U.S. patent application number 16/858387 was filed with the patent office on 2020-09-24 for assay for exo-site binding molecules.
This patent application is currently assigned to President and Fellows of Harvard College. The applicant listed for this patent is President and Fellows of Harvard College. Invention is credited to David R. Liu, Juan Pablo Maianti.
Application Number | 20200299685 16/858387 |
Document ID | / |
Family ID | 1000004882142 |
Filed Date | 2020-09-24 |
![](/patent/app/20200299685/US20200299685A1-20200924-D00000.png)
![](/patent/app/20200299685/US20200299685A1-20200924-D00001.png)
![](/patent/app/20200299685/US20200299685A1-20200924-D00002.png)
![](/patent/app/20200299685/US20200299685A1-20200924-D00003.png)
![](/patent/app/20200299685/US20200299685A1-20200924-D00004.png)
![](/patent/app/20200299685/US20200299685A1-20200924-D00005.png)
![](/patent/app/20200299685/US20200299685A1-20200924-D00006.png)
![](/patent/app/20200299685/US20200299685A1-20200924-D00007.png)
![](/patent/app/20200299685/US20200299685A1-20200924-D00008.png)
![](/patent/app/20200299685/US20200299685A1-20200924-D00009.png)
![](/patent/app/20200299685/US20200299685A1-20200924-D00010.png)
View All Diagrams
United States Patent
Application |
20200299685 |
Kind Code |
A1 |
Maianti; Juan Pablo ; et
al. |
September 24, 2020 |
ASSAY FOR EXO-SITE BINDING MOLECULES
Abstract
Methods for the identification of agents the bind to exo-sites
of proteins are provided. Agents identified by the methods
described herein and pharmaceutical compositions comprising the
identified agents are also provided. Methods of using an identified
agent for the treatment or prevention of a disease, disorder, or
condition are also provided, including methods of treating or
preventing a disease associated with reduced, elevated, or ectopic
expression or aberrant activity of a protein comprising an
exo-site.
Inventors: |
Maianti; Juan Pablo;
(Cambridge, MA) ; Liu; David R.; (Cambridge,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College |
Cambridge |
MA |
US |
|
|
Assignee: |
President and Fellows of Harvard
College
Cambridge
MA
|
Family ID: |
1000004882142 |
Appl. No.: |
16/858387 |
Filed: |
April 24, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16177141 |
Oct 31, 2018 |
10640767 |
|
|
16858387 |
|
|
|
|
PCT/US2017/058722 |
Oct 27, 2017 |
|
|
|
16177141 |
|
|
|
|
62414640 |
Oct 28, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C40B 40/10 20130101;
C12N 15/1093 20130101; C40B 20/04 20130101; C40B 40/14
20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10; C40B 40/10 20060101 C40B040/10; C40B 40/14 20060101
C40B040/14 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
number GM065865 awarded by National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method of identifying an agent that binds an exo-site of a
protein, the method comprising: providing a first variant of the
protein, wherein the protein comprises an exo-site; providing a
second variant of the protein, wherein the exo-site of the second
variant comprises at least one different amino acid than the
exo-site of the first variant; contacting a library of candidate
agents with each of the first and second variants; determining an
enrichment-dependent parameter of each candidate agent to each of
the first and second variants by a library binding or enrichment
assay; comparing, for each candidate agent, the binding to the
first variant with the binding to the second variant, wherein if
the enrichment-based parameter using the first protein variant is
greater than the enrichment-based parameter using the second
protein variant, then the candidate agent is identified as an agent
that binds an exo-site of the protein.
2. The method of claim 1, wherein the exo-site comprises a binding
pocket that modulates the interactions of the protein with one or
more substrates, one or more metabolites, or one or more native
partners of the protein when an agent is bound to the exo-site.
3. The method of claim 1 or 2, wherein the exo-site comprises a
binding pocket that modulates the substrate selectivity or binding
preferences of the protein when an agent is bound to the
exo-site.
4. The method of any one of claims 1-3, wherein the exo-site
comprises a binding pocket defined by amino acids that are at least
about 5 angstroms away from the amino acids of the protein
catalytic site.
5. The method of any one of claims 1-3, wherein the exo-site
comprises a binding pocket defined by amino acids that are at least
about 10 angstroms away from the amino acids of the protein
catalytic site.
6. The method of any one of claims 1-3, wherein the exo-site
comprises a binding pocket defined by amino acids that are at least
about 15 angstroms away from the amino acids of the protein
catalytic site.
7. The method of any one of claims 1-3, wherein the exo-site
comprises a binding pocket defined by amino acids that are at least
about 20 angstroms away from the amino acids of the protein
catalytic site.
8. The method of any one of claims 1-3, wherein the exo-site
comprises a binding pocket defined by amino acids that are at least
about 25 angstroms away from the amino acids of the protein
catalytic site.
9. The method of any one of claims 1-3, wherein the exo-site
comprises a binding pocket defined by amino acids that are at least
about 30 angstroms away from the amino acids of the protein
catalytic site.
10. The method of any one of claims 1-3, wherein the exo-site
comprises a binding pocket defined by amino acids that are at least
about 35 angstroms away from the amino acids of the protein
catalytic site.
11. The method of any one of claims 1-3, wherein the exo-site
comprises a binding pocket defined by amino acids that are at least
about 40 angstroms away from the amino acids of the protein
catalytic site.
12. The method of any one of claims 1-3, wherein the exo-site
comprises a binding pocket defined by amino acids that are at least
about 50 angstroms away from the amino acids of the protein
catalytic site.
13. The method of any one of claims 4-12, wherein the amino acids
of the protein catalytic site are the amino acids that participate
in chemical steps at the protein active site.
14. The method of any one of claims 1-13, wherein the exo-site
comprises a binding pocket defined by amino acids that can be
replaced with different amino acids without significantly altering
the activity of the protein.
15. The method of claim 14, wherein the activity of the protein is
not significantly altered if the K.sub.m or V.sub.max is altered by
a factor of between about 1.1-fold and about 10-fold for the same
substrate and under the same assay conditions.
16. The method of any one of claims 1-15, wherein the first variant
is a wild-type protein or a truncated domain of a wild-type
protein.
17. The method of any one of claims 1-16, wherein the second
variant comprises a mutant of the first variant, wherein one amino
acid of the exo-site in the first variant is replaced with a
different amino acid in the second variant.
18. The method of claim 17, wherein the replacement amino acid in
the second variant comprises a side chain with a higher number of
non-hydrogen atoms than the replaced amino acid in the first
variant.
19. The method of any one of claims 1-18, wherein the first and
second variants are two different isoforms of the protein, or
truncated domains from two different isoforms.
20. The method of any one of claims 1-19, wherein the library is a
library of small molecules.
21. The method of any one of claim 1-19, wherein the library is
library of polynucleotides, polypeptides, or polysaccharides.
22. The method of any one of claims 1-21, wherein the library is an
encoded library.
23. The method of claim 22, wherein the step of contacting
comprises incubating the encoded candidate agents with each of the
first and second variants in parallel.
24. The method of claim 22, wherein the step of contacting
comprises incubating the encoded candidate agents with each of the
first and second variants in series.
25. The method of any one of claims 22-24, wherein the library is a
DNA-encoded library.
26. The method of claim 25 further comprising amplifying the DNA
codons of DNA encoded agents with PCR, optionally wherein the PCR
primers are encoded for the first or second variant.
27. The method of claim 26 further comprising sequencing the PCR
amplified DNA.
28. The method of claim 27, wherein the step of determining binding
comprises measuring the relative or absolute library member
sequence abundance for the candidate agent DNA coding
sequences.
29. The method of claim 28, wherein the step of determining binding
comprises measuring an enrichment of postselection sequence
abundance for the candidate agent codons over preselection sequence
abundance.
30. The method of claim 29, wherein the step of comparing comprises
comparing the measured enrichment in sequence abundance for each
candidate agent after incubation with the first variant with the
measured enrichment in sequence abundance after incubation with the
second variant.
31. The method of any one of claims 1-30, wherein the protein is
insulin degrading enzyme (IDE).
32. The method of claim 31, wherein the second variant is IDE with
a mutation at Ala479, Leu201, Glu205, Tyr302, Thr316, Val360,
Gly361, Gly362, Leu364, Ala198, Trp199, Phe202, Tyr314, Ile374, or
a combination thereof.
33. The method of claim 31, wherein the second variant is IDE with
the mutation A479L, V360Q, G362Q, A198T, W199F, F202R, Y314F,
I374Q, or a combination thereof.
34. An agent that binds an exo-site of a protein identified by the
method of any one of claims 1-33.
35. A pharmaceutical composition comprising an agent of claim 34,
or a pharmaceutically acceptable salt thereof, and a
pharmaceutically acceptable excipient.
36. A method of treating a disease in a subject in need thereof,
the method comprising administering to the subject a
therapeutically effective amount of an agent of claim 34, or a
pharmaceutically acceptable salt thereof, or a pharmaceutical
composition of claim 35.
37. The method of claim 36, wherein the disease is associated with
reduced, elevated, or ectopic expression of a protein comprising an
exo-site.
38. The method of claim 36, wherein the disease is associated with
aberrant activity of a protein comprising an exo-site.
39. A method of modulating the substrate selectivity and
processivity of a protein comprising an exo-site in a biological
sample, the method comprising contacting the biological sample with
an agent of claim 34, or a pharmaceutically acceptable salt
thereof, or a pharmaceutical composition of claim 35.
40. A method of modulating the substrate selectivity and
processivity of a protein comprising an exo-site in a subject, the
method comprising administering to the subject an agent of claim 34
or a pharmaceutically acceptable salt thereof, or a pharmaceutical
composition of claim 35.
41. A method of inhibiting the activity on one or more substrates
by a protein comprising an exo-site in a biological sample, the
method comprising contacting the biological sample with an agent of
claim 34, or a pharmaceutically acceptable salt thereof, or a
pharmaceutical composition of claim 35.
42. A method of inhibiting the activity on one or more substrates
by a protein comprising an exo-site in a subject, the method
comprising administering to the subject an agent of claim 34, or a
pharmaceutically acceptable salt thereof, or a pharmaceutical
composition of claim 35.
43. An agent that binds an exo-site of IDE identified by the method
of any one of claims 31-33.
44. A pharmaceutical composition comprising an agent of claim 43,
or a pharmaceutically acceptable salt thereof, and a
pharmaceutically acceptable excipient.
45. A method of treating a metabolic disorder in a subject in need
thereof, the method comprising administering to the subject a
therapeutically effective amount of an agent of claim 34, or
pharmaceutically acceptable salt thereof, or a pharmaceutical
composition of claim 35.
46. The method of claim 45, wherein the metabolic disorder is
diabetes.
47. The method of claim 45, wherein the metabolic disorder is
hyperglycemia, impaired glucose tolerance, obesity, sodium
imbalance, or hypertension.
48. A method of inhibiting the degradation of insulin in a
biological sample, the method comprising contacting the biological
sample with an agent of claim 34, or a pharmaceutically acceptable
salt thereof, or a pharmaceutical composition of claim 35.
49. A method of inhibiting the degradation of insulin in a subject,
the method comprising administering to the subject an agent of
claim 34, or a pharmaceutically acceptable salt thereof, or a
pharmaceutical composition of claim 35.
50. The method of claim 48 or 49, wherein inhibiting the
degradation of insulin is selective over inhibiting the degradation
of glucagon.
51. The method of claim 50, wherein the selectivity is between
about 2-fold and about 10-fold, between about 10-fold and about
50-fold, between about 50-fold and about 100-fold, between about
100-fold and about 500-fold, or between about 500-fold and about
1000-fold.
Description
RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C.
.sctn..sctn. 120 and 365(c) to and is a continuation of
international PCT Application, PCT/US2017/058722, filed Oct. 27,
2017, which claims priority under 35 U.S.C. .sctn. 119(e) to U.S.
Provisional Patent Application Ser. No. 62/414,640, filed Oct. 28,
2016, the entire content of each are incorporated herein by
reference.
BACKGROUND
[0003] Screening methods for the identification of drug compounds
are central to effective drug discovery. Accordingly, the
efficiency and cost effectiveness of drug discovery can be improved
by selection-based methods with higher throughput and lower
infrastructure requirements as compared with many screening
methods.
SUMMARY
[0004] The traditional approach for identification of agents that
inhibit or otherwise modulate proteins relies on assays based on
the protein's activity. For example, a library of inhibitors may be
screened against a protease in a multi-well plate format through
the detection of cleavage of a fluorogenic internally-quenched
substrate that mimics the natural substrate. The identified
inhibitors of the protease's cleavage activity typically interact,
covalently or non-covalently, at or near the catalytic site and may
outcompete native substrates by having a higher binding affinity
for the protein. Identification of compounds that bind to sites
other than the catalytic site, such as exo-sites (e.g., allosteric
sites, distal binding pockets, regulatory sites, and non-catalytic
domains), is significantly less likely using an activity-based
assay reported by a small substrate mimic. Additionally, if an
enzyme is responsible for the processing of multiple substrates in
vivo, the outcomes that favorably modulate a disease by affecting
activity with regard to one substrate cannot easily be separated
from adverse effects due to the enzyme's pleiotropic action on
other substrates. For example, small molecule inhibitors of insulin
degrading enzyme (IDE) may improve insulin signaling by inhibiting
proteolysis of insulin but may cause adverse effects such as
increased blood sugar levels by inhibiting proteolysis of glucagon
by IDE. Screening assays relying on a fluorogenic insulin or
glucagon mimic may be unsuitable for identifying a selective
inhibitor for insulin degradation that does not also inhibit
glucagon degradation, or inhibits it to a lesser extent.
Protein-substrate interactions at sites other than the catalytic
site may modulate the activity or selectivity of proteins in the
case of proteins that process multiple substrates. Such sites may
be referred to as exo-sites, distal binding pockets, allosteric
sites, regulatory sites, or non-catalytic domains. Herein we use
the term exo-site as the most general definition that embodies all
binding sites for molecules that are typically distinct from the
catalytic site, but binding of a compound at the site may affect
the activity of the protein, particularly the catalytic activity
and/or binding of substrates near the pocket.
[0005] Affinity based selection methods may also be more suitable
than screens employing activity based assays for discovery of
agents that interact with proteins away from the active site, such
as at exo-sites. Such methods may be suitable for discovery of
agents that are intended to modulate protein selectivity rather
than to behave as competitive inhibitors for native substrates.
High-throughput selection on diverse libraries can identify novel
exo-site binding site agents or scaffolds, and may also be used to
identify unknown protein-agent interactions at previously unknown
exo-sites and other orphan binding pockets beyond the active
site.
[0006] In one aspect, the present disclosure provides methods,
compositions, and systems useful for identifying agents that
interact with exo-sites of a protein, distal binding pockets,
allosteric sites, or non-catalytic domains. Agents identified by
the methods and systems described herein may be used as
pharmaceutical agents for the treatment of a disease associated
with the protein or may be used in the design and development of a
pharmaceutical agent. Agents identified may also be used as
development leads for further drug design and discovery of
pharmaceuticals that target exo-sites of a protein, distal binding
pockets, allosteric sites, or non-catalytic domains. The method
typically involves performing a screen of a library of agents using
two variants of a protein, wherein the two variants have a
structural difference that fills or changes the shape of the
three-dimensional shape of the exo-site pocket of the protein.
Typically one protein variant includes a wild type exo-site, and
the other variant includes a mutated exo-site. For example, in
certain embodiments, a first variant may be the wild-type protein,
and a second variant may be a mutant with a "bump" due to a
sequence variation at the exo-site. The "bump" in the exo-site will
influence the binding of candidate agents at the exo-site. By
comparing the binding of the candidate agents to the exo-site of
the wild-type protein versus that of the mutant, candidate agents
that have greater binding for the wild-type protein can be
identified as agents that bind in the exo-site. Alternatively, the
binding or enrichment measurement results for the mutant variant
can be applied as a mathematical penalty for calculations using the
binding or enrichment measurement results from the wild-type
variant. The mathematical penalty may be derived from subtraction,
division, multiplication, or similar operation with the data. The
outcome of applying the calculation for all library members is to
preferentially highlight the exo-site binders, or conversely lower
the signal of the non-exo-site binders, improve the noise-signal
ratio of non-specific binders, or lower the stochastic background
noise to facilitate hit-calling of exo-site binders.
[0007] Agents identified using the methods described herein may be
further characterized using any assays and methods known in the
art, for example, biochemical assays, southwestern blotting,
western blotting, activity-based probes, capillary electrophoresis,
affinity chromatography, competition studies, siRNA studies, in
vitro cytotoxicity studies, in vivo animal model studies, etc. The
methods of identifying agents that bind to an exo-site are amenable
to high-throughput screening techniques including robotic assisted
fluid delivery, combinatorial chemistry, microfluidics, and
computer analysis of the resulting data. In certain embodiments, a
collection of compounds such as a combinatorial library may be
provided for screening. In other embodiments, a historical
collection of chemical compounds may screened using the inventive
methods. The methods may also be used to test one candidate
compound at a time.
[0008] Provided herein are methods of identifying and facilitating
the de novo discovery of agents that binds an exo-site of a
protein, the method comprising providing a first variant of the
protein, wherein the protein comprises an exo-site; providing a
second variant of the protein, wherein the exo-site of the second
variant comprises at least one different amino acid (e.g.,
substitution, deletion, or addition) than the exo-site of the first
variant; contacting a candidate agent with each of the first and
second variants; determining an enrichment-dependent parameter of
the candidate agent to each of the first and second variants
through a one-pot library binding or enrichment assay; comparing
(e.g., by visual or mathematic procedures) the results of a
candidate agent binding to the first variant with the binding to
the second variant, wherein if the enrichment-based parameter using
the first protein variant is greater than the enrichment-based
parameter using the second protein variant, then the candidate
agent is identified as an agent that binds an exo-site of the
protein. In certain embodiments, the protein is an enzyme (e.g.,
protease). In certain embodiments, the method is performed on a
library of candidate agents. The agents may be screened in a
high-throughput format that allows for the screening of tens,
hundreds, thousands, millions, or even billions of candidate agents
in parallel. In certain embodiments, the method involves
computation, mathematical operations, additive smoothing, or
statistical treatments to compare the enrichment-based parameters
from the two variants. In certain embodiments, the mathematical
operation involves subtraction, division, or multiplication of a
library member's enrichment-based parameter obtained the presence
of one variant (E.sub.1) versus the second variant (E.sub.2). In
certain embodiments the mathematical operations are:
E.sub.1-E.sub.2; or E.sub.1/E.sub.2; or x.E.sub.1-y.E.sub.2; or
x.E.sub.1/y.E.sub.2; or x.E.sub.1.sup.a-y.E.sub.2.sup.b; or
x.E.sub.1.sup.a/y.E.sub.2.sup.b; or
(x.E.sub.1.sup.a-y.E.sub.2.sup.b).sup.c; or
(E.sub.1.sup.a/E.sub.2.sup.b).sup.c, where x, y, a, b and c
represent variable coefficients.
[0009] In certain embodiments, the exo-site is a binding pocket
that modulates the interactions of the protein with one or more
substrates, one or more metabolites, one or more binding partners,
or one or more native partners of the protein when an agent is
bound to the exo-site. In certain embodiments, the exo-site
comprises a binding pocket that modulates the substrate selectivity
or binding preferences of the protein when an agent is bound to the
exo-site. In certain embodiments, the exo-site comprises a binding
pocket defined by amino acids that are at least about 2, 3, 5, 10,
15, 20, 25, 30, 35, 40, 45, or 50 angstroms away from the amino
acids of the protein's active site. In some embodiments, the amino
acids of the protein active site are the amino acids that
participate in catalysis at the protein's active site (e.g.,
catalytic site). In certain embodiments, the exo-site comprises a
binding pocket defined by amino acids that can be replaced with
different amino acids without significantly altering the activity
of the protein, for example, wherein the activity of the protein is
not significantly altered if the K.sub.m or V.sub.max is altered by
a factor of between about 1.1-fold and about 10-fold for the same
substrate and under the same assay conditions. In certain
embodiments, the amino acids of the exo-site are typically not
directly involved in catalysis of biochemical steps, transition
state stabilization, substrate, or cofactor binding. In certain
embodiments, the stability and activity over time for the variants
is similar by a factor of between about 1.1-fold and about
10-fold.
[0010] In certain embodiments, the first variant of the protein is
a wild-type protein or truncated variant of the wild-type protein.
In certain embodiments, the first variant includes a wild-type
exo-site. In certain embodiments, the second variant comprises a
mutant of the first variant, wherein one amino acid of the exo-site
in the first variant is replaced with a different amino acid in the
second variant. In some embodiments, the replacement amino acid in
the second variant comprises an amino acid side chain with a higher
number of non-hydrogen atoms (e.g., C, N, O, S) than the replaced
amino acid in the first variant. For example, an alanine in the
exo-site is replaced with a leucine in the second variant. In
certain embodiments, the first and second variants are two
different isoforms of a protein or truncated variant of two
different isoforms.
