U.S. patent application number 12/413241 was filed with the patent office on 2009-10-01 for coating compositions having improved performance.
This patent application is currently assigned to Affinergy. Invention is credited to Mohmed Anwer, Paul Hamilton, Shrikumar A. Nair, Guy Orgambide.
Application Number | 20090246250 12/413241 |
Document ID | / |
Family ID | 41114771 |
Filed Date | 2009-10-01 |
United States Patent
Application |
20090246250 |
Kind Code |
A1 |
Orgambide; Guy ; et
al. |
October 1, 2009 |
COATING COMPOSITIONS HAVING IMPROVED PERFORMANCE
Abstract
The presently disclosed subject matter provides compositions
comprising a first substrate-binding domain (a peptide or a
polymer) having binding affinity for a tissue or a medical device,
a second substrate-binding domain having binding affinity for a
target molecule, and the target molecule. In some embodiments, the
first and second substrate-binding domains are covalently linked.
The first and second substrate-binding domains are covalently
coupled to at least one hydrophobic interaction tag, negatively
charged interaction tag, or positively charged interaction tag.
When the substrate-binding domains are combined and coated onto the
tissue or medical device, the hydrophobic interaction tags interact
with each other and the charged interaction tags interact with the
oppositely charged interaction tags or the oppositely charged
substrate binding polymers, to form a macromolecular network of
non-covalently coupled substrate-binding domains to load the target
molecule onto the tissue or medical device.
Inventors: |
Orgambide; Guy;
(Morrisville, NC) ; Anwer; Mohmed; (Cary, NC)
; Nair; Shrikumar A.; (Cary, NC) ; Hamilton;
Paul; (Cary, NC) |
Correspondence
Address: |
Laura L.Kiefer;AFFINERGY, INC.
P.O. BOX 14650
Research Triangle Park
NC
27709-4650
US
|
Assignee: |
Affinergy
Research Triangle Park
NC
|
Family ID: |
41114771 |
Appl. No.: |
12/413241 |
Filed: |
March 27, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61039946 |
Mar 27, 2008 |
|
|
|
Current U.S.
Class: |
424/423 ;
424/93.1; 514/1.1 |
Current CPC
Class: |
A61K 38/16 20130101;
A61K 38/16 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/423 ;
424/93.1; 514/12; 514/13; 514/14; 514/15; 514/16; 514/17;
514/18 |
International
Class: |
A61F 2/00 20060101
A61F002/00; A61K 35/00 20060101 A61K035/00; A61K 38/16 20060101
A61K038/16; A61K 38/10 20060101 A61K038/10; A61K 38/08 20060101
A61K038/08; A61K 38/07 20060101 A61K038/07; A61K 38/06 20060101
A61K038/06 |
Goverment Interests
GRANT STATEMENT
[0002] This invention was made in part from government support
under Grant No. 2R44AR051264-02 from the National Institute of
Arthritis and Musculoskeletal and Skin Diseases. Thus, the U.S.
Government has certain rights in the invention.
Claims
1. A composition comprising: (a) a plurality of a first
substrate-binding peptide comprising 3 to 40 amino acids, wherein
the first substrate is a tissue or medical device and the first
substrate-binding peptide has binding affinity for the tissue or
medical device; (b) a plurality of a second substrate-binding
peptide comprising 3 to 40 amino acids, wherein the second
substrate is a target molecule and the second substrate-binding
peptide has binding affinity for the target molecule, wherein the
first and second substrate-binding peptides are covalently linked;
and (c) a plurality of the target molecule, wherein the plurality
of covalently linked first and second substrate-binding peptides
are covalently coupled to at least one interaction tag selected
from the group consisting of a hydrophobic interaction tag, a
positively charged interaction tag, and a negatively charged
interaction tag, wherein the hydrophobic interaction tags interact
with each other and the positively charged interaction tags
interact with the negatively charged interaction tags to form a
macromolecular network comprising the plurality of non-covalently
coupled substrate-binding peptides.
2. The composition of claim 1, wherein the first substrate-binding
peptide, the second substrate-binding peptide, and the target
molecule are present in a pharmaceutically acceptable solution.
3. The composition of claim 2, wherein the pharmaceutically
acceptable solution is in the form of a gel.
4. The composition of claim 1, wherein the first substrate tissue
or medical device comprises a material selected from the group
consisting of an animal tissue, an autologous tissue, an allogeneic
tissue, a transplanted tissue, an organ tissue, a bone tissue, a
skin tissue, a connective tissue, a muscle tissue, a nervous
tissue, a polymer, a silk, a collagen, a synthetic polymer, a
polyester, a polyurethane, a nylon, a polylactic acid, a
polyglycolic acid, poly(lactic acid-co-glycolic acid), a plastic, a
silicone material, a metal, a metal oxide, a non-metal oxide, a
ceramic material, a calcium phosphate based material, a
carbon-based material, a metallo-carbon composite, and combinations
thereof.
5. The composition of claim 1, wherein the target molecule is
selected from the group consisting of a cell, a protein, a
polypeptide, a growth factor, a growth differentiation factor
(GDF), a platelet derived growth factor (PDGF), a transforming
growth factor (TGF), an osteogenic protein, a bone morphogenic
protein (BMP), a hormone, a protein hormone, a parathyroid hormone
(PTH), a drug, a drug carrier, an antibiotic, a vancomycin
antibiotic, a steroid, a dexamethasone, and combinations
thereof.
6. The composition of claim 1, wherein the charged interaction tag
is selected from the group consisting of polylysine, polyarginine,
polyamines, polyimines, polyethylamines, polyethylenimines (PEI),
polyaspartic acid, polyglutamic acid, polystyrene sulfonate,
poly(styrenesulfonic-maleic acid), and combinations and copolymers
thereof.
7. The composition of claim 1, wherein the hydrophobic interaction
tag is selected from the group consisting of fatty acids,
undecanoic acid, poly-undecanoic acid, myristic acid, amino
hexanoic acid, capric acid, lauric acid, palmitic acid, stearic
acid, aromatic compounds, and combinations and copolymers
thereof.
8. The composition of claim 1, wherein the first and second
substrate-binding peptides are covalently linked by a peptide
bond.
9. The composition of claim 1, wherein the first and second
substrate-binding peptides are covalently linked through any one of
the hydrophobic interaction tag, the charged interaction tag, amino
acids, polymers, synthetic polymers, polyethers, poly(ethylene
glycol) ("PEG"), a 10 unit polyethylene glycol ("P10"), and a 6
unit polyethylene glycol ("MP").
10. The composition of claim 1, wherein the first substrate is a
metal medical device, the second substrate target molecule is
vancomycin, the first and second substrate binding peptides are
covalently linked through a polyethylene glycol, the hydrophobic
interaction tag is poly-undecanoic acid, the hydrophobic
interaction tag is covalently coupled to the first substrate
binding peptide either directly, through a polyethylene glycol, or
through an aminohexanoic acid, and the charged interaction tag is
absent.
11. The composition of claim 1, wherein the first substrate is a
metal medical device, the second substrate target molecule is
vancomycin, the first and second substrate binding peptides are
covalently linked through a polyethylene glycol, the positively
charged interaction tag is covalently coupled to a portion of the
plurality of second substrate binding peptide, the negatively
charged interaction tag is covalently coupled to a portion of the
second substrate binding peptide, and the hydrophobic interaction
tag is absent.
12. The composition of claim 1, wherein the first substrate medical
device is a synthetic polymer, the second substrate target molecule
is a growth factor, the hydrophobic interaction tag is
poly-undecanoic acid, the first and second substrate binding
peptides are covalently linked through the poly-undecanoic acid
hydrophobic interaction tag, and the charged interaction tag is
absent.
13. The composition of claim 1, wherein the first substrate medical
device is a synthetic polymer, the second substrate target molecule
is a growth factor, the first and second substrate binding peptides
are covalently linked through a polyethylene glycol, the
hydrophobic interaction tag is poly-undecanoic acid, the
poly-undecanoic acid is covalently coupled to the second substrate
binding peptides, and the charged interaction tag is absent.
14. A method for coating a tissue or a medical device, the method
comprising: (a) contacting a composition with the tissue or medical
device, the composition comprising: (i) a plurality of a first
substrate-binding peptide comprising 3 to 40 amino acids, wherein
the first substrate is a tissue or medical device and the first
substrate-binding peptide has binding affinity for the tissue or
medical device; (ii) a plurality of a second substrate-binding
peptide comprising 3 to 40 amino acids, wherein the second
substrate is a target molecule and the second substrate-binding
peptide has binding affinity for the target molecule, wherein the
first and second substrate-binding peptides are covalently linked;
and (iii) a plurality of the target molecule, wherein the plurality
of covalently linked first and second substrate-binding peptides
are covalently coupled to at least one interaction tag selected
from the group consisting of a hydrophobic interaction tag, a
positively charged interaction tag, and a negatively charged
interaction tag, wherein the hydrophobic interaction tags interact
with each other and the positively charged interaction tags
interact with the negatively charged interaction tags to form a
macromolecular network comprising the plurality of non-covalently
coupled substrate-binding peptides.
15. The method of claim 14, wherein the first substrate tissue or
medical device comprises a material selected from the group
consisting of an animal tissue, an autologous tissue, an allogeneic
tissue, a transplanted tissue, an organ tissue, a bone tissue, a
skin tissue, a connective tissue, a muscle tissue, a nervous
tissue, a polymer, a silk, a collagen, a synthetic polymer, a
polyester, a polyurethane, a nylon, a polylactic acid, a
polyglycolic acid, poly(lactic acid-co-glycolic acid), a plastic, a
silicone material, a metal, a metal oxide, a non-metal oxide, a
ceramic material, a calcium phosphate based material, a
carbon-based material, a metallo-carbon composite, and combinations
thereof.
16. The method of claim 14, wherein the target molecule is selected
from the group consisting of a cell, a protein, a polypeptide, a
growth factor, a growth differentiation factor (GDF), a platelet
derived growth factor (PDGF), a transforming growth factor (TGF),
an osteogenic protein, a bone morphogenic protein (BMP), a hormone,
a protein hormone, a parathyroid hormone (PTH), a drug, a drug
carrier, an antibiotic, a vancomycin antibiotic, a steroid, a
dexamethasone, and combinations thereof.
17. The method of claim 14, wherein the charged interaction tag is
selected from the group consisting of polylysine, polyarginine,
polyamines, polyimines, polyethylamines, polyethylenimines (PEI),
polyaspartic acid, polyglutamic acid, polystyrene sulfonate,
poly(styrenesulfonic-maleic acid), and combinations and copolymers
thereof.
18. The method of claim 14, wherein the hydrophobic interaction tag
is selected from the group consisting of fatty acids, undecanoic
acid, poly-undecanoic acid, myristic acid, amino hexanoic acid,
capric acid, lauric acid, palmitic acid, stearic acid, aromatic
compounds, and combinations and copolymers thereof.
19. The method of claim 14, wherein the first and second
substrate-binding peptides are covalently linked by a peptide
bond.
20. The method of claim 14, wherein the first and second
substrate-binding peptides are covalently linked through any one of
the hydrophobic interaction tag, the charged interaction tag, amino
acids, polymers, synthetic polymers, polyethers, poly(ethylene
glycol) ("PEG"), a 10 unit polyethylene glycol ("P10"), and a 6
unit polyethylene glycol ("MP").
21. The method of claim 14, wherein the first substrate is a metal
medical device, the second substrate target molecule is vancomycin,
the first and second substrate binding peptides are covalently
linked through a polyethylene glycol, the hydrophobic interaction
tag is poly-undecanoic acid, the hydrophobic interaction tag is
covalently coupled to the first substrate binding peptide either
directly, through a polyethylene glycol, or through an
aminohexanoic acid, and the charged interaction tag is absent.
22. The method of claim 14, wherein the first substrate is a metal
medical device, the second substrate target molecule is vancomycin,
the first and second substrate binding peptides are covalently
linked through a polyethylene glycol, the positively charged
interaction tag is covalently coupled to a portion of the plurality
of second substrate binding peptides, the negatively charged
interaction tag is covalently coupled to a portion of the second
substrate binding peptides, and the hydrophobic interaction tag is
absent.
23. The method of claim 14, wherein the first substrate medical
device is a synthetic polymer, the second substrate target molecule
is a growth factor, the hydrophobic interaction tag is
poly-undecanoic acid, the first and second substrate binding
peptides are covalently linked through the poly-undecanoic acid
hydrophobic interaction tag, and the charged interaction tag is
absent.
24. The method of claim 14, wherein the first substrate medical
device is a synthetic polymer, the second substrate target molecule
is a growth factor, the first and second substrate binding peptides
are covalently linked through a polyethylene glycol, the
hydrophobic interaction tag is poly-undecanoic acid, the
poly-undecanoic acid is covalently coupled to the second substrate
binding peptides, and the charged interaction tag is absent.
25. A coated medical device, wherein at least a portion of the
medical device is coated with a composition comprising: (a) a
plurality of a first substrate-binding peptide comprising 3 to 40
amino acids, wherein the first substrate is a tissue or medical
device and the first substrate-binding peptide has binding affinity
for the tissue or medical device; (b) a plurality of a second
substrate-binding peptide comprising 3 to 40 amino acids, wherein
the second substrate is a target molecule and the second
substrate-binding peptide has binding affinity for the target
molecule, wherein the first and second substrate-binding peptides
are covalently linked; and (c) a plurality of the target molecule,
wherein the plurality of covalently linked first and second
substrate-binding peptides are covalently coupled to at least one
interaction tag selected from the group consisting of a hydrophobic
interaction tag, a positively charged interaction tag, and a
negatively charged interaction tag, wherein the hydrophobic
interaction tags interact with each other and the positively
charged interaction tags interact with the negatively charged
interaction tags to form a macromolecular network comprising the
plurality of non-covalently coupled substrate-binding peptides.
26. The coated medical device of claim 25, wherein the first
substrate tissue or medical device comprises a material selected
from the group consisting of an animal tissue, an autologous
tissue, an allogeneic tissue, a transplanted tissue, an organ
tissue, a bone tissue, a skin tissue, a connective tissue, a muscle
tissue, a nervous tissue, a polymer, a silk, a collagen, a
synthetic polymer, a polyester, a polyurethane, a nylon, a
polylactic acid, a polyglycolic acid, poly(lactic acid-co-glycolic
acid), a plastic, a silicone material, a metal, a metal oxide, a
non-metal oxide, a ceramic material, a calcium phosphate based
material, a carbon-based material, a metallo-carbon composite, and
combinations thereof.
27. The coated medical device of claim 25, wherein the target
molecule is selected from the group consisting of a cell, a
protein, a polypeptide, a growth factor, a growth differentiation
factor (GDF), a platelet derived growth factor (PDGF), a
transforming growth factor (TGF), an osteogenic protein, a bone
morphogenic protein (BMP), a hormone, a protein hormone, a
parathyroid hormone (PTH), a drug, a drug carrier, an antibiotic, a
vancomycin antibiotic, a steroid, a dexamethasone, and combinations
thereof.
28. The coated medical device of claim 25, wherein the charged
interaction tag is selected from the group consisting of
polylysine, polyarginine, polyamines, polyimines, polyethylamines,
polyethylenimines (PEI), polyaspartic acid, polyglutamic acid,
polystyrene sulfonate, poly(styrenesulfonic-maleic acid), and
combinations and copolymers thereof.
29. The coated medical device of claim 25, wherein the hydrophobic
interaction tag is selected from the group consisting of fatty
acids, undecanoic acid, poly-undecanoic acid, myristic acid, amino
hexanoic acid, capric acid, lauric acid, palmitic acid, stearic
acid, aromatic compounds, and combinations and copolymers
thereof.
30. The coated medical device of claim 25, wherein the first and
second substrate-binding peptides are covalently linked by a
peptide bond.
31. The coated medical device of claim 25, wherein the first and
second substrate-binding peptides are covalently linked through any
one of the hydrophobic interaction tag, the charged interaction
tag, amino acids, polymers, synthetic polymers, polyethers,
poly(ethylene glycol) ("PEG"), a 10 unit polyethylene glycol
("P10"), and a 6 unit polyethylene glycol ("MP").
32. The coated medical device of claim 25, wherein the first
substrate is a metal medical device, the second substrate target
molecule is vancomycin, the first and second substrate binding
peptides are covalently linked through a polyethylene glycol, the
hydrophobic interaction tag is poly-undecanoic acid, the
hydrophobic interaction tag is covalently coupled to the first
substrate binding peptide either directly, through a polyethylene
glycol, or through an aminohexanoic acid, and the charged
interaction tag is absent.
33. The coated medical device of claim 25, wherein the first
substrate is a metal medical device, the second substrate target
molecule is vancomycin, the first and second substrate binding
peptides are covalently linked through a polyethylene glycol, the
positively charged interaction tag is covalently coupled to a
portion of the plurality of second substrate binding peptides, the
negatively charged interaction tag is covalently coupled to a
portion of the second substrate binding peptides, and the
hydrophobic interaction tag is absent.
34. The coated medical device of claim 25, wherein the first
substrate medical device is a synthetic polymer, the second
substrate target molecule is a growth factor, the hydrophobic
interaction tag is poly-undecanoic acid, the first and second
substrate binding peptides are covalently linked through the
poly-undecanoic acid hydrophobic interaction tag, and the charged
interaction tag is absent.
35. The coated medical device of claim 25, wherein the first
substrate medical device is a synthetic polymer, the second
substrate target molecule is a growth factor, the first and second
substrate binding peptides are covalently linked through a
polyethylene glycol, the hydrophobic interaction tag is
poly-undecanoic acid, the poly-undecanoic acid is covalently
coupled to the second substrate binding peptides, and the charged
interaction tag is absent.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The presently disclosed subject matter claims the benefit of
U.S. Provisional Patent Application Ser. No. 61/039,946 filed Mar.
27, 2008; the disclosure of which is herein incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0003] The presently disclosed subject matter relates to
compositions comprising macromolecular networks comprised of
non-covalently coupled substrate binding domains for loading of a
target molecule to a tissue or medical device.
BACKGROUND OF THE INVENTION
[0004] To provide an efficacious dose of a therapeutic agent at the
site of treatment, systemic administration of the therapeutic can
often lead to adverse or toxic side effects to the patient. Local
delivery provides smaller total amounts of the therapeutic
minimizing adverse side effects and targets the therapeutic to the
site of treatment. One way to locally deliver a therapeutic agent
to a treatment site is to coat the therapeutic agent onto the
surface of an implantable medical device.
[0005] Many matrix systems have been developed to deliver a
bioactive molecule to a substrate, such as the surface of a medical
device. Typically, the bioactive molecule is covalently coupled to
the substrate, or more commonly, the substrate is coated with a
matrix containing bioactive molecule. The matrix may be composed of
a polymer into which is trapped the bioactive molecule, and as the
matrix degrades, released is the bioactive molecule. Thus, the
efficiency of release of the bioactive molecule from the polymer
matrix depends on individual matrix characteristics such as the
affinity of the matrix for the bioactive molecule; and the matrix
degradation rate, density, and pore size. Typically, materials used
in such matrix systems include polymers such as polylactides,
polyglycolides, polyanhydrides, polyorthoesters, polylactic and
polyglycolic acid copolymers, alginate, poly(ethylene glycol),
polyoxyethylene oxide, carboxyvinyl polymer, and poly(vinyl
alcohol). Natural matrix proteins/polymers used to encapsulate
entrap bioactive molecules for release include collagen,
glycosaminoglycans, and hyaluronic acid, which are enzymatically
digested in the body.
[0006] Recently described are biological coating compositions for
medical devices (see, e.g., published patent applications US
20060051395, US 20070160644, co-pending and commonly owned)
comprising a biofunctional composition. The biofunctional
composition comprises a peptide having binding specificity for a
surface material comprising the surface onto which is to be applied
the coating composition, and a peptide having binding specificity
for a therapeutic agent; wherein covalently coupled are the peptide
having binding specificity for a surface material and the peptide
having binding specificity for a therapeutic agent. The coating
composition may further comprise therapeutic agent non-covalently
bound to peptide having binding specificity for the therapeutic
agent. Peptide-based biomaterials have gained interest as novel
materials for biomedical applications (see Fairman R, Akerfeldt K
S. Curr Opin Struct Biol 2005; 15 (4): 453-63 and Rajagopal K,
Schneider J P. Curr Opin Struct Biol 2004; 14 (4): 480-6). A large
variety of synthetic advantages of peptide-based biomaterials
include their programmability, biodegradability, and
bioresorbability. In addition, peptides can be isolated that bind
to specific therapeutic agents or the surface of biomaterials
(Grinstaff et al. U.S. Patent Application 20060263830; Beyer et al.
U.S. Patent Application 20060051395).
[0007] Certain peptides are able to self assemble into gel like
membranes when incubated in the presence of low concentrations of
monovalent cations (U.S. Pat. Nos. 5,670,483; 6,548,630) or based
on the spatial matching of the complementary functional groups
(U.S. Pat. No. 7,399,831). Versatile side-chain functional groups
and non-covalent interactions of 20 amino acids enable one to
design peptides for numerous applications. Most designed
peptide-based biomaterials are amphipathic, with both hydrophilic
and hydrophobic amino acids in their sequence. The order and repeat
of these amino acids in the primary sequence determines the nature
of the secondary structure adopted by these peptides and, thereby,
the final morphology of the assembled biomaterials. Assembly of
these peptides is driven by the non-covalent interactions between
the side-chain functional groups and backbone amides, which mostly
involve hydrophobic, electrostatic, hydrogen bonding, and
.pi.-stacking interactions (Ramachandran, S, Yu, Y. B. Biodrugs
2006; 20 (5): 263-269). Designed proteins offer favorable
properties such as precision and tight regulation of self assembly
by using environmental cues such as pH, ionic strength and
temperature (Whitesides, et al. (1991) Science 254, 1312-1319;
Yeates, T. O. & Padilla, J. E. (2002) Curr. Opin. Struct. Biol.
