest has been constructed, it may be used in an in silico
assay for screening a plurality of compounds 200. The in silico
assay may comprise generating a library of three-dimensional
structures for potential therapeutic agents 210. For example, in
one embodiment a library of small high information density organic
molecules (i.e., a library, wherein each small molecule within the
library contains at least one functional group of interest) may be
prepared. Such a library is provided by TTProbes.RTM. (TransTech
Pharma., Inc., High Point, N.C.) which is a set of more than 51,000
pharmacophorically diverse molecules of high information density.
The in silico probes may then be docked with the three-dimensional
structure of the protein or polypeptide of interest as described
herein to determine the atomic interactions between the
protein/polypeptide and the compound 220. Optionally, the compound
may also be modified by adding or removing molecular fragments from
the compound 230, and then the modified compounds docked into the
three-dimensional structure of the protein or polypeptide of
interest 240 to determine how the changes to the structure of the
compound may affect the interaction of the compound with the
protein/polypeptide. Such molecular alterations may be made until
there is no longer an apparent improvement in the ability of the
compound to interact with the polypeptide/protein of interest. For
example, for GLP-1R, and using the TTProbes.RTM. in silico library,
over 3,000 candidate potential GLP-1R modulators were identified.
The method may include the option 299 of developing the compounds
identified by in silico screening or performing further testing of
the compounds by a biological assay.
[0104] Thus, still referring to FIG. 4, the putative therapeutic
agents (i.e., potential modulator compounds) identified by in
silico screening may then be evaluated by other types of assays for
biological activity 300. For example, a putative receptor ligand
may be evaluated using a binding assay. For modulators of GLP-1R,
the compounds may be evaluated to determine whether they modulate
the activity of the GLP-1R 310.
[0105] The results of the biological testing may indicate that
certain structures are of interest as displaying efficacy as
modulators of GLP-1R activity. Thus, there is the option 399 to
test at least some of these structures in additional in silico
assays to determine if additional chemical modifications may be
made to the structures to improve the therapeutic effects.
Alternatively, the compounds may be considered as optimized and,
thus, comprise lead compounds for additional animal studies and the
like 400.
Therapeutics
[0106] The invention further provides pharmaceutical compositions
comprising the modulator compounds of the invention. The
pharmaceutical compositions containing a compound of the invention
may be in a form suitable for oral use, for example, as tablets,
troches, lozenges, aqueous or oily suspensions, dispersible powders
or granules, emulsions, hard or soft capsules, or syrups or
elixirs. Compositions intended for oral use may be prepared
according to any known method, and such compositions may contain
one or more agents selected from the group consisting of sweetening
agents, flavoring agents, coloring agents, and preserving agents in
order to provide pharmaceutically elegant and palatable
preparations. Tablets may contain the active ingredient in
admixture with non-toxic pharmaceutically-acceptable excipients
which are suitable for the manufacture of tablets. These excipients
may be, for example, inert diluents, such as calcium carbonate,
sodium carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example corn starch or
alginic acid; binding agents, for example, starch, gelatin or
acacia; and lubricating agents, for example, magnesium stearate,
stearic acid or talc. The tablets may be uncoated or they may be
coated by known techniques to delay disintegration and absorption
in the gastrointestinal tract and thereby provide a sustained
action over a longer period. For example, a time delay material
such as glyceryl monostearate or glyceryl distearate may be
employed. They may also be coated by the techniques described in
U.S. Pat. Nos. 4,356,108; 4,166,452; and 4,265,874, to form osmotic
therapeutic tablets for controlled release.
[0107] Formulations for oral use may also be presented as hard
gelatin capsules where the active ingredient is mixed with an inert
solid diluent, for example, calcium carbonate, calcium phosphate or
kaolin, or a soft gelatin capsules wherein the active ingredient is
mixed with water or an oil medium, for example, peanut oil, liquid
paraffin, or olive oil.