[0011] Any type of molecules may be screened in a library format to
identify exo-site binders. For example, small molecules, nucleic
acids, saccharides, polysaccharides, peptides, proteins, organic
molecules, organometallic molecules, etc. may be screened using the
inventive methods and systems. In certain embodiments, a library of
agents is screened. In certain embodiments, the library is a
library of small molecules. In certain embodiments, the library is
a library of polynucleotides, polypeptides, or polysaccharides. In
certain embodiments, the library is an encoded library. In some
embodiments, the library is a DNA-encoded library. In some
embodiments, the step of contacting comprises incubating the
encoded candidate agents with each of the first and second variants
in series or in parallel. The method may further comprise
amplifying the DNA codons of DNA-encoded agents by PCR, optionally
wherein the PCR primers are encoded to identify the compound to
which it is attached. In some embodiments, the method further
comprises sequencing the PCR amplified DNA. In certain embodiments,
the step of determining binding comprises measuring the relative or
absolute library member sequence abundance for the candidate agent
DNA coding sequences. In certain embodiments, the step of
determining binding comprises measuring an enrichment of
post-selection sequence abundance for the candidate agent codons
over pre-selection sequence abundance. In certain embodiments, the
step of comparing comprises comparing the measured enrichment in
sequence abundance for each candidate agent after incubation with
the first variant with the measured enrichment in sequence
abundance after incubation with the second variant. In certain
embodiments, the method involves computation, mathematical
operations, additive smoothing, or statistical treatments to
compare the enrichment-based parameters from the two variants. In
certain embodiments, the mathematical operation involves
subtraction, division, or multiplication of a library member's
enrichment-based parameter obtained the presence of one variant
(E.sub.1) versus the second variant (E.sub.2). In certain
embodiments the mathematical operations are: E.sub.1-E.sub.2; or
E.sub.1/E.sub.2; or x.E.sub.1-y.E.sub.2; or x.E.sub.1/y.E.sub.2; or
x.E.sub.1.sup.a-y.E.sub.2.sup.b; or
x.E.sub.1.sup.a/y.E.sub.2.sup.b; or
(x.E.sub.1.sup.a-y.E.sub.2.sup.b).sup.c; or
(E.sub.1.sup.a/E.sub.2.sup.b).sup.c, where x, y, a, b and c
represent variable coefficients.
[0012] In another aspect, the present disclosure provides an agent
that binds an exo-site of a protein identified by a method
described herein. The disclosure also provides pharmaceutical
compositions comprising an agent that binds an exo-site of a
protein identified by a method described herein.
[0013] In another aspect, the present disclosure provides a method
of treating or preventing a disease, disorder, or condition in a
subject in need thereof, the method comprising administering to the
subject a therapeutically effective amount of an agent that binds
an exo-site of a protein as identified by a method described
herein, or a pharmaceutically acceptable salt, or pharmaceutical
composition thereof. In certain embodiments, the disease, disorder,
or condition is associated with reduced, elevated, or ectopic
expression/activity of a protein with an exo-site. In certain
embodiments, the disease, disorder, or condition is associated with
the aberrant activity of a protein with an exo-site.
[0014] Any protein such as an enzyme may be used in accordance with
the invention. The protein may or may not be known to have an
exo-site (e.g., distal binding pocket, allosteric site, or
non-catalytic domain) and its function may or may not be known or
assigned (orphan exo-site). In certain embodiments, the protein
being used in the inventive method is insulin degrading enzyme
(IDE). In certain embodiments, the agent binds an exo-site of IDE.
In certain embodiments, the method of treating a disease, disorder,
or condition is for a disease, disorder, or condition associated
with reduced, elevated, or ectopic expression/activity of IDE. In
certain embodiments, the method of treating a disease, disorder, or
condition is for a disease, disorder, or condition associated with
aberrant activity of IDE. In some embodiments, the protein is IDE,
and the disease is metabolic disorder. In some embodiments, the
metabolic disorder is diabetes. In some embodiments, the metabolic
disorder is hyperglycemia, impaired glucose tolerance, obesity,
sodium imbalance, or hypertension.
[0015] The details of certain embodiments of the invention are set
forth in the Detailed Description of Certain Embodiments, as
described below. Other features, objects, and advantages of the
invention will be apparent from the Definitions, Detailed
Description, Examples, Figures, and Claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The accompanying drawings, which constitute a part of this
specification, illustrate several embodiments of the invention and
together with the description, serve to explain the principles of
the invention.
[0017] FIG. 1A is a scheme showing an exemplary identification
strategy for the discovery of distal or exo-site binding agents of
insulin degrading enzyme (IDE) by comparison of in vitro selections
of DNA-encoded libraries performed against a wildtype variant
(e.g., N-His.sub.6-IDE) and an exo-site "bumped" mutant variant
(shown as an orange star, e.g., N-His.sub.6-A479L-IDE). FIG. 1B and
FIG. 1C show enrichment results for the strategy in FIG. 1A, which
reveal the DNA barcodes that correspond to the building blocks and
structures of the IDE exo-site inhibitor 6b (D5-A12-B8-C6 barcode,
red marker) and other structurally similar hits of the D5-A12-B8-Cx
barcode family (yellow markers).
[0018] FIG. 2A shows gel electrophoresis of N-His.sub.6-WT-IDE
(variant 1) and N-His.sub.6-A479L-IDE (variant 2) expressed in E.
coli and purified using Ni-NTA beads. FIG. 2B shows q-PCR
monitoring of library abundance following incubation of the
DNA-encoded library and variants 1 and 2 in subsequent washings,
imidazole elution, and heat elution. FIG. 2C shows DNA sequencing
reads for each sample analyzed by Illumina MiSeq single-end 50 read
run.
[0019] FIG. 3 shows structures of the building blocks comprising
the DNA-encoded library used for validation of the in vitro
selection method.
[0020] FIG. 4A and FIG. 4B show examples of an in vitro selection
of a DNA-encoded library (FIG. 3) displaying enrichment of
non-specific binders to wildtype N-His.sub.6-IDE (variant 1) and an
exo-site "bumped" mutant N-His.sub.6-A479L-IDE (variant 2). This
library also encodes the known hit 5b highlighted in red, which is
not enriched above the noise. FIG. 4C and FIG. 4D show a
demonstration of a mathematical penalty based on the
enrichment-parameter against A479L-IDE (variant 2) shown in FIG. 4B
applied to the enrichment results of the wildtype IDE (variant 1)
selection shown in FIG. 4A. After this calculation the hit 5b
appears segregated from other library members because it is a
specific exo-site binder of variant 1. FIG. 4E shows the structure
of exo-site IDE inhibitors 5b and 6b from the hit family
D5-A12-B8-Cx, and FIG. 4F shows example structures of a family of
hits that are non-specific binders of IDE
(Dx-Ax-B.sub.1/8-C.sub.10).
[0021] FIGS. 5A-5F show structural data and assay experiments that
demonstrate the structural basis for exo-site binding in IDE. FIG.
5A shows the X-ray co-crystal structure of IDE bound to macrocyclic
inhibitor 6b (2.7 .ANG. resolution, Protein Data Bank entry: 4LTE).
Macrocycle 6b is represented as a ball-and-stick model, and the
catalytic zinc atom is represented as a sphere in the indicated
catalytic active site. FIG. 5B shows the relative position of
macrocycle 6b bound 11 .ANG. from the catalytic zinc atom. FIG. 5C
shows the electron density map (composite omit map contoured at 1
.sigma.) and model of IDE-bound macrocycle 6b interacting with a 10
.ANG.-deep hydrophobic pocket. FIG. 5D and FIG. 5E show activity
assays for wild-type or mutant human IDE variants in the presence
of 6bK. FIG. 5F shows a view of the exo-site of IDE with macrocycle
6b bound with the mutated residues from FIG. 5D and FIG. 5E
labeled.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0022] The present disclosure provides methods, compositions, and
systems for identifying an agent that binds an exo-site of a
protein. An exo-site is a secondary binding site, remote from the
catalytic site, on a protein. Also provided are agents that bind an
exo-site of a protein as identified by the inventive method,
pharmaceutical compositions comprising the agent, methods of
treating a disease using the agent, and methods of modulating the
selectivity or activity of a protein using the agent, wherein the
agent is identified by a method described herein. In certain
embodiments, the protein is an enzyme.
[0023] In one aspect, the disclosure provides methods of
identifying an agent that binds an exo-site of a protein. The
method typically involves comparison of the binding of a candidate
agent to two (or more) variants of a protein: a first variant,
wherein the protein comprises an exo-site; and a second variant,
which also includes the exo-site, but the exo-site of the second
variant differs from the exo-site of the first variant. The second
variant may differ from the first variant in the presence of a
"bump" or other feature in the exo-site binding pocket. The "bump"
may consist of one or more amino acid side chains present in the
second variant that differ from side chains present in the first
variant. Without wishing to be bound by any particular theory, the
"bump" may impair or otherwise affect the binding of an agent to
the exo-site in the second variant relative to the binding of the
agent to the exo-site in the first variant. The "bump" may impair
binding by, for example, altering the steric environment of the
exo-site (e.g., by taking up volume in the exo-site binding pocket
which the agent would otherwise occupy), altering specific binding
interactions between the exo-site and an agent (e.g., by disrupting
hydrogen bonding that would otherwise form between an exo-site
residue and the agent), or by changing the hydrophobic/hydrophilic
character of the exo-site (e.g., by introducing a more or less
polar side chain in the exo-site).
[0024] The first and second variant may be variants of the same
protein or of related proteins. Typically the variants are variants
of the same protein. In some embodiments, the second variant is a
mutant of the first variant. First and second variants that are
isoforms of the same protein or truncated versions of different
isoforms of a protein are also contemplated. Without wishing to be
bound by a particular theory, different isoforms of a protein with
an exo-site may display high evolutionarily conservation for
sequences or residues that comprise the catalytic site, which are
required for biochemical steps, but less evolutionary conservation
for sequences or residues comprising the exo-site. Thus the method
may be useful for identifying binding agents that display high
specificity for the exo-site in one or more isoforms of a protein
among a family of evolutionarily related proteins. In some
embodiments, the first and second variants are different isoforms
of a protein or truncated variants of different isoforms of a
protein. Other differences between the first and second variant are
also contemplated, such as a second variant that is prepared by a
chemical modification of a first variant. In some embodiments, the
first variant is a wild-type protein or a truncated variant of a
wild-type protein. In some embodiments, the first variant is not a
wild-type protein or truncated variant of a wild-type protein. In
some embodiments, the second variant is a wild-type protein or
truncated variant of a wild-type protein. In some embodiments, the
second variant is not a wild-type protein or a truncated variant of
a wild-type protein. In some embodiments, the first and second
variants are proteins from the same species. In some embodiments,
the first and second variants are proteins from different species.
In some embodiments, the first and second variants are proteins
that result in different phenotypes, for example, a first variant
resulting in a healthy or normal phenotype, prevention or
improvement of a disease phenotype, and a second variant resulting
in a phenotype associated with a disease, disorder, adverse effect
or condition associated with the protein.
[0025] The first and second variant may only differ by one or more
changes in the exo-site (e.g., one or more amino acid mutations) or
may have additional differences. In some embodiments, the first and
second variant are identical or homologous except for the mutation
of a single amino acid of the exo-site. In some embodiments, the
first and second variant are identical except for the mutation of
between one and two, one and three, one and five, two and five, or
five and ten amino acids of the exo-site. In some embodiments, the
first and second variant have homologous protein sequences with at
least 80%, at least 85%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98%, at least 99%, or at least 99.5% sequence
identity.
[0026] A second variant that is a mutant may be provided by any
method known in the art, as the disclosure is not limited in that
way. For example, a mutant may be a naturally occurring variant of
the wild-type protein, may be prepared by chemical peptide
synthesis, chemical modification of a first variant, or may be
prepared by mutagenesis (e.g., site-directed mutagenesis). In some
embodiments, the mutant is provided by recombinant expression of a
mutant gene construct in host cells.
[0027] The second variant (e.g., mutant, isoform, homolog) has at
least one different amino acid in the exo-site compared with the
exo-site of the first variant (e.g., wild-type, isoform, homolog).
In some embodiments, one amino acid of the exo-site of the first
variant is replaced with a different amino acid in the second
variant. In some embodiments, two amino acids of the exo-site of
the first variant are replaced with different amino acids in the
second variant. In some embodiments, three or more amino acids of
the exo-site of the first variant are replaced with different amino
acids in the second variant.
[0028] The difference of the exo-site between the first and second
variant may also be due to a deletion or addition of an amino acid.
In some embodiments, one or more amino acids of the exo-site of the
first variant are removed from the exo-site in the second variant.
In some embodiments, one or more amino acids of the exo-site of the
first variant are added to the exo-site in the second variant. The
difference of the exo-site between the first and second variant may
also involve chemical modification of an amino acid of the
exo-site. For example, an exo-site amino acid with a reactive group
(e.g., carboxylic acid, hydroxyl, thiol, amide) may be
synthetically modified to a form a covalent bond with a small
molecule. In some embodiments, one or more amino acids of the
exo-site of the first variant are chemically modified in the second
variant. In some embodiments, the one or more modified amino acids
in the second variant are proteinogenic amino acids. In some
embodiments, one or more of the modified amino acids in the second
variant are non-proteinogenic amino acids. The term "proteinogenic"
refers to the 23 amino acids that can be incorporated into proteins
during translation, including the 20 amino acids of the standard
genetic code and selenocysteine, pyrrolysine, and
N-formylmethionine. In some embodiments, the proteinogenic amino
acid is one of the 20 amino acids of the standard genetic code.
[0029] The different amino acid(s) in the exo-site of the second
variant may be an amino acid with a side chain that is larger than
the side chain of the corresponding residue(s) in the first
variant. In some embodiments, the replacement amino acid in the
second variant comprises a side chain with a higher number of
non-hydrogen atoms (e.g., the total number of C, N, O, and S atoms)
than the replaced amino acid in the first variant. In some
embodiments, the replacement amino acid(s) in the second variant
comprises a side chain with a lower number of non-hydrogen atoms
than the replaced amino acid(s) in the first variant. The side
chain of the different amino acid may also differ from the side
chain of the corresponding amino acid in the first variant by
having a different charge or hydrophobicity. In some embodiments,
the replacement amino acid in the second variant comprises a side
chain with a higher ionic charge than the corresponding amino acid
in the first variant (e.g., a positively charged side chain in
place of a neutral or negatively charged side chain, a neutral side
chain in place of a negatively charged side chain). In some
embodiments, the replacement amino acid in the second variant
comprises a side chain with a lower ionic charge than the
corresponding amino acid in the first variant (e.g., a negatively
charged side chain in place of a neutral or positively charged side
chain, a neutral side chain in place of a positively charged side
chain). In some embodiments, the replacement amino acid in the
second variant comprises a polar side chain and the corresponding
amino acid in the first variant comprises a non-polar side chain.
In some embodiments, the replacement amino acid in the second
variant comprises a non-polar side chain and the corresponding
amino acid in the first variant comprises a polar side chain. Amino
acids with positively charged side chains include arginine,
histidine, and lysine. Amino acids with negatively charged side
chains amino acids include aspartate and glutamate. Neutral amino
acid side chains include those in serine, threonine, asparagine,
glutamine, cysteine, glycine, proline, alanine, leucine,
isoleucine, methionine, phenylalanine, tryptophan, tyrosine, and
valine. Polar amino side chains include those with positively and
negatively charged side chains and serine, threonine, asparagine,
glutamine, cysteine, methionine, and tyrosine. Amino acids with
non-polar or hydrophobic side chains include glycine, proline,
alanine, isoleucine, leucine, phenylalanine, valine, and
tryptophan.
[0030] The methods described herein provide a means of identifying
an agent that binds an exo-site. An exo-site refers to a site of a
protein that is not an active site or catalytic site, i.e., not a
site at which a substrate is modified. An exo-site may also be
referred to as a distal site or distal binding pocket. In some
embodiments, an exo-site is a distal binding pocket. In some
embodiments, an exo-site is not a distal binding pocket. Both
exo-sites and allosteric site may refer to binding sites that
regulate a protein and are distinct from the catalytic site. In
some embodiments, an exo-site is an allosteric site. In some
embodiments, an exo-site is not an allosteric site. In certain
embodiments, an exo-site is on the surface of a protein. In some
embodiments, a protein may adopt one or more conformations wherein
an exo-site is on the surface of the protein. In other embodiments,
a protein may adopt one or more conformations wherein the exo-site
is not on the surface of the protein.
[0031] Exo-site binding may modulate interactions of the protein
with one or more substrates, one or more metabolites, or one or
more native partners of the proteins (e.g., protein, peptide,
polynucleotide, small molecule, or carbohydrate). In some
embodiments, the binding of an agent to the exo-site imparts
selectivity to the protein between two or more substrates, two or
more metabolites, or two or more native partners of the protein. In
some embodiments, the binding of an agent to the exo-site alters
the selectivity of the protein for two or more substrates, two or
more metabolites, or two or more native partners of the
protein.
[0032] Exo-site binding may modulate the activity of a protein,
e.g., an enzyme. In some embodiments, an exo-site is a site that
modulates the activity of the enzyme, e.g., the catalytic activity
of the protein. In some embodiments, binding of an agent to an
exo-site inhibits activity of the enzyme. In some embodiments,
binding of an agent to an exo-site promotes activity of the enzyme.
An enzyme may require a molecule, peptide, protein, or other agent
to occupy one or more exo-sites in order for the enzyme to have
activity, e.g., activity for modification of a substrate at the
active site. In some embodiments, the enzyme is essentially
inactive if the exo-site is unoccupied. In some embodiments, the
activity of the enzyme is not dependent on whether the exo-site is
occupied. In some embodiments, binding of an agent to an exo-site
alters the K.sub.m, k.sub.cat, and/or V.sub.max of the enzyme as
compared to the exo-site unoccupied or bound to another agent. In
some embodiments, binding of an agent to an exo-site alters the
k.sub.cat/K.sub.m of the enzyme as compared to the exo-site
unoccupied or bound to another agent. In some embodiments, the
binding of an agent to the exo-site partially inhibits the activity
of the protein, e.g., an enzyme, by at least 10%, at least 25%, at
least 50%, at least 75%, at least 90%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99%, or at least 99.5%.
[0033] Exo-site binding may also affect the selectivity of a
protein (e.g., enzyme) for different substrates. The binding of an
agent to an exo-site may increase the selectivity of an enzyme for
the binding and/or modification (e.g., cleavage, oxidation,
reduction, coupling, isomerization) of a particular substrate
relative to one or more other substrates, or may decrease the
selectivity of an enzyme for binding and/or modification of a
particular substrate relative to one or more other substrates. For
example, binding of an agent to an exo-site of insulin degrading
enzyme (IDE) may increase the selectivity of IDE for catabolism of
insulin versus glucagon. IDE degrades several substrates including,
but not limited to, insulin, glucagon, amylin, calcitonin-gene
related peptide (CGRP), amyloid beta-peptide, TGF-alpha,
.beta.-endorphin, somatostatin, and atrial natriuretic peptide. In
some embodiments, the binding of an agent to the exo-site of IDE
increases the selectivity of IDE for catabolism of a first IDE
substrate over a second IDE substrate, wherein the substrates are
selected from the group consisting of insulin, glucagon, amylin,
calcitonin-gene related peptide (CGRP), amyloid beta-peptide,
TGF-alpha, .beta.-endorphin, somatostatin, and atrial natriuretic
peptide.
[0034] Possible substrates of a protein with an exo-site as
discussed herein include, but are not limited to, small molecules,
proteins, hormones, polypeptides, metabolites, amino acids, lipids,
signaling molecules, redox shuttles, nucleic acids,
polynucleotides, nucleotides, nucleobases, carbohydrates,
polysaccharides, monosaccharides, and co-factors. Substrate
selectivity may be between two substrates of the same type (e.g.,
two polypeptides, two peptides, two small molecules) or between
different kinds of substrates (e.g., a polypeptide and a small
molecule). Generally, substrates may differ with regard to several
characteristics, such as size, charge, conformation, or
hydrophobicity. Proteins and polypeptides may differ in, for
example, chain length, size, volume, charge, sequence, folding
stability, number or location of disulfide bonds, isoform, and/or
by one or more post-translational modifications. Lipids may differ
in, for example, chain length, size, degree of saturation,
head-group, charge, or the number, type, and/or location of
substituents. Nucleic acids and polynucleotides may differ in, for
example, sequence, sequence length, sugar backbone (e.g.,
deoxyribose, ribose), base modification (e.g., methylation), and/or
by differences in secondary structure (e.g., single strand vs.
double strand, conformation). Carbohydrates may differ in, for
example, chain length, substitution pattern, linkage pattern,
epimerization, and/or charge. Signaling molecules, metabolites,
amino acids, and other small molecules may differ in, for example,
molecular weight, size, volume, degree of saturation, charge,
degree of oxidation, degree of protonation, conformation, and/or
the number, type, and/or location of substituents.
[0035] An exo-site may be defined in various ways. For example, the
exo-site may be defined by the distance of the exo-site from the
protein's active site or catalytic site. The exo-site may be
defined by the amino acids surrounding a binding pocket. A binding
pocket may have or comprise several regions or features including,
but not limited to, hydrophobic patches, hydrophobic pockets,
hydrophilic patches, hydrophilic pockets, amphiphilic patches,
hydrogen bond donors, hydrogen bond acceptors, amphiphilic pockets,
metal ions, or post-translational modifications. In some
embodiments, the exo-site comprises a binding pocket defined by
amino acids. In some embodiments, the amino acids defining the
binding pocket are at least about 2 angstroms away from the amino
acids of the protein's catalytic site. In some embodiments, the
amino acids defining the binding pocket are at least about 3
angstroms away from the amino acids of the protein's catalytic
site. In some embodiments, the amino acids defining the binding
pocket are at least about 5 angstroms away from the amino acids of
the protein's catalytic site. In some embodiments, the amino acids
defining the binding pocket are at least about 10 angstroms away
from the amino acids of the protein's catalytic site. In some
embodiments, the amino acids defining the binding pocket are at
least about 15 angstroms away from the amino acids of the protein's
catalytic site. In some embodiments, the amino acids defining the
binding pocket are at least about 20 angstroms away from the amino
acids of the protein's catalytic site. In some embodiments, the
amino acids defining the binding pocket are at least about 25
angstroms away from the amino acids of the protein's catalytic
site. In some embodiments, the amino acids defining the binding
pocket are at least about 30 angstroms away from the amino acids of
the protein's catalytic site. In some embodiments, the amino acids
defining the binding pocket are at least about 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95 or 100 angstroms away from the amino
acids of the protein's catalytic site.