12, 464-470; MacPhee, C. E., Woolfson, D. N. (2004) Curr. Opin.
Solid State Mater. 8, 141-149).
[0008] Nature forms complex multicomponent three-dimensional
structures through spontaneous association of molecules termed
"molecular self-assembly" (Whitesides, et al. (1991) Science 254,
1312-1319). The self-assembly process is mediated through weak
intermolecular bonds, such as van der waals bonds, electrostatic
interactions, hydrogen bonds and stacking interactions. These
relatively low energy interactions are combined together to form
intact and well-ordered supramolecular structures. The
self-assembly of peptide amphiphiles into nanostructures creates a
dense hydrocarbon-like microenvironment within an aqueous gel. The
environment created locally upon assembly makes peptide amphiphile
nanostructures and other self-assembling systems potentially ideal
candidates for the delivery of hydrophobic or water-insoluble
molecules in vivo (Guler, et al. J Mater Chem 2005, 15, 4507-4512).
In addition, peptide sequences that bind to cells or other
biologics can be attached to self-assembling peptides to generate
peptide nanofibers that bind biologics (U.S. Pat. No. 7,399,831;
U.S. Patent Application 20050272662; U.S. Patent Application
20050209145).
[0009] Within the art, however, there still exists a need to
generate self-assembling peptides that both bind a therapeutic
agent and to the surface of a medical device. These dual
functional, self-assembling peptides could be used for controlled,
local deliver of a therapeutic agent from an implanted medical
device.
SUMMARY OF THE INVENTION
[0010] The presently disclosed subject matter provides a
composition comprising a plurality of a first substrate-binding
peptide comprising 3 to 40 amino acids, wherein the first substrate
is a tissue or a medical device and the first substrate-binding
peptide has binding affinity for the tissue or the medical device;
a plurality of a second substrate-binding peptide comprising of 3
to 40 amino acids, wherein the second substrate is a target
molecule and the second substrate-binding peptide has binding
affinity for the target molecule, wherein the first and second
substrate-binding peptides are not covalently linked; and a
plurality of the target molecule; wherein each of the first and
second substrate-binding peptides is covalently coupled to at least
one interaction tag selected from the group consisting of a
hydrophobic interaction tag, a positively charged interaction tag,
and a negatively charged interaction tag, wherein the hydrophobic
interaction tags interact with each other and the positively
charged interaction tags interact with the negatively charged
interaction tags to form a macromolecular network comprising the
plurality of non-covalently coupled first and second
substrate-binding peptides.
[0011] In another embodiment, the presently disclosed subject
matter provides a composition comprising a plurality of a first
substrate-binding polymer having a net negative or a net positive
charge, wherein the first substrate is a tissue or medical device
and the first substrate-binding polymer has binding affinity for
the tissue or medical device; a plurality of a second
substrate-binding peptide of 3 to 40 amino acids, wherein the
second substrate is a target molecule and the second
substrate-binding peptide has binding affinity for the target
molecule, wherein the first substrate-binding polymer and the
second substrate-binding peptide are not covalently linked; and a
plurality of the target molecule, wherein the plurality of second
substrate-binding peptides are covalently coupled to at least one
net positively or net negatively charged interaction tag, wherein
the charge of the interaction tag is opposite to the charge of the
first substrate-binding polymer, wherein each of the plurality of
first substrate-binding polymers and second substrate-binding
peptides is optionally covalently coupled to a hydrophobic
interaction tag, wherein the charged interaction tag interacts with
the first substrate-binding polymer and the optional hydrophobic
interaction tags interact with each other to form a macromolecular
network comprising the plurality of non-covalently coupled first
substrate-binding polymers and second substrate-binding
peptides.
[0012] In another embodiment, the presently disclosed subject
matter provides a composition comprising a plurality of a first
substrate-binding peptide comprising 3 to 40 amino acids, wherein
the first substrate is a tissue or medical device and the first
substrate-binding peptide has binding affinity for the tissue or
medical device; a plurality of a second substrate-binding peptide
comprising 3 to 40 amino acids, wherein the second substrate is a
target molecule and the second substrate-binding peptide has
binding affinity for the target molecule, wherein the first and
second substrate-binding peptides are covalently linked; and a
plurality of the target molecule, wherein the plurality of
covalently linked first and second substrate-binding peptides are
covalently coupled to at least one interaction tag selected from
the group consisting of a hydrophobic interaction tag, a positively
charged interaction tag, and a negatively charged interaction tag,
wherein the hydrophobic interaction tags interact with each other
and the positively charged interaction tags interact with the
negatively charged interaction tags to form a macromolecular
network comprising the plurality of non-covalently coupled
substrate-binding peptides.
[0013] In another embodiment, the presently disclosed subject
matter provides a composition comprising a composition comprising,
a plurality of first molecules comprising a first substrate-binding
peptide comprising 3 to 40 amino acids, wherein the first substrate
is a tissue or medical device and the first substrate-binding
peptide has binding affinity for the tissue or medical device; and
a second substrate-binding peptide comprising 3 to 40 amino acids,
wherein the second substrate is a target molecule and the second
substrate-binding peptide has binding affinity for the target
molecule, wherein the first and second substrate-binding peptides
are covalently linked; and a plurality of second molecules
comprising the second substrate-binding peptide, wherein the second
substrate binding peptide is not covalently linked to the first
substrate binding peptide; and a plurality of the target molecule,
wherein each of the plurality of first and second molecules are
covalently coupled to at least one interaction tag selected from
the group consisting of a hydrophobic interaction tag, a positively
charged interaction tag, and a negatively charged interaction tag,
wherein the hydrophobic interaction tags interact with each other
and the positively charged interaction tags interact with the
negatively charged interaction tags to form a macromolecular
network comprising the plurality of non-covalently coupled first
and second molecules.
[0014] In another embodiment, the presently disclosed subject
matter provides methods for coating a tissue or a medical device
with the presently disclosed compositions, and medical devices,
wherein at least a portion of the medical device is coated with a
composition of the presently disclosed subject matter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a bar graph measuring the retention of
substrate-binding peptide by itself (e.g., SEQ ID NO:122) or as
coupled to a molecule for promoting self assembly (e.g.,
conjugates, or compositions 119-124, 127-132; see Example 6 herein
for a description of both) to a substrate in assay conditions which
mimic the presence of human plasma.
[0016] FIG. 2 is a bar graph measuring the retention of
substrate-binding peptide by itself (e.g., SEQ ID NO:124-linker-SEQ
ID NO:122) or compositions comprising molecular network of the
invention (e.g., compositions 133, 134, 136, 137, 139; see Example
6 herein) to a substrate in assay conditions which mimic the
presence of human plasma.
[0017] FIG. 3 is a schematic diagram showing a composition of the
presently disclosed subject matter where a first substrate-binding
domain having a covalently coupled hydrophobic or charged
interaction tag is non-covalently bound to a tissue or medical
device and it is also non-covalently coupled to the interaction tag
on a second substrate-binding domain that is non-covalently bound
to a target molecule.
[0018] FIG. 4A-4C are schematic diagrams showing 3 separate
compositions of the presently disclosed subject matter. FIG. 4A
shows a composition comprising a first substrate-binding peptide
(SBD-1) having 2 covalently coupled positively charged interaction
tags (+++) (far left) associating through electrostatic
interactions with a second substrate-binding peptide (SBD-2) having
1 covalently coupled negatively charged interaction tag (---)
(left, and association shown in the middle). The diagram further
shows how a multitude of the first and second substrate binding
domains associate together (far right). FIG. 4B shows a composition
comprising a first substrate-binding peptide having 1 covalently
coupled positively charged interaction tag and 1 covalently coupled
hydrophobic interaction tag (zig zag line) (far left) associating
through electrostatic interactions with a second substrate-binding
peptide having 1 covalently coupled negatively charged interaction
tag (left, and association shown in the middle). The diagram
further shows how a multitude of the first and second substrate
binding domains associate together through both the charged
interaction tags and the hydrophobic interaction tags (far right).
FIG. 4C shows a composition comprising a first substrate-binding
peptide having 1 covalently coupled positively charged interaction
tag and 1 covalently coupled hydrophobic interaction tag (far left)
associating through electrostatic and hydrophobic interactions with
a second substrate-binding peptide having 1 covalently coupled
negatively charged interaction tag and 1 covalently coupled
hydrophobic interaction tag (left, and association shown in the
middle). The diagram further shows how a multitude of the first and
second substrate binding domains associate together through both
the charged interaction tags and the hydrophobic interaction tags
(far right).
[0019] FIG. 5A-5C are schematic diagrams showing 3 separate
compositions of the presently disclosed subject matter. FIG. 5A
shows a composition comprising a first substrate-binding polymer
(SBD-1) having a positive charge (far left) associating through
electrostatic interactions with a second substrate-binding peptide
(SBD-2) having 1 covalently coupled negatively charged interaction
tag (---) (left, and association shown in the middle). The diagram
further shows how a multitude of the first and second substrate
binding domains associate together (far right). FIG. 5B shows a
composition comprising a first substrate-binding polymer having 1
covalently coupled hydrophobic interaction tag (zig zag line) (far
left) associating through electrostatic interactions with a second
substrate-binding peptide having 1 covalently coupled negatively
charged interaction tag (left, and association shown in the
middle). The diagram further shows how a multitude of the first and
second substrate binding domains associate together through both
the charged interaction tags and the hydrophobic interaction tags
(far right). FIG. 5C shows a composition comprising a first
substrate-binding polymer having 1 covalently coupled hydrophobic
interaction tag (far left) associating through electrostatic and
hydrophobic interactions with a second substrate-binding peptide
having 1 covalently coupled negatively charged interaction tag and
1 covalently coupled hydrophobic interaction tag (left, and
association shown in the middle). The diagram further shows how a
multitude of the first and second substrate binding domains
associate together through both the charged interaction tags and
the hydrophobic interaction tags (far right).
[0020] FIG. 6A-6B are a schematic diagrams showing 3 separate
compositions of the presently disclosed subject matter. FIG. 6A
shows a composition starting with 2 molecules of a first
substrate-binding peptide (SBD-1) covalently linked to a second
substrate binding peptide (SBD-2), wherein the peptides are
covalently linked together through a hydrophobic interaction tag
(zig zag line) and there is a further covalently coupled positively
charged interaction tag (+++) on 1 of the molecules and a
negatively charged interaction tag (---) on the other molecule (far
left). The middle shows an association of the 2 molecules through
both electrostatic interactions of the charged interaction tags and
hydrophobic interactions of the hydrophobic interaction tags. The
diagram further shows (far right) how a multitude of the molecules
comprising first and second substrate binding domains and charged
and hydrophobic tags associate together. FIG. 6B shows a
composition starting with 2 molecules of a first substrate-binding
peptide covalently linked to a second substrate binding peptide,
wherein the peptides are covalently linked together through a
linker (L) and each of the molecules further comprise a covalently
coupled hydrophobic interaction tag (far left). The middle shows an
association of the 2 molecules through hydrophobic interactions of
the hydrophobic interaction tags. The diagram further shows (far
right) how a multitude of the molecules comprising first and second
substrate binding domains and hydrophobic tags associate
together.
[0021] FIG. 7 is a schematic diagram showing a composition of the
presently disclosed subject matter. FIG. 7 (far left) shows a first
top left molecule of a first substrate-binding peptide (SBD-1)
covalently linked to a second substrate binding peptide (SBD-2),
wherein the peptides are covalently linked together through a
hydrophobic interaction tag (zig zag line) and there is a further
positively charged interaction tag covalently coupled to the SBD-1
(+++). A second molecule on the bottom far left having a second
substrate binding peptide (SBD-2) with a covalently coupled
negatively charged interaction tag (---) is shown to interact with
the first molecule through electrostatic interactions of the
charged interaction tags (middle). The diagram further shows (far
right) how a multitude of the molecules comprising first and second
substrate binding domains and charged and hydrophobic interaction
tags associate together.
DETAILED DESCRIPTION OF THE INVENTION
[0022] Definition Section While the following terms are believed to
be well understood by one of ordinary skill in the art, the
following definitions are set forth to facilitate explanation of
the invention. Also, additional definitions may be provided in the
specification outside of this "Definition Section" to facilitate
explanation of the invention.
[0023] The term "macromolecular network" is used herein, for
purposes of the specification and claims, to mean a structure
formed by a plurality of molecules of compound, wherein the
structure is formed by non-covalent molecular interactions between
fatty acid molecules of the plurality of molecules of compound,
resulting in a molecular association between ("linking") two or
more molecules of compound together. It is intended to be clear
that the use of the term "linking" in this specific instance is
referring to a non-covalent molecular association between the fatty
acid molecules of two or more molecules of compound, and should not
be confused with use of the term "linking" in other places
throughout the presently disclosed specification and claims where
the term is used to refer to a covalent bond. The macromolecular
network, when applied to a substrate, may form at least a monolayer
(a layer that is at least one molecule of compound in thickness).
Whether a monolayer or multilayer (more than a monolayer) is formed
depends on such factors as the number of fatty acids per
substrate-binding peptide in each compound, external factors of the
surrounding environment (pH, hydrophobicity), concentration of
compound (e.g., how many molecules of compound are added together,
and relative to the chance of interaction between fatty acid
components of individual compounds), and the like. Non-covalent
molecular interactions between two or more fatty acid molecules
that may contribute to formation of the macromolecular network
include one or more of, but are not limited to, hydrogen bonding,
van der Waals interactions, hydrophobic interactions, and
electrostatic interactions.
[0024] The terms "first" and "second" are used herein for purposes
of the specification and claims for ease of explanation in
differentiating between two different molecules, and are not
intended to be limiting the scope of the present invention, nor
imply a spatial, sequential, or hierarchical order unless otherwise
specifically stated.
[0025] The term "non-biological substrate" is used herein for
purposes of the specification and claims to mean a substrate that
is not a quality or component of a living system. A non-biological
substrate can comprise any form suitable to its intended use
including, but not limited to, a container, reactor, device, array,
medical device, particle (e.g., microparticle, nanoparticle, and
the like), a surface of a non-biological substrate, a diagnostic
agent, a drug (e.g., synthesized small molecule drug), a chemical
catalyst, a formulation, and a combination thereof. Representative
non-biological substrates include, but are not limited to, plastic,
silicon, synthetic polymer, metal (including mixed metal alloys),
metal oxide (e.g., glass), non-metal oxide, ceramic, carbon-based
materials (e.g., graphite, carbon nanotubes, carbon "buckyballs",
and metallo-carbon composites), and combinations thereof. In
addition to medical devices, as described more in detail herein,
other non-biological substrates that may benefit from the present
invention include, but are not limited to, (a) medical supplies,
such as bandages, dressings, sponges, covers, and the like; (b)
laboratory equipment, such as bioreactors, fermentors, test tubes,
assay plates, arrays, culture containers, and the like; and (c)
packaging or product protection (e.g., packaging materials,
coverings (such as wraps)), such as applied to perishables such as
foods, drugs, and medical devices. Diagnostic agents include, but
are not limited to, radiolabels, radiopaque compounds, calorimetric
reagents, dyes, fluorophores, fluorescent molecules, fluorescent
nanocrystals, luminescent molecules, chromophores, and the like.
Catalysts can be selected from the group consisting of
heterogeneous catalysts, homogeneous catalysts, biocatalysts (e.g.,
enzymes in metabolic or biological pathways), electrocatalysts
(e.g., metal-rich catalysts used in fuel cells, or energy
generation), organocatalysts (simple organic molecules used as
catalysts in chemical reactions), as known to those skilled in the
art. A preferred non-biological substrate may be used in accordance
with the present invention to the exclusion of a non-biological
substrate other than the preferred non-biological substrate.
[0026] The term "metal" is used herein for purposes of the
specification and claims to mean one or more compounds or
compositions comprising a metal represented in the Periodic Table
(e.g., a transition metal, alkali metals, and alkaline earth
metals, each of these comprise metals related in structure and
function, as classified in the Periodic Table), and may further
refer to a metal alloy, a metal oxide, a silicon oxide, and
bioactive glass. Examples of preferred metals include, but are not
limited to, titanium, titanium alloy, stainless steel, aluminum,
zirconium alloy metal substrate (e.g., Oxinium.TM.), cobalt
chromium alloy, gold, silver, rhodium, zinc, tungsten, platinum,
rubidium, and copper. A preferred metal may be used in accordance
with the present invention to the exclusion of a metal other than
the preferred metal.
[0027] The term "polymer" is used herein for purposes of the
specification and claims to mean a molecule or material comprised
of repeating structural units (a structural unit typically referred
to as a monomer) connected by covalent chemical bonds. Depending on
its intended use, a polymer may be biodegradable (e.g., one or more
of self-dissolving, or bioresorbable, or degradable in vivo) or
non-biodegradable; or synthetic (manufactured, and not found in
nature) or natural (found in nature, as made in living tissues of
plants and/or animals).
[0028] Non-limiting examples of suitable synthetic polymers
described as being biodegradable include: poly-amino acids;
polyanhydrides including maleic anhydride polymers; polycarboxylic
acid; some polyethylenes including, but not limited to,
polyethylene glycol, polyethylene oxide; polypropylenes, including,
but not limited to, polypropylene glycol, polypropylene fumarate;
one or more of polylactic acid or polyglycolic acid (and copolymers
and mixtures thereof, e.g., poly(L-lactic acid) (PLLA),
poly(D,L,-lactide), poly(lactic acid-co-glycolic acid), 50/50
(DL-lactide-co-glycolide)); polyorthoesters; polydioxanone;
polyphosphazenes; polydepsipeptides; one or more of
polycaprolactone (and co-polymers and mixtures thereof, e.g.,
poly(D,L-lactide-co-caprolactone) or polycaprolactone
co-butylacrylate; polyhydroxybutyrate valerate and blends; some
polycarbonates (e.g., tyrosine-derived polycarbonates and
arylates), polyiminocarbonates, calcium phosphates; cyanoacrylate;
some polyamides (including nylon); polyurethane;
polydimethyltrimethylcarbonates; synthetic cellulosic polymers
(e.g, cellulose acetate, cellulose butyrate, cellophane); and
mixtures, combinations, and copolymers of any of the foregoing.
Representative natural polymers described as being biodegradable
include macromolecules (such as polysaccharides, e.g., alginate,
starch, chitosan, cellulose, or their derivatives (e.g.,
hydroxypropylmethyl cellulose); proteins and polypeptides, e.g.,
gelatin, collagen, albumin, fibrin, fibrinogen);
polyglycosaminoglycans (e.g. hyaluronic acid, chondroitin sulfate);
and mixtures, combinations, and copolymers of any of the
foregoing.
[0029] Non-limiting examples of suitable synthetic polymers
described as being non-biodegradable include: inert
polyaryletherketones, including polyetheretherketone ("PEEK"),
polyether ketone, polyetherketoneketone, and
polyetherketoneetherketoneketone; polyurethanes; polystyrene, and
styrene-ethylene/butylene-styrene block copolymers; polyisobutylene
copolymers and styrene-isobutylene-styrene block copolymers;
polyvinylpyrrolidone; polyvinyl alcohols; copolymers of vinyl
monomers; polyvinyl ethers; polyvinyl aromatics; polyethylene
oxides; polyesters including polyethylene terephthalate; some
polyamides; polyacrylamides; polyethers including polyether
sulfone; polyalkylenes including polypropylene, polyethylene;
copolymers of ethylene and polypropylene; some polycarbonates,
silicone and silicone rubber; siloxane polymers;
polytetrafluoroethylene; expanded polytetrafluoroethylene (e-PTFE);
nylons and related polyamide copolymers; nylon; fluorinated
ethylene propylene; hexafluoropropylene, polymethylmethacrylate
(PMMA); 2-hydroxyethyl methacrylate (PHEMA); polyimides;
polyethyleneterephthalate; polysulfone, and polysulfides; and
mixtures, combinations, and copolymers (including cross-linked
copolymers) of any of the foregoing.
[0030] The term "ceramic" is used herein for purposes of the
specification and claims to mean inorganic non-metallic materials
whose formation is due to the action of heat. Suitable ceramic
materials include but are not limited to silicon oxides, aluminum
oxides, alumina, silica, hydroxyapatites, glasses, quartz, calcium
oxides, calcium phosphates, indium tin oxide, polysilanols,
phosphorous oxide, and combinations thereof.
[0031] The term "effective amount" is used herein, in referring to
a composition according to the present invention and for purposes
of the specification and claims, to mean an amount sufficient of
the composition to promote a beneficial property resulting from the
compound, including but not limited to, improved biophysical
properties. In the case that the composition also has binding
specificity for a substrate, an "effective amount" may also
comprise an amount sufficient so as to mediate binding of the
composition to the substrate.
[0032] The term "individual", as used herein for purposes of the
specification and claims, refers to either a human or an
animal.