[0108] Aqueous suspensions may contain the active compounds in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example,
sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents may be a naturally-occurring phosphatide, such as
lecithin, or condensation products of an alkylene oxide with fatty
acids, for example, polyoxyethylene stearate, or condensation
products of ethylene oxide with long chain aliphatic alcohols, for
example, heptadecaethyl-eneoxycetanol, or condensation products of
ethylene oxide with partial esters derived from fatty acids and a
hexitol such as polyoxyethylene sorbitol monooleate, or
condensation products of ethylene oxide with partial esters derived
from fatty acids and hexitol anhydrides, for example, polyethylene
sorbitan monooleate. The aqueous suspensions may also contain one
or more coloring agents, one or more flavoring agents, and one or
more sweetening agents, such as sucrose or saccharin.
[0109] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
compound in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already mentioned above. Additional excipients, for example,
sweetening, flavoring, and coloring agents may also be present.
[0110] The pharmaceutical compositions of the invention may also be
in the form of oil-in-water emulsions. The oily phase may be a
vegetable oil, for example, olive oil or arachis oil, or a mineral
oil, for example a liquid paraffin, or a mixture thereof. Suitable
emulsifying agents may be naturally-occurring gums, for example,
gum acacia or gum tragacanth, naturally-occurring phosphatides, for
example, soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol anhydrides, for example, sorbitan
monooleate, and condensation products of said partial esters with
ethylene oxide, for example polyoxyethylene sorbitan monooleate.
The emulsions may also contain sweetening and flavoring agents.
[0111] Also, oily suspensions may be formulated by suspending the
active ingredient in a vegetable oil, for example, arachis oil,
olive oil, sesame oil or coconut oil, or in a mineral oil such as a
liquid paraffin. The oily suspensions may contain a thickening
agent, for example, beeswax, hard paraffin or cetyl alcohol.
Sweetening agents such as those set forth above, and flavoring
agents may be added to provide a palatable oral preparation. These
compositions may be preserved by the addition of an anti-oxidant
such as ascorbic acid.
[0112] Syrups and elixirs may be formulated with sweetening agents,
for example, glycerol, propylene glycol, sorbitol or sucrose. Such
formulations may also contain a demulcent, a preservative and
flavoring and coloring agents.
[0113] The pharmaceutical compositions may also be in the form of a
sterile injectible aqueous or oleaginous suspension. This
suspension may be formulated according to the known methods using
suitable dispersing or wetting agents and suspending agents as
described above. The sterile injectible preparation may also be a
sterile injectible solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conveniently employed as solvent or suspending medium. For this
purpose, any bland, fixed oil may be employed using synthetic mono-
or diglycerides. In addition, fatty acids such as oleic acid find
use in the preparation of injectibles.
[0114] The compositions may also be in the form of suppositories
for rectal administration of the compounds of the invention. These
compositions can be prepared by mixing the drug with a suitable
non-irritating excipient which is solid at ordinary temperatures
but liquid at the rectal temperature and will thus melt in the
rectum to release the drug. Such materials include cocoa butter and
polyethylene glycols, for example.
[0115] For topical use, as, for example, for treatment of
molluscipox virus, creams, ointments, jellies, solutions of
suspensions, etc., containing the compounds of the invention are
contemplated. For the purpose of this application, topical
applications shall include mouthwashes and gargles.
[0116] The compounds of the present invention may also be
administered in the form of liposome delivery systems, such as
small unilamellar vesicles, large unilamellar vesicles, and
multilamellar vesicles. Liposomes may be formed from a variety of
phospholipids, such as cholesterol, stearylamine, or
phosphatidylcholines.
[0117] Pharmaceutically acceptable salts of the compounds of the
present invention, where a basic or acidic group is present in the
structure, are also included within the scope of the invention. The
term "pharmaceutically acceptable salts" refers to non-toxic salts
of the compounds of this invention which are generally prepared by
reacting the free base with a suitable organic or inorganic acid or
by reacting the acid with a suitable organic or inorganic base.