[0036] The distance from the catalytic site may be measured as
nearest atom-to-atom distance between amino acid residues of the
exo-site and amino acid residues of the catalytic site, a metal ion
of the catalytic site, a cofactor, or a co-substrate. For the
purposes of the measurement the amino acids of the catalytic site
may be defined in different ways. In some embodiments, the amino
acids of the protein's catalytic site are the amino acids that
surround the active site binding pocket, e.g., the binding pocket
for one or more target substrates. In certain embodiments, the
amino acids of the protein's catalytic site bind a substrate. In
some embodiments, the amino acids of the protein's catalytic site
are the amino acids that assist in catalysis. In some embodiments,
the amino acids of the protein catalytic site are the amino acids
that bind a cofactor. In some embodiments, the amino acids of the
protein catalytic site are the amino acids that bind an active site
metal ion. In some embodiments, the amino acids of the protein
catalytic site are the amino acids that participate in substrate
modification. In some embodiments, the amino acids of the protein's
catalytic site are the amino acids that transfer protons,
electrons, atoms, or groups of atoms (e.g., a functional group) to
or from a substrate. In some embodiments, amino acids of the
protein's catalytic site are the amino acids that form covalent,
ionic, or hydrogen bonds with a substrate. In some embodiments,
amino acids of the protein's catalytic site are the amino acids
that catalyze chemical steps at the protein's catalytic site.
[0037] The distance from the active site may also be measured as
nearest atom to atom distance between atoms of the exo-site binding
agent and amino acid residues of the catalytic site, a metal ion of
the catalytic site, a cofactor, or a co-substrate. For the purposes
of the measurement the amino acids of the active site may be
defined in different ways, as described above. In some embodiments,
the atoms of the exo-site binding agent are at least about 2
angstroms away from the amino acids of the protein's catalytic
site. In some embodiments, the amino acids defining the binding
pocket are at least about 3 angstroms away from the amino acids of
the protein's catalytic site. In some embodiments, the atoms of the
exo-site binding agent are at least about 5 angstroms away from the
amino acids of the protein's catalytic site. In some embodiments,
the atoms of the exo-site binding agent are at least about 10
angstroms away from the amino acids of the protein's catalytic
site. In some embodiments, the atoms of the exo-site binding agent
are at least about 15 angstroms away from the amino acids of the
protein's catalytic site. In some embodiments, the atoms of the
exo-site binding agent are at least about 20 angstroms away from
the amino acids of the protein's catalytic site. In some
embodiments, the atoms of the exo-site binding agent are at least
about 25 angstroms away from the amino acids of the protein's
catalytic site. In some embodiments, the atoms of the exo-site
binding agent are at least about 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95 or 100 angstroms away from the amino acids
of the protein's catalytic site.
[0038] The protein's catalytic site may have a spherical or roughly
spherical volume that extends from its center and has a radius of
about 5 angstroms, about 6 angstroms, about 7 angstroms, about 8
angstroms, about 9 angstroms, about 10 angstroms, about 11
angstroms, about 12 angstroms, about 13 angstroms, about 14
angstroms, about 15 angstroms, about 16 angstroms, about 17
angstroms, about 18 angstroms, about 19 angstroms, about 20
angstroms, about 21 angstroms, about 22 angstroms, about 23
angstroms, about 24 angstroms, about 25 angstroms, about 26
angstroms, about 27 angstroms, about 28 angstroms, about 29
angstroms, or about 30 angstroms. The protein's active site may
have a spherical or roughly spherical volume that extends from its
center and has a radius of at least about 5 angstroms, at least
about 6 angstroms, at least about 7 angstroms, at least about 8
angstroms, at least about 9 angstroms, at least about 10 angstroms,
at least about 11 angstroms, at least about 12 angstroms, at least
about 13 angstroms, at least about 14 angstroms, at least about 15
angstroms, at least about 16 angstroms, at least about 17
angstroms, at least about 18 angstroms, at least about 19
angstroms, at least about 20 angstroms, at least about 21
angstroms, at least about 22 angstroms, at least about 23
angstroms, at least about 24 angstroms, at least about 25
angstroms, at least about 26 angstroms, at least about 27
angstroms, at least about 28 angstroms, at least about 29
angstroms, at least about 30 angstroms, at least about 40
angstroms, or at least about 50 angstroms.
[0039] In certain embodiments, a protein may adopt one or more
conformations that alter the distance between an exo-site of the
protein and the protein's active site or catalytic site. In some
embodiments, one conformation of a protein may result in an
exo-site of the protein being of closer proximity to the protein's
catalytic site (e.g., about 10 angstroms), while another
conformation results in the exo-site being more distal to the
protein's catalytic site (e.g., at least about 20 angstroms).
[0040] An exo-site may be fully distinct from the active site, for
example, the exo-site and active site may not share any space
within the protein, for example, the exo-site may be in a different
subunit of the protein. Alternatively, the exo-site or distal site
may be a smaller region or pocket of a space that also contains the
active site. In such a case, substrates of the protein may bind to
the region of the space associated with the active site, whereas
exo-site binding agents bind to a different region or regions of
the space. In some embodiments, the amino acids defining the
exo-site and the amino acids defining the active may share one or
more amino acid residues in common. In some embodiments, the amino
acids defining the exo-site and the amino acids defining the active
site do not share any amino acids.
[0041] Modifications or mutations to amino acids in a protein
active site typically reduce or eliminate catalytic activity of the
protein. Whereas, without wishing to be bound by theory,
modifications or mutation to amino acids in an exo-site may not
significantly alter the catalytic activity of a protein
particularly for proteins that do not require occupation of the
exo-site to be active. In certain embodiments, as would be
appreciated by one of skill in the art, the activity of the protein
may not be affected for a particular substrate but may be affected
for another substrate. In some embodiments, an exo-site comprises a
binding pocket defined by amino acids that can be replaced with
different amino acids without significantly altering the activity
of the protein. Altering includes increasing or decreasing protein
activity, as well as changes in protein selectivity with respect to
one or more substrates. In some embodiments, the activity of a
protein with at least one modified or mutated exo-site amino acid
is considered not significantly altered if the activity or
selectivity of the protein is the same as the activity or
selectivity of the protein before modification or mutation (e.g.,
the activities are not measurably different).
[0042] Significantly altered activity may be defined in terms of a
change in K.sub.m, k.sub.cat, and/or V.sub.max for the activity of
a protein on a given substrate under the same assay conditions for
the modified protein versus the unmodified protein or control. In
some embodiments, the activity of a protein with at least one
modified or mutated exo-site amino acid is considered not
significantly altered if K.sub.m, k.sub.cat, and/or V.sub.max is
altered by a factor of between about 1.1-fold and about 10-fold for
the same substrate and under the same assay conditions, in
comparison between the modified and unmodified protein. In some
embodiments, K.sub.m, k.sub.cat, and/or V.sub.max is increased by
between about 1.1 fold and about 10-fold, about 1.1 fold and about
8-fold, about 1.1 fold and about 6-fold, about 1.1 fold and about
4-fold, or about 1.1 fold and about 2-fold. In some embodiments,
K.sub.m, k.sub.cat, and/or V.sub.max is decreased by between about
1.1 fold and about 10-fold, about 1.1 fold and about 8-fold, about
1.1 fold and about 6-fold, about 1.1 fold and about 4-fold, or
about 1.1 fold and about 2-fold. In some embodiments, the activity
of a protein with at least one modified or mutated exo-site amino
acid is considered not significantly altered if the activity or
selectivity of the protein is at least about 95%, 90%, 80%, 70%,
60%, 50%, 25%, or 10% of the activity or selectivity of the protein
before modification or mutation. In some embodiments, the activity
of a protein with at least one modified or mutated exo-site amino
acid is considered not significantly altered if the activity or
selectivity of the protein is less than about 95%, 90%, 80%, 70%,
60%, 50%, 25%, or 10% of the activity or selectivity of the protein
before modification or mutation. In some embodiments, the activity
of a protein with at least one modified or mutated exo-site amino
acid is considered not significantly altered if the activity or
selectivity of the protein is at most about 110%, 150%, 200%, 300%,
400%, 500%, 750%, or 1000% of the activity or selectivity of the
protein before modification or mutation. Combinations of these
ranges are also contemplated, including protein activities between
about 10% and 1000%, 25% and 500%, 70% and 200%, and 90% and 150%,
though other ranges and endpoints are also considered.
[0043] The protein for which an exo-site binding molecule is to be
identified may be a protein with a known exo-site. The exo-site may
have been previously identified, and in some cases, other exo-site
binding agents may be known. In certain embodiments, it may not be
known whether the protein has an exo-site. The second variant of
the protein may be modified or mutated at one or more amino acids
in a known cavity on the surface of a protein or region of anactive
site which is suspected of being a possible exo-site. In certain
embodiments, no specific site of the protein is targeted, but there
are sequence differences between the variants (e.g., two isoforms,
random mutation). Thus, the method of identifying an exo-site
binding agent may also be a method of de novo discovery of an
exo-site. The location of a sequence difference may be used to
identify the location of a binding pocket for variants that
demonstrate different binding of one or more candidate agents.
[0044] Any kind of agent may be found to bind to an exo-site. In
certain embodiments, the agent is a small molecule or a salt
thereof. In some embodiments, the small molecule has a molecular
weight of between about 100 Da and about 500 Da. In some
embodiments, the small molecule has a molecular weight of between
about 500 Da and about 1000 Da. In some embodiments, the small
molecule has a molecular weight of between about 500 Da and about
2000 Da. In some embodiments, the small molecule has a molecular
weight of between about 500 Da and about 5000 Da. The small
molecules may be natural or synthesized in the laboratory. In some
embodiments, the collection of small molecules may comprise
compounds that are structurally related to one another, e.g., are
analogs of one another and/or of a common parent compound. In some
embodiments, the small molecules screened using the inventive
methods may be provided as a combinatorial library prepared by
technologies and methods known in the field of combinatorial
chemistry. In certain embodiments, the small molecules are
non-polymeric, non-oligomeric, and/or non-peptidic. In certain
embodiments, the small molecules are drugs, drug-like molecules, or
drug candidates.
[0045] In certain embodiments, the agent found to bind an exo-site
or screened for binding to an exo-site is a polynucleotide or
nucleic acid (e.g., DNA, RNA). In certain embodiments, the
polynucleotide is a sequence of between 3 and 10 bases, 10 and 20
bases, 20 and 50 bases, or more than 50 bases. In certain
embodiments, the agent is a polypeptide. In certain embodiments the
agent found to bind an exo-site or screened for binding to an
exo-site is a protein (e.g., enzyme, antibody). In certain
embodiments, the polypeptide contains between 3 and 10 amino acids,
10 and 20 amino acids, 20 and 50 amino acids, or greater than 50
amino acids. In certain embodiments, the agent found to bind an
exo-site or screened for binding to an exo-site is a carbohydrate
(e.g., polysaccharide). In certain embodiments, the polysaccharide
contains between 3 and 10 monosaccharides, 10 and 20
monosaccharides, 20 and 50 monosaccharides, or greater than 50
monosaccharides.
[0046] The library of candidate agents may be made of up of any
suitable kind of agent (e.g., small molecule, polynucleotide,
polypeptide, protein, carbohydrate). In some embodiments, all
candidate agents of a library are the same kind of agent (e.g., all
small molecules, all polypeptides). In some embodiments, a library
may comprise candidate agents which are different kinds of agents
(e.g., small molecules and polypeptides). In some embodiments, the
library comprises candidate agents that are based on a similar core
or scaffold. In some embodiments, the library comprises candidate
agents based on two or more distinct cores or scaffolds.
[0047] The library may be of any size suitable for the screening
method being employed. In some embodiments, the library has between
2 and 100 members. In some embodiments, the library has between 100
and 1000 members. In some embodiments, the library has between 1000
and 10000 members. In some embodiments, the library has between
10000 and 100000 members. In some embodiments, the library has
between 100000 and 1 million members. In some embodiments, the
library has between 1 million and 10 million members. In some
embodiments, the library has between 10 million and 100 million
members. In some embodiments, the library has between 100 million
and 1 billion members. In some embodiments, the library has between
1 billion and 10 billion members. In some embodiments, the library
has greater than 10 billion members.
[0048] The library may be selected based on a known or suspected
exo-site binding pocket of a protein. For example, if an exo-site
binding molecule for a particular protein exo-site is known the
library may comprise candidate agents of a similar kind (e.g.,
small molecule, polypeptide) or candidate agents based on a similar
core or scaffold. The library may also be selected without knowing
what type of agents or scaffolds are likely to bind in the
exo-site.
[0049] The binding of candidate agents to the variants of the
protein may be probed by any suitable screening or selection
techniques. Screening methods typically involve contacting each
candidate agent separately with a target protein, e.g., using a
multi-well plate wherein each well is loaded with a different
candidate agent. Alternatively selection methods involve contacting
all candidate agents of a library with the target protein
simultaneously in a one-pot experiment. The time and effort
required to test interaction of large libraries with a protein is
proportional to the number of library members for screening, but is
largely independent of library size for selections. In certain
embodiments, the binding of candidate agents to the first and
second variant is determined using a screening technique. In some
embodiments, the screening technique is a high-throughput screening
technique. In certain embodiments, the binding of candidate agents
to the first and second variant is determined using a selection
technique. Since candidate agents are mixed in one-pot during
selection methods, selection techniques require some means of
deconvolution the complex mixture, typically by encoding the
library members. In certain embodiments, the library is an encoded
library. In some embodiments, the library is encoded by attachment
of a biopolymer (e.g., nucleic acid, peptide) to candidate agents.
Selection or screens may also be run at different concentrations of
candidate agents, different concentrations of protein, or different
relative concentrations of candidate agents to protein.
[0050] In some embodiments, the library is a DNA encoded library.
DNA encoded libraries can encode millions of candidate agents with
sequence lengths of about 20 nucleotides. Polymerase chain reaction
(PCR) methods known in the art allow for the amplification of DNA
sequences from minute quantities (e.g., sub-femtomole) of DNA for
subsequent sequencing. This may allow for large libraries to be
tested for interactions with a protein without synthesis of larger
quantities of candidate agents that would be necessary for some
activity-based assays (e.g., competitive inhibition of an optically
detected probe).
[0051] In certain embodiments, a DNA encoded library comprises
DNA-encoded candidate agents synthesized by DNA-directed library
synthesis. In some embodiments, DNA encoded candidate agents are
synthesized by DNA-templated synthesis (DTS). See, e.g., Gartner et
al., Science (2004), 305:1601-1605; Tse et al., J. Am. Chem. Soc.
(2008) 130:15611-15626; Brudno et al., Nat. Chem. Biol. (2010)
6:148-155; U.S. patent application U.S. Ser. No. 10/101,030, filed
Mar. 19, 2002; and U.S. patent application U.S. Ser. No.
10/643,752, filed Aug. 19, 2003, each of which is incorporated
herein by reference. In some embodiments, DNA encoded candidate
agents are synthesized using a YoctoReactor system. See e.g.,
Hansen et al., J. Am. Chem. Soc. (2009) 1313:1322-1327, which is
incorporated herein by reference. In some embodiments, DNA encoded
candidate agents (e.g., polypeptides) are synthesized using a
DNA-display. See, e.g., Halpin et al., PLoS Biol. (2004),
2:1015-1021; Halpin et al., PLoS Biol. (2004) 2:1022-1030; Halpin
et al., PLoS Biol. (2004) 2:1031-1038, each of which is
incorporated herein by reference. In some embodiments, DNA encoded
candidate agents (e.g., polypeptides) are synthesized using an
encoded self-assembling combinatorial (ESAC) approach. See, e.g.,
Melkko et al., Nat. Biotechnol. (2004) 22:568-574, which is
incorporated by reference herein. In some embodiments, DNA encoded
candidate agents are synthesized as a DNA-recorded library. See,
e.g., Clark et al., Nat. Chem. Biol., 2009, 5:647-654; Buller et
al., Med. Chem. Lett. (2008) 18:5926-5931; Mannocci et al., Proc.
Natl. Acad. Sci. U.S.A. (2008) 105:17670-17675, each of which is
incorporated herein by reference.
[0052] Affinity selections may comprise incubating candidate agents
of a library with a variant of a protein with an exo-site. In
certain embodiments, the step of contacting comprises incubating a
library of candidate agents with each of the first and second
variants. In some embodiments, each candidate agent is separately
incubated with each of the first and second variants. In some
embodiments, the step of screening comprises incubating the encoded
candidate agents with each of the first and second variants in
parallel. In some embodiments, the step of screening comprises
incubating the encoded candidate agents with each of the first and
second variants in series. In some embodiments, all of the
candidate agents are incubated with each of the first and second
variants. In some embodiments, groups of one or more candidate
agents are incubated with each of the first and second variants,
for example, in the case where not all candidate agents in a
library are compatible with each other they may be separated into
groups for incubation. The first and second variant may be
immobilized on a solid support to facilitate separation of bound
molecules of candidate agents from unbound molecules of candidate
agents. After incubation the enriched library fraction may be
isolated by separation of the proteins attached to a solid support,
followed by washing and elution.
[0053] For DNA-encoded libraries, identification of the candidate
agents following affinity selection may be done using PCR
amplification and DNA sequencing. The identified DNA encoded
candidate agents may be exposed to DNA primers and then subjected
to PCR conditions to amplify the concentration of each codon
present following incubation. In some embodiments, the DNA primers
are barcoded to identify the specific experiment from which the
amplified codons originate. For example, the DNA primers may be
encoded to indicate with which variant the candidate agents were
incubated (See FIG. 1A), indicate a particular subset from a larger
library, or indicate another feature of the particular selection
experiment (e.g., agent and/or variant concentration, presence or
absence of a substrate or cofactor). In certain embodiments, the
candidate agents of a library are incubated with a first variant
and second variant; the agents incubated with the first variant are
amplified by PCR in the presence of a first set of DNA primers; and
the agents incubated with the second variant are amplified by PCR
in the presence of a second set of DNA primers.
[0054] Sequencing of the candidate agents codons after PCR
amplification can be accomplished by any suitable sequencing
technology (e.g., Sanger sequencing, microarray hybridization,
pyrosequencing, reversible dye-terminator (Solexa) sequencing).
See, e.g., Marguiles et al., Nature (2005) 437:376-380; Bentley et
al., Nature (2008) 456:53-59, each of which is incorporated herein
by reference. In some embodiments, the amplified DNA codons are
sequenced by a high-throughput sequencing method. In some
embodiments, the amplified DNA codons are sequenced by
pyrosequencing. In some embodiments, the amplified DNA codons are
sequenced by reversible dye-terminator (Solexa) sequencing. The
sequencing method may provide a sequence abundance or abundance
fraction for each candidate agent in the library for which a
selection was run. The sequence abundance or abundance fraction for
a candidate agent may be calculated by measuring the number of
sequence reads corresponding to the individual agent (or agent with
a specific primer encoding) and dividing by the measured number of
total interpretable sequence reads.
[0055] The binding of a candidate agent may be determined as
relative binding of a candidate agent compared to other agents in a
library. In certain embodiments, the binding is determined by a
library binding experiment. In some embodiments, the library
binding experiment is an affinity-based selection. In some
embodiments, the library binding experiment comprises comparing the
abundance of each library member after contacting the library with
each protein variant, with a control treatment (e.g., the input
preselection library). One or multiple binding experiments may be
run in series or in parallel to determine the differential binding
between the first and second protein variant.
[0056] For DNA encoded libraries, the binding of a candidate agent
may be determined as an enrichment factor for the post-selection
and pre-selection sequence abundance corresponding to each agent.
In some embodiments, the step of determining binding comprises
measuring a sequence abundance for the candidate agent codons. In
some embodiments, the step of determining binding comprises
measuring the relative or absolute library member abundance for the
candidate agent DNA coding sequences. In some embodiments, the step
of comparing, for each candidate agent, comprises comparing the
measured sequence abundance after incubation with the first variant
with the measured sequence abundance after incubation with the
second variant.
[0057] The pre-selection sequence abundance is the sequence
abundance measured for a codon corresponding to an individual agent
for a sequencing run on the unselected library (e.g., library that
has not been incubated with any target). In some embodiments, the
unselected library is sequenced multiple times to determine an
average background sequence abundance. The post-selection sequence
abundance is the sequence abundance measured for a codon
corresponding to an individual agent for a sequencing run on the
post-selection library (e.g., library that has been incubated with
a target and separated to isolate bound agents). The enrichment
factor may be calculated by dividing the post-selection sequence
abundance by the pre-selection sequence abundance. A significant
enrichment factor may indicate that a candidate agent binds to the
target protein variant. The binding may be to the exo-site, active
site, or another type of protein-agent interaction. In some
embodiments, a significant enrichment factor is greater than 1. In
some embodiments, a significant enrichment factor is greater than
about 2. In some embodiments, a significant enrichment factor is
greater than about 3. In some embodiments, a significant enrichment
factor is greater than about 4. In some embodiments, a significant
enrichment factor is greater than about 5. In some embodiments, a
significant enrichment factor is greater than about 10. The
significance of enrichment factors may be based not only on the
basis of their absolute value, but also by comparison to typical
enrichment factor ranges observed for sequences of similar
preselection abundance. The measurement of sequence abundance and
enrichment factors for analysis of DNA-encoded library selections
has also been described in Kleiner et al., J. Am. Chem. Soc. (2010)
132:11779-11791; U.S. patent application U.S. Ser. No. 14/130,336,
filed Mar. 3, 2014; U.S. patent application U.S. Ser. No.
14/786,185, filed Oct. 22, 2015; U.S. patent application U.S. Ser.
No. 13/812,431, filed Mar. 26, 2013, each of which is incorporated
herein by reference.