[0033] The terms "biological molecule" or "biological substrate"
(which may sometimes be used interchangeably herein), as used
herein for purposes of the specification and claims, refers to a
quality or component pertaining to living systems. As such, a
"biological substrate" can comprise an organ, a tissue, a cell,
components or structures thereof or associated therewith, or a
biological molecule. Thus, a biological substrate can comprise a
biological molecule including, but not limited, to a protein (e.g.,
an antibody, antibody chain, avimer, collagen, keratin or other
proteinaceous tissue component or structure, polypeptide, a
receptor, a glycoprotein, a lipoprotein, a hormone, a growth
factor, a cytokine, a chemical mediator, and the like), a peptide,
a lipid, a carbohydrate (e.g., a polysaccharide, starch,
monosaccharide), a nucleic acid molecule (e.g., an aptamer, DNA,
RNA, hybrid nucleic acid molecule, vectors, chemically modified
nucleic acid molecule), an oligomer, a small molecule (e.g., a
chemical compound; metabolites, such as sugars, folic acid, uric
acid, lactic acid), a drug (e.g., a biological-based drug, hormone,
antimicrobial compound, growth factor, signaling molecule, ligand,
etc.), a signaling molecule, a ligand, a nucleic acid-protein
fusion, fragments thereof, analogs thereof, and a combination
thereof. The term "biological substrate" also encompasses
substrates that have been isolated from a living system, and
substrates that have been recombinantly or synthetically produced
based on knowledge of a biological substrate such as found in a
living system, and biologically-active analogs thereof. While the
origin of the biological molecule or biological substrate is
preferably human, it may be originated from any biological source
or organism; e.g., any animal, plant, bacteria, virus, yeast, etc.
Typically, a biologically-active analog of a biological molecule
has a chemical composition having from about 1% to about 25%
difference, as compared to the chemical composition of the
biological molecule from which the analog was derived. A preferred
biological substrate or biological molecule may be used in
accordance with the present invention to the exclusion of a
biological substrate or biological molecule other than the
preferred biological substrate or biological molecule.
[0034] The term "time sufficient for binding" generally refers to a
temporal duration sufficient for specific binding of a composition
to a substrate for which the composition has binding specificity,
as known to those skilled in the art. For example, based on the
affinity/binding specificity of a substrate-binding peptide used in
a composition according to the present invention, generally a time
sufficient for binding a composition according to the present
invention to a substrate ranges from about 5 minutes to no more
than 60 minutes.
[0035] The term "compound" is used herein, in reference to a
composition of the present invention and for purposes of the
specification and claims, to refer to a molecule comprising fatty
acid covalently coupled to a substrate-binding peptide, either
directly or via a linker. Thus, the peptide is functionalized with
one or more molecules of fatty acid, the number may depend on the
improved biophysical property which is desired (e.g., see Examples
6-9 herein). In one embodiment, the compound of the invention may
be a pharmaceutically acceptable salt or cosmetically acceptable
salt of a molecule comprising fatty acid covalently coupled to a
substrate-binding peptide, either directly or via a linker. A
preferred compound may be used in accordance with the invention to
the exclusion of a compound other than the preferred compound.
[0036] The term "composition", as used herein for purposes of the
specification and claims, refers to a macromolecular network
comprised of a plurality of compound according to the invention,
wherein non-covalent interactions between fatty acid components of
the plurality of compound contribute to association between
individual molecules of compound in the composition, resulting in
formation of a macromolecular network, and while allowing the
substrate-binding peptide components of the plurality of compound
to bind to a substrate for which they have binding specificity. As
will be described herein in more detail, the molecular network
provides the compound with unexpected and beneficial properties,
including but not limited to one or more improved biophysical
properties. A composition of the invention comprises a
macromolecular network represented by general formula (I):
(SBP-FA-FA-SBP).sub.n [0037] wherein [0038] SBP comprises a
substrate-binding peptide, and more preferably a substrate-binding
peptide in a biofunctional composition comprising at least two
substrate-binding peptides covalently coupled to each other; [0039]
FA comprises fatty acid; [0040] wherein FA of one compound
associates with FA of one or more other compounds through
non-covalent interactions (as schematically represented by the "-"
in formula (I)) in forming a macromolecular network capable of
binding to a substrate via the substrate-binding peptide component;
[0041] n is an integer equal to or greater than 1; and [0042]
wherein the composition has improved biophysical properties as
compared to the substrate-binding peptide by itself. In one
preferred embodiment, FA comprises two or more molecules of fatty
acid covalently coupled to each other; or one large fatty acid of
greater or equal to 25 carbons in the carboxylic acid chain. The
term "macromolecular", when referring to a network of which is
comprised a composition of the invention, means that the network is
comprised of more than one monomeric, molecular unit; and also
refers to a network formed by aggregates of two or more molecules
held together by non-covalent interactions. Preferably, the
non-covalent interactions are sufficient in molecular association
so that the two or more molecules do not readily dissociate. A
preferred composition may be used in accordance with the invention
to the exclusion of a composition other than the preferred
composition.
[0043] In addition, the term "composition", as used herein for
purposes of the specification and claims, refers to a
macromolecular network comprised of a plurality of first and second
substrate binding domains that are non-covalently coupled at least
in part through one or more hydrophobic or charged interaction tags
according to the presently disclosed subject matter, wherein
non-covalent interactions between hydrophobic and/or charged
interaction tags of the plurality of substrate binding domain
comprising molecules contribute to association between individual
molecules in the composition resulting in formation of a
macromolecular network, and while allowing the substrate-binding
domain components of the plurality of molecules to bind to a
substrate for which they have binding specificity. In some
embodiments, the "compositions" of the presently disclosed subject
matter further comprise the target molecules to which the plurality
of second substrate binding molecules have binding affinity. When
the composition comprising the macromolecular network comprised of
a plurality of non-covalently coupled first and second substrate
binding domains is contacted with a tissue or a medical device in
the presence of the target molecule, the composition is useful for
loading the target molecule onto the tissue or medical device.
[0044] Fatty acids are known to those skilled in the art as
aliphatic monocarboxylic acids having a chain of no less than 5 and
no more than 30 carbons. The fatty acid may be branched,
unbranched, saturated, unsaturated, even-numbered carbons,
odd-numbered carbons, a monoacid, a di-acid. Preferred fatty acids
useful in this invention are fatty acid having a chain ranging from
9 carbons to 30 carbons. Also, as described in more detail herein,
one or more (and preferably two or more) molecules of fatty acid
may be covalently coupled to single molecule of substrate-binding
peptide to form a compound of the invention. Illustrative examples
of preferred fatty acids useful for producing a compound of the
invention include, but are not limited to, decanoic acid,
aminoundecanoic acid, lauric acid, myristic acid, palmitic acid,
aminohexanoic acid, and stearic acid. A preferred fatty acid may be
used in accordance with the invention to the exclusion of a fatty
acid other than the preferred fatty acid.
[0045] The term "charged interaction tag" is used, for purposes of
the specification and claims, to refer to a molecule, compound, or
moiety having a net positive or a net negative charge that can
non-covalently interact with another charged molecule, compound, or
moiety having a net opposite charge through electrostatic
interactions. With respect to the presently disclosed subject
matter, the charged interaction tags are used to non-covalently
couple one substrate binding peptide or polymer to another
substrate binding peptide or polymer. Thus, for example, a
positively charged interaction tag coupled to a substrate binding
peptide couples electrostatically with a negatively charged
interaction tag covalently coupled to another substrate binding
peptide or couples electrostatically with a negatively charged
substrate binding polymer. In another example, a negatively charged
interaction tag covalently coupled to a substrate binding peptide
couples electrostatically with a positively charged interaction tag
covalently coupled to another substrate binding peptide or couples
electrostatically with a positively charged substrate binding
polymer. In another example, a positively charged interaction tag
covalently coupled so as to link a first and a second substrate
binding peptide couples electrostatically with a negatively charged
interaction tag similarly linking the first and second substrate
binding peptides on a separate molecule. In this manner, the two
separate molecules, each having a covalently linked first and
second substrate binding peptide, are non-covalently coupled
through the electrostatic interaction. Through such non-covalent
electrostatic interactions, the charged interaction tags of the
presently disclosed subject matter contribute to formation of a
higher order macromolecular network of a plurality of molecules of
first and second substrate binding domains.
[0046] There is no particular size or content limitations for the
charged interaction tag so long as it has a net positive or a net
negative charge and can fulfill its purpose when covalently coupled
to a substrate-binding peptide or polymer to electrostatically
couple with an oppositely charged tag on another substrate binding
peptide or another oppositely charged substrate binding polymer. In
addition, the charged interaction tag must allow for the
substrate-binding activity of the peptides and polymers to be
substantially retained. In some embodiments of the presently
disclosed subject matter the charged interaction tags of the
presently disclosed subject matter have a molecular weight of less
than 10 kDa.
[0047] Examples of positively charged interaction tags include, for
example, but are not limited to, poly-amino acids including
polylysine and polyarginine and combinations and copolymers
thereof; and polyamines and polyimines including, for example,
polyethylamines, polyethylenimines (PEI), and combinations and
copolymers thereof. In one embodiment where the positively charged
interaction tag is a polyamino acid, the positively charged
interaction tag comprises a net positive charge of about +3 to
about +50, from about +3 to about +20, from about +4 to about +17,
from about +5 to about +14, from about +6 to about +10, from about
+6 to about +9, from about +6 to about +8, and from about +6 to
about +7. A positively charged interaction tag that is a polylysine
or a polyarginine, or a combination or copolymer thereof, ranges in
length from about 3 amino acids to about 50 amino acids, from about
3 amino acids to about 40 amino acids, from about 3 amino acids to
about 30 amino acids, from about 3 amino acids to about 20 amino
acids, from about 3 amino acids to about 11, 12, 13, 14, 15, 16,
17, 18, or 19 amino acids, from about 4 amino acids to about 10
amino acids, from about 4 amino acids to about 9 amino acids, from
about 5 amino acids to about 8 amino acids, and from about 6 amino
acids to about 7 amino acids.
[0048] Examples of negatively charged interaction tags include, for
example, but are not limited to, poly-amino acids including
polyglutamic acid and polyaspartic acid and combinations and
copolymers thereof; polylactic acid, polyglycolic acid, poly(lactic
acid-co-glycolic acid), polystyrene sulfonate (PSS), and
poly(styrenesulfonic-maleic acid), and combinations and copolymers
thereof. In one embodiment where the negatively charged interaction
tag is a polyamino acid, the negatively charged interaction tag
comprises a net negative charge of about -3 to about -50, from
about -3 to about -20, from about -4 to about -17, from about -5 to
about -14, from about -6 to about -10, from about -6 to about -9,
from about -6 to about -8, and from about -6 to about -7. A
negatively charged interaction tag that is a polyaspartic acid or a
polyglutamic acid, or a combination or copolymer thereof, ranges in
length from about 3 amino acids to about 50 amino acids, from about
3 amino acids to about 40 amino acids, from about 3 amino acids to
about 30 amino acids, from about 3 amino acids to about 20 amino
acids, from about 3 amino acids to about 11, 12, 13, 14, 15, 16,
17, 18, or 19 amino acids, from about 4 amino acids to about 10
amino acids, from about 4 amino acids to about 9 amino acids, from
about 5 amino acids to about 8 amino acids, and from about 6 amino
acids to about 7 amino acids.
[0049] The term "hydrophobic interaction tag" is used, for purposes
of the specification and claims, to refer to a molecule, compound,
or moiety that is hydrophobic in nature and non-covalently
interacts with another molecule, compound, or moiety that is
hydrophobic in nature. With respect to the presently disclosed
subject matter, the hydrophobic interaction tags are used to
non-covalently couple one substrate binding peptide or substrate
binding polymer domain to other substrate binding peptide or
polymer domains through interactions including, but not limited to,
all non-electrostatic interactions such as hydrophobic
interactions, van der Waals interactions, and pi-stacking
interactions.
[0050] Thus, for example, a hydrophobic interaction tag covalently
coupled to a first substrate binding peptide or polymer can
non-covalently couple with another hydrophobic interaction tag
covalently coupled to a second substrate binding peptide or
polymer. In another example, a hydrophobic interaction tag
covalently coupled so as to link a first and a second substrate
binding peptide non-covalently couples with a hydrophobic
interaction tag covalently coupled to link the first and second
substrate binding peptides on a separate molecule. In this manner,
the two separate molecules, each having a linked first and second
substrate binding peptide are non-covalently coupled together.
Through such non-covalent interactions the hydrophobic interaction
tags of the presently disclosed subject matter contribute to
formation of a higher order macromolecular network of a plurality
of first and second substrate binding domains. There is no
particular size or content limitations for the hydrophobic
interaction tag so long as it is hydrophobic in nature and can
fulfill its purpose to non-covalently couple separate substrate
binding peptides and/or polymers, and the substrate-binding
activity of the peptides/polymers is substantially retained. In
some embodiments of the presently disclosed subject matter the
hydrophobic interaction tags of the presently disclosed subject
matter have a molecular weight of less than 10 kDa.
[0051] Examples of hydrophobic interaction tags include, for
example, but are not limited to, poly-amino acids (natural and
non-natural and D- and L-isomers) including combinations, strings,
and copolymers of very hydrophobic amino acids such as valine,
leucine, isoleucine, methionine, tryptophan, phenylalanine,
biphenylalanine, N-methylisoleucine; N-methylvaline; norvaline;
norleucine; and less hydrophobic amino acids such as alanine, and
tyrosine. Another example of hydrophobic interaction tags of the
presently disclosed subject matter is fatty acids. The fatty acids
of the presently disclosed subject matter include saturated and
unsaturated fatty acids such as but not limited to butyric acid,
caproic acid (or amino hexanoic acid ("Ahx")), caprylic acid,
capric acid, undecanoic acid, aminoundecanoic acid (AU D),
poly-aminoundecanoic acid, lauric acid, myristic acid, palmitic
acid, stearic acid, arachidic acid, behenic acid, lignoceric acid,
eicosanoic acid, docosanoic acid, tetracosanoic acid, myristoleic
acid, palmitoleic acid, oleic acid, linoleic acid,
.alpha.-linolenic acid, arachidonic acid, eicosapentaenoic acid,
erucic acid, and docosahexaenoic acid. Another example of
hydrophobic interaction tags of the presently disclosed subject
matter are aromatic groups including pyrene or pi-stacking
interactions such as with combinations of tyrosine and
tryptophan.
[0052] The term "first substrate-binding peptide" is herein used
interchangeably with the term "first substrate-binding domain" is
used for purposes of the specification and claims, to refer to a
peptide having ranging in length from 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or more amino
acids in length that has binding affinity for the tissue or medical
device that is the first substrate of the presently disclosed
subject matter.
[0053] The term "second substrate-binding peptide" is herein used
interchangeably with the term "second substrate-binding domain" and
is used for purposes of the specification and claims, to refer to a
peptide having ranging in length from 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or more amino
acids in length that has binding affinity for the tissue or medical
device that is the first substrate of the presently disclosed
subject matter.
[0054] The phrase "first substrate-binding polymer having a net
positive charge" is herein used interchangeably with the term
"first substrate-binding domain", and is used, for purposes of the
specification and claims, to refer to a polymer having a net
positive charge that has binding affinity for the tissue or medical
device that is the first substrate of the presently disclosed
subject matter. With respect to the presently disclosed subject
matter, the substrate-binding polymer having a net positive charge
includes those polymers that non-covalently couple to the tissue or
medical device. There is no particular size or content limitations
for the substrate-binding polymer having a net positive charge so
long as it fulfills its purpose of electrostatically coupling with
the tissue or medical device. In some embodiments of the presently
disclosed subject matter, the positively charged substrate-binding
polymers of the presently disclosed subject matter have a molecular
weight ranging from more than 1 kDa to about 700 kDa, from about 5
kDa to about 700 kDa, from about 5 kDa to about 100 kDa, from about
6 kDa, 7 kDa, 8 kDa, 9 kDa, or 10 kDa to about 50 kDa, from about
10 kDa to about 20 kDa, 30 kDa, or 40 kDa. The substrate-binding
polymer having a net positive charge includes but is not limited to
polymers such as, for example, poly-amino acids including
polylysine and polyarginine and combinations and copolymers
thereof; some polyamides including, for example, nylon and silk;
polyamines and polyimines including, for example, polyethylamines,
branched and linear polyethylenimines (PEI) and mixtures,
combinations, and copolymers thereof. The positively charged
substrate-binding polymers of the presently disclosed subject
matter have a molecular weight ranging from more than 1 kDa to
about 700 kDa, from about 5 kDa to about 700 kDa, from about 5 kDa
to about 100 kDa, from about 6 kDa, 7 kDa, 8 kDa, 9 kDa, or 10 kDa
to about 50 kDa, from about 10 kDa to about 20 kDa, 30 kDa, or 40
kDa.
[0055] The phrase "first substrate-binding polymer having a net
negative charge" is used, for purposes of the specification and
claims, to refer to a polymer having a net negative charge that has
binding affinity for the tissue or medical device that is the first
substrate of the presently disclosed subject matter. With respect
to the presently disclosed subject matter, the negatively charged
substrate binding polymer includes those polymers that
non-covalently couple to the tissue or medical device. There is no
particular size or content limitations for the substrate-binding
polymer having a net negative charge so long as it fulfills its
purpose of electrostatically coupling with the tissue or medical
device. In some embodiments of the presently disclosed subject
matter, the negatively charged substrate-binding polymers of the
presently disclosed subject matter have a molecular weight ranging
from more than 1 kDa to about 700 kDa, from about 5 kDa to about
700 kDa, from about 5 kDa to about 100 kDa, from about 6 kDa, 7
kDa, 8 kDa, 9 kDa, or 10 kDa to about 50 kDa, from about 10 kDa to
about 20 kDa, 30 kDa, or 40 kDa. The negatively charged first
substrate-binding polymer includes but is not limited to polymers
such as, for example, poly-amino acids including polyglutamic acid,
polyaspartic acid and combinations and copolymers thereof,
polycarboxylic acids; polylactic acid, polyglycolic acid,
poly(lactic acid-co-glycolic acid), polymannuronic acid,
polygalacturonic acid, polyglucuronic acid, polyguluronic acid,
polystyrenesulfonic acids and combinations and copolymers thereof;
polysaccharides, e.g., alginate, starch, chitin, carrageenan
(sulfated polysaccharides), heparin, and pectin and their
derivatives; cellulose and cellulosic polymers including, for
example, carboxy methyl cellulose ("CMC"), hydroxypropylmethyl
cellulose, cellulose acetate, cellulose butyrate, and cellophane;
polyglycosaminoglycans including, for example, hyaluronic acid,
chondroitin sulfate; and mixtures, combinations, and copolymers
thereof.
[0056] The first substrate-binding peptide or polymer is also
referred to herein for purposes of simplicity as the first
substrate-binding domain. Similarly, the second substrate-binding
peptide is also referred to herein as a substrate-binding domain.
Accordingly, all of the first and second substrate-binding peptides
and polymers can be referred to herein as substrate-binding
domains.
[0057] The term "linker" is used, for purposes of the specification
and claims, to refer to a compound or moiety that acts as a
molecular bridge to couple at least two different molecules (e.g.,
with respect to the present invention, coupling a fatty acid to a
peptide, coupling one substrate binding peptide or polymer to
another substrate binding peptide or polymer, coupling a charged
interaction tag or a hydrophobic interaction tag to a substrate
binding peptide or polymer). Thus, for example, coupling at least
one fatty acid to an amino acid of a peptide may involve one
portion of the linker binding to the at least one fatty acid, and
another portion of the linker binding to a chemical moiety of the
amino acid of the peptide to be functionalized with the at least
one fatty acid. As apparent to those skilled in the art, and using
methods known in the art, two different molecules may be coupled to
the linker in a step-wise manner, or may be coupled simultaneously
to the linker. There is no particular size or content limitations
for the linker so long as it can fulfill its purpose as a molecular
bridge, and that the binding specificity of a substrate-binding
peptide or a substrate binding-domain in a coating composition is
substantially retained.
[0058] Linkers are known to those skilled in the art to include,
but are not limited to, chemical compounds (e.g., chemical chains,
compounds, reagents, and the like). The linkers may include, but
are not limited to, homobifunctional linkers and heterobifunctional
linkers. Heterobifunctional linkers, well known to those skilled in
the art, contain one end having a first reactive functionality (or
chemical moiety) to specifically link a first molecule, and an
opposite end having a second reactive functionality to specifically
link to a second molecule. It will be evident to those skilled in
the art that a variety of bifunctional or polyfunctional reagents,
both homo- and hetero-functional (such as those described in the
catalog of the Pierce Chemical Co., Rockford, Ill.), amino acid
linkers (typically, a short peptide of between 3 and 15 amino
acids, and often containing amino acids such as glycine, and/or
serine), and polymers (e.g., polyethylene glycol) may be employed
as a linker with respect to the present invention. In one
embodiment, representative peptide linkers comprise multiple
reactive sites to be coupled to a binding domain (e.g.,
polylysines, polyornithines, polycysteines, polyglutamic acid and
polyaspartic acid) or comprise substantially inert peptide linkers
(e.g., polyglycine, polyserine, polyproline, polyalanine, and other
oligopeptides comprising alanyl, serinyl, prolinyl, or glycinyl
amino acid residues.