Representative salts include the following salts: Acetate,
Benzenesulfonate, Benzoate, Bicarbonate, Bisulfate, Bitartrate,
Borate, Bromide, Calcium Edetate, Camsylate, Carbonate, Chloride,
Clavulanate, Citrate, Dihydrochloride, Edetate, Edisylate,
Estolate, Esylate, Fumarate, Gluceptate, Gluconate, Glutamate,
Glycollylarsanilate, Hexylresorcinate, Hydrabamine, Hydrobromide,
Hydrocloride, Hydroxynaphthoate, Iodide, Isethionate, Lactate,
Lactobionate, Laurate, Malate, Maleate, Mandelate,
Methanesulfonate, Methylbromide, Methylnitrate, Methylsulfate,
Monopotassium Maleate, Mucate, Napsylate, Nitrate,
N-methylglucamine, Oxalate, Pamoate (Embonate), Palmitate,
Pantothenate, Phosphate/diphosphate, Polygalacturonate, Potassium,
Salicylate, Sodium, Stearate, Subacetate, Succinate, Tannate,
Tartrate, Teoclate, Tosylate, Triethiodide, Trimethylammonium and
Valerate. When an acidic substituent is present, such as --COOH,
there can be formed the ammonium, morpholinium, sodium, potassium,
barium, calcium salt, and the like, of the compounds of the present
invention, for use as the dosage form. When a basic group is
present, such as amino or a basic heteroaryl radical, such as
pyridyl, an acidic salt, such as hydrochloride, hydrobromide,
phosphate, sulfate, trifluoroacetate, trichloroacetate, acetate,
oxalate, male ate, private, malamute, succinct, citrate, tartarate,
fumarate, mandelate, benzoate, cinnamate, methanesulfonate,
ethanesulfonate, picrate and the like. Other salts, which are not
pharmaceutically acceptable, may be useful in the preparation of
compounds of the invention; these form a further aspect of the
invention.
[0118] Thus, in another embodiment of the present invention, there
is provided a pharmaceutical composition comprising a
therapeutically effective amount of a compound identified as
binding to or modulating the GLP-1R, or a pharmaceutically
acceptable salt thereof, and one or more pharmaceutically
acceptable carriers, excipients, or diluents. In an embodiment of
the pharmaceutical composition, the compound identified as binding
to or modulating the GLP-1R is an agonist of the human GLP-1R.
[0119] In another embodiment, the present invention provides a
pharmaceutical composition comprising a therapeutically effective
amount of the compound identified as binding to or modulating the
GLP-1R, and one or more pharmaceutically acceptable carriers,
excipients, or diluents.
[0120] In another embodiment, the present invention provides a
pharmaceutical composition comprising a therapeutically effective
amount of the compound identified as binding to, or modulating the
GLP-1R, and one or more pharmaceutically acceptable carriers,
excipients, or diluents, and further comprising one or more
additional therapeutic agents.
[0121] The compound identified as binding to, or modulating the
GLP-1R, may be administered in an amount sufficient to modulate
GLP-1R activity in a subject. The compound identified as binding to
or modulating the GLP-1R may be administered in the form of an oral
dosage or parenteral dosage unit. In alternative embodiments, the
compound identified as binding to, or modulating the GLP-1R, is
administered as a dose in a range from about 0.01 to 1,000 mg/kg of
body weight per day, or as a dose in a range from about 0.1 to 100
mg/kg of body weight per day, or as a dose in a range from about
0.5 to 10 mg/kg of body weight per day.
EXAMPLES
Example 1
Materials and Methods
[0122] Small organic compound stocks may be prepared as described
in U.S. Provisional Patent applications 61/034,599 and
61/034,606.