[0058] Comparison of binding for a candidate agent to a first
protein variant with binding of the candidate agent to a second
protein variant can be used to identify the candidate agent as an
agent that binds an exo-site of the protein. Candidate agents that
have a greater binding to the first variant than the second variant
may be exo-site binders. For DNA-encoded libraries, the comparison
can be made by using the enrichment factors for each candidate
agent. As described above, a significant enrichment factor may
indicate that a candidate agent binds to the target protein
variant. Differences in the enrichment factor for a selection run
with the first variant and a selection run with the second variant
indicate that the structural differences between variants have
influenced binding of the candidate agent. For example, a candidate
agent may have an enrichment factor greater than one with a
wild-type protein, but have an enrichment factor less than one or
about one for a mutant of the protein with an exo-site "bump,"
which may mean that the mutation inhibits or interferes with a
protein-agent interaction that is present for the wild-type (See
FIG. 1B and FIG. 1C). Alternatively, a candidate agent may have
similar enrichment factors with both the wild-type and mutant
variants, which may indicate that the exo-site is not involved in
the protein-agent interaction. The protein variants may also have
additional structural differences than just amino acids at the
exo-site, thus, in some embodiments, a candidate agent with greater
binding to one variant may bind to a non-exo-site position of the
protein. In certain embodiments, an agent that binds an exo-site of
the protein is identified by determining the binding of the
candidate agent is greater for binding to the first variant than
binding to the second variant. In certain embodiments, an agent
that binds an exo-site of the protein is identified by determining
the enrichment factor for the sequence corresponding to the
candidate agent is greater for selection with the first variant
than selection with the second variant.
Identification of Agents that Bind an Exo-Site of IDE
[0059] In certain embodiments, the protein comprising an exo-site
is insulin degrading enzyme (IDE). Insulin-degrading enzyme, also
referred to as insulysin, insulinase, or insulin protease, is a 110
kDa zinc-binding protease of the M16A metalloprotease subfamily (EC
3.4.24.56). IDE was first identified by its ability to degrade the
.beta. chain of insulin and has since been shown to target
additional substrates, including, but not limited to, glucagon,
amylin, amyloid beta, TGF alpha, beta-endorphin, bradykinin,
kallidin, calcitonin-gene related peptide (CGRP), somatostatin, and
atrial natriuretic peptide. Non-selective inhibition of IDE may
lead to increased levels of both insulin and glucagon, whereas the
selective inhibition of IDE to block insulin degradation but allow
IDE to continue to catabolize glucagon could enhance insulin
concentrations without effecting glucagon concentrations. The
agents identified by a method provided herein may be selective or
non-selective inhibitors of IDE. In certain embodiments, the
selectivity is between insulin and glucagon. In certain
embodiments, the selectivity is between insulin and another IDE
substrate, between glucagon and another IDE substrate, or between
two IDE substrates other than either insulin or glucagon.
[0060] Inhibitors of IDE are described in U.S. patent application
U.S. Ser. No. 14/130,336, filed Mar. 3, 2014, which is incorporated
herein by reference. IDE has an exo-site or distal binding pocket
at the interface of IDE domains 1 and 2. The distal binding pocket
comprises a deep hydrophobic pocket about 10 .ANG. in length,
defined by the residues Leu201, Glu205, Tyr302, Thr316, and Ala479.
(See FIGS. 2A-2C) The binding of selective inhibitors to the distal
binding pocket of IDE has been described in Maianti et al., Nature
(2014), 511, 94-98, which is incorporated herein by reference. The
exo-site also comprises a first hydrophobic patch defined by IDE
residues Val360, Gly361, Gly362, Lys364, and Ile374, and a second
hydrophobic patch defined by IDE residues Ala198, Trp199, and
Phe202. An agent identified herein may interact with at least one
of the deep hydrophobic pocket, first hydrophobic patch, or second
hydrophobic patch. Unless otherwise specified, specific residues of
IDE referred to herein are residues in the protein sequence of
human insulin-degrading enzyme isoform 1 (see, e.g., SEQ ID NO: 1),
though the methods described herein are not limited to human
isoform 1 and contemplate using IDE's from other species, other
isoforms of IDE, and naturally occurring and synthetic IDE sequence
variants and mutations.
[0061] In certain embodiments, the first variant of IDE is a
wild-type IDE or at least includes a wild type (unmutated)
exo-site, and the second variant of IDE is a mutant of IDE. In some
embodiments, the mutant is provided by site directed mutagenesis.
In some embodiments, the mutation is at a residue of the exo-site
(e.g., a residue of the deep hydrophobic pocket, first hydrophobic
patch, or second hydrophobic patch). In some embodiments, the
mutation is at Ala479, Leu201, Glu205, Tyr302, Thr316, Val360,
Gly361, Gly362, Leu364, Ala198, Trp199, Phe202, Tyr314, or Ile374,
or is a mutation at a combination of residues thereof. In some
embodiments, the mutation is at Leu201, Glu205, Tyr302, Thr316, or
Ala479. In some embodiments, the mutation is at Val360, Gly361,
Gly362, Lys364, or Ile374. In some embodiments, the mutation is at
Ala198, Trp199, and Phe202. In some embodiments, the mutation is
A479L, V360Q, G362Q, A198T, W199F, F202R, Y314F, or I374Q, or a
combination thereof. In some embodiments, the mutation is
A479L.
Pharmaceutical Compositions and Administration
[0062] The present disclosure also provides pharmaceutical
compositions comprising an agent identified by a method described
herein, or a pharmaceutically acceptable salt, solvate, hydrate,
polymorph, co-crystal, tautomer, stereoisomer, or prodrug thereof,
and optionally a pharmaceutically acceptable excipient. In certain
embodiments, the agent described herein is provided in an effective
amount in the pharmaceutical composition. In certain embodiments,
the effective amount is a therapeutically effective amount. In
certain embodiments, the effective amount is a prophylactically
effective amount.
[0063] In certain embodiments, the effective amount of an agent is
an amount effective for treating a metabolic disorder (e.g.,
diabetes, hyperglycemia, impaired glucose tolerance, insulin
resistance, obesity, sodium imbalance, hypertension) in a subject
in need thereof. In certain embodiments, the effective amount of an
agent is an amount effective for preventing a metabolic disorder
(e.g., diabetes, hyperglycemia, impaired glucose tolerance, insulin
resistance, obesity, sodium imbalance, hypertension) in a subject
in need thereof. In certain embodiments, the effective amount of an
agent is an amount effective for reducing the risk of developing a
metabolic disorder (e.g., diabetes, hyperglycemia, impaired glucose
tolerance, insulin resistance, obesity, sodium imbalance,
hypertension) in a subject in need thereof. In certain embodiments,
the effective amount of an agent is an amount effective for
inhibiting the activity of a protease (e.g., IDE) in a subject or
biological sample.
[0064] In certain embodiments, the subject being treated is an
animal. The animal may be of either sex and may be at any stage of
development. In certain embodiments, the subject described herein
is a human. In certain embodiments, the subject is a non-human
animal. In certain embodiments, the subject is a mammal. In certain
embodiments, the subject is a non-human mammal. In certain
embodiments, the subject is a domesticated animal, such as a dog,
cat, cow, pig, horse, sheep, or goat. In certain embodiments, the
subject is a companion animal, such as a dog or cat. In certain
embodiments, the subject is a livestock animal, such as a cow, pig,
horse, sheep, or goat. In certain embodiments, the subject is a zoo
animal. In another embodiment, the subject is a research animal,
such as a rodent (e.g., mouse, rat), dog, pig, or non-human
primate. In certain embodiments, the animal is a genetically
engineered animal. In certain embodiments, the animal is a
transgenic animal (e.g., transgenic mice and transgenic pigs). In
certain embodiments, the subject is a fish or reptile.
[0065] In certain embodiments, the effective amount of an agent is
an amount effective for inhibiting the activity on one or more
substrates by a protein by at least about 10%, at least about 20%,
at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least
about 90%, at least about 95%, or at least about 98%. In certain
embodiments, the effective amount is an amount effective for
inhibiting the activity on one or more substrates by a protein by
not more than 10%, not more than 20%, not more than 30%, not more
than 40%, not more than 50%, not more than 60%, not more than 70%,
not more than 80%, not more than 90%, not more than 95%, or not
more than 98%. In certain embodiments, the effective amount is an
amount effective for inhibiting the activity of IDE by at least
about 10%, at least about 20%, at least about 30%, at least about
40%, at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least about 90%, at least about 95%, or at
least about 98%. In certain embodiments, the effective amount is an
amount effective for inhibiting the activity of IDE by not more
than about 10%, not more than about 20%, not more than about 30%,
not more than about 40%, not more than about 50%, not more than
about 60%, not more than about 70%, not more than about 80%, not
more than about 90%, not more than about 95%, or not more than
about 98%. In certain embodiments, the effective amount is an
amount effective for inhibiting the activity of IDE on insulin by
at least about 10%, at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80%, at least about 90%, at least about
95%, or at least about 98%. In certain embodiments, the effective
amount is an amount effective for inhibiting the activity of IDE on
insulin by not more than about 10%, not more than about 20%, not
more than about 30%, not more than about 40%, not more than about
50%, not more than about 60%, not more than about 70%, not more
than about 80%, not more than about 90%, not more than about 95%,
or not more than about 98%. In certain embodiments, the effective
amount is an amount effective for inhibiting the activity of IDE on
glucagon by at least about 10%, at least about 20%, at least about
30%, at least about 40%, at least about 50%, at least about 60%, at
least about 70%, at least about 80%, at least about 90%, at least
about 95%, or at least about 98%. In certain embodiments, the
effective amount is an amount effective for inhibiting the activity
of IDE on glucagon by not more than about 10%, not more than about
20%, not more than about 30%, not more than about 40%, not more
than about 50%, not more than about 60%, not more than about 70%,
not more than about 80%, not more than about 90%, not more than
about 95%, or not more than about 98%. In certain embodiments, the
effective amount is an amount effective for a range of inhibition
between a percentage described in this paragraph and another
percentage described in this paragraph, inclusive.
[0066] Pharmaceutical compositions described herein can be prepared
by any method known in the art of pharmacology. In general, such
preparatory methods include bringing the agent described herein
(i.e., the "active ingredient") into association with a carrier or
excipient, and/or one or more other accessory ingredients, and
then, if necessary and/or desirable, shaping, and/or packaging the
product into a desired single- or multi-dose unit.
[0067] Pharmaceutical compositions can be prepared, packaged,
and/or sold in bulk, as a single unit dose, and/or as a plurality
of single unit doses. A "unit dose" is a discrete amount of the
pharmaceutical composition comprising a predetermined amount of the
active ingredient. The amount of the active ingredient is generally
equal to the dosage of the active ingredient which would be
administered to a subject and/or a convenient fraction of such a
dosage, such as one-half or one-third of such a dosage.
[0068] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a pharmaceutical composition described herein will
vary, depending upon the identity, size, and/or condition of the
subject treated and further depending upon the route by which the
composition is to be administered. The composition may comprise
between 0.1% and 100% (w/w) active ingredient.
[0069] Pharmaceutically acceptable excipients used in the
manufacture of provided pharmaceutical compositions include inert
diluents, dispersing and/or granulating agents, surface active
agents and/or emulsifiers, disintegrating agents, binding agents,
preservatives, buffering agents, lubricating agents, and/or oils.
Excipients such as cocoa butter and suppository waxes, coloring
agents, coating agents, sweetening, flavoring, and perfuming agents
may also be present in the composition.
[0070] Exemplary diluents include calcium carbonate, sodium
carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate,
calcium hydrogen phosphate, sodium phosphate lactose, sucrose,
cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,
inositol, sodium chloride, dry starch, cornstarch, powdered sugar,
and mixtures thereof.
[0071] Exemplary granulating and/or dispersing agents include
potato starch, corn starch, tapioca starch, sodium starch
glycolate, clays, alginic acid, guar gum, citrus pulp, agar,
bentonite, cellulose, and wood products, natural sponge,
cation-exchange resins, calcium carbonate, silicates, sodium
carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone),
sodium carboxymethyl starch (sodium starch glycolate),
carboxymethyl cellulose, cross-linked sodium carboxymethyl
cellulose (croscarmellose), methylcellulose, pregelatinized starch
(starch 1500), microcrystalline starch, water insoluble starch,
calcium carboxymethyl cellulose, magnesium aluminum silicate
(Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and
mixtures thereof.
[0072] Exemplary surface active agents and/or emulsifiers include
natural emulsifiers (e.g., acacia, agar, alginic acid, sodium
alginate, tragacanth, chondrux, cholesterol, xanthan, pectin,
gelatin, egg yolk, casein, wool fat, cholesterol, wax, and
lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and
Veegum (magnesium aluminum silicate)), long chain amino acid
derivatives, high molecular weight alcohols (e.g., stearyl alcohol,
cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene
glycol distearate, glyceryl monostearate, and propylene glycol
monostearate, polyvinyl alcohol), carbomers (e.g., carboxy
polymethylene, polyacrylic acid, acrylic acid polymer, and
carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.,
carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl
cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,
methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene
sorbitan monolaurate (Tween.RTM. 20), polyoxyethylene sorbitan
(Tween.RTM. 60), polyoxyethylene sorbitan monooleate (Tween.RTM.
80), sorbitan monopalmitate (Span.RTM. 40), sorbitan monostearate
(Span.RTM. 60), sorbitan tristearate (Span.RTM. 65), glyceryl
monooleate, sorbitan monooleate (Span.RTM. 80), polyoxyethylene
esters (e.g., polyoxyethylene monostearate (Myrj.RTM. 45),
polyoxyethylene hydrogenated castor oil, polyethoxylated castor
oil, polyoxymethylene stearate, and Solutol.RTM.), sucrose fatty
acid esters, polyethylene glycol fatty acid esters (e.g.,
Cremophor.RTM.), polyoxyethylene ethers, (e.g., polyoxyethylene
lauryl ether (Brij.RTM. 30)), poly(vinyl-pyrrolidone), diethylene
glycol monolaurate, triethanolamine oleate, sodium oleate,
potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium
lauryl sulfate, Pluronic.RTM. F-68, poloxamer P-188, cetrimonium
bromide, cetylpyridinium chloride, benzalkonium chloride, docusate
sodium, and/or mixtures thereof.
[0073] Exemplary binding agents include starch (e.g., cornstarch
and starch paste), gelatin, sugars (e.g., sucrose, glucose,
dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.),
natural and synthetic gums (e.g., acacia, sodium alginate, extract
of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks,
carboxymethylcellulose, methylcellulose, ethylcellulose,
hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl
methylcellulose, microcrystalline cellulose, cellulose acetate,
poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum.RTM.),
and larch arabogalactan), alginates, polyethylene oxide,
polyethylene glycol, inorganic calcium salts, silicic acid,
polymethacrylates, waxes, water, alcohol, and/or mixtures
thereof.
[0074] Exemplary preservatives include antioxidants, chelating
agents, antimicrobial preservatives, antifungal preservatives,
antiprotozoan preservatives, alcohol preservatives, acidic
preservatives, and other preservatives. In certain embodiments, the
preservative is an antioxidant. In other embodiments, the
preservative is a chelating agent.
[0075] Exemplary antioxidants include alpha tocopherol, ascorbic
acid, acorbyl palmitate, butylated hydroxyanisole, butylated
hydroxytoluene, monothioglycerol, potassium metabisulfite,
propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite,
sodium metabisulfite, and sodium sulfite.
[0076] Exemplary chelating agents include
ethylenediaminetetraacetic acid (EDTA) and salts and hydrates
thereof (e.g., sodium edetate, disodium edetate, trisodium edetate,
calcium disodium edetate, dipotassium edetate, and the like),
citric acid and salts and hydrates thereof (e.g., citric acid
monohydrate), fumaric acid and salts and hydrates thereof, malic
acid and salts and hydrates thereof, phosphoric acid and salts and
hydrates thereof, and tartaric acid and salts and hydrates thereof.
Exemplary antimicrobial preservatives include benzalkonium
chloride, benzethonium chloride, benzyl alcohol, bronopol,
cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol,
chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin,
hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol,
phenylmercuric nitrate, propylene glycol, and thimerosal.
[0077] Exemplary antifungal preservatives include butyl paraben,
methyl paraben, ethyl paraben, propyl paraben, benzoic acid,
hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium
benzoate, sodium propionate, and sorbic acid.
[0078] Exemplary alcohol preservatives include ethanol,
polyethylene glycol, phenol, phenolic compounds, bisphenol,
chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.
[0079] Exemplary acidic preservatives include vitamin A, vitamin C,
vitamin E, beta carotene, citric acid, acetic acid, dehydroacetic
acid, ascorbic acid, sorbic acid, and phytic acid.
[0080] Other preservatives include tocopherol, tocopherol acetate,
deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA),
butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl
sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium
bisulfite, sodium metabisulfite, potassium sulfite, potassium
metabisulfite, Glydant.RTM. Plus, Phenonip.RTM., methylparaben,
German.RTM. 115, Germaben.RTM. II, Neolone.RTM., Kathon.RTM., and
Euxyl.RTM..
[0081] Exemplary buffering agents include citrate buffer solutions,
acetate buffer solutions, phosphate buffer solutions, ammonium
chloride, calcium carbonate, calcium chloride, calcium citrate,
calcium glubionate, calcium gluceptate, calcium gluconate,
D-gluconic acid, calcium glycerophosphate, calcium lactate,
propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium
phosphate, phosphoric acid, tribasic calcium phosphate, calcium
hydroxide phosphate, potassium acetate, potassium chloride,
potassium gluconate, potassium mixtures, dibasic potassium
phosphate, monobasic potassium phosphate, potassium phosphate
mixtures, sodium acetate, sodium bicarbonate, sodium chloride,
sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic
sodium phosphate, sodium phosphate mixtures, tromethamine,
magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free
water, isotonic saline, Ringer's solution, ethyl alcohol, and
mixtures thereof.
[0082] Exemplary lubricating agents include magnesium stearate,
calcium stearate, stearic acid, silica, talc, malt, glyceryl
behanate, hydrogenated vegetable oils, polyethylene glycol, sodium
benzoate, sodium acetate, sodium chloride, leucine, magnesium
lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.
[0083] Exemplary natural oils include almond, apricot kernel,
avocado, babassu, bergamot, black current seed, borage, cade,
camomile, canola, caraway, carnauba, castor, cinnamon, cocoa
butter, coconut, cod liver, coffee, corn, cotton seed, emu,
eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd,
grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui
nut, lavandin, lavender, lemon, litsea cubeba, macademia nut,
mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,
orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed,
pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,
sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,
soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut,
and wheat germ oils. Exemplary synthetic oils include, but are not
limited to, butyl stearate, caprylic triglyceride, capric
triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,
isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,
silicone oil, and mixtures thereof.
[0084] Liquid dosage forms for oral and parenteral administration
include pharmaceutically acceptable emulsions, microemulsions,
solutions, suspensions, syrups and elixirs. In addition to the
active ingredients, the liquid dosage forms may comprise inert
diluents commonly used in the art such as, for example, water or
other solvents, solubilizing agents and emulsifiers such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ,
olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl
alcohol, polyethylene glycols and fatty acid esters of sorbitan,
and mixtures thereof. Besides inert diluents, the oral compositions
can include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, and perfuming agents. In
certain embodiments for parenteral administration, the conjugates
described herein are mixed with solubilizing agents such as
Cremophor.RTM., alcohols, oils, modified oils, glycols,
polysorbates, cyclodextrins, polymers, and mixtures thereof.
[0085] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions can be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation can be a
sterile injectable solution, suspension, or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that can be employed are water, Ringer's solution, U.S.P.,
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or di-glycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables.
[0086] The injectable formulations can be sterilized, for example,
by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0087] In order to prolong the effect of a drug, it is often
desirable to slow the absorption of the drug from subcutaneous or
intramuscular injection. This can be accomplished by the use of a
liquid suspension of crystalline or amorphous material with poor
water solubility. The rate of absorption of the drug then depends
upon its rate of dissolution, which, in turn, may depend upon
crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally administered drug form may be
accomplished by dissolving or suspending the drug in an oil
vehicle.
[0088] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active ingredient is mixed with at least one inert,
pharmaceutically acceptable excipient or carrier such as sodium
citrate or dicalcium phosphate and/or (a) fillers or extenders such
as starches, lactose, sucrose, glucose, mannitol, and silicic acid,
(b) binders such as, for example, carboxymethylcellulose,
alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia,
(c) humectants such as glycerol, (d) disintegrating agents such as
agar, calcium carbonate, potato or tapioca starch, alginic acid,
certain silicates, and sodium carbonate, (e) solution retarding
agents such as paraffin, (f) absorption accelerators such as
quaternary ammonium compounds, (g) wetting agents such as, for
example, cetyl alcohol and glycerol monostearate, (h) absorbents
such as kaolin and bentonite clay, and (i) lubricants such as talc,
calcium stearate, magnesium stearate, solid polyethylene glycols,
sodium lauryl sulfate, and mixtures thereof. In the case of
capsules, tablets, and pills, the dosage form may include a
buffering agent.
[0089] Solid compositions of a similar type can be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings and other
coatings well known in the art of pharmacology. They may optionally
comprise opacifying agents and can be of a composition that they
release the active ingredient(s) only, or preferentially, in a
certain part of the intestinal tract, optionally, in a delayed
manner. Examples of encapsulating compositions which can be used
include polymeric substances and waxes. Solid compositions of a
similar type can be employed as fillers in soft and hard-filled
gelatin capsules using such excipients as lactose or milk sugar as
well as high molecular weight polethylene glycols and the like.