[0059] Suitable polymeric linkers are known in the art, and can
comprise a synthetic polymer or a natural polymer. Representative
synthetic polymers include but are not limited to polyethers (e.g.,
poly(ethylene glycol) ("PEG")), poly(propylene glycol),
poly(butylene glycol), polyesters (e.g., polylactic acid (PLA) and
polyglycolic acid (PGA)), polyamines, polyamides (e.g., nylon),
polyurethanes, polymethacrylates (e.g., polymethylmethacrylate;
PMMA), polyacrylic acids, polystyrenes, polyhexanoic acid, flexible
chelators such as EDTA, EGTA, and other synthetic polymers which
preferably have a molecular weight of about 20 Daltons to about
1,000 kiloDaltons. Representative natural polymers include but are
not limited to hyaluronic acid, alginate, chondroitin sulfate,
fibrinogen, fibronectin, albumin, collagen, calmodulin, and other
natural polymers which preferably have a molecular weight of about
200 Daltons to about 20,000 kiloDaltons (for constituent monomers).
Polymeric linkers can comprise a diblock polymer, a multi-block
copolymer, a comb polymer, a star polymer, a dendritic or branched
polymer, a hybrid linear-dendritic polymer, a branched chain
comprised of lysine, or a random copolymer. A linker can also
comprise a mercapto(amido)carboxylic acid, an acrylamidocarboxylic
acid, an acrylamido-amidotriethylene glycolic acid, 7-aminobenzoic
acid, and derivatives thereof. In another example, a linker can be
the charged or the hydrophobic interaction tag of the presently
disclosed subject matter. Linkers may also be generated during the
coupling process such a `trizole nucleus` that is generated as a
linker during the copper-catalyzed azide-alkyne cycloaddition
(e.g., "click chemistry") or any other methods such as
chemoselective ligation chemistry well known in the art.
[0060] If desired, a predetermined amount of the plurality of
compound in the macromolecular network may be synthesized so as to
be susceptible to cleavage (e.g., so as to promote biodegraDation
after the macromolecular network has served its intended purpose),
e.g., by choice of a particular linker between the fatty acid
component and the peptide component of the compound. Cleavable
linkers are known in the art that to be cleaved by a number of
mechanisms (e.g., by heat, by natural enzymes found in or on the
body of an individual, by pH sensitivity). Examples of pH-sensitive
materials useful as linkers may include, but are not limited to,
cellulose acetate phthalate, cellulose acetate trimellitate,
polyvinyl acetate phthalate, hydroxypropyl methylcellulose
phthalate, and hydroxypropyl methylcellulose acetate succinate. An
example of a linker cleaved by natural enzymes may comprise an
amino acid linker comprised of a short chain of (e.g., 3 to 8)
amino acids, with a C-terminal amino acid residue comprising lysine
or arginine, and cleavage of the linker is via serum
carboxypeptiDases (N or R or both) which cleave C-terminal lysine
or arginine residues.
[0061] Depending on such factors as the molecules to be linked, and
the conditions in which the linking is performed, the linker may
vary in length and composition for optimizing such properties as
preservation of biological function, stability, resistance to
certain chemical, enzymatic, and/or temperature parameters, and of
sufficient stereo-selectivity or size. For example, where the
compound of the invention comprises a fatty acid linked to a
substrate-binding peptide, the linker should not significantly
interfere with the ability of a compound according to the present
invention to sufficiently bind specifically, with appropriate
avidity for the purpose, to a substrate for which the
substrate-binding peptide has the ability to bind. A preferred
linker may be a molecule which may have activities which enhance or
complement the effect of a compound or composition of the present
invention. A preferred linker may be used in the present invention
to the exclusion of a linker other than the preferred linker.
[0062] The interaction tags of the presently disclosed subject
matter are covalently coupled (or covalently linked) to the
substrate binding peptides and polymers of the presently disclosed
subject matter. The terms "covalently coupled", "covalently
linked", and "linked" are for the purposes of the specification and
claims to have the same meaning and are herein used
interchangeably. In one embodiment of the presently disclosed
subject matter, the covalent coupling between the interaction tag
and the substrate binding domain is a direct coupling between a
chemical group on the hydrophobic or charged interaction tag to a
chemical group on the substrate binding peptide or polymer. In
another embodiment, the covalent coupling is an indirect coupling
through another group. For example, in some embodiments the charged
and hydrophobic interaction tags of the presently disclosed subject
matter are linked in a manner including, but not limited to, linked
directly, linked through one or more amino acids, linked through a
proline amino acid residue, linked through a polymer, linked
through a polyethylene glycol ("PEG") polymer, linked through a 10
unit polyethylene glycol ("P10") polymer, linked through a 6 unit
polyethylene glycol ("MP") polymer, linked through one or more
fatty acid molecules, and linked through one or more aminohexanoic
acid molecules.
[0063] The terms "binds specifically" or "binding specificity" or
"binding affinity" and like terms used herein, are interchangeably
used, for the purposes of the specification and claims, to refer to
the ability of a peptide and (as a substrate-binding domain is
described herein) to have a binding affinity that is greater for
one target substrate selected to be bound over another substrate
other than the target substrate; e.g., an affinity for a given
substrate in a heterogeneous population of other substrates which
is greater than, for example, that attributable to non-specific
adsorption. For example, a peptide has binding specificity for
metal when the peptide demonstrates preferential binding to metal,
as compared to binding to another non-biological substrate such as
a polymer or a biological substrate (e.g., a cell). Such
preferential binding may be dependent upon the presence of a
particular conformation, structure, and/or charge on or within the
peptide and/or material for which it has binding specificity.
[0064] In some embodiments, a peptide that binds specifically to a
particular substrate, material or composition binds at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 200%, 300%, 400%,
500%, or a higher percentage, than the peptide binds to an
appropriate control such as, for example, a different substrate, or
a protein typically used for such comparisons such as bovine serum
albumin. For example, binding specificity can determined by an
assay in which quantitated is a signal (e.g., fluorescence, or
calorimetric) representing the relative amount of binding between a
peptide and a substrate. In a preferred embodiment, a peptide has a
binding specificity that is characterized by a relative binding
affinity as measured by an EC50 of 10 .mu.M or less, preferably
less than 1 .mu.M, and more preferably less than 0.1 .mu.M. The
EC50 can be determined using any number of methods known in the
art, such as by generating a concentration response curve from a
binding assay in which the concentration of the peptide is titered
with a known amount of the substrate for which the peptide has
binding specificity. In such case, the EC50 represents the
concentration of peptide producing 50% of the maximal binding
observed for that peptide in the assay.
[0065] The term "peptide" is used herein, for the purposes of the
specification and claims to refer to chain of contiguous amino
acids comprising no less than about 3 amino acids and no more than
about 100 amino acid residues in length, and more preferably from
about 8 amino acids to about 60 amino acids. The amino acid chain
may include naturally occurring amino acids, synthetic amino acids,
genetically encoded amino acids, non-genetically encoded amino
acids, one or more enantiomers of an amino acid, and combinations
thereof; an oligomer of the peptide (as previously described
herein); a peptide derivative (including, for example, peptide
conjugate, cyclized peptide, polymerized peptide, chemically
modified peptide, and a peptide mimetic). As known to those skilled
in the art, polypeptide (also known as a "protein") comprises an
amino acid chain larger than a peptide. As used herein, the term
"peptide" also encompasses a peptide wherein one or more of the
peptide bonds are replaced by pseudopeptide bonds including but not
limited to a carba bond (CH.sub.2--CH.sub.2), a depsi bond (CO--O),
a hydroxyethylene bond (CHOH--CH.sub.2), a ketomethylene bond
(CO--CH.sub.2), a methylene-oxy bond (CH.sub.2--O), a reduced bond
(CH.sub.2NH), a thiomethylene bond (CH.sub.2--S), an N-modified
bond (--NRCO), and a thiopeptide bond (CS--NH). A peptide or
polypeptide (protein) used in accordance with the present invention
may be produced by chemical synthesis, recombinant expression,
biochemical or enzymatic fragmentation of a larger molecule,
chemical cleavage of larger molecule, biological assembly, a
combination of the foregoing or, in general, made by any other
method in the art, and preferably isolated. A preferred peptide may
be used in the present invention to the exclusion of a peptide
other than the preferred peptide.
[0066] A peptide, used as a component of the compound according to
the invention, may also comprise an oligomer (e.g., dimer,
multimer) of the same peptide amino acid sequence or comprised of
two or more different amino acid sequences. For example, two or
more substrate-binding peptides are coupled together (e.g., by one
or more of physically, chemically, synthetically, or biologically
(e.g., via recombinant expression)) in such a way that each retains
its respective function to bind to the respective substrate for
which each has binding specificity. Such coupling may include
forming a multimeric molecule having two or more peptides having
binding specificity the same substrate (e.g., two or more polymer
binders), two or more peptides having binding specificity for
different substrates (e.g., one or more metal binders, and one or
more polymer binders), and a combination thereof. For example,
using standard reagents and methods known in the art of peptide
chemistry, two peptides may be coupled via a side chain-to-side
chain bond (e.g., where each of the peptides has a side chain amine
(e.g., such as the epsilon amine of lysine)), a side chain-to-N
terminal bond (e.g., coupling the N-terminal amine of one peptide
with the side chain amine of the other peptide), a side
chain-to-C-terminal bond (e.g., coupling the C-terminal chemical
moiety (e.g., carboxyl) of one peptide with the side chain amine of
the other peptide), an N-terminal-to-N-terminal bond, an N-terminal
to C-terminal bond, a C-terminal to C-terminal bond, or a
combination thereof. In synthetic or recombinant expression, two or
more peptides can be coupled directly to a peptide by synthesizing
or expressing the two or more peptides as a single peptide. The
coupling of two or more peptides may also be via a linker to form
substrate-binding peptide used in the composition according to the
present invention.
[0067] The term "isolated" means that a molecule (e.g., compound of
the invention) is substantially free of components which have not
become part of the integral structure of the molecule itself; e.g.,
such as substantially free of cellular material or culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically synthesized
or produced using biochemical, enzymatic, recombinant, or chemical
processes.
[0068] The term "amino acid" is used herein, for the purposes of
the specification and claims to refer to one or more of: an L-form
amino acid, D-form amino acid, natural amino acid (genetically
encoded amino acid), non-genetically encoded amino acid, and a
chemically-modified amino acid (e.g. containing one or more
protecting groups, or chemical end group, as will be described
herein in more detail). Representative non-genetically encoded
amino acids include but are not limited to 2-aminoadipic acid;
3-aminoadipic acid; .beta.-aminopropionic acid; 2-aminobutyric
acid; 4-aminobutyric acid (piperidinic acid); 6-aminocaproic acid;
2-aminoheptanoic acid; 2-aminoiso-butyric acid; 3-aminoisobutyric
acid; 2-aminopimelic acid; 2,4-diaminobutyric acid;
biphenylalanine, desmosine; 2,2'-diaminopimelic acid;
2,3-diaminopropionic acid; N-ethylglycine; N-ethylasparagine;
hydroxylysine; allo-hydroxylysine; 3-hydroxyproline;
4-hydroxyproline; isodesmosine; allo-isoleucine;
N-methylglycine(sarcosine); N-methylisoleucine; N-methylvaline;
norvaline; norleucine; ornithine; and
3-(3,4-dihydroxyphenyl)-L-alanine ("DOPA"). Representative
chemically modified amino acids include, for example, those
molecules in which free amino groups have been derivatized to form
amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy
groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl
groups. Free carboxyl groups can be derivatized to form salts,
methyl and ethyl esters or other types of esters or hydrazides.
Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl
derivatives. The imidazole nitrogen of histidine can be derivatized
to form N-im-benzylhistidine. Also, a chemically-modified amino
acid, for example, comprises a chemical moiety (an "N-terminal
group") added to an amino acid, such as an N-terminal amino acid on
a peptide, to block chemical reactivity of that amino terminus.
Peptides containing amino acids protected by chemical modification
are termed "modified peptides". Such N-terminal groups for
protecting the amino terminus of a peptide are well known in the
art, and include, but are not limited to, lower alkanoyl groups,
acyl groups, sulfonyl groups, and carbamate forming groups.
Preferred N-terminal groups may include acetyl, Fmoc, and Boc. A
chemical moiety, added to the C-terminal amino acid of a peptide to
block chemical reactivity of that carboxy terminus, comprises a
C-terminal group. Such C-terminal groups for protecting the carboxy
terminus are well known in the art, and include, but are not
limited to, an ester or amide group. Such terminal modifications
are often useful to reduce susceptibility by proteinase digestion,
and to therefore prolong a half-life of amino acids and peptides in
the presence of biological fluids where proteases can be present.
Optionally, a chemically modified amino acid may be one that is
modified to contain one or more chemical moieties (e.g., reactive
functionalities such as fluorine, bromine, or iodine) to facilitate
linking the peptide to a linker molecule or fatty acid.
[0069] The term "carrier medium" is herein used interchangeably
with the term "pharmaceutically acceptable solution", when used
herein for purposes of the specification and claims, means a medium
to which is added compound according to the present invention. In
one embodiment, a composition of the invention may be formed by
adding compound and the carrier medium together under sufficient
conditions to form macromolecular network of which is comprised the
composition. As known to those skilled in the art, components
included in a carrier medium will often depend on the intended use
of the resultant composition. Examples of such a carrier medium
include, but are not limited to, a liquid, a pharmaceutically
acceptable carrier, a cosmetically acceptable carrier, aqueous
solution, aqueous or non-aqueous solvent, suspension, emulsion,
gel, paste, formulation, cream, lotion, powder, serum, and a
combination thereof. As known to those skilled in the art, a
carrier medium may comprise one or substances, including but not
limited to, water, buffered water, medical parenteral vehicles,
saline, 0.3% glycine, aqueous alcohols, isotonic aqueous buffer;
and may further include one or more substances such as alginic
acid, water-soluble polymer, glycerol, glycols (e.g., polyethylene
glycol), polyols (e.g., glycerin, sorbitol, etc.), oils, salts
(such as sodium, potassium, magnesium and ammonium, phosphonates),
esters (e.g., carbonate esters, ethyl oleate, ethyl laurate, etc.),
fatty acids, vitamins, protein, carbohydrates, polysaccharides,
starches, glycoproteins (for enhanced stability), buffering agents
(e.g., magnesium hydroxide, aluminum hydroxide, and the like),
bulking agents, excipients, wetting agents, and preservatives
(including, but not limited to, ascorbic acid, cysteine
hydrochloride, sodium bisulfite, ascorbyl palmitate, tocopherol),
and/or stabilizers (to increase shelf-life or as necessary and
suitable for manufacture and distribution of the composition).
[0070] The term "medical device" is used herein, as used herein for
purposes of the specification and claims, refers to a structure
that is positioned or positionable into or onto an individual's
body to prevent, treat, modulate or ameliorate damage or a disorder
or disease or condition, repair or restore a function of a damaged
tissue; or to provide a new function. In a preferred embodiment in
which applied to a medical device is a compound or composition
according to the invention, the medical device comprises at least
one substrate or surface with which is contacted a compound or
composition according to the invention. Representative medical
devices include, but are not limited to: hip endoprostheses,
artificial joints, jaw or facial implants, dental implants, tendon
and ligament replacements, skin replacements, metal replacements
and metal screws, metal nails or pins, metal graft devices,
polymer-containing grafts, vascular prostheses, heart pacemakers,
artificial heart valves, blood filters, closure devices (e.g., for
closure of wounds, incisions, or defects in tissues, including but
not limited to skin and other organs (heart, stomach, liver,
etc.)), sutures, breast implants, penile implants, stents,
catheters, shunts, nerve growth guides, leads for battery-powered
medical devices, intraocular lenses, wound dressings, tissue
sealants, aneurismal coils, prostheses (e.g., cochlear implants,
visual prostheses (including, but not limited to, contact lenses,
and other visual aid devices), neurostimulators, muscular
stimulators, joint prosthesis, a spinal cord implant (e.g., an
implant for bridging a gap in a severed spinal cord or nerve,
typically used to promote nerve regeneration), dental prosthesis,
etc.), ophthalmic devices (glaucoma shunts, ophthalmic inserts,
intraocular lenses, overlay lenses, ocular inserts, optical
inserts), and nebulizers. Medical devices may be comprised of one
or more non-biological substrates, one or more biological
substrates, and a combination thereof. A preferred medical device
may be used in accordance with the present invention to the
exclusion of a medical device other than the preferred medical
device.
[0071] The phrase "substrate is a tissue or a medical device" is
used herein for purposes of the specification and claims to mean a
substrate that is a tissue or a medical device as the term "tissue"
and the term "medical device" is defined herein. The term "tissue"
is herein meant to comprise living animal tissue or a tissue
isolated or extracted from a living animal. The term "tissue"
comprises a material selected from the group consisting of an
animal tissue, an autologous tissue, an allogeneic tissue, a
transplanted tissue, an organ tissue, a bone tissue, a skin tissue,
a connective tissue, a muscle tissue, a nervous tissue, a polymer,
a collagen, and a calcium phosphate based material, and
combinations thereof. In addition to the definition of the term
"medical device" herein above, the term "medical device" comprises
a material selected from the group consisting an allogeneic tissue,
a transplanted tissue, a polymer, a silk, a collagen, a synthetic
polymer, a polyester, a polyurethane, a nylon, a polylactic acid, a
polyglycolic acid, poly(lactic acid-co-glycolic acid), a plastic, a
silicone material, a metal, a metal oxide, a non-metal oxide, a
ceramic material, a calcium phosphate based material, a
carbon-based material, a metallo-carbon composite, and combinations
thereof. In some aspects the term "medical device" comprises
certain "non-biological substrates" as used herein. For example,
the phrase "substrate is a medical device" further includes, but is
not limited to, a container, reactor, device, array, medical
device, particle (e.g., microparticle, nanoparticle, and the like),
and a combination thereof. The phrase "substrate is a medical
device" further includes, but is not limited to (a) medical
supplies, such as bandages, dressings, sponges, covers, and the
like; (b) laboratory equipment, such as bioreactors, fermentors,
test tubes, assay plates, arrays, culture containers, and the like;
and (c) packaging or product protection (e.g., packaging materials,
coverings (such as wraps)), such as applied to perishables such as
foods, drugs, and medical devices.
[0072] The term ""target molecule" is used herein for purposes of
the specification and claims to mean the target molecule is
selected from the group consisting of a cell, a protein, a
polypeptide, a growth factor, a growth differentiation factor
(GDF), a platelet derived growth factor (PDGF), a transforming
growth factor (TGF), an osteogenic protein, a bone morphogenic
protein (BMP), a hormone, a protein hormone, a parathyroid hormone
(PTH), a drug, a drug carrier, an antibiotic, a vancomycin
antibiotic, a steroid, a dexamethasone, and combinations thereof.
In some aspects the term "target molecule" comprises certain
"biological substrates" as used herein. For example, a target
molecule can comprise a biological molecule including, but not
limited, to a protein (e.g., an antibody, antibody chain, avimer,
collagen, keratin or other proteinaceous tissue component or
structure, polypeptide, a receptor, a glycoprotein, a lipoprotein,
a hormone, a growth factor, a cytokine, a chemical mediator, and
the like), a peptide, a lipid, a carbohydrate (e.g., a
polysaccharide, starch, monosaccharide), a nucleic acid molecule
(e.g., an aptamer, DNA, RNA, hybrid nucleic acid molecule, vectors,
chemically modified nucleic acid molecule), an oligomer, a small
molecule (e.g., a chemical compound; metabolites, such as sugars,
folic acid, uric acid, lactic acid), a drug (e.g., a
biological-based drug, hormone, antimicrobial compound, growth
factor, signaling molecule, ligand, etc.), a signaling molecule, a
ligand, a nucleic acid-protein fusion, fragments thereof, analogs
thereof, and a combination thereof.
[0073] The term "drug delivery vehicle", when used herein for
purposes of the specification and claims, means a carrier for one
or more biologically active agents; preferably, the carrier
comprising a microparticle, liposome, polymer, carrier structure
(e.g., matrix formed of biological substrate or a non-biological
substrate or a combination thereof), or combination thereof, and
generally in the size range of nanometers to microns.
[0074] The terms "covalent coupling", "covalently coupled" and like
terms, refer to a covalent bond being formed between two molecules.
Covalent coupling may be achieved by any means known in the art.
For example, a first molecule comprises a reactive functionality
comprising a chemical group which can covalently bond with a
chemical-reactive group (reactive with the chemical group of the
first molecule) of a second molecule. Free chemical groups include,
but are not limited to, a thiol carboxyl, hydroxyl, amino, amine,
sulfo, phosphate, or the like; whereas chemical-reactive groups
include, but are not limited to, thiol-reactive group,
carboxyl-reactive group, hydroxyl-reactive group, amino-reactive
group, amine-reactive group, sulfo-reactive group, or the like.
[0075] The terms "pharmaceutically acceptable salt" and
"cosmetically acceptable salt", when used herein for purposes of
the specification and claims, is known in the art to mean that the
compound or composition according to the invention may also be in
the form of a salt. Preferably, the salt form retains one or more
beneficial properties of the compound or composition of the
invention. Typically, salts are formed with inorganic acids (e.g.,
phosphoric acid, hydrochloric acid, sulfuric acid, and the like),
organic acids (e.g., acetic acid, benzoic acid, propionic acid,
maleic acid, glycolic acid, succinic acid, N-acetylcysteine, and
the like), and other salts known to those skilled in the art which
can be readily adapted for use as a compound or composition
according to the invention.
[0076] In one embodiment, the presently disclosed subject matter
provides compositions comprising a first substrate-binding peptide
(or a first substrate-binding polymer having a net positive or a
net negative charge) having binding affinity for a tissue or a
medical device, a second substrate-binding peptide having binding
affinity for a target molecule, wherein the first and second
substrate-binding domains are not covalently linked, and the target
molecule (see, for example FIG. 3). Each of the first and second
substrate-binding peptides/polymers is covalently coupled to at
least one interaction tag selected from the group consisting of a
hydrophobic interaction tag, a negatively charged interaction tag,
and a positively charged interaction tag (see FIG. 3). When the
substrate-binding peptide/polymer molecules are combined, the
hydrophobic interaction tags interact with each other and the
charged interaction tags interact with the oppositely charged
interaction tags or the oppositely charged substrate binding
polymers, to form a macromolecular network of non-covalently
coupled first and second substrate-binding peptides/polymers (see,
for example, FIGS. 4A-C (peptides) & 5A-5C (polymer/peptide)).