Example 2
Computer Modeling
[0123] TransTech Pharma's Translational Technology.TM., described
in U.S. Patent Publications 2003/0125315, 2004/0019432 and
2004/0010515, each of which are incorporated by reference in their
entireties, may be used to model the GLP-1R domain, to discover
specific small molecule inhibitors, and to optimize GLP-1R binding
agents into preclinical drug candidates. TransTech Pharma's
Translational Technology.TM. was designed and developed for rapid
lead generation and optimization of drug candidates. The system
consists of two subtechnologies: TTProbes.TM. and TTPredict.TM..
TTProbes.TM. is a set of greater than 51,000 pharmacologically
diverse molecules. TTPredict.TM. is a computer-based technology
that automates high-throughput three-dimensional target model
building, binding site identification, and conformational analysis.
The TTPredict computer program is used to dock, score, and rank
members of TTProbes set with a target binding site.
[0124] To develop putative modulator compounds, TTPredict.TM. was
used to construct threading and homology models for the GLP-1R.
Example 3
In Silico Assay
[0125] TTProbes molecules may be docked into the putative ligand
binding site of GLP-1R (FIGS. 2A and 2B). The fit of every docked
TTProbes molecule may be computed using several scoring functions.
High-scoring molecules may be identified, and the highest ranking
TTProbes molecules may be submitted for in vivo screening.
[0126] For the GLP-1R, the amino acid residues Ser31, Thr35, Trp39,
Arg43, Thr65, Phe66, Glu68, Asp67, Tyr69, Trp91, Leu89, Tyr88,
Arg121, Arg102, Leu123, Glu127, Trp110 and Glu128 are predicted to
be important in binding to substrates within Site 1, as described
above. In addition, residues: Lys38, Glu34, Trp87, Gln45, Ser49,
Asp53, Pro55, Pro73, Val 83, Arg40, Tyr42, Leu50, Ser93, Pro86,
His99, Val81 and Ser84 are predicted to be important in binding to
substrates within Site 2, as described above.
[0127] The 51,389 probe molecules comprising TTProbes.TM. database
may be docked into the binding site. The fit of every docked
TTProbes molecule may be computed using several scoring functions.
Prior to docking the TTProbes molecules into the GLP-1R active
site, 1000 low energy conformers per molecule may be generated
using Monte-Carlo procedures. TTPredict.TM. may be used to dock in
silico every conformer with the predicted site (e.g., Site 1 and
Site 2) of the GLP-1R. Individual or consensus scoring functions
including LUDI (Bohm, H. J., 1994, J. Comp. Aided Molec. Design,
8:243-256), PLP (Gehlhaar et al, 1995, Chem. Bio., 2:317-324), DOCK
(Meng, E. C., et al., 1992, J. Comp. Chem. 13:505-524), LigFit,
(Accelrys, San Diego, Calif.), JAIN (Jain, A. N, 1996, J. Comp.
Aided Molec. Design 10:427-440), and Poisson-Boltzmann (Honig, B.
et al., 1995, Science, 268:1144-9) may be used. High consensus
scoring TTProbes molecules may be identified and the
highest-ranking probes may be submitted for in vitro (i.e.,
biological) testing.
Biological Assay
[0128] The following assay methods may be utilized to identify
compounds that are effective in showing modulation of the
GLP-1R.
Receptor Binding Assay
[0129] The affinity of compounds for GLP-1 receptor may be studied
in an [.sup.125I]GLP-1(aa 7-36) equilibrium radioligand binding
assay. Membranes from CHO cells expressing the human GLP-1R may be
used in the GLP-1R binding assay. Reactions may be carried out in
96-well plates. Compound may be diluted in 20% DMSO/water. A final
binding assay concentration of the compound ranging from 0.1 nM to
100 uM in 2% DMSO concentration may be used. The final binding
assay conditions may be 25 mM Tris-HCL, pH 7.4 buffer containing 10
mM MgCl.sub.2, 1 mM DTT, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% BSA, 1-10
ug membrane, 20-200 pM [.sup.125I]GLP-1 (aa 7-36) (SA=2200
Ci/mmol.), (Perkin Elmer part no. NEX308), and compound in final
DMSO concentration of 2% (final assay volume of 100 uL). Positive
control wells (C+) may lack compound, and negative control wells
(C-) may lack compound and may contain excess non-radio-labeled
GLP-1 (1 .mu.M). Non-specific binding (NSB) may be determined for
each compound concentration by addition of cold excess GLP-1 (1
.mu.M). The reaction may be carried out at room temp for 120 min.