[0090] The active ingredient can be in a micro-encapsulated form
with one or more excipients as noted above. The solid dosage forms
of tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings, release
controlling coatings, and other coatings well known in the
pharmaceutical formulating art. In such solid dosage forms the
active ingredient can be admixed with at least one inert diluent
such as sucrose, lactose, or starch. Such dosage forms may
comprise, as is normal practice, additional substances other than
inert diluents, e.g., tableting lubricants and other tableting aids
such a magnesium stearate and microcrystalline cellulose. In the
case of capsules, tablets and pills, the dosage forms may comprise
buffering agents. They may optionally comprise opacifying agents
and can be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain part of the
intestinal tract, optionally, in a delayed manner. Examples of
encapsulating agents which can be used include polymeric substances
and waxes.
[0091] Although the descriptions of pharmaceutical compositions
provided herein are principally directed to pharmaceutical
compositions which are suitable for administration to humans, it
will be understood by the skilled artisan that such compositions
are generally suitable for administration to animals of all sorts.
Modification of pharmaceutical compositions suitable for
administration to humans in order to render the compositions
suitable for administration to various animals is well understood,
and the ordinarily skilled veterinary pharmacologist can design
and/or perform such modification with ordinary experimentation.
[0092] Agents provided herein are typically formulated in dosage
unit form for ease of administration and uniformity of dosage. It
will be understood, however, that the total daily usage of the
compositions described herein will be decided by a physician within
the scope of sound medical judgment. The specific therapeutically
effective dose level for any particular subject or organism will
depend upon a variety of factors including the disease being
treated and the severity of the disorder; the activity of the
specific active ingredient employed; the specific composition
employed; the age, body weight, general health, sex, and diet of
the subject; the time of administration, route of administration,
and rate of excretion of the specific active ingredient employed;
the duration of the treatment; drugs used in combination or
coincidental with the specific active ingredient employed; and like
factors well known in the medical arts.
[0093] The agents and compositions provided herein can be
administered by any route, including enteral (e.g., oral),
parenteral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, subcutaneous, intraventricular,
transdermal, interdermal, rectal, intravaginal, intraperitoneal,
topical (as by powders, ointments, creams, and/or drops), mucosal,
nasal, bucal, sublingual; by intratracheal instillation, bronchial
instillation, and/or inhalation; and/or as an oral spray, nasal
spray, and/or aerosol. Specifically contemplated routes are oral
administration, intravenous administration (e.g., systemic
intravenous injection), regional administration via blood and/or
lymph supply, and/or direct administration to an affected site. In
general, the most appropriate route of administration will depend
upon a variety of factors including the nature of the agent (e.g.,
its stability in the environment of the gastrointestinal tract),
and/or the condition of the subject (e.g., whether the subject is
able to tolerate oral administration). In certain embodiments, the
agent or pharmaceutical composition described herein is suitable
for topical administration to the eye of a subject.
[0094] The exact amount of an agent required to achieve an
effective amount will vary from subject to subject, depending, for
example, on species, age, and general condition of a subject,
severity of the side effects or disorder, identity of the
particular agent, mode of administration, and the like. An
effective amount may be included in a single dose (e.g., single
oral dose) or multiple doses (e.g., multiple oral doses). In
certain embodiments, when multiple doses are administered to a
subject or applied to a tissue or cell, any two doses of the
multiple doses include different or substantially the same amounts
of an agent described herein. In certain embodiments, when multiple
doses are administered to a subject or applied to a tissue or cell,
the frequency of administering the multiple doses to the subject or
applying the multiple doses to the tissue or cell is three doses a
day, two doses a day, one dose a day, one dose every other day, one
dose every third day, one dose every week, one dose every two
weeks, one dose every three weeks, or one dose every four weeks. In
certain embodiments, the frequency of administering the multiple
doses to the subject or applying the multiple doses to the tissue
or cell is one dose per day. In certain embodiments, the frequency
of administering the multiple doses to the subject or applying the
multiple doses to the tissue or cell is two doses per day. In
certain embodiments, the frequency of administering the multiple
doses to the subject or applying the multiple doses to the tissue
or cell is three doses per day. In certain embodiments, when
multiple doses are administered to a subject or applied to a tissue
or cell, the duration between the first dose and last dose of the
multiple doses is one day, two days, four days, one week, two
weeks, three weeks, one month, two months, three months, four
months, six months, nine months, one year, two years, three years,
four years, five years, seven years, ten years, fifteen years,
twenty years, or the lifetime of the subject, tissue, or cell. In
certain embodiments, the duration between the first dose and last
dose of the multiple doses is three months, six months, or one
year. In certain embodiments, the duration between the first dose
and last dose of the multiple doses is the lifetime of the subject,
tissue, or cell.
[0095] In certain embodiments, a dose (e.g., a single dose, or any
dose of multiple doses) described herein includes independently
between 0.1 .mu.g and 1 .mu.g, between 0.001 mg and 0.01 mg,
between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg
and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between
30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and
1,000 mg, or between 1 g and 10 g, inclusive, of an agent described
herein. In certain embodiments, a dose described herein includes
independently between 1 mg and 3 mg, inclusive, of an agent
described herein. In certain embodiments, a dose described herein
includes independently between 3 mg and 10 mg, inclusive, of an
agent described herein. In certain embodiments, a dose described
herein includes independently between 10 mg and 30 mg, inclusive,
of an agent described herein. In certain embodiments, a dose
described herein includes independently between 30 mg and 100 mg,
inclusive, of an agent described herein.
[0096] Dose ranges as described herein provide guidance for the
administration of provided pharmaceutical compositions to an adult.
The amount to be administered to, for example, a child or an
adolescent can be determined by a medical practitioner or person
skilled in the art and can be lower or the same as that
administered to an adult.
[0097] An agent or composition, as described herein, can be
administered in combination with one or more additional
pharmaceutical agents (e.g., therapeutically and/or
prophylactically active agents). The agents or compositions can be
administered in combination with additional pharmaceutical agents
that improve their activity (e.g., activity (e.g., potency and/or
efficacy) in treating a disease in a subject in need thereof, in
preventing a disease in a subject in need thereof, in reducing the
risk to develop a disease in a subject in need thereof, and/or in
inhibiting the activity of a protease in a subject or cell),
improve bioavailability, improve safety, reduce drug resistance,
reduce and/or modify metabolism, inhibit excretion, and/or modify
distribution in a subject or cell. It will also be appreciated that
the therapy employed may achieve a desired effect for the same
disorder, and/or it may achieve different effects. In certain
embodiments, a pharmaceutical composition described herein
including an agent described herein and an additional
pharmaceutical agent shows a synergistic effect that is absent in a
pharmaceutical composition including one of the agent and the
additional pharmaceutical agent, but not both.
Methods of Treatment and Uses
[0098] The present disclosure also provides methods that may be
useful for the treatment or prevention of a disease, disorder, or
condition. In certain embodiments, the disease is associated with a
protein comprising an exo-site. In certain embodiments, the disease
is associated with reduced expression of a protein comprising an
exo-site. In certain embodiments, the disease is associated with
elevated expression of a protein comprising an exo-site. In certain
embodiments, the disease is associated with ectopic expression of a
protein comprising an exo-site. In certain embodiments, the disease
is associated with the aberrant activity of a protein comprising an
exo-site. In certain embodiments, the disease is associated with
the increased activity of a protein comprising an exo-site. In
certain embodiments, the disease is associated with the reduced
activity of a protein comprising an exo-site. In some embodiments,
the disease is associated with the reduced, elevated, or ectopic
expression or reduced, elevated, or aberrant activity of a protein
comprising an exo-site, wherein the binding of an agent to the
exo-site modulates the activity of the protein (e.g., the activity
of the protein with respect to a particular substrate). In some
embodiments, the disease is associated with the reduced, elevated,
or ectopic expression or reduced, elevated, or aberrant activity of
a protein comprising an exo-site, wherein the binding of an agent
to the exo-site modulates the selectivity of the protein. Aberrant
activity refers to activity that is abnormal or undesirable and
includes deficient activity of a protein and overactivity of a
protein. In some embodiments, deficient activity or overactivity
may be caused by reduced, elevated, or ectopic expression of the
gene encoding the protein. Aberrant activity may be a pathological
level of activity, and may be a cause of a pathological condition
or a symptom of a pathological condition. In certain embodiments,
the disease is associated with the aberrant selectivity of a
protein comprising an exo-site. Aberrant selectivity refers to a
protein's selectivity as amongst two or more substrates that is
abnormal or undesirable. For example, the protein may have exhibit
increased activity toward one substrate versus another, and this
activity may be undesired.
[0099] In certain embodiments, the protein comprising an exo-site
is IDE, and the disease is a metabolic disorder. In certain
embodiments, the disorder is a diabetic condition. In certain
embodiments, the disorder is diabetes (e.g., type I diabetes
mellitus, type II diabetes mellitus, gestational diabetes). In
certain embodiments, the disorder is type I diabetes mellitus. In
certain embodiments, the disorder is type II diabetes mellitus. In
certain embodiments, the disorder is gestational diabetes. In
certain embodiments, the disorder is congenital diabetes,
cystic-fibrosis-related diabetes, steroid diabetes, or a monogenic
diabetes (e.g., mature onset diabetes of the young). In certain
embodiments, the disorder is hyperglycemia, impaired glucose
tolerance, or insulin resistance. In certain embodiments, the
disorder is hypoglycemia or hyperinsulinemia. In certain
embodiments, the disorder is an obesity-related condition. In
certain embodiments, the disorder is obesity. In certain
embodiments, the disorder is class I obesity, class II obesity,
class III obesity, or pre-obesity. In certain embodiments, the
disorder is undesired weight gain or an over-eating disorder. In
some embodiments, the disorder is impaired insulin signaling or
insulin resistance. In some embodiments, the disorder is an
electrolyte imbalance. In some embodiments, the disorder is sodium
imbalance. In some embodiments, the disorder is hypertension.
[0100] In certain embodiments, the method of treating a disease
comprises administering to a subject in need thereof a
therapeutically effective amount of an agent identified by a method
described herein, or a pharmaceutically acceptable salt, solvate,
hydrate, polymorph, co-crystal, tautomer, stereoisomer, or prodrug
thereof. In certain embodiments, the method of treating a disease
comprises administering to a subject in need thereof a
therapeutically effective amount of an agent identified by a method
described herein, or a pharmaceutically acceptable salt thereof. In
certain embodiments, the method of treating a disease comprises
administering to a subject in need thereof a therapeutically
effective amount of a pharmaceutical composition comprising an
agent identified by a method described herein, or a
pharmaceutically acceptable salt thereof. In certain embodiments,
the method of preventing a disease comprises administering to a
subject in need thereof a prophylactically effective amount of an
agent identified by a method described herein, or a
pharmaceutically acceptable salt, solvate, hydrate, polymorph,
co-crystal, tautomer, stereoisomer, or prodrug thereof. In certain
embodiments, the method of preventing a disease comprises
administering to a subject in need thereof a prophylactically
effective amount of an agent identified by a method described
herein, or a pharmaceutically acceptable salt thereof. In certain
embodiments, the method of preventing a disease comprises
administering to a subject in need thereof a prophylactically
effective amount of a pharmaceutical composition comprising an
agent identified by a method described herein, or a
pharmaceutically acceptable salt thereof.
[0101] The agents identified by a method described herein may
exhibit selective inhibition of a protein with an exo-site for
modification of one substrate over another substrate. In certain
embodiments, the agent identified by a method described herein
exhibits selective inhibition of a protein with an exo-site for
modification of one or more first substrates over one or more
second substrates. Exemplary modifications of a substrate include,
but are not limited to, oxidation, reduction, degradation,
cleavage, proteolysis, dephosphorylation, phosphorylation,
ligation, hydrogenation, dehydrogenation, hydration, dehydration,
confirmation change, protein folding, protein aggregation,
dimerization, isomerization, and other types of atom or functional
group transfer. In certain embodiments, the selectivity for
inhibiting modification of one substrate over another is between
about 1.1-fold and about 2-fold, between about 2-fold and about
5-fold, between about 5-fold and about 10-fold, between about
10-fold and about 50-fold, between about 50-fold and about
100-fold, or greater than about 100-fold. In certain embodiments,
there is no selectivity for one substrate over another
substrate.
[0102] In certain embodiments, the protein is IDE, and the
modification is degradation of an IDE substrate (e.g., insulin,
glucagon, amylin, TGF alpha, beta-endorphin, amyloid beta,
bradykinin, kallidin, calcitonin-gene related peptide (CGRP),
somatostatin, atrial natriuretic peptide). In some embodiments, the
first substrate is insulin, and the second substrate is glucagon or
amylin. In some embodiments, the first substrate is insulin, and
the second substrate is TGF alpha, beta-endorphin, amyloid beta,
bradykinin, kallidin, calcitonin-gene related peptide (CGRP),
somatostatin, or atrial natriuretic peptide. In some embodiments,
the first substrate is glucagon, and the second substrate is
insulin or amylin. In some embodiments, the first substrate is
glucagon, and the second substrate is TGF alpha, beta-endorphin,
amyloid beta, bradykinin, kallidin, calcitonin-gene related peptide
(CGRP), somatostatin, or atrial natriuretic peptide. In some
embodiments, the first substrate is amylin, and the second
substrate is insulin or glucagon. In some embodiments, the first
substrate is amylin, and the second substrate is TGF alpha,
beta-endorphin, amyloid beta, bradykinin, kallidin, calcitonin-gene
related peptide (CGRP), somatostatin, or atrial natriuretic
peptide. In some embodiments, the first substrate is TGF alpha,
beta-endorphin, amyloid beta, bradykinin, kallidin, calcitonin-gene
related peptide (CGRP), somatostatin, or atrial natriuretic
peptide, and the second substrate is insulin. In some embodiments,
the first substrate is TGF alpha, beta-endorphin, amyloid beta,
bradykinin, kallidin, calcitonin-gene related peptide (CGRP),
somatostatin, or atrial natriuretic peptide, and the second
substrate is glucagon or amylin.
[0103] Selectivity of an agent to inhibit modification of specific
substrates of a protein with an exo-site may be measured by
comparing an assay for modification of a first substrate by the
protein in the presence of the agent with an assay for modification
of a second substrate by the protein in the presence of the
agent.
[0104] An agent described herein may interact with a protein with
an exo-site to inhibit modification of a first substrate but not
modification of a second substrate. The agent may bind near the
active site for modification of the first substrate and impede
binding of the first substrate but not impede binding of the second
substrate or impede binding of the second substrate to a lesser
extent. The selectivity of the agent will be particular to the size
and shape of the agent, and the location and orientation of binding
to the protein exo-site. In some embodiments, the protein is IDE,
the first substrate is insulin, and the second substrate is
glucagon.
[0105] The present disclosure provides methods that may be useful
for the treatment of a metabolic disorder by administering an agent
identified by a method described herein, or a pharmaceutically
acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer,
stereoisomer, or prodrug thereof, or pharmaceutical composition
thereof, to a subject in need thereof. In certain embodiments, the
agent is administered as a pharmaceutically acceptable salt,
stereoisomer, or tautomer thereof. In certain embodiments, the
agent is administered as a pharmaceutically acceptable salt of the
agent. In certain embodiments, the agent is administered as a
specific stereoisomer or mixture of stereoisomers of the agent. In
certain embodiments, the agent is administered as a specific
tautomer or mixture of tautomers of the agent. In certain
embodiments, the agent is administered as a pharmaceutical
composition as described herein comprising the agent.
[0106] The present disclosure also provides uses of the inventive
agents, and pharmaceutically acceptable salts, solvates, hydrates,
polymorphs, co-crystals, tautomers, stereoisomers, prodrugs, and
pharmaceutical compositions thereof, in the manufacture of
medicaments for the treatment or prevention of diseases, disorders,
or conditions.
[0107] In another aspect, the present disclosure provides methods
that may be useful for modulating the activity on one or more
substrates by a protein comprising an exo-site in a subject in need
thereof, by administering to the subject an agent described herein
(e.g., an identified exo-site binding agent), or a pharmaceutically
acceptable salt, stereoisomer, or tautomer thereof, or a
pharmaceutical composition thereof. In some embodiments, the method
is useful for inhibiting the activity of a protein comprising an
exo-site. In some embodiments, the method is useful for enhancing
the activity of a protein comprising an exo-site.
[0108] In another aspect, the present disclosure provides methods
that may be useful for modulating the activity on one or more
substrates by a protein comprising an exo-site in a biological
sample, by contacting the sample with an agent described herein
(e.g., an identified exo-site binding agent), or a pharmaceutically
acceptable salt, stereoisomer, or tautomer thereof, or a
pharmaceutical composition thereof. In some embodiments, the method
is useful for inhibiting the activity of a protein comprising an
exo-site. In some embodiments, the method is useful for enhancing
the activity of a protein comprising an exo-site.
[0109] In another aspect, the present disclosure provides methods
that may be useful for modulating the substrate selectivity and/or
processivity of a protein comprising an exo-site in a subject in
need thereof, by administering to the subject an agent described
herein (e.g., an identified exo-site binding agent), or a
pharmaceutically acceptable salt, stereoisomer, or tautomer
thereof, or a pharmaceutical composition thereof. In some
embodiments, the method is useful for increasing the selectivity of
a protein for modification of a first substrate over modification
over a second substrate. In some embodiments, the method is useful
for decreasing the selectivity of a protein for modification of a
first substrate over modification over a second substrate. In
certain embodiments, the selectivity is between about 2-fold and
about 5-fold. In certain embodiments, the selectivity is between
about 5-fold and about 10-fold. In certain embodiments, the
selectivity is between about 10-fold and about 20-fold. In certain
embodiments, the selectivity is between about 20-fold and about
50-fold. In certain embodiments, the selectivity is between about
50-fold and about 100-fold. In certain embodiments, the selectivity
is between about 100-fold and about 200-fold. In certain
embodiments, the selectivity is between about 200-fold and about
500-fold. In certain embodiments, the selectivity is between about
500-fold and about 1000-fold. In certain embodiments, the
selectivity is at least about 1000-fold.
[0110] In another aspect, the present disclosure provides methods
that may be useful for modulating the substrate selectivity and/or
processivity of a protein comprising an exo-site in a biological
sample, by contacting the sample with an agent described herein
(e.g., an identified exo-site binding agent), or a pharmaceutically
acceptable salt, stereoisomer, or tautomer thereof, or a
pharmaceutical composition thereof. In some embodiments, the method
is useful for increasing the selectivity of a protein for
modification of a first substrate over modification over a second
substrate. In some embodiments, the method is useful for decreasing
the selectivity of a protein for modification of a first substrate
over modification over a second substrate. In certain embodiments,
the selectivity is between about 2-fold and about 5-fold. In
certain embodiments, the selectivity is between about 5-fold and
about 10-fold. In certain embodiments, the selectivity is between
about 10-fold and about 20-fold. In certain embodiments, the
selectivity is between about 20-fold and about 50-fold. In certain
embodiments, the selectivity is between about 50-fold and about
100-fold. In certain embodiments, the selectivity is between about
100-fold and about 200-fold. In certain embodiments, the
selectivity is between about 200-fold and about 500-fold. In
certain embodiments, the selectivity is between about 500-fold and
about 1000-fold. In certain embodiments, the selectivity is at
least about 1000-fold.
[0111] In another aspect, the present disclosure provides methods
that may be useful for modulating the activity of IDE in a subject
in need thereof, by administering to the subject an agent described
herein (e.g., an identified exo-site binding agent), or a
pharmaceutically acceptable salt, stereoisomer, or tautomer
thereof, or a pharmaceutical composition thereof. In some
embodiments, the method is useful for inhibiting the activity of
IDE. In some embodiments, the method is useful for enhancing the
activity of IDE.
[0112] In another aspect, the present disclosure provides methods
that may be useful for modulating the activity of IDE in a
biological sample in need thereof, by contacting the sample with an
agent described herein (e.g., an identified exo-site binding
agent), or a pharmaceutically acceptable salt, stereoisomer, or
tautomer thereof, or a pharmaceutical composition thereof. In some
embodiments, the method is useful for inhibiting the activity of
IDE toward a particular substrate. In some embodiments, the method
is useful for enhancing the activity of IDE toward a particular
substrate.
[0113] In another aspect, the present disclosure provides methods
that may be useful for inhibiting the degradation of insulin in a
subject in need thereof, by administering to the subject a an agent
described herein (e.g., an identified exo-site binding agent), or a
pharmaceutically acceptable salt, stereoisomer, or tautomer
thereof, or a pharmaceutical composition thereof. In some
embodiments, the degradation of insulin is selectively inhibited
over the degradation of glucagon. In certain embodiments, the
selectivity is between about 2-fold and about 5-fold. In certain
embodiments, the selectivity is between about 5-fold and about
10-fold. In certain embodiments, the selectivity is between about
10-fold and about 20-fold. In certain embodiments, the selectivity
is between about 20-fold and about 50-fold. In certain embodiments,
the selectivity is between about 50-fold and about 100-fold. In
certain embodiments, the selectivity is between about 100-fold and
about 200-fold. In certain embodiments, the selectivity is between
about 200-fold and about 500-fold. In certain embodiments, the
selectivity is between about 500-fold and about 1000-fold. In
certain embodiments, the selectivity is at least about
1000-fold.
[0114] In another aspect, the present disclosure provides methods
that may be useful for inhibiting the degradation of insulin in a
biological sample (e.g., cells, tissues, biopsied tissues, purified
or partially purified IDE), by contacting the sample with an agent
described herein (e.g., an identified exo-site binding agent), or a
pharmaceutically acceptable salt, stereoisomer, or tautomer
thereof, or a pharmaceutical composition thereof. In some
embodiments, the degradation of insulin is selectively inhibited
over the degradation of glucagon. In certain embodiments, the
selectivity is between about 2-fold and about 5-fold. In certain
embodiments, the selectivity is between about 5-fold and about
10-fold. In certain embodiments, the selectivity is between about
10-fold and about 20-fold. In certain embodiments, the selectivity
is between about 20-fold and about 50-fold. In certain embodiments,
the selectivity is between about 50-fold and about 100-fold. In
certain embodiments, the selectivity is between about 100-fold and
about 200-fold. In certain embodiments, the selectivity is between
about 200-fold and about 500-fold. In certain embodiments, the
selectivity is between about 500-fold and about 1000-fold. In
certain embodiments, the selectivity is at least about
1000-fold.