In this manner, the substrate-binding peptide/polymer molecules are
useful when combined with the target molecule for coating onto the
tissue or medical device to achieve loading of the target molecule
onto the tissue or medical device (see, e.g., Examples 17-19). In
some embodiments, the first substrate-binding peptide/polymer, the
second substrate-binding peptide, and the target molecule are
present in a pharmaceutically acceptable solution. In some
embodiments, the pharmaceutically acceptable solution is in the
form of a gel. In some embodiments, the tissue or medical device is
first coated with one or more of the first or second substrate
binding peptide/polymer, rather than being coated after the three
components are mixed together. The order of coating the tissue or
medical device with the composition comprising a first and a second
substrate-binding peptide/polymer and a target molecule can be
varied.
[0077] The first substrate-binding peptide or polymer is also
referred to herein for purposes of simplicity as the first
substrate-binding domain. Similarly, the second substrate-binding
peptide is also referred to herein as a substrate-binding domain.
Accordingly, all of the first and second substrate-binding peptides
and polymers can be referred to herein as substrate-binding
domains. In addition, the substrate binding molecules depicted in
FIGS. 4A-4C and 5A-5C are not meant to attempt to describe every
possible combination of covalently coupled interaction tag on a
substrate binding domain. For example, the first and second
substrate binding domains can comprise any combination of one or
more hydrophobic and charged interaction tag, as long as the
combination allows for a plurality of first and second substrate
binding molecules to form a non-covalent coupling with each other
according to the rules of hydrophobic tags interacting with each
other and charged interaction tags interacting with other
oppositely charged interaction tags. One embodiment, for example,
that is not depicted in either FIG. 4A-4C or FIG. 5A-5C is the
embodiment where the charged interaction tags are absent and each
of the first and second binding domains comprises a covalently
coupled hydrophobic interaction tag.
[0078] In some embodiments of the presently disclosed subject
matter a composition is provided comprising a plurality of a first
substrate-binding peptide comprising 3 to 40 amino acids, wherein
the first substrate is a tissue or a medical device and the first
substrate-binding peptide has binding affinity for the tissue or
the medical device; a plurality of a second substrate-binding
peptide comprising of 3 to 40 amino acids, wherein the second
substrate is a target molecule and the second substrate-binding
peptide has binding affinity for the target molecule, wherein the
first and second substrate-binding peptides are not covalently
linked; and a plurality of the target molecule; wherein each of the
first and second substrate-binding peptides is covalently coupled
to at least one interaction tag selected from the group consisting
of a hydrophobic interaction tag, a positively charged interaction
tag, and a negatively charged interaction tag, wherein the
hydrophobic interaction tags interact with each other and the
positively charged interaction tags interact with the negatively
charged interaction tags to form a macromolecular network
comprising the plurality of non-covalently coupled first and second
substrate-binding peptides.
[0079] In some embodiments, the first substrate tissue or medical
device comprises a material selected from the group consisting of
an animal tissue, an autologous tissue, an allogeneic tissue, a
transplanted tissue, an organ tissue, a bone tissue, a skin tissue,
a connective tissue, a muscle tissue, a nervous tissue, a polymer,
a silk, a collagen, a synthetic polymer, a polyester, a
polyurethane, a nylon, a polylactic acid, a polyglycolic acid,
poly(lactic acid-co-glycolic acid), a plastic, a silicone material,
a metal, a metal oxide, a non-metal oxide, a ceramic material, a
calcium phosphate based material, a carbon-based material, a
metallo-carbon composite, and combinations thereof.
[0080] In some embodiments, the target molecule is selected from
the group consisting of a cell, a protein, a polypeptide, a growth
factor, a growth differentiation factor (GDF), a platelet derived
growth factor (PDGF), a transforming growth factor (TGF), an
osteogenic protein, a bone morphogenic protein (BMP), a hormone, a
protein hormone, a parathyroid hormone (PTH), a drug, a drug
carrier, an antibiotic, a vancomycin antibiotic, a steroid, a
dexamethasone, and combinations thereof.
[0081] In some embodiments, the charged interaction tag is selected
from the group consisting of polylysine, polyarginine, polyamines,
polyimines, polyethylamines, polyethylenimines (PEI), polyaspartic
acid, polyglutamic acid, polystyrene sulfonate,
poly(styrenesulfonic-maleic acid), and combinations and copolymers
thereof.
[0082] In some embodiments, the hydrophobic interaction tag is
selected from the group consisting of fatty acids, undecanoic acid,
poly-undecanoic acid, myristic acid, amino hexanoic acid, capric
acid, lauric acid, palmitic acid, stearic acid, aromatic compounds,
and combinations and copolymers thereof.
[0083] In some embodiments, the first substrate is a metal medical
device, the second substrate target molecule is vancomycin, the
positively charged interaction tag covalently coupled to the first
substrate binding peptide is polyarginine and the negatively
charged interaction tag covalently coupled to the second substrate
binding peptide is polyglutamic acid or polyaspartic acid, the
positively and negatively charged interaction tags are coupled to
the substrate binding peptide directly or coupled through a
polyethylene glycol, and the hydrophobic interaction tag is absent.
In some embodiments, the first substrate binding polymer having a
positive charge is polyethyleneimine of various molecular
weights.
[0084] In some embodiments, the first substrate is a metal medical
device, the second substrate target molecule is vancomycin, the
positively charged interaction tag covalently coupled to the first
substrate binding peptide is polyarginine and the negatively
charged interaction tag covalently coupled to the second substrate
binding peptide is polyglutamic acid or polyaspartic acid, the
positively and negatively charged interaction tags are coupled to
the substrate binding peptides directly by a peptide bond or
coupled through a polyethylene glycol, the hydrophobic interaction
tag is poly-undecanoic acid and is covalently coupled to either the
first or the second substrate binding peptide directly, through a
polyethylene glycol, or through an aminohexanoic acid.
[0085] In some embodiments, the presently disclosed subject matter
provides a composition comprising a plurality of a first
substrate-binding polymer having a net negative or a net positive
charge, wherein the first substrate is a tissue or medical device
and the first substrate-binding polymer has binding affinity for
the tissue or medical device; a plurality of a second
substrate-binding peptide of 3 to 40 amino acids, wherein the
second substrate is a target molecule and the second
substrate-binding peptide has binding affinity for the target
molecule, wherein the first substrate-binding polymer and the
second substrate-binding peptide are not covalently linked; and a
plurality of the target molecule, wherein the plurality of second
substrate-binding peptides are covalently coupled to at least one
net positively or net negatively charged interaction tag, wherein
the charge of the interaction tag is opposite to the charge of the
first substrate-binding polymer, wherein each of the plurality of
first substrate-binding polymers and second substrate-binding
peptides is optionally covalently coupled to a hydrophobic
interaction tag, wherein the charged interaction tag interacts with
the first substrate-binding polymer and the optional hydrophobic
interaction tags interact with each other to form a macromolecular
network comprising the plurality of non-covalently coupled first
substrate-binding polymers and second substrate-binding
peptides.
[0086] In some embodiments, the first substrate tissue or medical
device comprises a material selected from the group consisting of
an autologous tissue, an allogeneic tissue, a transplanted tissue,
an organ tissue, a bone tissue, a skin tissue, a connective tissue,
a muscle tissue, a polymer, a synthetic polymer, a plastic, a
metal, a metal oxide, a non-metal oxide, a ceramic material, a
calcium phosphate based material, and combinations thereof. In some
embodiments, the target molecule is selected from the group
consisting of a cell, a protein, a polypeptide, a growth factor, a
growth differentiation factor (GDF), a platelet derived growth
factor (PDGF), a transforming growth factor (TGF), an osteogenic
protein, a bone morphogenic protein (BMP), a hormone, a protein
hormone, a parathyroid hormone (PTH), a drug, a drug carrier, an
antibiotic, a vancomycin antibiotic, a steroid, a dexamethasone,
and combinations thereof.
[0087] In some embodiments, the first substrate-binding polymer
having a net negative charge is selected from the group consisting
of polystyrene sulfonate, polyglutamic acid, polylactic acid,
polyglycolic acid, poly(lactic acid-co-glycolic acid), heparin, and
combinations and copolymers thereof. In some embodiments, the first
substrate-binding polymer having a net positive charge is selected
from the group consisting of polyimines, polyamines,
polyethylenimines, polyethylamines, and polylysine, and
combinations and copolymers thereof. In some embodiments, the
charged interaction tag is selected from the group consisting of
polylysine, polyarginine, polyamines, polyimines, polyethylamines,
polyethylenimines (PEI), polyaspartic acid, polyglutamic acid,
polystyrene sulfonate, poly(styrenesulfonic-maleic acid), and
combinations and copolymers thereof. In some embodiments, the
hydrophobic interaction tag is selected from the group consisting
of fatty acids, undecanoic acid, poly-undecanoic acid, myristic
acid, amino hexanoic acid, capric acid, lauric acid, palmitic acid,
stearic acid, aromatic compounds, and combinations and copolymers
thereof.
[0088] In some embodiments, the first substrate is a metal medical
device, the second substrate target molecule is vancomycin, the
first substrate binding polymer having a positive charge is
polyethylenimine, the negatively charged interaction tag covalently
coupled to the second substrate binding peptide is polyglutamic
acid or polyaspartic acid, the charged interaction tag is coupled
to the substrate binding peptide directly or coupled through a
polyethylene glycol, and the optional hydrophobic interaction tag
is absent.
[0089] In some embodiments of the presently disclosed subject
matter, the compositions of the presently disclosed subject matter
comprise a first substrate-binding peptide having binding affinity
for a tissue or medical device covalently linked to a second
substrate-binding peptide having binding affinity for a target
molecule, and the target molecule. Each of the covalently linked
first and second substrate-binding peptides is covalently coupled
to at least one interaction tag selected from the group consisting
of a hydrophobic interaction tag, a positively charged interaction
tag, and a negatively charged interaction tag. The covalently
linked substrate-binding peptide molecules and the target molecules
are combined resulting in the hydrophobic interaction tags
interacting with each other and the charged interaction tags
interacting with oppositely charged interaction tags (see, for
example, FIG. 6A-6B). In this manner, a macromolecular network is
formed comprising the linked substrate-binding domain molecules
non-covalently coupled together, and when combined with the target
molecule and coated onto a tissue or medical device, the
composition loads the target molecule onto the tissue or medical
device. (see, e.g., Examples 17-19). In some embodiments, the first
and second substrate-binding domains and the target molecule are
present in a pharmaceutically acceptable solution. In some
embodiments, the pharmaceutically acceptable solution is in the
form of a gel. In some embodiments, the tissue or medical device is
first coated with one or more of the first or second substrate
binding domains, rather than being coated after all the components
are mixed together. The order of coating the tissue or medical
device with the compositions comprising a first and a second
substrate-binding domain and a target molecule can be varied.
[0090] The substrate binding molecules depicted in FIG. 6A-4B are
not meant to attempt to describe every possible combination of
covalently coupled interaction tag on a molecule comprising a
covalently linked first and second substrate binding domain. For
example, the molecule comprising the linked substrate binding
domains can comprise any combination of one or more hydrophobic and
charged interaction tag, as long as the combination allows for a
plurality of molecules comprising the linked first and second
substrate binding domains to form a non-covalent coupling with each
other according to the rules of hydrophobic tags interacting with
each other and charged interaction tags interacting with other
oppositely charged interaction tags. One embodiment, for example,
that is not depicted in FIG. 6A-6C is the embodiment where each
molecule comprising the linked first and second binding domains has
both a charged and a hydrophobic interaction tag.
[0091] In some embodiments, the presently disclosed subject matter
provides a composition comprising a plurality of a first
substrate-binding peptide comprising 3 to 40 amino acids, wherein
the first substrate is a tissue or medical device and the first
substrate-binding peptide has binding affinity for the tissue or
medical device; a plurality of a second substrate-binding peptide
comprising 3 to 40 amino acids, wherein the second substrate is a
target molecule and the second substrate-binding peptide has
binding affinity for the target molecule, wherein the first and
second substrate-binding peptides are covalently linked; and a
plurality of the target molecule, wherein the plurality of
covalently linked first and second substrate-binding peptides are
covalently coupled to at least one interaction tag selected from
the group consisting of a hydrophobic interaction tag, a positively
charged interaction tag, and a negatively charged interaction tag,
wherein the hydrophobic interaction tags interact with each other
and the positively charged interaction tags interact with the
negatively charged interaction tags to form a macromolecular
network comprising the plurality of non-covalently coupled
substrate-binding peptides.
[0092] In some embodiments, the first substrate tissue or medical
device comprises a material selected from the group consisting of
an animal tissue, an autologous tissue, an allogeneic tissue, a
transplanted tissue, an organ tissue, a bone tissue, a skin tissue,
a connective tissue, a muscle tissue, a nervous tissue, a polymer,
a silk, a collagen, a synthetic polymer, a polyester, a
polyurethane, a nylon, a polylactic acid, a polyglycolic acid,
poly(lactic acid-co-glycolic acid), a plastic, a silicone material,
a metal, a metal oxide, a non-metal oxide, a ceramic material, a
calcium phosphate based material, a carbon-based material, a
metallo-carbon composite, and combinations thereof.
[0093] In some embodiments, the target molecule is selected from
the group consisting of a cell, a protein, a polypeptide, a growth
factor, a growth differentiation factor (GDF), a platelet derived
growth factor (PDGF), a transforming growth factor (TGF), an
osteogenic protein, a bone morphogenic protein (BMP), a hormone, a
protein hormone, a parathyroid hormone (PTH), a drug, a drug
carrier, an antibiotic, a vancomycin antibiotic, a steroid, a
dexamethasone, and combinations thereof.
[0094] In some embodiments, the charged interaction tag is selected
from the group consisting of polylysine, polyarginine, polyamines,
polyimines, polyethylamines, polyethylenimines (PEI), polyaspartic
acid, polyglutamic acid, polystyrene sulfonate,
poly(styrenesulfonic-maleic acid), and combinations and copolymers
thereof. In some embodiments, the hydrophobic interaction tag is
selected from the group consisting of fatty acids, undecanoic acid,
poly-undecanoic acid, myristic acid, amino hexanoic acid, capric
acid, lauric acid, palmitic acid, stearic acid, aromatic compounds,
and combinations and copolymers thereof.
[0095] In some embodiments, the first and second substrate-binding
peptides are covalently linked by a peptide bond. In some
embodiments, the first and second substrate-binding domains are
covalently linked through any one of the hydrophobic interaction
tag, the charged interaction tag, amino acids, polymers, synthetic
polymers, polyethers, poly(ethylene glycol) ("PEG"), a 10 unit
polyethylene glycol ("P10"), and a 6 unit polyethylene glycol
("MP").
[0096] In some embodiments, the first substrate is a metal medical
device, the second substrate target molecule is vancomycin, the
first and second substrate binding peptides are covalently linked
through a polyethylene glycol, the hydrophobic interaction tag is
poly-undecanoic acid, the hydrophobic interaction tag is covalently
coupled to the first substrate binding peptide either directly,
through a polyethylene glycol, or through an aminohexanoic acid,
and the charged interaction tag is absent.
[0097] In some embodiments, the first substrate is a metal medical
device, the second substrate target molecule is vancomycin, the
first and second substrate binding peptides are covalently linked
through a polyethylene glycol, the positively charged interaction
tag is covalently coupled to a portion of the plurality of second
substrate binding peptide, the negatively charged interaction tag
is covalently coupled to a portion of the second substrate binding
peptide, and the hydrophobic interaction tag is absent.
[0098] In some embodiments, the first substrate medical device is a
synthetic polymer, the second substrate target molecule is a growth
factor, the hydrophobic interaction tag is poly-undecanoic acid,
the first and second substrate binding peptides are covalently
linked through the poly-undecanoic acid hydrophobic interaction
tag, and the charged interaction tag is absent.
[0099] In some embodiments, the first substrate medical device is a
synthetic polymer, the second substrate target molecule is a growth
factor, the first and second substrate binding peptides are
covalently linked through a polyethylene glycol, the hydrophobic
interaction tag is poly-undecanoic acid, the poly-undecanoic acid
is covalently coupled to the second substrate binding peptides, and
the charged interaction tag is absent.
[0100] In another embodiment, the compositions used in the
presently disclosed subject matter comprise a first
substrate-binding peptide having binding affinity for a tissue or
medical device covalently linked to a second substrate-binding
peptide having binding affinity for a target molecule, another
additional second substrate-binding peptide, and the target
molecule vancomycin. Each of the covalently linked first and second
substrate-binding peptide comprising molecules and the second
substrate binding peptide molecules are covalently coupled to at
least one interaction tag selected from the group consisting of a
hydrophobic interaction tag, a positively charged interaction tag,
and a negatively charged interaction tag. The covalently linked
substrate-binding peptide molecules, the additional second
substrate-binding domain comprising molecule, and the target
molecules are combined resulting in the hydrophobic interaction
tags interacting with each other and the charged interaction tags
interacting with oppositely charged interaction tags. (see, for
example, FIG. 7). In this manner, a macromolecular network is
formed comprising the substrate-binding domain molecules
non-covalently coupled together, and when combined with the target
molecule and coated onto a tissue or medical device, the
composition loads the target molecule onto the tissue or medical
device. (see, e.g., FIG. 7 & Example 17). In some embodiments,
the linked first and second substrate-binding domains, the
additional second substrate binding domain, and the target molecule
are present in a pharmaceutically acceptable solution. In some
embodiments, the pharmaceutically acceptable solution is in the
form of a gel. In some embodiments, the tissue or medical device is
first coated with one or more of the linked first and second
substrate binding domain comprising molecules, the additional
second substrate binding domain comprising molecules, and the
target molecules, rather than being coated after all the components
are mixed together. The order of coating the tissue or medical
device with the compositions comprising the substrate-binding
domains and the target molecules can be varied.
[0101] The substrate binding molecules depicted in FIG. 7 are not
meant to be an attempt to describe every possible combination of
covalently coupled interaction tag on the molecules comprising a
covalently linked first and second substrate binding domains and
the additional second substrate comprising domains. For example,
the hydrophobic interaction tag covalently coupled to the molecule
comprising the linked substrate binding domains can be coupled at a
terminus rather than coupled so as to link the two
substrate-binding domains as depicted. The molecule comprising the
additional second substrate binding domain can also further
comprise a hydrophobic interaction tag. In another example, both
molecules can comprise hydrophobic interaction tags with the
charged interaction tags being absent. Any combination of
hydrophobic and/or charged interaction tags is acceptable, as long
as the combination allows for a plurality of molecules comprising
the linked first and second substrate binding domains and the
additional second substrate binding domains to form a non-covalent
coupling with each other according to the rules of hydrophobic tags
interacting with each other and charged interaction tags
interacting with other oppositely charged interaction tags.
[0102] In another embodiment, the presently disclosed subject
matter provides a composition comprising a composition comprising,
a plurality of first molecules comprising a first substrate-binding
peptide comprising 3 to 40 amino acids, wherein the first substrate
is a tissue or medical device and the first substrate-binding
peptide has binding affinity for the tissue or medical device; and
a second substrate-binding peptide comprising 3 to 40 amino acids,
wherein the second substrate is a target molecule and the second
substrate-binding peptide has binding affinity for the target
molecule, wherein the first and second substrate-binding peptides
are covalently linked; and a plurality of second molecules
comprising the second substrate-binding peptide, wherein the second
substrate binding peptide is not covalently linked to the first
substrate binding peptide; and a plurality of the target molecule,
wherein each of the plurality of first and second molecules are
covalently coupled to at least one interaction tag selected from
the group consisting of a hydrophobic interaction tag, a positively
charged interaction tag, and a negatively charged interaction tag,
wherein the hydrophobic interaction tags interact with each other
and the positively charged interaction tags interact with the
negatively charged interaction tags to form a macromolecular
network comprising the plurality of non-covalently coupled first
and second molecules.
[0103] In some embodiments, the first substrate tissue or medical
device comprises a material selected from the group consisting of
an animal tissue, an autologous tissue, an allogeneic tissue, a
transplanted tissue, an organ tissue, a bone tissue, a skin tissue,
a connective tissue, a muscle tissue, a nervous tissue, a polymer,
a silk, a collagen, a synthetic polymer, a polyester, a
polyurethane, a nylon, a polylactic acid, a polyglycolic acid,
poly(lactic acid-co-glycolic acid), a plastic, a silicone material,
a metal, a metal oxide, a non-metal oxide, a ceramic material, a
calcium phosphate based material, a carbon-based material, a
metallo-carbon composite, and combinations thereof.
[0104] In some embodiments, the target molecule is selected from
the group consisting of a cell, a protein, a polypeptide, a growth
factor, a growth differentiation factor (GDF), a platelet derived
growth factor (PDGF), a transforming growth factor (TGF), an
osteogenic protein, a bone morphogenic protein (BMP), a hormone, a
protein hormone, a parathyroid hormone (PTH), a drug, a drug
carrier, an antibiotic, a vancomycin antibiotic, a steroid, a
dexamethasone, and combinations thereof.
[0105] In some embodiments, the charged interaction tag is selected
from the group consisting of polylysine, polyarginine, polyamines,
polyimines, polyethylamines, polyethylenimines (PEI), polyaspartic
acid, polyglutamic acid, polystyrene sulfonate,
poly(styrenesulfonic-maleic acid), and combinations and copolymers
thereof. In some embodiments, the hydrophobic interaction tag is
selected from the group consisting of fatty acids, undecanoic acid,
poly-undecanoic acid, myristic acid, amino hexanoic acid, capric
acid, lauric acid, palmitic acid, stearic acid, aromatic compounds,
and combinations and copolymers thereof.