Membranes containing bound [.sup.125I]GLP-1 (aa 7-36) ligand may be
isolated following filtration onto Unifilter-96 GF/C filter plates
(PerkinElmer part no. 6005177) using a cell harvester instrument.
Plates may be washed 5 times with cold 25 mM Tris-HCL, pH 7.5
containing 0.05% bovine serum albumin (BSA). Following filtration,
50 uL of Microscint PS (Packard part no. 6013631) may be added and
plates may be sealed with TopSeal-A adhesive seals (Packard part
no. 6005185). .sup.125I isotope bound to the Unifilter-96 GF/C
plates may be counted using a TopCount instrument (Packard).
[0130] Percent inhibition of [.sup.125I] GLP-1 (aa 7-36) binding
may be calculated according to the equation
100.times.1-{(Sample.sub.cpm-NSB.sub.cpm)/C+.sub.cpm-C-.sub.cpm)}.
Percent inhibition of [.sup.125I]GLP-1 binding (Y) vs compound
concentration (X) data may be generated. The IC.sub.50 values may
be calculated by fitting the data using parameters for a sigmoidal
dose response, variable slope nonlinear regression (GraphPAD Prizm,
San Diego, Calif.) according to the equation:
Y=Bottom+(Top-Bottom)/(1+10 ((Log EC50-X)*HillSlope))
X is the logarithm of concentration. Y is the response. Y starts at
Bottom and goes to Top with a sigmoid shape.
Functional Cell-Based Assay
[0131] The efficacy of GLP-1R agonists may be studied in a cAMP
production functional assay using HEK-293 cells expressing the
cloned human GLP-1R. GLP-1R-expressing cells (10,000 cells per 0.1
mL) may be plated in 96-well plates in Dulbecco's Modified Eagles
Media (DMEM) containing 10% fetal bovine serum and
penicillin-streptomycin. Following overnight incubation of cells,
media may be removed, and compounds (at concentrations ranging from
0.0001 to 100 .mu.M) may be added to monolayer cells in Iscove's
Modified Dulbecco's Medium (IMDM), 100 .mu.M RO 20-1724 PDE
inhibitor, 0.1% BSA, 2% DMSO in a final volume of 100 .mu.L, and
incubated for 30 min at 37.degree. C. 95% O.sub.2, 5% CO.sub.2 in a
humidified incubator. cAMP production may be quantitated using a
homogenous time-resolved fluorescence detection system (cAMP
Dynamic, CIS bio International). GLP-1 may typically produce cAMP
production dose response curves with EC.sub.50 values ranging from
about 0.01 .mu.M-100 .mu.M, typically ranging from about 0.02 .mu.M
to about 10 .mu.M. Receptor activation may be expressed as
percentage relative to maximal GLP-1-induced cAMP accumulation.
Percent GLP-1 activation vs compound concentration dose response
curves may be generated by fitting the data using a sigmoidal dose
response curve-fitting program (GraphPAD Prizm). Several compounds
may exhibit an EC-50 from about 20 nM to about 500 nM with GLP-1R
activation ranging from about 32% to about 64%.
[0132] The specificity of GLP-1 agonists for the GLP-1R may be
confirmed by performing the assay with vector-control mock cells
which lack the cloned human GLP-1R. All compounds may be devoid of
cAMP accumulation in the mock-transfected cell lines.
[0133] While the invention has been described and illustrated with
reference to certain preferred embodiments thereof, those skilled
in the art will appreciate that various changes, modifications and
substitutions can be made therein without departing from the spirit
and scope of the invention.
* * * * *