Definitions
[0115] The term "pharmaceutically acceptable salt" refers to those
salts which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of humans and lower
animals without undue toxicity, irritation, allergic response, and
the like, and are commensurate with a reasonable benefit/risk
ratio. Pharmaceutically acceptable salts are well known in the art.
For example, Berge et al. describe pharmaceutically acceptable
salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19,
incorporated herein by reference. Pharmaceutically acceptable salts
of the agents of this disclosure include those derived from
suitable inorganic and organic acids and bases. Examples of
pharmaceutically acceptable, nontoxic acid addition salts are salts
of an amino group formed with inorganic acids, such as hydrochloric
acid, hydrobromic acid, phosphoric acid, sulfuric acid, and
perchloric acid or with organic acids, such as acetic acid, oxalic
acid, maleic acid, tartaric acid, citric acid, succinic acid, or
malonic acid or by using other methods known in the art such as ion
exchange. Other pharmaceutically acceptable salts include adipate,
alginate, ascorbate, aspartate, benzenesulfonate, benzoate,
bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Salts derived from appropriate bases include alkali metal, alkaline
earth metal, ammonium, and N.sup.+(C.sub.1-4 alkyl).sub.4.sup.-
salts. Representative alkali or alkaline earth metal salts include
sodium, lithium, potassium, calcium, magnesium, and the like.
Further pharmaceutically acceptable salts include, when
appropriate, nontoxic ammonium, quaternary ammonium, and amine
cations formed using counterions such as halide, hydroxide,
carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate,
and aryl sulfonate.
[0116] The term "solvate" refers to forms of the agent, or a salt
thereof, that are associated with a solvent, usually by a
solvolysis reaction. This physical association may include hydrogen
bonding. Conventional solvents include water, methanol, ethanol,
acetic acid, DMSO, THF, diethyl ether, and the like. The agents
described herein may be prepared, e.g., in crystalline form, and
may be solvated. Suitable solvates include pharmaceutically
acceptable solvates and further include both stoichiometric
solvates and non-stoichiometric solvates. In certain instances, the
solvate will be capable of isolation, for example, when one or more
solvent molecules are incorporated in the crystal lattice of a
crystalline solid. "Solvate" encompasses both solution-phase and
isolatable solvates. Representative solvates include hydrates,
ethanolates, and methanolates.
[0117] The term "hydrate" refers to an agent that is associated
with water. Typically, the number of the water molecules contained
in a hydrate of an agent is in a definite ratio to the number of
the agent molecules in the hydrate. Therefore, a hydrate of an
agent may be represented, for example, by the general formula Rx
H.sub.2O, wherein R is the agent, and x is a number greater than 0.
A given an agent may form more than one type of hydrate, including,
e.g., monohydrates (x is 1), lower hydrates (x is a number greater
than 0 and smaller than 1, e.g., hemihydrates (R0.5 H.sub.2O)), and
polyhydrates (x is a number greater than 1, e.g., dihydrates (R2
H.sub.2O) and hexahydrates (R6 H.sub.2O)).
[0118] The term "tautomers" or "tautomeric" refers to two or more
interconvertible agents or compounds resulting from at least one
formal migration of a hydrogen atom and at least one change in
valency (e.g., a single bond to a double bond, a triple bond to a
single bond, or vice versa). The exact ratio of the tautomers
depends on several factors, including temperature, solvent, and pH.
Tautomerizations (i.e., the reaction providing a tautomeric pair)
may catalyzed by acid or base. Exemplary tautomerizations include
keto-to-enol, amide-to-imide, lactam-to-lactim, enamine-to-imine,
and enamine-to-(a different enamine) tautomerizations.
[0119] It is also to be understood that agents or compounds that
have the same molecular formula but differ in the nature or
sequence of bonding of their atoms or the arrangement of their
atoms in space are termed "isomers". Isomers that differ in the
arrangement of their atoms in space are termed "stereoisomers".
[0120] Stereoisomers that are not mirror images of one another are
termed "diastereomers" and those that are non-superimposable mirror
images of each other are termed "enantiomers". When an agent or
compound has an asymmetric center, for example, it is bonded to
four different groups, a pair of enantiomers is possible. An
enantiomer can be characterized by the absolute configuration of
its asymmetric center and is described by the R- and S-sequencing
rules of Cahn and Prelog, or by the manner in which the molecule
rotates the plane of polarized light and designated as
dextrorotatory or levorotatory (i.e., as (+) or (-)-isomers
respectively). A chiral agent or compound can exist as either
individual enantiomer or as a mixture thereof. A mixture containing
equal proportions of the enantiomers is called a "racemic
mixture".
[0121] The term "polymorph" refers to a crystalline form of an
agent (or a salt, hydrate, or solvate thereof). All polymorphs have
the same elemental composition. Different crystalline forms usually
have different X-ray diffraction patterns, infrared spectra,
melting points, density, hardness, crystal shape, optical and
electrical properties, stability, and solubility. Recrystallization
solvent, rate of crystallization, storage temperature, and other
factors may cause one crystal form to dominate. Various polymorphs
of an agent can be prepared by crystallization under different
conditions.
[0122] The term "co-crystal" refers to a crystalline structure
composed of at least two components. In certain embodiments, a
co-crystal contains an agent of the present disclosure and one or
more other component, including but not limited to, atoms, ions,
molecules, or solvent molecules. In certain embodiments, a
co-crystal contains an agent of the present disclosure and one or
more solvent molecules. In certain embodiments, a co-crystal
contains an agent of the present disclosure and one or more acid or
base. In certain embodiments, a co-crystal contains an agent of the
present disclosure and one or more components related to said
agent, including not limited to, an isomer, tautomer, salt,
solvate, hydrate, synthetic precursor, synthetic derivative,
fragment or impurity of said agent.
[0123] The term "prodrug" refers to agents or compounds that have
cleavable groups and become by solvolysis or under physiological
conditions the agents described herein, which are pharmaceutically
active in vivo. Such examples include, but are not limited to,
choline ester derivatives and the like, N-alkylmorpholine esters
and the like. Other derivatives of the agents described herein have
activity in both their acid and acid derivative forms, but in the
acid sensitive form often offer advantages of solubility, tissue
compatibility, or delayed release in the mammalian organism (see,
Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier,
Amsterdam 1985). Prodrugs include acid derivatives well known to
practitioners of the art, such as, for example, esters prepared by
reaction of the parent acid with a suitable alcohol, or amides
prepared by reaction of the parent acid agent or compound with a
substituted or unsubstituted amine, or acid anhydrides, or mixed
anhydrides. Simple aliphatic or aromatic esters, amides, and
anhydrides derived from acidic groups pendant on the agents or
compounds described herein are particular prodrugs. In some cases
it is desirable to prepare double ester type prodrugs such as
(acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkylesters. C1-C8
alkyl, C2-C8 alkenyl, C2-C8 alkynyl, aryl, C7-C12 substituted aryl,
and C7-C12 arylalkyl esters of the agents or compounds described
herein may be preferred.
[0124] The terms "composition" and "formulation" are used
interchangeably.
[0125] A "subject" to which administration is contemplated refers
to a human (i.e., male or female of any age group, e.g., pediatric
subject (e.g., infant, child, or adolescent) or adult subject
(e.g., young adult, middle-aged adult, or senior adult)) or
non-human animal. In certain embodiments, the non-human animal is a
mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey),
commercially relevant mammal (e.g., cattle, pig, horse, sheep,
goat, cat, or dog), or bird (e.g., commercially relevant bird, such
as chicken, duck, goose, or turkey)). In certain embodiments, the
non-human animal is a fish, reptile, or amphibian. The non-human
animal may be a male or female at any stage of development. The
non-human animal may be a transgenic animal or genetically
engineered animal. The term "patient" refers to a human subject in
need of treatment of a disease.
[0126] The term "biological sample" refers to any sample including
tissue samples (such as tissue sections and needle biopsies of a
tissue); cell samples (e.g., cytological smears (such as Pap or
blood smears) or samples of cells obtained by microdissection);
samples of whole organisms (such as samples of yeasts or bacteria);
or cell fractions, fragments or organelles (such as obtained by
lysing cells and separating the components thereof by
centrifugation or otherwise). Other examples of biological samples
include blood, serum, urine, semen, fecal matter, cerebrospinal
fluid, interstitial fluid, mucous, tears, sweat, pus, biopsied
tissue (e.g., obtained by a surgical biopsy or needle biopsy),
nipple aspirates, milk, vaginal fluid, saliva, swabs (such as
buccal swabs), or any material containing biomolecules that is
derived from a first biological sample.
[0127] The term "administer," "administering," or "administration"
refers to implanting, absorbing, ingesting, injecting, inhaling, or
otherwise introducing an agent described herein, or a composition
thereof, in or on a subject.
[0128] The terms "treatment," "treat," and "treating" refer to
reversing, alleviating, delaying the onset of, or inhibiting the
progress of a disease described herein. In some embodiments,
treatment may be administered after one or more signs or symptoms
of the disease have developed or have been observed. In other
embodiments, treatment may be administered in the absence of signs
or symptoms of the disease. For example, treatment may be
administered to a susceptible subject prior to the onset of
symptoms (e.g., in light of a history of symptoms and/or in light
of exposure to a pathogen). Treatment may also be continued after
symptoms have resolved, for example, to delay or prevent
recurrence.
[0129] The terms "condition," "disease," and "disorder" are used
interchangeably.
[0130] As used herein the term "inhibit" or "inhibition" in the
context of proteins, for example, in the context of IDE, refers to
a reduction in the activity of the protein. In some embodiments,
the term refers to a reduction of the level of protein activity,
e.g., IDE activity, to a level that is statistically significantly
lower than an initial level, which may, for example, be a baseline
level of protein activity. In some embodiments, the term refers to
a reduction of the level of protein activity, e.g., IDE activity,
to a level that is less than 75%, less than 50%, less than 40%,
less than 30%, less than 25%, less than 20%, less than 10%, less
than 9%, less than 8%, less than 7%, less than 6%, less than 5%,
less than 4%, less than 3%, less than 2%, less than 1%, less than
0.5%, less than 0.1%, less than 0.01%, less than 0.001%, or less
than 0.0001% of an initial level, which may, for example, be a
baseline level of protein activity.
[0131] As used herein, the term "insulin degrading enzyme" or "IDE"
refers to the enzyme primarily responsible for insulin catabolism
in vivo which is also referred to as insulysin, insulinase, or
insulin protease. IDE is a 110 kDa zinc-binding protease of the
M16A metalloprotease subfamily (EC 3.4.24.56), which degrades the
beta chain of insulin and is also known to process additional
substrates including, but not limited to, glucagon, amylin, amyloid
TGF alpha, beta-endorphin, amyloid beta, bradykinin, kallidin,
calcitonin-gene related peptide (CGRP), somatostatin, and atrial
natriuretic peptide. IDE enzymes (also referred to herein as IDE
proteins) and their respective encoding RNA and DNA sequences
according to some aspects of this disclosure include human IDE
protein and encoding sequences, as well as, in some embodiments,
IDE proteins and encoding sequences from other species, for
example, from other mammals (e.g., IDE proteins and encoding
sequences from mouse, rat, cat, dog, cattle, goat, sheep, pig, or
primate), from other vertebrates, and from insects. In some
embodiments, an IDE inhibitor provided herein is specific for an
IDE from a species, e.g., for human IDE, mouse IDE, rat IDE, and so
on. In some embodiment, an IDE provided herein inhibits IDEs from
more than one species, e.g., human IDE and mouse IDE. In some
embodiments, an IDE provided herein exhibits equipotent inhibition
of IDEs from more than one species, e.g., equipotent inhibition of
human and mouse IDEs. The term IDE further includes, in some
embodiments, sequence variants and mutations (e.g., naturally
occurring or synthetic IDE sequence variants or mutations), and
different IDE isoforms. In some embodiments, the term IDE includes
protein or encoding sequences that are homologous to an IDE protein
or encoding sequence, for example, a protein or encoding sequence
having at least 80%, at least 85%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, at least 99%, or at least 99.5%
sequence identity with an IDE sequence, for example, with an IDE
sequence provided herein. In some embodiments, the term IDE refers
to a protein exhibiting IDE activity, for example, a protein
exhibiting insulin-targeted protease activity, or a nucleic acid
sequence encoding such a protein. In some embodiments, the term IDE
included proteins that exhibit at least 50%, at least 60%, at least
70%, at least 80%, at least 90%, at least 95%, or at least 100%
insulin-targeting protease activity as compared to a known IDE
protein or encoding sequence, for example, as compared to an IDE
sequence provided herein. IDE protein and encoding gene sequences
are well known to those of skill in the art, and exemplary protein
sequences include, but are not limited to, the following sequences.
Additional IDE sequences will be apparent to those of skill in the
art, and the disclosure is not limited to the exemplary sequences
provided herein.
TABLE-US-00001 >gi|155969707|ref|NP_004960.2|insulin-degrading
enzyme isoform 1 [Homo sapiens] (SEQ ID NO: 1)
MRYRLAWLLHPALPSTFRSVLGARLPPPERLCGFQKKTYSKMNNPAIKRL
GNHITKSPEDKREYRGLELANGIKVLLISDPTTDKSSAALDVHIGSLSDP
PNIAGLSHFCEHMLFLGTKKYPKENEYSQFLSEHAGSSNAFTSGEHTNYY
FDVSHEHLEGALDRFAQFFLCPLFDESCKDREVNAVDSEHEKNVMNDAWR
LFQLEKATGNPKHPFSKFGTGNKYTLETRPNQEGIDVRQELLKFHSAYYS
SNLMAVCVLGRESLDDLTNLVVKLFSEVENKNVPLPEFPEHPFQEEHLKQ
LYKIVPIKDIRNLYVTFPIPDLQKYYKSNPGHYLGHLIGHEGPGSLLSEL
KSKGWVNTLVGGQKEGARGFMFFIINVDLTEEGLLHVEDIILHMFQYIQK
LRAEGPQEWVFQECKDLNAVAFRFKDKERPRGYTSKIAGILHYYPLEEVL
TAEYLLEEFRPDLIEMVLDKLRPENVRVAIVSKSFEGKTDRTEEWYGTQY
KQEAIPDEVIKKWQNADLNGKFKLPTKNEFIPTNFEILPLEKEATPYPAL
IKDTAMSKLWFKQDDKFFLPKACLNFEFFSPFAYVDPLHCNMAYLYLELL
KDSLNEYAYAAELAGLSYDLQNTIYGMYLSVKGYNDKQPILLKKIIEKMA
TFEIDEKRFEIIKEAYMRSLNNFRAEQPHQHAMYYLRLLMTEVAWTKDEL
KEALDDVTLPRLKAFIPQLLSRLHIEALLHGNITKQAALGIMQMVEDTLI
EHAHTKPLLPSQLVRYREVQLPDRGWFVYQQRNEVHNNCGIEIYYQTDMQ
STSENMFLELFCQIISEPCFNTLRTKEQLGYIVFSGPRRANGIQGLRFII
QSEKPPHYLESRVEAFLITMEKSIEDMTEEAFQKHIQALAIRRLDKPKKL
SAECAKYWGEIISQQYNFDRDNTEVAYLKTLTKEDIIKFYKEMLAVDAPR
RHKVSVHVLAREMDSCPVVGEFPCQNDINLSQAPALPQPEVIQNMTEFKR
GLPLFPLVKPHINFMAAKL
>gi|260099676|ref|NP_001159418.1|insulin-degrading enzyme
isoform 2 [Homo sapiens] (SEQ ID NO: 2)
MSKLWFKQDDKFFLPKACLNFEFFSPFAYVDPLHCNMAYLYLELLKDSLN
EYAYAAELAGLSYDLQNTIYGMYLSVKGYNDKQPILLKKIIEKMATFEID
EKRFEIIKEAYMRSLNNFRAEQPHQHAMYYLRLLMTEVAWTKDELKEALD
DVTLPRLKAFIPQLLSRLHIEALLHGNITKQAALGIMQMVEDTLIEHAHT
KPLLPSQLVRYREVQLPDRGWFVYQQRNEVHNNCGIEIYYQTDMQSTSEN
MFLELFCQIISEPCFNTLRTKEQLGYIVFSGPRRANGIQGLRFIIQSEKP
PHYLESRVEAFLITMEKSIEDMTEEAFQKHIQALAIRRLDKPKKLSAECA
KYWGEIISQQYNFDRDNTEVAYLKTLTKEDIIKFYKEMLAVDAPRRHKVS
VHVLAREMDSCPVVGEFPCQNDINLSQAPALPQPEVIQNMTEFKRGLPLF PLVKPHINFMAAKL
>gi|121583922|ref|NP_112419.2|insulin-degrading enzyme [Mus
musculus] (SEQ ID NO: 3)
MRNGLVWLLHPALPGTLRSILGARPPPAKRLCGFPKQTYSTMSNPAIQRI
EDQIVKSPEDKREYRGLELANGIKVLLISDPTTDKSSAALDVHIGSLSDP
PNIPGLSHFCEHMLFLGTKKYPKENEYSQFLSEHAGSSNAFTSGEHTNYY
FDVSHEHLEGALDRFAQFFLCPLFDASCKDREVNAVDSEHEKNVMNDAWR
LFQLEKATGNPKHPFSKFGTGNKYTLETRPNQEGIDVREELLKFHSTYYS
SNLMAICVLGRESLDDLTNLVVKLFSEVENKNVPLPEFPEHPFQEEHLRQ
LYKIVPIKDIRNLYVTFPIPDLQQYYKSNPGHYLGHLIGHEGPGSLLSEL
KSKGWVNTLVGGQKEGARGFMFFIINVDLTEEGLLHVEDIILHMFQYIQK
LRAEGPQEWVFQECKDLNAVAFRFKDKERPRGYTSKIAGKLHYYPLNGVL
TAEYLLEEFRPDLIDMVLDKLRPENVRVAIVSKSFEGKTDRTEQWYGTQY
KQEAIPEDIIQKWQNADLNGKFKLPTKNEFIPTNFEILSLEKDATPYPAL
IKDTAMSKLWFKQDDKFFLPKACLNFEFFSPFAYVDPLHCNMAYLYLELL
KDSLNEYAYAAELAGLSYDLQNTIYGMYLSVKGYNDKQPILLKKITEKMA
TFEIDKKRFEIIKEAYMRSLNNFRAEQPHQHAMYYLRLLMTEVAWTKDEL
KEALDDVTLPRLKAFIPQLLSRLHIEALLHGNITKQAALGVMQMVEDTLI
EHAHTKPLLPSQLVRYREVQLPDRGWFVYQQRNEVHNNCGIEIYYQTDMQ
STSENMFLELFCQIISEPCFNTLRTKEQLGYIVFSGPRRANGIQGLRFII
QSEKPPHYLESRVEAFLITMEKAIEDMTEEAFQKHIQALAIRRLDKPKKL
SAECAKYWGEIISQQYNYDRDNIEVAYLKTLTKDDIIRFYQEMLAVDAPR
RHKVSVHVLAREMDSCPVVGEFPSQNDINLSEAPPLPQPEVIHNMTEFKR
GLPLFPLVKPHINFMAAKL
[0132] As used herein, use of the phrase "at least one instance"
refers to 1, 2, 3, 4, or more instances, but also encompasses a
range, e.g., for example, from 1 to 4, from 1 to 3, from 1 to 2,
from 2 to 4, from 2 to 3, or from 3 to 4 instances, inclusive.
[0133] The term "carbohydrate" or "saccharide" refers to an
aldehydic or ketonic derivative of polyhydric alcohols.
Carbohydrates include compounds with relatively small molecules
(e.g., sugars) as well as macromolecular or polymeric substances
(e.g., starch, glycogen, and cellulose polysaccharides). The term
"sugar" refers to monosaccharides, disaccharides, or
polysaccharides. Monosaccharides are the simplest carbohydrates in
that they cannot be hydrolyzed to smaller carbohydrates. Most
monosaccharides can be represented by the general formula
C.sub.yH.sub.2yO.sub.y (e.g., C.sub.6H.sub.12O.sub.6 (a hexose such
as glucose)), wherein y is an integer equal to or greater than 3.
Certain polyhydric alcohols not represented by the general formula
described above may also be considered monosaccharides. For
example, deoxyribose is of the formula C.sub.5H.sub.10O.sub.4 and
is a monosaccharide. Monosaccharides usually consist of five or six
carbon atoms and are referred to as pentoses and hexoses,
receptively. If the monosaccharide contains an aldehyde it is
referred to as an aldose; and if it contains a ketone, it is
referred to as a ketose. Monosaccharides may also consist of three,
four, or seven carbon atoms in an aldose or ketose form and are
referred to as trioses, tetroses, and heptoses, respectively.
Glyceraldehyde and dihydroxyacetone are considered to be aldotriose
and ketotriose sugars, respectively. Examples of aldotetrose sugars
include erythrose and threose; and ketotetrose sugars include
erythrulose. Aldopentose sugars include ribose, arabinose, xylose,
and lyxose; and ketopentose sugars include ribulose, arabulose,
xylulose, and lyxulose. Examples of aldohexose sugars include
glucose (for example, dextrose), mannose, galactose, allose,
altrose, talose, gulose, and idose; and ketohexose sugars include
fructose, psicose, sorbose, and tagatose. Ketoheptose sugars
include sedoheptulose. Each carbon atom of a monosaccharide bearing
a hydroxyl group (--OH), with the exception of the first and last
carbons, is asymmetric, making the carbon atom a stereocenter with
two possible configurations (R or S). Because of this asymmetry, a
number of isomers may exist for any given monosaccharide formula.