[0106] In some embodiments, the first and second substrate-binding
peptides are covalently linked through a peptide bond. The
composition of claim 5, wherein the first and second
substrate-binding domains are covalently linked by any one of the
hydrophobic interaction tag, the charged interaction tag, amino
acids, polymers, synthetic polymers, polyethers, poly(ethylene
glycol) ("PEG"), a 10 unit polyethylene glycol ("P10"), and a 6
unit polyethylene glycol ("MP").
[0107] In some embodiments, the first substrate is a metal medical
device, the second substrate target molecule is vancomycin, the
first and second substrate-binding peptides are covalently linked
through the poly-undecanoic acid hydrophobic interaction tag, and
each of the first and the second molecules comprise a covalently
coupled charged interaction tag wherein the charged interaction tag
on the first molecules is oppositely charged to the charged
interaction tag on the second molecules.
[0108] In some embodiments, the first substrate is a metal medical
device, the second substrate target molecule is vancomycin, the
hydrophobic interaction tag is poly-undecanoic acid and is
covalently coupled to the first molecules, and each of the first
and the second molecules comprise a covalently coupled charged
interaction tag, wherein the charged interaction tag on the first
molecules is oppositely charged to the charged interaction tag on
the second molecules. Also provided in the presently disclosed
subject matter are methods for applying the compositions of
presently disclosed subject matter to a substrate that is tissue or
a medical device, the methods comprising contacting the composition
with the substrate so that the composition binds the substrate,
such as in forming a coating on the substrate which has one or more
improved biophysical properties. Also provided in the presently
disclosed subject matter are medical devices coated with the
compositions of the presently disclosed subject matter, wherein at
least a portion of the medical device is coated with the
composition.
EXAMPLE 1
[0109] While substrate-binding peptides can be identified using any
one of several methods known to those skilled in the art,
Illustrated in this example are various methods for utilizing phage
display technology to produce a substrate-binding peptide having
binding specificity for substrate, such substrate-binding peptide
useful as a component in producing a compound according to the
present invention.
Phage Screening and Selections.
[0110] Phage display technology is well-known in the art, and can
be used to identify additional peptides for use as binding domains
in the compositions according to the present invention. In general,
using phage display, a library of diverse peptides can be presented
to a target substrate, and peptides that specifically bind to the
substrate can be selected for use as binding domains. Multiple
serial rounds of selection, called "panning," may be used. As is
known in the art, any one of a variety of libraries and panning
methods can be employed to identify a binding domain that is useful
in a composition according to the present invention. Panning
methods can include, for example, solution phase screening, solid
phase screening, or cell-based screening. Once a candidate binding
domain is identified, directed or random mutagenesis of the
sequence may be used to optimize the binding properties (including
one or more of specificity and avidity) of the binding domain.
[0111] For example, a variety of different phage display libraries
were screened for peptides that bind to a selected target substrate
(e.g., a substrate selected to find a binding domain useful in the
present invention). The substrate was either bound to or placed in
(depending on the selected substrate) a container (e.g., wells of a
96 well microtiter plate, or a microfuge tube). Nonspecific binding
sites on the surfaces of the container were blocked with a buffer
containing bovine serum albumin ("BSA"; e.g., in a range of from 1%
to 10%). The containers were then washed 5 times with a buffer
containing buffered saline with Tween.TM. 20 ("buffer-T"). Each
library was diluted in buffer-T and added at a concentration of
10.sup.10 pfu/ml in a total volume of 100 .mu.l. After incubation
(in a range of from 1 to 3 hours) at room temperature with shaking
at 50 rpm, unbound phage were removed by multiple washes with
buffer-T. Bound phage were used to infect E. coli cells in growth
media. The cell and phage-containing media was cultured by
incubation overnight at 37.degree. C. in a shaker at 200 rpm.
Phage-containing supernatant was harvested from the culture after
centrifuging the culture. Second and third rounds of selection were
performed in a similar manner to that of the first round of
selection, using the amplified phage from the previous round as
input. To detect phage that specifically bind to the selected
substrate, enzyme-linked immunosorbent (ELISA-type) assays were
performed using an anti-phage antibody conjugated to a detector
molecule, followed by the detection and quantitation of the amount
of detector molecule bound in the assay. The DNA sequences encoding
peptides from the phage that specifically bind to the selected
substrate were then determined; i.e., the sequence encoding the
peptide is located as an insert in the phage genome, and can be
sequenced to yield the corresponding amino acid sequence displayed
on the phage surface.
EXAMPLE 2
[0112] As summarized previously herein, the compound useful in
making a composition of the invention is comprised of fatty acid
covalently coupled to substrate-binding peptide. The composition
may comprise substrate-binding peptide of a single type (e.g.,
"type" defined by the substrate for which the substrate-binding
peptide has binding specificity), or may comprise more than one
type of substrate-binding peptide. Further, as illustrated and
described in more detail in Examples 4 & 5 herein, the peptide
component of the compound may comprise a peptide comprised of a
single binding specificity (see, e.g., Example 4); or may comprise
two or more binding domains, with each binding domain comprised of
a substrate-binding peptide, and with the two or more binding
domains covalently coupled (directly or via a linker) (see, e.g.,
Example 5).
[0113] In one example, the substrate, for which a substrate
comprising a material comprising a surface of a device, and more
preferably a medical device; wherein the material is selected from
the group consisting of a metal, a polymer, a non-metal oxide, and
a ceramic. As a specific illustrative example for developing
substrate-binding peptides using the methods outlined in Example 1,
and to develop substrate-binding peptides having binding
specificity for polymer, various polymers were used as a substrate
for performing phage selection using several different libraries of
phage. Table 1 illustrates exemplary substrate-binding peptides,
which may be used in the compounds and compositions according to
the present invention, having binding specificity for a polymer,
and comprise: SEQ ID NOs:1-22 that have binding specificity for
polystyrene; SEQ ID NO:23 that has binding specificity for
polyurethane; SEQ ID NOs: 24-37 that have binding specificity for
polyglycolic acid; SEQ ID NOs: 38-43 that have binding specificity
for polycarbonate; SEQ ID NOs: 44-52 that have binding specificity
for nylon; and SEQ ID NOs: 53 and 54 that have binding specificity
for teflon. Such peptides may be used as substrate-binding peptides
having binding specificity for non-biological substrate comprising
a polymer to which they having binding specificity.
TABLE-US-00001 TABLE 1 SEQ ID NO: Amino acid sequence (single
letter code) Binding specificity for polystyrene 1 FLSFVFPASAWGG 2
FYMPFGPTWWQHV 3 LFSWFLPTDNYPV 4 FMDIWSPWHLLGT 5 FSSLFFPHWPAQL 6
SCAMAQWFCDRAEPHHVIS 7 SCNMSHLTGVSLCDSLATS 8 SCVYSFIDGSGCNSHSLGS 9
SCSGFHLLCESRSMQRELS 10 SCGILCSAFPFNNHQVGAS 11 SCCSMFFKNVSYVGASNPS
12 SCPIWKYCDDYSRSGSIFS 13 SCLFNSMKCLVLILCFVS 14 SCYVNGHNSVWVVVFWGVS
15 SCDFVCNVLFNVNHGSNMS 16 SCLNKFFVLMSVGLRSYTS 17
SCCNHNSTSVKDVQFPTLS 18 FFPSSWYSHLGVL 19 FFGFDVYDMSNAL 20
LSFSDFYFSEGSE 21 FSYSVSYAHPEGL 22 LPHLIQYRVLLVS Binding specificity
for polyurethane 23 SCYVNGHNSVWVVVFWGVS Binding specificity of
polyglycolic acid 24 SCNSFMFINGSFKETGGCS 25 SCFGNLGNLIYTCDRLMPS 26
SCSFFMPWCNFLNGEMAVS 27 SCFGNVFCVYNQFAAGLFS 28 SCCFINSNFSVMNHSLFKS
29 SCDYFSFLECFSNGWSGAS 30 SCWMGLFECPDAWLHDWDS 31
SCFWYSWLCSASSSDALIS 32 SCFGNFLSFGFNCESALGS 33 SCLYCHLNNQFLSWVSGNS
34 SCFGFSDCLSWFVQPSTAS 35 SCNHLGFFSSFCDRLVENS 36
SCGYFCSFYNYLDIGTASS 37 SCNSSSYSWYCWFGGSSPS Binding specificity for
polycarbonate 38 FGHGWLNTLNLGW 39 FSPFSANLWYDMF 40 VFVPFGNWLSTSV 41
FWNVNYNPWGWNY 42 FYWDRLNVGWGLL 43 LYSTMYPGMSWLV Binding specificity
for nylon 44 SCFYQNVISSSFAGNPWEC 45 SCNMLLNSLPLPSEDWSAC 46
SCPFTHSLALNTDRASPGC 47 SCFESDFPNVRHHVLKQSC 48 SCVFDSKHFSPTHSPHDVC
49 SCGDHMTDKNMPNSGISGC 50 SCDFFNRHGYNSGCEHSVC 51
SCGDHMTDKNMPNSGISGC 52 SCYYNGLVVHHSNSGHKDC Binding specificity for
Teflon 53 CWSRFRLFMLFCMFYLVS 54 CIKYPFLYCCLLSLFLFS
[0114] As a specific illustrative example for developing
substrate-binding peptides using the methods outlined in Example 1,
and to develop substrate-binding peptides having binding
specificity for metal, metal (e.g., titanium or stainless steel)
was used as a substrate for performing phage selection using
several different libraries of phage. Titanium beads and stainless
steel beads of approximately 5/32-inch diameter were individually
prepared for selections by sequentially washing the beads with 70%
ethanol, 40% nitric acid, distilled water, 70% ethanol and,
finally, acetone, to remove any surface contaminants. After drying,
one metal bead was placed per well of a 96-well polypropylene
plate. Non-specific binding sites on the metal beads and the
surface of the polypropylene plate were blocked with 1% bovine
serum albumin (BSA) in phosphate-buffered saline (PBS). The plate
was incubated for 1 hour at room temperature with shaking at 50
rpm. The wells were then washed 5 times with 300 .mu.L of
buffer-T.
[0115] Each library was diluted in buffer-T and added at a
concentration of 10.sup.10 pfu/mL in a total volume of 100 .mu.L.
After 3 hours of incubation at room temperature and shaking at 50
rpm, unbound phage were removed by 5 washes of buffer-T. The phage
were added directly to E. coli DH5.alpha.F' cells in 2.times. YT
media, and the phage-infected cells were transferred to a fresh
tube containing 2.times. YT media and incubated overnight at
37.degree. C. in a shaker incubator. Phage supernatant was
harvested by centrifugation at 8500.times.g for 10 minutes. Second
and third rounds of selection were performed in a similar manner to
the first round, using the amplified phage from the previous round
as input. Each round of selection was monitored for enrichment of
metal binding peptides using ELISA-like assays performed using an
anti-M13 phage antibody conjugated to horseradish-peroxidase,
followed by the addition of chromogenic agent ABTS
(2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), and
determining a read-out at 405 nm. Libraries that showed enrichment
of phage displaying metal binding peptides were plated on a lawn of
E. coli cells, and individual plaques were picked and tested for
binding to metals (e.g., titanium, stainless steel, etc.). Relative
binding strengths of the phage can also be determined by testing
serial dilutions of the phage for binding to a metal substrate in
an ELISA. For example, serial dilutions of the display-selected
clones were exposed to titanium or steel in an ELISA. The higher
dilutions represent more stringent assays for affinity; therefore,
phage that yield a signal at higher dilutions represent peptides
with higher relative affinity for the particular target metal.
Primers against the phage vector sequence that flank the insertion
site were used to determine the DNA sequence encoding the peptide
for the phage in each group. The sequence encoding the peptide
insert was translated to yield the corresponding amino acid
sequence displayed on the phage surface. Similar procedures were
used to develop surface-binding peptides that have binding
specificity for polymers.
[0116] The DNA sequences encoding peptides isolated on either
polymer substrates or metal substrates were determined. While
typically such phage amino acids adjoining the peptide displayed
had no significant contribution to the binding specificity of the
peptide, the peptides useful in the present invention may also
comprise, in their amino acid sequence, such phage amino acids
adjoining the peptide at the N-terminus and at the C-terminus
(e.g., denoted as ss and sr in Table 2).
Binding Specificity Characterizations
[0117] Relative binding strengths (affinities) of the
substrate-binding peptides to a substrate, also used as a measure
of binding specificity, were determined by testing serial dilutions
of the substrate-binding peptides for binding to a target substrate
(e.g., comprising either metal or polymer, depending on the
substrate-binding peptide's binding specificity being
characterized). Plotting the absorbance observed across the
concentration range for each peptide sequence yielded a binding
curve of the peptides to its target substrate from which can be
determined an EC50 (e.g., the concentration of peptide that gives
50% of the maximum signal in the binding curve is used as an
estimate of the affinity of the peptide for the target). Preferred
for use in a compound or composition according to the present
invention are substrate-binding peptides that bind to the selected
substrate with binding specificity, preferably with an EC50 of less
than or equal to about 1 .mu.M, and more preferably, in the
nanomolar range (e.g., <0.1 .mu.M). A typical binding assay for
titanium (note, a different substrate may be substituted for
titanium in the assay, depending on the binding specificity of the
substrate-binding peptide) may be performed according to the
following procedure.
[0118] Briefly, 5/32-inch diameter Grade 200 titanium beads were
washed by sonication in acetone for 15 minutes, and the beads were
allowed to dry. One bead was added to each well of a 96-well
polypropylene plate. Two hundred fifty (250) .mu.L of 1% BSA in PBS
was added to each well of the plate. The surface of the wells and
the beads were blocked by incubation for 1 hour at 20.degree. C.
with shaking at 500 rpm. The plate was washed three times with 250
.mu.L of buffer-T per well. A 1:3 dilution series of each of the
peptides was prepared using PBS as a diluent, starting at a peptide
concentration of 20 .mu.M, and going down to 0.0001 .mu.M. A 200
.mu.L sample of each dilution was added to wells of the plate. The
plate was incubated for 1 hour at 20.degree. C. with shaking at 500
rpm. The beads were washed three times with 250 .mu.L of buffer-T
per well. Two hundred (200) .mu.L of streptavidin-alkaline
phosphatase ("streptavidin AP") reagent, at a dilution of 1:2000 in
buffer+1% BSA, was added to each well. The plate was incubated for
30 minutes at room temperature. The beads were washed three times
with 250 .mu.L of buffer-T per well. Two hundred (200) .mu.L of
color development reagent (PNPP, p-nitrophenol phosphate) was added
to each well. After color had developed (10 minutes), the samples
were transferred to a clear 96-well plate and the absorbance at 405
nm determined. A binding curve was generated by plotting the
absorbance at 405 nm against the peptide concentration (.mu.M).
Table 2 illustrates exemplary substrate-binding peptides, which may
be used in producing a compound or composition according to the
present invention, having binding specificity for a metal
(including a metal alloy, a metal oxide, or a non-metal oxide), and
comprising: SEQ ID NOs:55-82 that specifically bind to titanium;
and SEQ ID NOs: 83-102 that specifically bind to stainless
steel.
TABLE-US-00002 TABLE 2 SEQ ID NO: Amino acid sequence (single
letter code) Binding specificity for titanium 55
SCFWFLRWSLFIVLFTCCS 56 SCESVDCFADSRMAKVSMS 57 SCVGFFCITGSDVASVNSS
58 SCSDCLKSVDFIPSSLASS 59 SCAFDCPSSVARSPGEWSS 60 SCVDVMHADSPGPDGLNS
61 SCSSFEVSEMFTCAVSSYS 62 SCGLNFPLCSFVDFAQDAS 63
SCMLFSSVFDCGMLISDLS 64 SCVDYVMHADSPGPDGLNS 65 SCSENFMFNMYGTGVCTES
66 HKHPVTPRFFVVE 67 CNCYVTPNLLKHKCYKIC 68 CSHNHHKLTAKHQVAHKC 69
CDQNDIFYTSKKSHKSHC 70 SSDVYLVSHKHHLTRHNS 71 SDKCHKHWYCYESKYGGS 72
SDKSHKHWYSYESKYGGS 73 HHKLKHQMLHLNGG 74 GHHHKKDQLPQLGG 75
ssHKHPVTPRFFVVEsr 76 ssCNCYVTPNLLKHKCYKICsr 77
ssCSHNHHKLTAKHQVAHKCsr 78 ssCDQNDIFYTSKKSHKSHCsr 79
ssSSDVYLVSHKHHLTRHNSsr 80 ssSDKCHKHWYCYESKYGGSsr 81 HHKLKHQMLHLNGG
82 GHHHKKDQLPQLGG Binding specificity for steel 83 CFVLNCHLVLDRP 84
SCFGNFLSFGFNCEYALGS 85 DGFFILYKNPDVL 86 NHQNQTN 87 ATHMVGS 88
GINPNFI 89 TAISGHF 90 LYGTPEYAVQPLR 91 CFLTQDYCVLAGK 92
VLHLDSYGPSVPL 93 VVDSTGYLRPVST 94 VLQNATNVAPFVT 95 WWSSMPYVGDYTS 96
SSYFNLGLVKHNHVRHHDS 97 CHDHSNKYLKSWKHQQNC 98 SCKHDSEFIKKHVHAVKKC 99
SCHHLKHNTHKESKMHHEC 100 VNKMNRLWEPL 101 SSHRTNHKKNNPKKKNKTR 102
NHTISKNHKKKNKNSNKTR
[0119] While these exemplary peptide sequences are disclosed
herein, one skilled in the art will appreciate that the deletions,
additions or substitutions of these peptides may be made using
methods known in the art, provided the resultant amino acid
sequence retains substantially the binding properties as the
exemplary peptide disclosed herein. For example, based on the amino
acid sequences of substrate-binding peptides illustrated by SEQ ID
NOs:75-82 in Table 2, shown in Table 3 is a series of synthetic,
second-generation peptides which were synthesized, some of which
had improved binding specificities as compared to the binding
specificity of the peptide from which it was derived.
TABLE-US-00003 TABLE 3 SEQ ID NO: Amino acid sequence 103
SKKHGGKKHGSSGK 104 SKHKGGKHKGSSGK 105 SHKHGGHKHGGHKHGSSGK 106
SKHKGGHKHGSSGK 107 SHKHGGKHKGSSGK 108 SKHKGGGGKHKGSSGK 109
SHKHGGGGHKHGSSGK 110 SHKHGGHKHGSSGK 111 SHHKGGHHKGSSGK 112
SKHKGGKHKGGKHKGSSGK
[0120] Several oligomers (also referred to as "multimers") of
different substrate-binding peptides were synthesized. Briefly, the
oligomers were built on a lysine MAP core and comprised of two and
four peptide modules, respectively, of a substrate-binding peptide.
In an illustrative example, this core matrix was used to generate a
peptide dimer and peptide tetramer using, in each branch, a
monomeric peptide consisting essentially of the amino acid sequence
of SEQ ID NO:112. The oligomers were synthesized sequentially using
solid phase chemistry on a peptide synthesizer. The synthesis was
carried out at a 0.05 mmol scale which ensures maximum coupling
yields during synthesis. The biotin reporter moiety was placed at
the C-terminus of the molecule, and was appended by a short linker
containing glycine and serine residues to the lysine core. Standard
Fmoc/t-Bu chemistry was employed using AA/HBTU/HOBt/NMM (1:1:1:2)
as the coupling reagents (AA is amino acid; HOBt is O-Pfp
ester/1-hydroxybenzotriazole; HBTU is
N-[1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium
hexafluorophosphate N-oxide; NMM is N-methylmorpholine). Amino
acids were used in 5-10 fold excess in the synthesis cycles, and
all residues were doubly, triply or even quadruply coupled
depending upon the complexity of residues coupled. The coupling
reactions were monitored by Kaiser ninhydrin test. The Fmoc
deprotection reactions was carried out using 20% piperidine in
dimethyl-formamide. Peptide cleavage from the resin was
accomplished using trifluoracetic acid
(TFA:H.sub.2O:Triisopropylsilane=95:2.5:2.5) at room temperature
for 4 hours. The crude product was precipitated in cold ether. The
pellet obtained after centrifugation was washed thrice with cold
ether and lyophilized to give a white solid as crude desired
product. The crude products were analyzed by analytical high
performance liquid chromatography (HPLC) on a C-18 column using
mobile eluants (A=H.sub.2O/TFA (0.1% TFA) and B=Acetonitrile/TFA
(0.1% TFA). The polymers were also further analyzed by mass
spectrometry for before subjecting each to final purification by
HPLC. The fractions containing the desired product were pooled and
lyophilized to obtain a fluffy white powder (>98% purity).