The aldohexose D-glucose, for example, has the formula
C.sub.6H.sub.12O.sub.6, of which all but two of its six carbons
atoms are stereogenic, making D-glucose one of the 16 (i.e.,
2.sup.4) possible stereoisomers. The assignment of D or L is made
according to the orientation of the asymmetric carbon furthest from
the carbonyl group: in a standard Fischer projection if the
hydroxyl group is on the right the molecule is a D sugar, otherwise
it is an L sugar. The aldehyde or ketone group of a straight-chain
monosaccharide will react reversibly with a hydroxyl group on a
different carbon atom to form a hemiacetal or hemiketal, forming a
heterocyclic ring with an oxygen bridge between two carbon atoms.
Rings with five and six atoms are called furanose and pyranose
forms, respectively, and exist in equilibrium with the
straight-chain form. During the conversion from the straight-chain
form to the cyclic form, the carbon atom containing the carbonyl
oxygen, called the anomeric carbon, becomes a stereogenic center
with two possible configurations: the oxygen atom may take a
position either above or below the plane of the ring. The resulting
possible pair of stereoisomers is called anomers. In an a anomer,
the --OH substituent on the anomeric carbon rests on the opposite
side (trans) of the ring from the --CH.sub.2OH side branch. The
alternative form, in which the --CH.sub.2OH substituent and the
anomeric hydroxyl are on the same side (cis) of the plane of the
ring, is called a .beta. anomer. A carbohydrate including two or
more joined monosaccharide units is called a disaccharide or
polysaccharide (e.g., a trisaccharide), respectively. The two or
more monosaccharide units bound together by a covalent bond known
as a glycosidic linkage formed via a dehydration reaction,
resulting in the loss of a hydrogen atom from one monosaccharide
and a hydroxyl group from another. Exemplary disaccharides include
sucrose, lactulose, lactose, maltose, isomaltose, trehalose,
cellobiose, xylobiose, laminaribiose, gentiobiose, mannobiose,
melibiose, nigerose, or rutinose. Exemplary trisaccharides include,
but are not limited to, isomaltotriose, nigerotriose, maltotriose,
melezitose, maltotriulose, raffinose, and kestose. The term
carbohydrate also includes other natural or synthetic stereoisomers
of the carbohydrates described herein.
[0134] A "protein," "peptide," or "polypeptide" comprises a polymer
of amino acid residues linked together by peptide bonds. The term
refers to proteins, polypeptides, and peptides of any size,
structure, or function. Typically, a protein will be at least three
amino acids long. A protein may refer to an individual protein or a
collection of proteins. Inventive proteins preferably contain only
natural amino acids, although non-natural amino acids (i.e.,
compounds that do not occur in nature but that can be incorporated
into a polypeptide chain) and/or amino acid analogs as are known in
the art may alternatively be employed. Also, one or more of the
amino acids in a protein may be modified, for example, by the
addition of a chemical entity such as a carbohydrate group, a
hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl
group, a fatty acid group, a linker for conjugation or
functionalization, or other modification. A protein may also be a
single molecule or may be a multi-molecular complex. A protein may
be a fragment of a naturally occurring protein or peptide. A
protein may be naturally occurring, recombinant, synthetic, or any
combination of these.
[0135] Polynucleotides described herein may be synthesized by
standard methods known in the art, e.g., by use of an automated DNA
synthesizer (such as those that are commercially available from
Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein et al.,
Nucl. Acids Res., 16, 3209, (1988), methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A. 85,
7448-7451, (1988)). A number of methods have been developed for
delivering antisense DNA or RNA to cells, e.g., antisense molecules
can be injected directly into the tissue site, or modified
antisense molecules, designed to target the desired cells
(antisense linked to peptides or antibodies that specifically bind
receptors or antigens expressed on the target cell surface) can be
administered systemically. Alternatively, RNA molecules may be
generated by in vitro and in vivo transcription of DNA sequences
encoding the antisense RNA molecule. Such DNA sequences may be
incorporated into a wide variety of vectors that incorporate
suitable RNA polymerase promoters such as the T7 or SP6 polymerase
promoters. Alternatively, antisense cDNA constructs that synthesize
antisense RNA constitutively or inducibly, depending on the
promoter used, can be introduced stably into cell lines. However,
it is often difficult to achieve intracellular concentrations of
the antisense sufficient to suppress translation of endogenous
mRNAs. Therefore a preferred approach utilizes a recombinant DNA
construct in which the antisense oligonucleotide is placed under
the control of a strong promoter. The use of such a construct to
transfect target cells in the patient will result in the
transcription of sufficient amounts of single stranded RNAs that
will form complementary base pairs with the endogenous target gene
transcripts and thereby prevent translation of the target gene
mRNA. For example, a vector can be introduced in vivo such that it
is taken up by a cell and directs the transcription of an antisense
RNA. Such a vector can remain episomal or become chromosomally
integrated, as long as it can be transcribed to produce the desired
antisense RNA. Such vectors can be constructed by recombinant DNA
technology methods standard in the art. Vectors can be plasmid,
viral, or others known in the art, used for replication and
expression in mammalian cells. Expression of the sequence encoding
the antisense RNA can be by any promoter known in the art to act in
mammalian, preferably human, cells. Such promoters can be inducible
or constitutive. Any type of plasmid, cosmid, yeast artificial
chromosome, or viral vector can be used to prepare the recombinant
DNA construct that can be introduced directly into the tissue
site.
[0136] The polynucleotides may be flanked by natural regulatory
(expression control) sequences or may be associated with
heterologous sequences, including promoters, internal ribosome
entry sites (IRES) and other ribosome binding site sequences,
enhancers, response elements, suppressors, signal sequences,
polyadenylation sequences, introns, 5'- and 3'-non-coding regions,
and the like. The nucleic acids may also be modified by many means
known in the art. Non-limiting examples of such modifications
include methylation, "caps", substitution of one or more of the
naturally occurring nucleotides with an analog, and internucleotide
modifications, such as, for example, those with uncharged linkages
(e.g., methyl phosphonates, phosphotriesters, phosphoroamidates,
carbamates, etc.) and with charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.). Polynucleotides may
contain one or more additional covalently linked moieties, such as,
for example, proteins (e.g., nucleases, toxins, antibodies, signal
peptides, poly-L-lysine, etc.), intercalators (e.g., acridine,
psoralen, etc.), chelators (e.g., metals, radioactive metals, iron,
oxidative metals, etc.), and alkylators. The polynucleotides may be
derivatized by formation of a methyl or ethyl phosphotriester or an
alkyl phosphoramidate linkage. Furthermore, the polynucleotides
herein may also be modified with a label capable of providing a
detectable signal, either directly or indirectly. Exemplary labels
include radioisotopes, fluorescent molecules, isotopes (e.g.,
radioactive isotopes), biotin, and the like.
[0137] The term "distal binding pocket" refers to a substrate
binding pocket distinct from the active site of an enzyme. The
distal binding pocket may be at least about 2 angstroms, at least
about 3 angstroms, at least about 5 angstroms, at least about 10
angstroms, at least about 15 angstroms, at least about 20
angstroms, at least about 25 angstroms, at least about 30
angstroms, at least about 35 angstroms, at least about 40
angstroms, at least about 45 angstroms, at least about 50
angstroms, at least about 55 angstroms, at least about 60
angstroms, at least about 65 angstroms, at least about 70
angstroms, at least about 75 angstroms, at least about 80
angstroms, at least about 85 angstroms, at least about 90
angstroms, at least about 95 angstroms, or at least about 100
angstroms from the active site of the enzyme.
[0138] The term "exo-site" is a secondary binding site, remote from
the active site, on a protein, such as an enzyme. It may be
characterized by any of the embodiments described herein. The
exo-site may be at least about 2 angstroms, at least about 3
angstroms, at least about 5 angstroms, at least about 10 angstroms,
at least about 15 angstroms, at least about 20 angstroms, at least
about 25 angstroms, at least about 30 angstroms, at least about 35
angstroms, at least about 40 angstroms, at least about 45
angstroms, at least about 50 angstroms, at least about 55
angstroms, at least about 60 angstroms, at least about 65
angstroms, at least about 70 angstroms, at least about 75
angstroms, at least about 80 angstroms, at least about 85
angstroms, at least about 90 angstroms, at least about 95
angstroms, or at least about 100 angstroms from the active site of
the enzyme.
[0139] The term "allosteric site" refers to a binding site on a
protein that is distinct from the active site and binds a substrate
molecule that regulates the enzyme. Allosteric sites allow
substrates to bind to the protein, often resulting in a
conformational change involving protein dynamics. Substrates that
enhance the protein's activity are referred to as allosteric
activators, whereas those that decrease the protein's activity are
called allosteric inhibitors. The distal binding pocket may be at
least about 2 angstroms, at least about 3 angstroms, at least about
5 angstroms, at least about 10 angstroms, at least about 15
angstroms, at least about 20 angstroms, at least about 25
angstroms, at least about 30 angstroms, at least about 35
angstroms, at least about 40 angstroms, at least about 45
angstroms, at least about 50 angstroms, at least about 55
angstroms, at least about 60 angstroms, at least about 65
angstroms, at least about 70 angstroms, at least about 75
angstroms, at least about 80 angstroms, at least about 85
angstroms, at least about 90 angstroms, at least about 95
angstroms, or at least about 100 angstroms from the active site of
the enzyme.
[0140] The term "isoform" refers to protein isoforms or protein
variants, describing either several different forms of protein
coded from the same gene, or proteins with amino acid sequence and
functional similarities, even when they are products of different
genes.
[0141] The term "active site" is the region of an enzyme where
substrate molecules bind and undergo a chemical reaction. The
active site comprises residues that form temporary bonds with the
substrate, known as the "binding site," and residues that catalyze
a reaction of that substrate, known as the "catalytic site."
[0142] The term "substrate" or "enzyme substrate" refers to a
molecule upon which an enzyme acts. Enzymes catalyze chemical
reactions involving the substrate(s). In some embodiments, a
substrate may bind to the active site and be subsequently
transformed into one or more products followed by release from the
active site. In some embodiments, a substrate may be unchanged
after binding to an enzyme. In some embodiments, a substrate may
bind to an exo-site of an enzyme, as defined herein.
EXAMPLES
[0143] In order that the invention described herein may be more
fully understood, the following examples are set forth. The
examples described in this application are offered to illustrate
the agents, pharmaceutical compositions, and methods provided
herein and are not to be construed in any way as limiting their
scope.
Dual In Vitro Selection of a DNA-Encoded Library with Immobilized
Wildtype Enzyme and Exo-Site Mutant
Site-Directed Mutagenesis, Expression, and Purification of Human
Wildtype IDE and IDE-A479L Exo-Site Mutant.
[0144] The reported N-His.sub.6-tagged human IDE.sub.42_1019
construct (isoform containing the amino acids 42-1019 of the IDE
sequence) was introduced in the expression plasmid pTrcHis-A
(Invitrogen) using primers for uracil-specific excision reactions
(USER) by Taq (NEB) and Pfu polymerases (PfuTurbo CX.RTM.,
Agilent). The IDE gene was amplified with the primers
TABLE-US-00002 (SEQ ID NO: 4)
5'-ATCATCATATGAATAATCCAGCCA-dU-CAAGAGAATAGG and (SEQ ID NO: 5)
5'-ATGCTAGCCATACCTCAGAG-dU-TTTGCAGCCATGAAG
(underlined sequences represent overhangs, and italics highlight
the PCR priming sequence). Similarly, the pTrcHis-A vector was
amplified for USER cloning with the primers
TABLE-US-00003 (SEQ ID NO: 6)
5'-ATGGCTGGATTATTCATATGATGA-dU-GATGATGATGAGAACCC and (SEQ ID NO: 7)
5'-ACTCTGAGGTATGGCTAGCA-dU-GACTGGTG.
The IDE-A479L mutation was introduced by amplifying the wild-type
IDE pTrcHis-A vector construct with USER cloning primers carrying
the mutation in the overhang region:
TABLE-US-00004 (SEQ ID NO: 8)
5'-ATGTCCGGGTTCTGATAGTTTCTAAA-dU-CTTTTGAAGGAAAAAC TG and (SEQ ID
NO: 9) 5'-ATTTAGAAACTATCAGAACCCGGACA-dU-TTTCTGGTCTGAG.
[0145] All PCR products were purified on microcentrifuge membrane
columns (MinElute.RTM., Qiagen) and quantified by UV absorbance
(NanoDrop). Each fragment (0.2 pmol) was combined in a 10 .mu.L
reaction mixture containing 20 units DpnI (NEB), 0.75 units of USER
mix (Endonuclease VIII and Uracil-DNA Glycosylase, NEB), 20 mM
Tris-acetate, 50 mM potassium acetate, 10 mM magnesium acetate, 1
mM dithiothreitol at pH 7.9 (1.times.NEBuffer 4). The reactions
were incubated at 37.degree. C. for 45 min, followed by heating to
80.degree. C. and slow cooling to 30.degree. C. (0.2.degree. C./s).
The hybridized constructs were directly used for heat-shock
transformation of chemically competent NEB turbo E. coli cells
according to the manufacturer's instructions. Transformants were
selected on carbenicillin LB agar, and isolated colonies were
cultured overnight in 2 mL LB.
[0146] The plasmids were extracted using microcentrifuge membrane
column kits (Miniprep.RTM., Qiagen), and the sequence of genes and
vector junctions were confirmed by Sanger sequencing. The plasmid
constructs were transformed by heat-shock into chemically-competent
expression strain Rosetta 2 (DE3) pLysS E. coli cells (EMD
Millipore), and selected on carbenicillin/chloramphenicol LB agar.
Cells transformed with IDE pTrcHis A constructs were cultured
overnight at 37.degree. C. in 2 XYT media (31 g in 1 L) containing
100 .mu.g/mL ampicillin and 34 .mu.g/mL chloramphenicol. Expression
of His.sub.6-tagged IDE proteins was induced when the culture
measured OD600.about.0.6 by addition of
isopropyl-.beta.-D-1-thiogalactopyranoside (IPTG) to 1 mM final
concentration, incubated overnight at 37.degree. C., followed by
centrifugation at 10,000 g for 30 min at 4.degree. C.
[0147] Recombinant His.sub.6-tagged IDE and IDE-A479L were purified
by Ni(II)-affinity chromatography (IMAC sepharose beads, GE
Healthcare.RTM.) according to the manufacturer's instructions. The
cell pellets were resuspended in pH 8.0 buffer containing 50 mM
phosphate, 300 mM NaCl, 10 mM imidazole, 1% Triton X-100 and 1 mM
tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and were lysed
by probe sonication for 4 min at <4.degree. C., followed by
clearing of cell debris by centrifugation at 10,000 g for 25 min at
4.degree. C. The supernatant was incubated with Ni(II)-doped IMAC
resin (2 mL) for 3 h at 4.degree. C. The resin was washed twice
with the cell resuspension/lysis buffer, and three times with pH
8.0 buffer containing 50 mM phosphate, 300 mM NaCl, 50 mM imidazole
and 1 mM TCEP. Elution was performed in 2 mL aliquots by raising
the imidazole concentration to 250 mM and subsequently to 500 mM in
the previous buffer. The fractions were combined and the buffer was
exchanged to the recommended IDE buffer (R&D) using spin
columns with 100 KDa molecular weight cut off membranes
(Millipore). Protein yields were typically .about.10 .mu.g/L, and
>90% purity based on gel electrophoresis analysis (Coomassie
stained). IDE-specific protease activity was >95% as assessed by
inhibition of degradation of peptide substrate
Mca-RPPGFSAFK(Dnp)-OH (R&D) by inhibitor 6bK (20 .mu.M final),
and compared with pre-quantitated commercially available human IDE
(R&D).
Dual In Vitro Selection of a DNA-Templated Library with Immobilized
Human IDE and IDE-A479L Exo-Site Mutant.
[0148] Recombinant N-His.sub.6-tagged human IDE.sub.42-1019 and
A479L-IDE.sub.42-1019 (.about.10 .mu.g) were immobilized on cobalt
magnetic beads (Dynabeads.RTM. His-Tag Isolation & Pulldown,
Invitrogen.RTM.) by incubating the protein solutions with beads (30
.mu.L) at 4.degree. C. for 30 min in 300 .mu.L of pH 8.0 buffer
containing 50 mM phosphate, 300 mM NaCl and 0.01% Tween-20 (PBST
buffer), and washed twice with the same buffer. The two
individually prepared protein-bead suspensions were incubated for
30 min with 5 pmol of the Liu lab 13,824-membered DNA-templated
macrocycle library.sup.1 at 4.degree. C., in pH 7.4 buffer
containing 50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20 (TBST
buffer) supplemented with 0.01% BSA and 3 mg/mL yeast RNA
(Ambion.RTM.). The beads were washed three times with 200 .mu.L
TBST buffer. The enriched library fraction was eluted by treatment
with 200 mM imidazole in PBST buffer (50 .mu.L) for 5 min.
[0149] PCR amplification of the enriched pool of library barcodes,
and input library, was performed in two steps. The first
amplification used a set of primers differentiated by addition of
extra bases as a diversity element (N.sub.0-5) for sequencing
purposes, and secondly using primers that append adaptors for
Illumina sequencing and a 7-base identifier (XXXXXX). The first set
of primers were
5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT(N.sub.0-5)GAGTGGGATG (SEQ ID
NO: 10) and TGGAGTTCAGACGTGTGCTCTTCCGATCTCCCTGTACAC (SEQ ID NO:
11). The adaptor primers were
5'-CAAGCAGAAGACGGCATACGAGATXXXXXXXGTGACTGGAGTTCAGACGTGTGC TCTTC
(SEQ ID NO: 12) and 5'-AATGATACGGCGACCACCGAGATCTACACXXXXXXX
ACACTCTTTCCCTACACGAC (SEQ ID NO: 13). The PCR amplicons were
purified by polyacrylamide gel electrophoresis, extracted, and
quantified using UV absorbance (NanoDrop) and qPCR (KAPA
Biosystems).
[0150] High-throughput DNA sequencing was performed on an Illumina
MiSeq instrument to yield an average of .about.1 million sequence
reads for each selection, untreated bead control and pre-selection
library. Deconvolution of library barcodes and enrichment
calculations were performed with custom software as described
previously..sup.1 Variations in library member abundance as a
result of binding to immobilized IDE was revealed by calculating
fold-enrichment over the pre-selection library for the two
independent selection experiments.
REFERENCES
[0151] 1. Kleiner, R. E., Dumelin, C. E., Tiu, G. C., Sakurai, K.
& Liu, D. R. In vitro selection of a DNA-templated
small-molecule library reveals a class of macrocyclic kinase
inhibitors. J. Am. Chem. Soc. (2010) 132, 11779-11791.
EQUIVALENTS AND SCOPE
[0152] In the claims articles such as "a," "an," and "the" may mean
one or more than one unless indicated to the contrary or otherwise
evident from the context. Claims or descriptions that include "or"
between one or more members of a group are considered satisfied if
one, more than one, or all of the group members are present in,
employed in, or otherwise relevant to a given product or process
unless indicated to the contrary or otherwise evident from the
context. The invention includes embodiments in which exactly one
member of the group is present in, employed in, or otherwise
relevant to a given product or process. The invention includes
embodiments in which more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process.
[0153] Furthermore, the invention encompasses all variations,
combinations, and permutations in which one or more limitations,
elements, clauses, and descriptive terms from one or more of the
listed claims is introduced into another claim. For example, any
claim that is dependent on another claim can be modified to include
one or more limitations found in any other claim that is dependent
on the same base claim. Where elements are presented as lists,
e.g., in Markush group format, each subgroup of the elements is
also disclosed, and any element(s) can be removed from the group.
It should it be understood that, in general, where the invention,
or aspects of the invention, is/are referred to as comprising
particular elements and/or features, certain embodiments of the
invention or aspects of the invention consist, or consist
essentially of, such elements and/or features. For purposes of
simplicity, those embodiments have not been specifically set forth
in haec verba herein. It is also noted that the terms "comprising"
and "containing" are intended to be open and permits the inclusion
of additional elements or steps. Where ranges are given, endpoints
are included. Furthermore, unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or sub-range within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates
otherwise.
[0154] This application refers to various issued patents, published
patent applications, journal articles, and other publications, all
of which are incorporated herein by reference. If there is a
conflict between any of the incorporated references and the instant
specification, the specification shall control. In addition, any
particular embodiment of the present invention that falls within
the prior art may be explicitly excluded from any one or more of
the claims. Because such embodiments are deemed to be known to one
of ordinary skill in the art, they may be excluded even if the
exclusion is not set forth explicitly herein. Any particular
embodiment of the invention can be excluded from any claim, for any
reason, whether or not related to the existence of prior art.
[0155] Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation many
equivalents to the specific embodiments described herein. The scope
of the present embodiments described herein is not intended to be
limited to the above Description, but rather is as set forth in the
appended claims. Those of ordinary skill in the art will appreciate
that various changes and modifications to this description may be
made without departing from the spirit or scope of the present
invention, as defined in the following claims.