EXAMPLE 3
[0121] Substrate-binding peptides, which bind to a biological
substrate, can be used to produce a compound according to the
invention. Thus, a fatty acid may be covalently coupled to a
substrate-binding peptide having binding specificity for a
biological substrate, whether it is a substrate-binding peptide by
itself, or forms part of a biofunctional composition comprised of
two or more substrate-binding peptides, wherein two or more
substrate-binding peptides are covalently coupled together to form
the biofunctional composition. For example, the biological
substrate may comprise a biological molecule. In an illustrative
example, wherein the biological molecule is a protein, and may
further comprise a growth factor, disclosed is a substrate-binding
peptide having binding specificity for BMP. For example, disclosed
in commonly owned U.S. patent application US 20060051396 are
families of peptides having binding specificity for BMP; one
example being a peptide comprising the consensus amino acid
sequence of GGALGFPLKGEVVEGWA (SEQ ID NO:113). In another example,
wherein the biological substrate comprises a tissue, disclosed in
commonly owned U.S. application 60/914,341 are bone-trophic
peptides; one example being a peptide comprising the amino acid
sequence of FDIDWSGMRSWWG (SEQ ID NO:114). In another embodiment,
wherein the biological substrate comprises a tissue, disclosed in
published US applications US20030152976, US20050249682, and PCT
application WO2006/094093 are skin-binding peptides, with one
example of a peptide given as LSPSRMK (SEQ ID NO:115). In a further
embodiment, wherein the biological substrate comprises a tissue,
disclosed in commonly owned U.S. 60/972,277 are families of
hair-binding peptides, with one example as a peptide comprising an
amino acid sequence of SRKSSQKNPHHPKPPKKPTAR (SEQ ID NO:116). In
another embodiment, wherein the biological substrate comprises a
cell (preferably, cells of a cell type), a peptide having a
sequence of ALPSTSSQMPQL (SEQ ID NO:117) has been described as
binding to stem cells; and a peptide comprising the amino acid
sequence of SSSCQHVSLLRPSAALGPDNCSR (SEQ ID NO:118) has binding
specificity for human adipose-derived stem cells and endothelial
cells (disclosed in commonly owned U.S. application Ser. No.
11/649,950).
[0122] In another embodiment, the substrate (either biological or
non-biological, as the case may be) comprises a therapeutic drug.
For example, it has been reported that by use of phage display to
screen for peptides that bind to paclitaxel (trade name
Taxol.RTM.), identified was a peptide having the amino acid
sequence of HTPHPDASIQGV (SEQ ID NO: 119). In another embodiment
where the substrate comprises a therapeutic drug, the therapeutic
drug may comprise an antimicrobial. For example, vancomycin and
vancomycin analogs bind to bacterial cell wall peptides ending with
D-Ala-D-Ala (two D-alanine residues). A peptide that mimics
bacterial cell wall peptide binding to vancomycin, and therefore
binds to vancomycin and its analogs, comprises an amino acid
sequence of Lys-Ala-Ala (L-Lys-D-Ala-D-Ala). In another embodiment,
the biological substrate comprises a hormone. Thus, a
substrate-binding peptide may having binding specificity for a
hormone. For example, peptides having a core amino acid sequence of
VMNV (SEQ ID NO: 120) have been described as binding to human
growth hormone. In another embodiment, the biological molecule
comprises a nucleic acid molecule, and more preferably, a nucleic
acid molecule encoding a protein. For example, peptide having the
amino acid sequence of AEDG (SEQ ID NO: 121) complexes with duplex
DNA comprising [poly(dA-dT):poly(dA-dT)].
EXAMPLE 4
[0123] Using the methods of the present invention described herein,
a compound according to the present invention may be formed by
covalently coupling one or more molecules of fatty acid to a
substrate-binding peptide. In this example, illustrated are
compounds formed by covalently coupling fatty acid to
substrate-binding peptides having binding specificity for a
non-biological substrate. Shown in Table 4 are illustrative
compounds of the invention, synthesized by the methods described
herein. The compounds are listed as a linear sequence, with "AUD"
representing aminoundecanoic acid, "MYR" representing myristic
acid; "PALM" representing palmitic acid; "LAU" representing lauric
acid; "K" is single letter designation for lysine; "Y" is single
letter designation for tyrosine; "R" is single letter designation
for arginine; brackets "[ ]" around a fatty acid indicate the fatty
acid is branched on a lysine; and "Ac" means a modified N-terminal
amino acid which has been acetylated. A peptide comprising an amino
acid sequence of SEQ ID NO:101, and having binding specificity for
metal, was synthesized to further include a linker at the
C-terminal end to be biotinylated to facilitate detection during
functional studies. Such peptide is represented by the amino acid
sequence SSHRTNHKKNNPKKKNKTRGSSGK (SEQ ID NO:122).
TABLE-US-00004 TABLE 4 Compound Ref. # Compound linear sequence 122
AUD-AUD-AUD-AUD-SEQ ID NO:122 123 AUD-AUD-K-AUD-AUD-AUD-AUD-SEQ ID
NO:122 124 AUD-AUD-AUD-AUD-AUD-AUD-SEQ ID NO:122-RRRR RRR 125
MYR-SEQ ID NO:122 126 LAU-SEQ ID NO:122 127 MYR-linker-SEQ ID
NO:122 128 [MYR].sub.2-K-linker-SEQ ID NO:122 129
PALM-PALM-PALM-linker-SEQ ID NO:122 130 AUD-AUD-AUD-AUD-AUD-AUD-SEQ
ID NO:122 131 AUD-AUD-AUD-AUD-AUD-AUD-AUD-AUD-SEQ ID NO:122 132
Ac-Y-AUD-AUD-AUD-AUD-AUD-AUD-SEQ ID NO:122
The following acronyms are used in the description of methods for
making compounds of the invention (see, e.g., Examples 4 & 5).
Mtt is 4-methyltrityl; TATU is
2-(7-Aza-1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate; DIEA is diisopropylethylamine; NMP is
1-Methyl-2-pyrrolidone; DCM is dichloromethane; DMF is
dimethylformamide; TFA is trifluoracetic acid; TIS is
triisopropylsilane; TBTU is
O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate; HOBt is O-Pfp ester/1-hydroxybenzotriazole; NMM
is N-methylmorpholine; RP-HPLC is reverse phase high performance
liquid chromatography; Fmoc is 9-fluorenylmethoxycarbonyl; tBU is
t-butyl; mini-PEG is Fmoc-8-Amino-3,6-Dioxaoctanoic Acid; MALDI-TOF
is matrix-assisted laser desorption ionization-time of flight mass
spectrometry; Reagent A is water/TFA (0.1% TFA); Reagent B is
Acetonotrile/TFA (0.1% TFA); Fmoc-PAL-PEG resin is
[5-(4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy)valeric
acid]-polyethylene glycol-polystryrene resin.
[0124] Compound 125 and compound 126 were synthesized by coupling
150 mg of a `universal` SSHRTNHKKNNPKKKNKTRGSSGK(Mtt)-resin (the
amino acid sequence being that of SEQ ID NO:122) with 1 mmol of
myristic and lauric acid, respectively, using TATU/2 mmol DIEA in
NMP for 2 hours. Following coupling, the resin was washed with NMP
(3 times) and DCM (5 times) and dried under vacuum overnight. The
dried resin was subjected to cleavage using TFA cocktail (3 mL)
comprised of 2.5% (v/v) each of water and TIS in TFA. After
cleavage for 1.5 hours, the reaction was filtered into 25 mL of
cold ether. The pellet obtained was separated by centrifugation,
and then washed with chilled ether (3.times.). The crude product
was air-dried and purified by RP-HPLC using the following
conditions: Column: C-18 (250.times.21.2 mm). Flow: 10 mL/min.
Gradient: 0-20% Reagent B in 1 minute; 20-60% Reagent B in 30
minutes. The fractions containing the desired product were pooled
and lyophilized to obtain a fluffy white powder (>95% purity).
Compound 125 was purified (HPLC retention time of 15.32 minutes (0
to 60% Reagent B in 30 minutes @ 0.075 mL/min at 220 nm)); and
characterized by MALDI-TOF (observed mass=2929.2; expected
mass=2929.39). Compound 126 was purified (HPLC retention time of
12.63 min (0 to 60% Reagent B in 30 minutes @ 0.075 mL/min at 220
nm)), and characterized by MALDI-TOF (observed mass=2901; expected
mass=2901.34).
[0125] Compounds 127, 128 and 129 were synthesized using standard
Fmoc/tBu chemistry. Foc-Lys(Biotin) was introduced at the
C-terminus by coupling to Fmoc-PAL-PEG-PS resin. Linear synthesis
was performed to synthesize the peptide component of the compound
(SSHRTNHKKN--NPKKKNKTRGSSGK; SEQ ID NO:122). Amino acids were used
in 5 fold excess in the synthesis cycles, and all residues were
doubly or triply coupled. The coupling reactions were monitored by
Kaiser ninhydrin test or chloranil test. Fmoc deprotection
reactions were carried out using 20% piperidine in DMF. A mini-PEG
linker was introduced between the peptide and fatty acid moieties,
in covalently coupling fatty acid to peptide. Myristic acid was
pre-activated with HOBt, and double coupled to the resin using the
TBTU/HOBt/NMM method. For compound 128 synthesis, Fmoc-Lys(Fmoc)-OH
was coupled to mini-PEG linker. The two terminal Fmoc groups were
removed, followed by coupling with 10 equivalents of myristic acid
using the TBTU method. For compound 129 synthesis, 5 fold excess of
palmitoyl-Cys((RS)-2,3-di(palmitoyloxy)-propyl)-OH was double
coupled to mini-PEG-peptide resin using TBTU activation.
[0126] Following synthesis, the resin containing the compound was
cleaved using TFA cocktail (2.5% water; 2.5% TIS; 95% TFA) for 4
hours. The cleavage reaction mixture was filtered into cold ether.
The pellet obtained was further washed thrice with cold ether, and
dried under vacuum. The crude products were purified using RP-HPLC
(column: C-4, 250.times.21.2 mm; Flow: 10 mL/minute Gradient: 0-20%
Reagent B in 1 minute; 20-80% Reagent B in 30 minutes. Compound 127
was purified (HPLC retention time of 14.63 minutes (30 to 60% B in
30 minutes @ 1 mL/min at 220 nm)) and characterized by MALDI-TOF
(observed mass=3300; expected mass=3300.86). Compound 128 was
purified (HPLC retention time of 24.25 minutes (40 to 65% Reagent B
in 30 minutes @ 1 mL/min at 220 nm)), and characterized by
MALDI-TOF (observed mass=3638.5; expected mass=3639.32). Compound
129 was characterized by MALDI-TOF (observed mass=3981.8; expected
mass=3982.96).
[0127] Compounds 122-124, and 130-132 were synthesized using
similar methods and reagents as described herein for compounds
125-129.
EXAMPLE 5
[0128] Using the methods of the present invention described herein,
a compound according to the present invention may be formed by
covalently coupling one or more molecules of fatty acid to a
substrate-binding peptide comprising a biofunctional composition.
In this example, illustrated are compounds formed by covalently
coupling fatty acid to a biofunctional composition comprising a
substrate-binding peptide having binding specificity for a
non-biological substrate, and a substrate-binding peptide having
binding specificity for a biological substrate, with the two
respective substrate binding peptides being covalently coupled to
each other. Also illustrated is a compound comprised of fatty acid
coupled to a biofunctional composition comprised of a first
substrate-binding peptide having binding specificity for a
non-biological substrate, and a second substrate-binding peptide
having binding specificity for a non-biological substrate, with the
two respective substrate-binding peptides being covalently coupled
to each other. It is apparent to one skilled in the art, that
additional embodiments of the 2 substrate-binding peptides of a
biofunctional composition can include, but is not limited to, each
being substrate-binding peptide having binding specificity for a
biological substrate.
[0129] Shown in Table 5 are illustrative compounds of the
invention, synthesized by the methods described herein. The
compounds are listed as a linear sequence, with same abbreviations
used in Example 4, as well as "Ahx" representing fatty acid
comprising aminohexanoic acid; and "NH2" means a modified
C-terminal amino acid which has been amidated. A first and second
representative substrate-binding peptide having binding specificity
for a non-biological substrate (comprising metal), and further
including a linker at the C-terminal end to be biotinylated to
facilitate detection during functional studies, are represented by
the amino acid sequence of SEQ ID NO:122 and HKKNNPKKKNKTRGSSK (SEQ
ID NO:123) (a shortened version of SEQ ID NO:122). A third and
fourth representative substrate-binding peptide having binding
specificity for a non-biological substrate (comprising vancomycin
and related analogs) are represented by the amino acid sequence of
SSSCLIDMYGVCHNFDGAYDSSRG (SEQ ID NO:124), SSCLIDIYGVCHNFDAY (SEQ ID
NO:125) (shortened version of SEQ ID NO:124), and SSCLIDIYGKCHNPLR
(SEQ ID NO:126) (shortened version of SEQ ID NO:124). A
representative substrate-binding peptide having binding activity
for a biological substrate (a known antimicrobial peptide binding
to a bacterial surface component) is represented by the amino acid
sequence of KWKLFKKIGAVLKVLK (SEQ ID NO:127).
TABLE-US-00005 TABLE 5 Compound Ref. # Compound linear sequence 133
MYR-Ahx-SEQ ID NO:124-linker-SEQ ID NO:122 134 AUD-AUD-AUD-AUD-SEQ
ID NO:124-linker-SEQ ID NO:122 135 MYR-Ahx-SEQ ID NO:125-linker-SEQ
ID NO:123 136 [MYR-Ahx].sub.2-K-SEQ ID NO:125-linker-SEQ ID NO:123
137 MYR-Ahx-SEQ ID NO:127-linker-SEQ ID NO:122 138 SEQ ID
NO:126-AUD-AUD-AUD-AUD-AUD-AUD-SEQ ID NO:122-NH2 139 SEQ ID
NO:126-AUD-AUD-AUD-AUD-AUD-SEQ ID NO:122-NH2
[0130] Standard Fmoc/t-Bu chemistry using AA/TBTU/HOBt/NMM
(1:1:1:2) as the coupling reagents was employed to synthesize
compound 133. The base resin, Fmoc-PAL-PEG-PS (.about.0.20 mmol/g)
was used for synthesis of an amino acid sequence comprising SEQ ID
NO:122, followed by two mini-PEG linkers, followed by an amino acid
sequence comprising SEQ ID NO:124. Amino acids were used in 5 fold
excess in the synthesis cycles and all residues were doubly or
triply coupled. The coupling reactions were monitored by Kaiser
ninhydrin test or chloranil test. In order to suppress peptide
aggregation, pseudoproline dipeptides Fmoc-Ser-Ser(PsiMe,Me pro)-OH
were employed, and were double coupled in 5 fold excess.
Fmoc-Lys(Biotin)-OH and Fmoc-Mini-PEG-CO.sub.2H were double coupled
manually using the above coupling conditions. Fmoc deprotection
reactions were carried out using 20% piperidine in DMF with 0.1 M
HOBt. Aminohexanoic acid (Ahx) was introduced at the N-terminus of
the resin-bound peptide followed by double coupling of myristic
acid using TBTU activation method.
[0131] The compound was cleaved from the resin using Reagent K
(TFA:EDT:H.sub.2O:phenol:thioanisole=82.5:2.5:5:5:5) at room
temperature for 4 hours. The crude products were precipitated in
cold ether. The pellet obtained after centrifugation was washed
thrice with cold ether, and lyophilized to give white solid as
crude peptide. The crude linear peptides were cyclized using 3%
DMSO in 10 mM PBS (pH 7.4) buffer for 48 hours (peptide
concentration.about.0.065-0.075 mM) The crude cyclic peptide was
purified on an RP-HPLC column (C18; 250.times.21.2 mm) using mobile
eluants (A=H.sub.2O/TFA (0.1% TFA) and B=Acetonitrile/TFA (0.1%
TFA) using a gradient of 15% B to 55% B in 50 min at 10 mL/min @
220 nm. The fractions containing the desired product were pooled
and lyophilized to obtain a fluffy white powder (>95% purity) in
10% overall yield. Compound 133 was purified (HPLC retention time
of 13.61 minutes (25 to 65% B in 30 min @ 1 mL/min at 220 nm), and
characterized by MALDI-TOF (observed mass=6131; expected
mass=6122.7).
[0132] Compounds 134-139 were synthesized using similar methods and
reagents as described herein for compounds 133.
EXAMPLE 6
[0133] In this example, illustrated are unexpected beneficial
properties of the composition of the invention, including but not
limited to, one or more improved biophysical properties. Such one
or more unexpected beneficial properties of a composition of the
invention may comprise an increase in stability. An increase in
stability may comprise any one or more of resistance to chemical
denaturation, resistance to proteolytic degradation, improved
retention to a substrate (e.g., resistance to being competed off a
substrate to which it is bound, such as by proteins or other
biomolecules found in body tissues). Thus, to ascertain a
beneficial property of a composition of the invention, it is
compared to the properties of a component of the
composition-namely, a substrate-binding peptide by itself (e.g.,
without fatty acid covalently coupled thereto). An assay was
developed that, from our direct comparisons (see, e.g., discussion
of FIG. 2 below, and Example 7 herein), mimicked the effect of
incubation of substrate-binding peptide with human plasma on
stability of binding of a substrate-binding peptide to its
substrate. The assay utilized incubations in the presence of 10%
bovine serum album (BSA) with 10 mM guanidinium chloride. The high
BSA concentration mimics the elevated albumin content of human
plasma, and the guanidinium chloride is used to compete with any
charged interactions involved in binding of the substrate-binding
peptide to its substrate.
[0134] In this assay, two immunoassay plates were prepared for a
"side-by-side" comparison of binding of a test sample
(substrate-binding peptide or composition of the invention) to its
substrate with or without the presence of 10% BSA with 10 mM
guanidinium chloride. The wells of each 96-well polypropylene plate
were incubated with 350 .mu.l BSA 1% in PBS for 30 minutes at
20.degree. C. with 500 rpm shaking. To each well was added one
acetone-cleaned 3/32'' 316LVM stainless steel bead, followed by
addition of dilutions in PBS of the test sample in a range of
concentrations starting from 10 .mu.M. Final volume in each well
was 200 .mu.l. The plates were incubated for 1 hour at 20.degree.
C. with 500 rpm shaking to allow for the binding to occur. The
beads were washed 3 times with 250 .mu.l PBS using a plate washer.
At this point the method differed in further steps to complete
depending on whether the assay was in the presence of 10% BSA with
10 mM guanidinium chloride or without the presence of 10% BSA with
10 mM guanidinium chloride.
[0135] In the immunoassay for detecting binding without the
presence of 10% BSA with 10 mM guanidinium chloride, added to each
well was 200 .mu.l of streptavidin AP (AP is alkaline phosphatase)
at 1/200 in TBS+1% BSA. The plate was then incubated at room
temperature for 20 minutes with 500 rpm shaking. The wells were
then washed 3 times with 250 .mu.l of a buffer containing
Tween.RTM. using a plate washer. The beads were transferred to a
clean polypropylene plate, and the added to each well was 200 .mu.l
of pNPP (p-Nitrophenyl Phosphate) substrate. When color has
developed, each well was read at OD405 nm in endpoint mode using a
plate reader. In the comparator assay for detecting the effect of
10% BSA with 10 mM guanidinium chloride, to each well was added 350
.mu.l of 10% BSA with 10 mM guanidinium chloride. The plate was
then incubated for 18 hours at 37.degree. C. with 250 rpm shaking.
The wells were washed three times with 250 .mu.l PBS using a plate
washer. Added to each well was 200 .mu.l of streptavidin AP at
1/200 dilution in buffer+1% BSA. The plate was then incubated at
room temperature for 20 minutes with 500 rpm shaking. The wells
were washed three times with 250 .mu.l buffer containing Tween.RTM.
using a plate washer. The beads were transferred to a clean
polypropylene plate, and added to the wells was 200 .mu.l of pNPP.
When color has developed, the wells were read at OD405 nm in
endpoint mode using a plate reader. From these two assays, a
concentration of peptide or composition was chosen as a comparison
point, and calculated was the percent of peptide or composition
remaining bound to its substrate (in this case, a metal surface)
after an 18 hour incubation in the 10% BSA+10 mM guanidinium
chloride ("% retention", see FIGS. 1 & 2).
[0136] With respect to this example and as shown FIG. 1, some
additional test samples were included in the comparison assay.
Included was a substrate-binding peptide by itself (having the
amino acid sequence of SEQ ID NO:122). Also, in the development of
the invention, molecules which are know to self assemble (e.g.,
PEG, hydrophobic amino acids, and the amino acid sequence RADARADA
(SEQ ID NO:128)) were each covalently coupled to substrate-binding
peptide. For example, using the methods described herein and
methods known in the art, Fmoc-NH-(PEG).sub.27-CO.sub.2H was
covalently coupled to a peptide having the amino acid sequence of
SEQ ID NO:122 (compound 119, FIG. 1), amino acid sequence YWAWAYAW
(SEQ ID NO:129) was covalently coupled to a peptide having the
amino acid sequence of SEQ ID NO:122 (compound 120, FIG. 1). As
shown in FIG. 1, and as compared to the substrate-binding peptide
alone (FIG. 1, "SEQ ID NO: 122") in this assay, surprisingly the
aforementioned molecules that are known to promote self assembly
failed to promote retention of binding of the substrate-binding
peptide component to its substrate (see, e.g., compounds 119 &
120 in FIG. 1). Generally speaking, even using a compound having
one or two molecules of fatty acid attached (e.g., less than 25
carbons in total) had little effect in promoting retention.
However, unexpectedly, a composition of the invention comprising
compound having more than 2 fatty acids covalently coupled to
substrate-binding peptide significantly improved retention of
binding of substrate-binding peptide component to its substrate
(see, e.g., compositions 122, 123, 129-132 in FIG. 1), and in some
cases approaching retention of 100% of the substrate binding
peptide to its substrate. Significant retention of binding, as
compared to the substrate-peptide control, is a measure of an
improvement of or promotion in stability.
[0137] This is surprising and unexpected for the following reasons.