Sequence CWU 1
1
1311019PRTHomo sapiens 1Met Arg Tyr Arg Leu Ala Trp Leu Leu His Pro
Ala Leu Pro Ser Thr1 5 10 15Phe Arg Ser Val Leu Gly Ala Arg Leu Pro
Pro Pro Glu Arg Leu Cys 20 25 30Gly Phe Gln Lys Lys Thr Tyr Ser Lys
Met Asn Asn Pro Ala Ile Lys 35 40 45Arg Ile Gly Asn His Ile Thr Lys
Ser Pro Glu Asp Lys Arg Glu Tyr 50 55 60Arg Gly Leu Glu Leu Ala Asn
Gly Ile Lys Val Leu Leu Ile Ser Asp65 70 75 80Pro Thr Thr Asp Lys
Ser Ser Ala Ala Leu Asp Val His Ile Gly Ser 85 90 95Leu Ser Asp Pro
Pro Asn Ile Ala Gly Leu Ser His Phe Cys Glu His 100 105 110Met Leu
Phe Leu Gly Thr Lys Lys Tyr Pro Lys Glu Asn Glu Tyr Ser 115 120
125Gln Phe Leu Ser Glu His Ala Gly Ser Ser Asn Ala Phe Thr Ser Gly
130 135 140Glu His Thr Asn Tyr Tyr Phe Asp Val Ser His Glu His Leu
Glu Gly145 150 155 160Ala Leu Asp Arg Phe Ala Gln Phe Phe Leu Cys
Pro Leu Phe Asp Glu 165 170 175Ser Cys Lys Asp Arg Glu Val Asn Ala
Val Asp Ser Glu His Glu Lys 180 185 190Asn Val Met Asn Asp Ala Trp
Arg Leu Phe Gln Leu Glu Lys Ala Thr 195 200 205Gly Asn Pro Lys His
Pro Phe Ser Lys Phe Gly Thr Gly Asn Lys Tyr 210 215 220Thr Leu Glu
Thr Arg Pro Asn Gln Glu Gly Ile Asp Val Arg Gln Glu225 230 235
240Leu Leu Lys Phe His Ser Ala Tyr Tyr Ser Ser Asn Leu Met Ala Val
245 250 255Cys Val Leu Gly Arg Glu Ser Leu Asp Asp Leu Thr Asn Leu
Val Val 260 265 270Lys Leu Phe Ser Glu Val Glu Asn Lys Asn Val Pro
Leu Pro Glu Phe 275 280 285Pro Glu His Pro Phe Gln Glu Glu His Leu
Lys Gln Leu Tyr Lys Ile 290 295 300Val Pro Ile Lys Asp Ile Arg Asn
Leu Tyr Val Thr Phe Pro Ile Pro305 310 315 320Asp Leu Gln Lys Tyr
Tyr Lys Ser Asn Pro Gly His Tyr Leu Gly His 325 330 335Leu Ile Gly
His Glu Gly Pro Gly Ser Leu Leu Ser Glu Leu Lys Ser 340 345 350Lys
Gly Trp Val Asn Thr Leu Val Gly Gly Gln Lys Glu Gly Ala Arg 355 360
365Gly Phe Met Phe Phe Ile Ile Asn Val Asp Leu Thr Glu Glu Gly Leu
370 375 380Leu His Val Glu Asp Ile Ile Leu His Met Phe Gln Tyr Ile
Gln Lys385 390 395 400Leu Arg Ala Glu Gly Pro Gln Glu Trp Val Phe
Gln Glu Cys Lys Asp 405 410 415Leu Asn Ala Val Ala Phe Arg Phe Lys
Asp Lys Glu Arg Pro Arg Gly 420 425 430Tyr Thr Ser Lys Ile Ala Gly
Ile Leu His Tyr Tyr Pro Leu Glu Glu 435 440 445Val Leu Thr Ala Glu
Tyr Leu Leu Glu Glu Phe Arg Pro Asp Leu Ile 450 455 460Glu Met Val
Leu Asp Lys Leu Arg Pro Glu Asn Val Arg Val Ala Ile465 470 475
480Val Ser Lys Ser Phe Glu Gly Lys Thr Asp Arg Thr Glu Glu Trp Tyr
485 490 495Gly Thr Gln Tyr Lys Gln Glu Ala Ile Pro Asp Glu Val Ile
Lys Lys 500 505 510Trp Gln Asn Ala Asp Leu Asn Gly Lys Phe Lys Leu
Pro Thr Lys Asn 515 520 525Glu Phe Ile Pro Thr Asn Phe Glu Ile Leu
Pro Leu Glu Lys Glu Ala 530 535 540Thr Pro Tyr Pro Ala Leu Ile Lys
Asp Thr Ala Met Ser Lys Leu Trp545 550 555 560Phe Lys Gln Asp Asp
Lys Phe Phe Leu Pro Lys Ala Cys Leu Asn Phe 565 570 575Glu Phe Phe
Ser Pro Phe Ala Tyr Val Asp Pro Leu His Cys Asn Met 580 585 590Ala
Tyr Leu Tyr Leu Glu Leu Leu Lys Asp Ser Leu Asn Glu Tyr Ala 595 600
605Tyr Ala Ala Glu Leu Ala Gly Leu Ser Tyr Asp Leu Gln Asn Thr Ile
610 615 620Tyr Gly Met Tyr Leu Ser Val Lys Gly Tyr Asn Asp Lys Gln
Pro Ile625 630 635 640Leu Leu Lys Lys Ile Ile Glu Lys Met Ala Thr
Phe Glu Ile Asp Glu 645 650 655Lys Arg Phe Glu Ile Ile Lys Glu Ala
Tyr Met Arg Ser Leu Asn Asn 660 665 670Phe Arg Ala Glu Gln Pro His
Gln His Ala Met Tyr Tyr Leu Arg Leu 675 680 685Leu Met Thr Glu Val
Ala Trp Thr Lys Asp Glu Leu Lys Glu Ala Leu 690 695 700Asp Asp Val
Thr Leu Pro Arg Leu Lys Ala Phe Ile Pro Gln Leu Leu705 710 715
720Ser Arg Leu His Ile Glu Ala Leu Leu His Gly Asn Ile Thr Lys Gln
725 730 735Ala Ala Leu Gly Ile Met Gln Met Val Glu Asp Thr Leu Ile
Glu His 740 745 750Ala His Thr Lys Pro Leu Leu Pro Ser Gln Leu Val
Arg Tyr Arg Glu 755 760 765Val Gln Leu Pro Asp Arg Gly Trp Phe Val
Tyr Gln Gln Arg Asn Glu 770 775 780Val His Asn Asn Cys Gly Ile Glu
Ile Tyr Tyr Gln Thr Asp Met Gln785 790 795 800Ser Thr Ser Glu Asn
Met Phe Leu Glu Leu Phe Cys Gln Ile Ile Ser 805 810 815Glu Pro Cys
Phe Asn Thr Leu Arg Thr Lys Glu Gln Leu Gly Tyr Ile 820 825 830Val
Phe Ser Gly Pro Arg Arg Ala Asn Gly Ile Gln Gly Leu Arg Phe 835 840
845Ile Ile Gln Ser Glu Lys Pro Pro His Tyr Leu Glu Ser Arg Val Glu
850 855 860Ala Phe Leu Ile Thr Met Glu Lys Ser Ile Glu Asp Met Thr
Glu Glu865 870 875 880Ala Phe Gln Lys His Ile Gln Ala Leu Ala Ile
Arg Arg Leu Asp Lys 885 890 895Pro Lys Lys Leu Ser Ala Glu Cys Ala
Lys Tyr Trp Gly Glu Ile Ile 900 905 910Ser Gln Gln Tyr Asn Phe Asp
Arg Asp Asn Thr Glu Val Ala Tyr Leu 915 920 925Lys Thr Leu Thr Lys
Glu Asp Ile Ile Lys Phe Tyr Lys Glu Met Leu 930 935 940Ala Val Asp
Ala Pro Arg Arg His Lys Val Ser Val His Val Leu Ala945 950 955
960Arg Glu Met Asp Ser Cys Pro Val Val Gly Glu Phe Pro Cys Gln Asn
965 970 975Asp Ile Asn Leu Ser Gln Ala Pro Ala Leu Pro Gln Pro Glu
Val Ile 980 985 990Gln Asn Met Thr Glu Phe Lys Arg Gly Leu Pro Leu
Phe Pro Leu Val 995 1000 1005Lys Pro His Ile Asn Phe Met Ala Ala
Lys Leu 1010 10152464PRTHomo sapiens 2Met Ser Lys Leu Trp Phe Lys
Gln Asp Asp Lys Phe Phe Leu Pro Lys1 5 10 15Ala Cys Leu Asn Phe Glu
Phe Phe Ser Pro Phe Ala Tyr Val Asp Pro 20 25 30Leu His Cys Asn Met
Ala Tyr Leu Tyr Leu Glu Leu Leu Lys Asp Ser 35 40 45Leu Asn Glu Tyr
Ala Tyr Ala Ala Glu Leu Ala Gly Leu Ser Tyr Asp 50 55 60Leu Gln Asn
Thr Ile Tyr Gly Met Tyr Leu Ser Val Lys Gly Tyr Asn65 70 75 80Asp
Lys Gln Pro Ile Leu Leu Lys Lys Ile Ile Glu Lys Met Ala Thr 85 90
95Phe Glu Ile Asp Glu Lys Arg Phe Glu Ile Ile Lys Glu Ala Tyr Met
100 105 110Arg Ser Leu Asn Asn Phe Arg Ala Glu Gln Pro His Gln His
Ala Met 115 120 125Tyr Tyr Leu Arg Leu Leu Met Thr Glu Val Ala Trp
Thr Lys Asp Glu 130 135 140Leu Lys Glu Ala Leu Asp Asp Val Thr Leu
Pro Arg Leu Lys Ala Phe145 150 155 160Ile Pro Gln Leu Leu Ser Arg
Leu His Ile Glu Ala Leu Leu His Gly 165 170 175Asn Ile Thr Lys Gln
Ala Ala Leu Gly Ile Met Gln Met Val Glu Asp 180 185 190Thr Leu Ile
Glu His Ala His Thr Lys Pro Leu Leu Pro Ser Gln Leu 195 200 205Val
Arg Tyr Arg Glu Val Gln Leu Pro Asp Arg Gly Trp Phe Val Tyr 210 215
220Gln Gln Arg Asn Glu Val His Asn Asn Cys Gly Ile Glu Ile Tyr
Tyr225 230 235 240Gln Thr Asp Met Gln Ser Thr Ser Glu Asn Met Phe
Leu Glu Leu Phe 245 250 255Cys Gln Ile Ile Ser Glu Pro Cys Phe Asn
Thr Leu Arg Thr Lys Glu 260 265 270Gln Leu Gly Tyr Ile Val Phe Ser
Gly Pro Arg Arg Ala Asn Gly Ile 275 280 285Gln Gly Leu Arg Phe Ile
Ile Gln Ser Glu Lys Pro Pro His Tyr Leu 290 295 300Glu Ser Arg Val
Glu Ala Phe Leu Ile Thr Met Glu Lys Ser Ile Glu305 310 315 320Asp
Met Thr Glu Glu Ala Phe Gln Lys His Ile Gln Ala Leu Ala Ile 325 330
335Arg Arg Leu Asp Lys Pro Lys Lys Leu Ser Ala Glu Cys Ala Lys Tyr
340 345 350Trp Gly Glu Ile Ile Ser Gln Gln Tyr Asn Phe Asp Arg Asp
Asn Thr 355 360 365Glu Val Ala Tyr Leu Lys Thr Leu Thr Lys Glu Asp
Ile Ile Lys Phe 370 375 380Tyr Lys Glu Met Leu Ala Val Asp Ala Pro
Arg Arg His Lys Val Ser385 390 395 400Val His Val Leu Ala Arg Glu
Met Asp Ser Cys Pro Val Val Gly Glu 405 410 415Phe Pro Cys Gln Asn
Asp Ile Asn Leu Ser Gln Ala Pro Ala Leu Pro 420 425 430Gln Pro Glu
Val Ile Gln Asn Met Thr Glu Phe Lys Arg Gly Leu Pro 435 440 445Leu
Phe Pro Leu Val Lys Pro His Ile Asn Phe Met Ala Ala Lys Leu 450 455
46031019PRTMus musculus 3Met Arg Asn Gly Leu Val Trp Leu Leu His
Pro Ala Leu Pro Gly Thr1 5 10 15Leu Arg Ser Ile Leu Gly Ala Arg Pro
Pro Pro Ala Lys Arg Leu Cys 20 25 30Gly Phe Pro Lys Gln Thr Tyr Ser
Thr Met Ser Asn Pro Ala Ile Gln 35 40 45Arg Ile Glu Asp Gln Ile Val
Lys Ser Pro Glu Asp Lys Arg Glu Tyr 50 55 60Arg Gly Leu Glu Leu Ala
Asn Gly Ile Lys Val Leu Leu Ile Ser Asp65 70 75 80Pro Thr Thr Asp
Lys Ser Ser Ala Ala Leu Asp Val His Ile Gly Ser 85 90 95Leu Ser Asp
Pro Pro Asn Ile Pro Gly Leu Ser His Phe Cys Glu His 100 105 110Met
Leu Phe Leu Gly Thr Lys Lys Tyr Pro Lys Glu Asn Glu Tyr Ser 115 120
125Gln Phe Leu Ser Glu His Ala Gly Ser Ser Asn Ala Phe Thr Ser Gly
130 135 140Glu His Thr Asn Tyr Tyr Phe Asp Val Ser His Glu His Leu
Glu Gly145 150 155 160Ala Leu Asp Arg Phe Ala Gln Phe Phe Leu Cys
Pro Leu Phe Asp Ala 165 170 175Ser Cys Lys Asp Arg Glu Val Asn Ala
Val Asp Ser Glu His Glu Lys 180 185 190Asn Val Met Asn Asp Ala Trp
Arg Leu Phe Gln Leu Glu Lys Ala Thr 195 200 205Gly Asn Pro Lys His
Pro Phe Ser Lys Phe Gly Thr Gly Asn Lys Tyr 210 215 220Thr Leu Glu
Thr Arg Pro Asn Gln Glu Gly Ile Asp Val Arg Glu Glu225 230 235
240Leu Leu Lys Phe His Ser Thr Tyr Tyr Ser Ser Asn Leu Met Ala Ile
245 250 255Cys Val Leu Gly Arg Glu Ser Leu Asp Asp Leu Thr Asn Leu
Val Val 260 265 270Lys Leu Phe Ser Glu Val Glu Asn Lys Asn Val Pro
Leu Pro Glu Phe 275 280 285Pro Glu His Pro Phe Gln Glu Glu His Leu
Arg Gln Leu Tyr Lys Ile 290 295 300Val Pro Ile Lys Asp Ile Arg Asn
Leu Tyr Val Thr Phe Pro Ile Pro305 310 315 320Asp Leu Gln Gln Tyr
Tyr Lys Ser Asn Pro Gly His Tyr Leu Gly His 325 330 335Leu Ile Gly
His Glu Gly Pro Gly Ser Leu Leu Ser Glu Leu Lys Ser 340 345 350Lys
Gly Trp Val Asn Thr Leu Val Gly Gly Gln Lys Glu Gly Ala Arg 355 360
365Gly Phe Met Phe Phe Ile Ile Asn Val Asp Leu Thr Glu Glu Gly Leu
370 375 380Leu His Val Glu Asp Ile Ile Leu His Met Phe Gln Tyr Ile
Gln Lys385 390 395 400Leu Arg Ala Glu Gly Pro Gln Glu Trp Val Phe
Gln Glu Cys Lys Asp 405 410 415Leu Asn Ala Val Ala Phe Arg Phe Lys
Asp Lys Glu Arg Pro Arg Gly 420 425 430Tyr Thr Ser Lys Ile Ala Gly
Lys Leu His Tyr Tyr Pro Leu Asn Gly 435 440 445Val Leu Thr Ala Glu
Tyr Leu Leu Glu Glu Phe Arg Pro Asp Leu Ile 450 455 460Asp Met Val
Leu Asp Lys Leu Arg Pro Glu Asn Val Arg Val Ala Ile465 470 475
480Val Ser Lys Ser Phe Glu Gly Lys Thr Asp Arg Thr Glu Gln Trp Tyr
485 490 495Gly Thr Gln Tyr Lys Gln Glu Ala Ile Pro Glu Asp Ile Ile
Gln Lys 500 505 510Trp Gln Asn Ala Asp Leu Asn Gly Lys Phe Lys Leu
Pro Thr Lys Asn 515 520 525Glu Phe Ile Pro Thr Asn Phe Glu Ile Leu
Ser Leu Glu Lys Asp Ala 530 535 540Thr Pro Tyr Pro Ala Leu Ile Lys
Asp Thr Ala Met Ser Lys Leu Trp545 550 555 560Phe Lys Gln Asp Asp
Lys Phe Phe Leu Pro Lys Ala Cys Leu Asn Phe 565 570 575Glu Phe Phe
Ser Pro Phe Ala Tyr Val Asp Pro Leu His Cys Asn Met 580 585 590Ala
Tyr Leu Tyr Leu Glu Leu Leu Lys Asp Ser Leu Asn Glu Tyr Ala 595 600
605Tyr Ala Ala Glu Leu Ala Gly Leu Ser Tyr Asp Leu Gln Asn Thr Ile
610 615 620Tyr Gly Met Tyr Leu Ser Val Lys Gly Tyr Asn Asp Lys Gln
Pro Ile625 630 635 640Leu Leu Lys Lys Ile Thr Glu Lys Met Ala Thr
Phe Glu Ile Asp Lys 645 650 655Lys Arg Phe Glu Ile Ile Lys Glu Ala
Tyr Met Arg Ser Leu Asn Asn 660 665 670Phe Arg Ala Glu Gln Pro His
Gln His Ala Met Tyr Tyr Leu Arg Leu 675 680 685Leu Met Thr Glu Val
Ala Trp Thr Lys Asp Glu Leu Lys Glu Ala Leu 690 695 700Asp Asp Val
Thr Leu Pro Arg Leu Lys Ala Phe Ile Pro Gln Leu Leu705 710 715
720Ser Arg Leu His Ile Glu Ala Leu Leu His Gly Asn Ile Thr Lys Gln
725 730 735Ala Ala Leu Gly Val Met Gln Met Val Glu Asp Thr Leu Ile
Glu His 740 745 750Ala His Thr Lys Pro Leu Leu Pro Ser Gln Leu Val
Arg Tyr Arg Glu 755 760 765Val Gln Leu Pro Asp Arg Gly Trp Phe Val
Tyr Gln Gln Arg Asn Glu 770 775 780Val His Asn Asn Cys Gly Ile Glu
Ile Tyr Tyr Gln Thr Asp Met Gln785 790 795 800Ser Thr Ser Glu Asn
Met Phe Leu Glu Leu Phe Cys Gln Ile Ile Ser 805 810 815Glu Pro Cys
Phe Asn Thr Leu Arg Thr Lys Glu Gln Leu Gly Tyr Ile 820 825 830Val
Phe Ser Gly Pro Arg Arg Ala Asn Gly Ile Gln Gly Leu Arg Phe 835 840
845Ile Ile Gln Ser Glu Lys Pro Pro His Tyr Leu Glu Ser Arg Val Glu
850 855 860Ala Phe Leu Ile Thr Met Glu Lys Ala Ile Glu Asp Met Thr
Glu Glu865 870 875 880Ala Phe Gln Lys His Ile Gln Ala Leu Ala Ile
Arg Arg Leu Asp Lys 885 890 895Pro Lys Lys Leu Ser Ala Glu Cys Ala
Lys Tyr Trp Gly Glu Ile Ile 900 905 910Ser Gln Gln Tyr Asn Tyr Asp
Arg Asp Asn Ile Glu Val Ala Tyr Leu 915 920 925Lys Thr Leu Thr Lys
Asp Asp Ile Ile Arg Phe Tyr Gln Glu Met Leu 930 935 940Ala Val Asp
Ala Pro Arg Arg His Lys Val Ser Val His Val Leu Ala945 950 955
960Arg Glu Met Asp Ser Cys Pro Val Val Gly Glu Phe Pro Ser Gln Asn
965 970 975Asp Ile Asn Leu Ser Glu Ala Pro Pro Leu Pro Gln
Pro Glu Val Ile 980 985 990His Asn Met Thr Glu Phe Lys Arg Gly Leu
Pro Leu Phe Pro Leu Val 995 1000 1005Lys Pro His Ile Asn Phe Met
Ala Ala Lys Leu 1010 1015437DNAArtificial SequenceSynthetic
Polynucleotidemisc_feature(25)..(25)U is deoxyuridine 4atcatcatat
gaataatcca gccaucaaga gaatagg 37536DNAArtificial SequenceSynthetic
Polynucleotidemisc_feature(21)..(21)U is deoxyuridine 5atgctagcca
tacctcagag utttgcagcc atgaag 36642DNAArtificial SequenceSynthetic
Polynucleotidemisc_feature(25)..(25)U is deoxyuridine 6atggctggat
tattcatatg atgaugatga tgatgagaac cc 42729DNAArtificial
SequenceSynthetic Polynucleotidemisc_feature(21)..(21)U is
deoxyuridine 7actctgaggt atggctagca ugactggtg 29845DNAArtificial
SequenceSynthetic Polynucleotidemisc_feature(27)..(27)U is
deoxyuridine 8atgtccgggt tctgatagtt tctaaauctt ttgaaggaaa aactg
45940DNAArtificial SequenceSynthetic
Polynucleotidemisc_feature(27)..(27)U is deoxyuridine 9atttagaaac
tatcagaacc cggacauttt ctggtctgag 401048DNAArtificial
SequenceSynthetic polynucleotidemisc_feature(34)..(38)N may be
absent 10acactctttc cctacacgac gctcttccga tctnnnnnga gtgggatg
481139DNAArtificial SequenceSynthetic polynucleotide 11tggagttcag
acgtgtgctc ttccgatctc cctgtacac 391252DNAArtificial
SequenceSynthetic polynucleotidemisc_feature(24)..(25)modified by a
7-base identifier 12caagcagaag acggcatacg agatgtgact ggagttcaga
cgtgtgctct tc 521349DNAArtificial SequenceSynthetic
polynucleotidemisc_feature(29)..(30)modified by a 7-base identifier
13aatgatacgg cgaccaccga gatctacaca cactctttcc ctacacgac 49
* * * * *