First, as shown in FIG. 1 and discussed above, molecules which are
known in the art to promote self-assembly failed to promote
stability of a substrate-binding peptide. Secondly, not only must
the molecules of fatty acid (which are covalently coupled to
substrate-binding peptide) be able to associate with each other in
forming the macromolecular network, but the fatty acid must also be
able to self-associate without negatively affecting the binding of
the substrate-binding peptide to its substrate. Additionally, the
self-association in forming the macromolecular network formation
must also present a conformation that promotes or increases
stability of the substrate-binding peptide. Thus, these and other
results show that in vitro and in vivo stability of a
substrate-binding peptide can be unexpectedly improved or increased
by covalently coupling two or more fatty acid molecules to the
substrate-binding peptide in forming a compound which is then mixed
with a carrier medium to form a composition of the invention
comprising a macromolecular network.
[0138] As shown in FIG. 2, and as compared to the substrate-binding
peptide alone (biofunctional composition; FIG. 2, "SEQ ID
NO:124-linker-SEQ ID NO:122"), generally speaking relative to a
biofunctional composition as a substrate-binding peptide, a
composition formed of compound having one or more molecules of
fatty acid attached to substrate-binding peptide can increase or
promote stability of the substrate-binding peptide component to its
substrate (see, e.g., compositions 133, 134, 136, 137 & 139 in
FIG. 2), and in some cases approaching retention of 100% of the
substrate binding peptide to its substrate. This is surprising and
unexpected for the same and similar reasons discussed above in
reference to FIG. 1. As shown in FIG. 2, it is noted that in this
assay assessing stability, composition 133 promotes stability as
measured by about 75% retention of the substrate-binding peptide to
it substrate. Using a radiolabeled compound 133 and measuring
exposure for periods ranging from 18 hours to 10 days in human
plasma in assaying amount of compound 133 retained to a substrate
for which the substrate-binding peptide component has binding
specificity, demonstrated is stability comprising between about 80%
to about 100% retention of compound 133 at the highest
concentrations of compound 133 tested.
EXAMPLE 7
[0139] This example further illustrates the ability of a
composition according to the invention to promote stability of a
substrate-binding peptide to a substrate. In this example, an in
vitro model for in vivo stability on a medical device comprising a
stent was performed. The stent flow model included a "circulatory
system" comprising a peristaltic pump and clear silicone tubing
into which is placed a stent (8 mm, stainless steel) coated with a
composition according to the invention. Coating of the stents was
accomplished by incubating the stents with radiolabelled
composition 132 (specific activity 2,400 cpm/pmole at 20 .mu.M in
PBS) for 1 hour at 20.degree. C., followed by extensive washing
with buffer. In one variation of the stent flow model, human plasma
was circulated through the circulatory system at a flow rate of 5
ml/min for 7 days at 37.degree. C. In another variation of the
stent flow model, 10% BSA+10 mM guanidinium chloride was circulated
through the circulatory system at a flow rate of 5 ml/min for 18
hours at 37.degree. C. After circulation in the respective
variations of the stent flow model, the coated stents were then
counted for radioactivity. To serve as an assay "control", some
coated stents were not placed in the stent flow model, but rather
counted for radioactive counts to provide a reference from which
percent retention could be calculated for those coated stents
included in the stent flow model. In both variations of the stent
flow model (exposure to human plasma or 10% BSA+10 mM guanidinium
chloride), over 90% of the composition remained bound to the coated
stents.
EXAMPLE 8
[0140] In this example, included is another illustration of a
composition of the invention having improved beneficial properties
(including, but not limited to, one or more improved biophysical
properties), as compared to a substrate-binding peptide by itself.
The unexpected benefit illustrated in this example relates to
loading capacity for a biomolecule of which a substrate-binding
peptide has binding specificity. As described in Example 5, an
illustrative biofunctional composition ("SEQ ID NO:124-linker-SEQ
ID NO:122") comprises a substrate-binding peptide having binding
specificity for vancomycin ("SEQ ID NO:124") linked to a
substrate-binding peptide having binding specificity for metal
("SEQ ID NO:122"). In this illustration, compared at equal
concentrations was the ability of the biofunctional composition to
bind vancomycin and the ability of composition 133 to bind
vancomycin.
[0141] A substrate comprising a metal (as represented by stainless
steel bead) was placed into wells of a 96 well plate. To each well
was added a test sample comprising either the biofunctional
composition alone, or composition 133 according to the invention,
at concentrations ranging from about 0.1 .mu.M to about 2 .mu.M (in
PBS, in a total volume of 150 .mu.l). The plate was incubated for
30 minutes, and then the wells and beads were washed three times
with buffer. Added to each well was 150 .mu.l of a stock solution
of BODIPY-FL vancomycin, a commercially available,
green-fluorescent analog of vancomycin having antibiotic activity
comparable to vancomycin. The plates were incubated for 30 minutes,
and then the wells and beads were washed three times with buffer.
By adding 200 .mu.l of 10 mM HCl per well, any BODIPY-FL vancomycin
specifically bound to the composition or the control is eluted from
the metal bead. The fluorescent signal from BODIPY-FL vancomycin
was then measured by detecting emission of light of wavelength 530
nm following excitation with light of wavelength 490 nm, and
plotted against the concentrations of the test sample to generate a
binding curve. At a concentration of 1 .mu.M, the fluorescent
intensity for the biofunctional composition by itself was
quantified as about 12,000 counts per second (cps), whereas at the
same concentration, the fluorescent intensity for composition 133
was quantified as about 22,000 cps. Thus, unexpectedly, a
composition of the invention resulted in almost 2 fold increase in
the loading capacity of a biomolecule (in this case, vancomycin) by
the biofunctional composition, as compared to the biofunctional
composition itself (not in the form of a composition of the
invention).
EXAMPLE 9
[0142] This example further illustrates the beneficial properties
of a composition according to the invention to promote stability of
a substrate-binding peptide to a substrate, as well as to increase
loading of a biomolecule for which a substrate-binding peptide has
binding specificity. In this assay, titanium pins were used as a
model for tibia pins. Briefly, sterile pins were first coated with
biofunctional composition ("SEQ ID NO:124-linker-SEQ ID NO:122")
comprising a substrate-binding peptide having binding specificity
for vancomycin ("SEQ ID NO:124") linked to a substrate-binding
peptide having binding specificity for metal ("SEQ ID NO:122"), or
a composition of the invention formed from compound comprising the
biofunctional composition covalently coupled to fatty acid. The
respective coatings also include vancomycin bound thereto. The
coated pins were then placed in a silicone tube containing liquid
bacterial growth medium and inoculated with bacteria. After
incubation, the pins were removed, and then the liquid growth
medium was serially diluted. The serial dilutions were inoculated
onto bacterial culture plates, the plates were incubated, and
counted on the plates were bacterial colonies.
[0143] In this assay, test samples (biofunctional composition
(Table 6, "BC") or compositions 133, 134, 135, 136 139) each were
used to coat a pin by incubating the test sample (in a range of
from about 0.8 mM to about 1 mM) with 2.5 .mu.l of a 10 mM
vancomycin solution and PBS to a final volume of 250 .mu.l in a
microtube (3 pins per tube) for 60 minutes at room temperature with
occasional agitation. In a piece of silicone tubing (1.5 mm inner
diameter, 50 mm long), added is tryptic soy broth+0.2% glucose, the
coated pin, and 10.sup.3 colony forming units (cfu) of
Staphylococcus aureus strain MZ100 in either a 20 .mu.l inoculum,
40 .mu.l inoculum, or 60 .mu.l inoculum. The tubing is clamped
closed, and the tubing is incubated for 37.degree. C. for 3 hours.
After 3 hours, serial dilutions (1:10, 1:100, 1:1000) were made of
the culture media from each tubing, and 10 .mu.l of the undiluted
culture medium and of each dilution were spotted onto the bacterial
culture plate. The bacterial culture plate was incubated overnight
at 37.degree. C., and then the cfus were counted. The results, a
composite of different assay runs, are shown in Table 6 ("-" means
no cfus; "many" means too many cfus to count, as they converge into
one spot; "NT" means not tested).
TABLE-US-00006 TABLE 6 Test cfu undiluted cfu 1:10 cfu 1:100 cfu
1:1000 Sample Volume (.mu.l) 12+ 4 -- -- BC 20 -- -- -- -- 134 20
-- -- -- -- 135 20 -- -- -- -- 133 20 2 1 -- -- 136 20 -- -- -- --
139 20 many 16 7 -- BC 40 many 6 1 -- 134 40 1 1 -- -- 135 40 many
10 -- -- 133 40 many 15 -- -- 136 40 13 -- -- -- 139 40 many 24 7
-- BC 60 many 17 5 -- 134 60 60 1 1 -- 135 60 NT NT NT NT 133 60 NT
NT NT NT 136 60 NT NT NT NT 139 60
[0144] From the tibia pin assay results shown in Table 6,
compositions 135 and 139 clearly show improved beneficial
properties over the biofunctional composition alone. The benefit
illustrated by this example may be attributable to both (a) an
increase in stability of the substrate-binding peptide, having
binding specificity for metal, to its metal substrate; and (b)
increased loading capacity of the substrate-binding peptide, having
binding specificity for vancomycin, to vancomycin.
EXAMPLE 10
[0145] In this example, illustrated is an embodiment relating to
formation of a composition according to the invention. Basically,
to form a composition of the invention, compound of the invention
is mixed with a carrier medium. For example, compound of the
invention may be reconstituted with a pharmaceutically-acceptable
carrier, as known to those skilled in the art. Typically, a
preferred carrier medium is an aqueous solution which is contacted
and mixed with the compound of the invention to form a composition
according to the invention.
[0146] Formation of a composition of the invention, and evidence of
macromolecular network formation, may be monitored or quantified by
any means known in the art. In this example, macromolecular
formation was detected using a standard assay for determining
critical micelle concentration ("CMC"). In this assay, a solution
containing the composition of the invention (in a range of
concentrations in pH 7 phosphate buffered saline ("PBS")) was mixed
with a solution of methyl orange (0.04 mM in PBS), and the
absorbance of the mixture was measured at 484 nm (A.sub.484). CMC
is the concentration at which a sharp decrease in absorbance at
A.sub.484 is observed, a change in the optical properties of methyl
orange when trapped in a hydrophobic phase, such as caused by
self-association as a macromolecular network (Table 7, "CMC"). Also
included in this assay were an assay control peptide having an
amino acid sequence of SEQ ID NO:122 (Table 7, "Control"), and
compound 119 (a PEGylated peptide, the peptide having an amino acid
sequence of SEQ ID NO:122, as described in more detail in Example 6
herein). As shown by the results illustrated in Table 7, only
compounds of the invention demonstrated a CMC of less than 1 .mu.M,
an indicator of macromolecular network formation at such
concentrations.
TABLE-US-00007 TABLE 7 Compound Ref. # CMC (.mu.M) Control
>>10 119 >>10 122 0.123 123 0.04 125 0.123 126 0.37 127
0.123 128 0.123 129 0.123 130 0.041 131 0.37 132 0.123
EXAMPLE 11
[0147] In this example, illustrated is a method of applying a
composition of the invention to a substrate, the method comprising
contacting the composition with the substrate under conditions
suitable so that the composition binds to the substrate. In one
example, wherein the substrate is a medical device, a composition
of the invention is applied to the medical device as a coating
before positioning the medical device in situ. In another example,
a composition according to the invention is applied to a medical
device in situ. For example, if the medical device is exposed
through an open site in the body (e.g., such as in surgery), or is
positioned at a site openly accessible outside the body (e.g., a
dental implant accessible through an open mouth), a physician may
spray or otherwise apply the composition to the medical device in
situ. In another example wherein the medical device is not readily
accessible by applications such as a spray coating, a composition
according to the invention may be administered by injection at the
site of the medical device such that the composition comes in
contact with the medical device so as to bind to the medical
device.
[0148] To facilitate formation of the composition and application
of the composition (e.g., by spray, soaking, or injection) to a
substrate, the composition comprises a pharmaceutically acceptable
carrier. Conventional processes known in the art may be used to
apply a composition according to the present invention to one or
more surfaces of a substrate to be coated (in contacting the
composition with the one or more surfaces in forming a coating
thereon). Depending on the nature of the substrate to which the
composition is to be applied, such processes are known to include,
but are not limited to, soaking, mixing, dipping, brushing,
spraying, and vapor deposition. For example, a solution or
suspension comprising the composition may be applied through the
spray nozzle of a spraying device, creating droplets that coat the
surface of the substrate to be coated. The coated substrate is
allowed to dry. If desired, the coated substrate may be further
processed prior to use (e.g., washed in a solution (e.g., water or
isotonic buffer) to remove excess composition not specifically
bound to the substrate; if for in vivo use, by sterilizing using
any one or methods known in the art for sterilization).
Alternatively, the composition and the substrate may each be
separately sterilized prior to the process of combining them, and
then performed under sterile conditions is the applying of the
composition to one or more surfaces of the substrate.
[0149] In another process for applying the composition to one or
more surfaces of a substrate to be coated, a surface of the
substrate to be coated is dipped into a liquid (e.g., solution or
suspension, aqueous or solvent) containing the composition
according to the invention in an amount effective to coat the
surface of the substrate. For example, the surface is dipped or
immersed into a bath containing the composition. Suitable
conditions for applying the composition as a coating composition
include allowing the surface to be coated to remain in contact with
the carrier medium containing the composition for a suitable period
of time (e.g., ranging from about 5 minutes to about 5 hours; more
preferably, ranging from 5 minutes to 60 minutes), at a suitable
temperature (e.g., ranging from 10.degree. C. to about 50.degree.
C.; more preferably, ranging from room temperature to 37.degree.
C.). If desired, the coated substrate may be further processed, as
necessary for use (e.g., one or more of drying, washing,
sterilization, and the like). These illustrative processes for
applying a composition to a substrate are not exclusive, as other
coating and stabilization methods may be employed (as one of skill
in the art will be able to select the compositions and methods used
to fit the needs of the particular surface material of which a
substrate is comprised, substrate, or purpose).
[0150] Additionally, in a method according to the present
invention, a coat on a substrate surface comprising the composition
may be stabilized, for example, by air drying. However, these
treatments are not exclusive, and other coating and stabilization
methods may be employed. Suitable coating and stabilization methods
are known in the art. For example, the at least one surface of the
substrate to be coated with the composition of the present
invention may be pre-treated prior to the coating step so as to
enhance one or more of the binding of the composition to the
surface, and the consistency and uniformity of the coating.
EXAMPLE 12
Chemistry for Coupling Interaction Tags to Substrate Binding
Domains
[0151] One or more hydrophobic interaction tags comprising fatty
acid residues can be covalently coupled to the substrate binding
peptides of the presently disclosed subject matter according to the
procedures described herein above, for example, at Example 5. The
hydrophobic interaction tags can be coupled at one or both the
N-terminus or C-terminus. Similarly, one or more charged
interaction tags comprising amino acid residues can be covalently
coupled at one or both the N-terminus or C-terminus of the
substrate binding peptides of the presently disclosed subject
matter.
[0152] Briefly, a method for coupling either a hydrophobic
interaction tag or a charged interaction tag is as follows. For
example, a single aminoundecanoic acid (AUD) or a polymer of 2-10
AUDs (poly-aminoundecanoic acid ("polyAUD)") or a single amino acid
or a poly-amino acid of the appropriate length is first assembled
separately as a building block using standard solid phase methods.
The appropriately protected fatty acid hydrophobic tags (e.g.,
Fmoc-polyAUD, Fmoc-Myristic acid, etc.) and the appropriately
protected charged amino acid interaction tags (e.g,
Fmoc-polyLys(Boc)-OH, Fmoc-polyArg(Mtr)-OH, Fmoc-polyAsp(OtBu)-OH,
Fmoc-polyGlu(OtBu)-OH, etc.) are coupled sequentially using the
standard Fmoc/t-Bu chemistry using AA/HBTU/HOBt/NMM (1:1:1:2) as
the coupling reagents (AA is amino acid; HOBt is O-Pfp
ester/1-hydroxybenzotriazole; H BTU is
N-[1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium
hexafluorophosphate N-oxide; NMM is N-methylmorpholine). The amino
acids and fatty acids are used in 5-10 fold excess in the synthesis
cycles, and all residues are doubly, triply or even quadruply
coupled depending upon the complexity of residues coupled. The
coupling reactions are monitored by Kaiser ninhydrin test. The Fmoc
deprotection reactions are carried out using 20% piperidine in
dimethyl-formamide. Peptide cleavage from the resin is accomplished
using Reagent K (TFA (trifluoroacetic acid):EDT
(1,2-ethanedithiol):H.sub.2O:phenol:thioanisole=82.5:2.5:5:5:5) at
room temperature for 4 hours. The crude product is precipitated in
cold ether. The pellet obtained after centrifugation is washed with
cold ether and lyophilized to give a white solid as crude desired
product. The crude products are analyzed by analytical high
performance liquid chromatography (HPLC) on a C-18 column using
mobile eluants (A=H.sub.2O/TFA (0.1% TFA) and B=Acetonitrile/TFA
(0.1% TFA). The peptides are also further analyzed by mass
spectrometry before subjecting each to final purification by
preparative HPLC. The fractions containing the desired product are
pooled and lyophilized to obtain a fluffy white powder.
[0153] One or more hydrophobic interaction tags comprising fatty
acid residues can be covalently coupled to the substrate binding
polymers of the presently disclosed subject matter. For example, a
single aminoundecanoic acid (AUD) or a polymer of 2-10 AUDs
(poly-aminoundecanoic acid; ("polyAUD)") of the appropriate length
is first assembled separately as a building block using standard
solid phase methods. The polyAUD is deprotected and purified by
HPLC. The free acid is activated using carbodiimide chemistry. The
polyethylenimine (PEI) polymer is dissolved in appropriate buffer
having pH between 7 and 9 (0.1 M sodium phosphate, pH 7.5). Amine
containing buffers like TRIS are avoided. The activated polyAUD
acid is dissolved in an acetonitrile-buffer mix and added to the
PEI solution in at least 5-10 molar excess with stirring. The
reaction is allowed to proceed for a few hours at room temperature
until completion. The PEI polymer-AUD conjugate is purified by gel
filtration of dialysis.
EXAMPLE 13
Substrate Binding Peptides having Binding Affinity for Target
Molecule Vancomycin
[0154] This Example describes the generation of substrate binding
peptides having binding affinity a target molecule vancomycin
according to the methods for utilizing phage display technology
outlined previously in Example 1. More specifically, the following
subject matter for discovering substrate binding peptides having
binding affinity for the target molecule, vancomycin, and
generation of the vancomycin binding peptides is taken from PCT
International Patent Application No. PCT/US2008/080321 having PCT
International Patent Application Publication No. ______, which is
herein incorporated by reference in its entirety.
[0155] As an illustrative example of methods used in development of
this presently disclosed subject matter, an aliquot of biotinylated
vancomycin (100 pmoles) in buffer-T (200 .mu.l, 0.05 M
Tris-buffered saline, with TWEEN-20 at a final concentration of
0.05%) was dispensed in a series of microfuge tubes. Added per tube
was 25 .mu.l of a mixture of phage libraries to be screened (e.g.,
at a concentration of 10.sup.10 pfu/ml each), and the mixture was
incubated at room temperature for 2 hours. To the mixture was added
streptavidin-labeled metal beads which had been blocked with 1%
bovine serum albumin (BSA) in buffer-T, and the bead-containing
mixture was gently mixed for 2 hours at room temperature. The tubes
were then washed 3 times with 1 ml of buffer-T+0.5 mM biotin, using
magnetism to pull down the metal beads each time. The supernatant
was removed, and phage was eluted from the metal beads by
competition with vancomycin. In the elution process, added to each
tube containing the beads was 20 .mu.l of 0.1 mM vancomycin, and
the bead-containing mixture was incubated at room temperature for
20 minutes. The phage-containing supernatant was then transferred
to cultures of E. coli cells susceptible to phage infection, and
incubated overnight at 37.degree. C. in a shaker incubator. Phage
supernatant was harvested by centrifugation of culture medium at
8500.times.g for 10 minutes. Second and third rounds of selection
were performed in a similar manner to the first round, using the
amplified phage from the previous round as input.
[0156] For determining phage binding, an ELISA (enzyme-linked
immunoassay) was performed as follows. Wells of a microtiter plate
were coated with streptavidin by incubating 50 .mu.l of a 10
.mu.g/ml solution per well for 16 hours and at 4.degree. C.
Non-specific binding sites on the well surfaces of the microtiter
plate were blocked with 250 .mu.l 1% BSA in 0.1 M NaHCO.sub.3. The
plate was incubated for at least 2 hours at room temperature. After
washing the wells 3 times with buffer-T, to each well was added
biotinylated vancomycin (0.1 .mu.M) in 100 .mu.l buffer-T and
incubated for 30 minutes at room temperature. Biotin (0.1 .mu.M) in
100 .mu.l buffer-T was then added to each well, to block any
available streptavidin sites. The plate was incubated for 30
minutes at room temperature, followed by 5 washes with buffer-T. To
each well was added 175 .mu.l of buffer-T and 25 .mu.l of the phage
solution being tested, followed by incubation at room temperature
for 2 hours. Following several washes with buffer-T, added was
anti-M13 phage antibody conjugated to horseradish-peroxidase,
followed by incubation, and washing. Added was chromogenic agent
ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), and
determined was a read-out at 405 nm at 15 minutes. The resultant
absorbance value for each well correlates to the amount of phage
bound to vancomycin.
[0157] Primers against the phage vector sequence that flank the
insertion site were used to determine the DNA sequence encoding the
peptide for the phage in each group. The sequence encoding the
peptide insert was translated to yield the corresponding amino acid
sequence displayed on the phage surface. The amino acid sequences,
encoding peptides isolated using vancomycin as the representative
glycopeptide antibiotic, were determined and are shown in Table 1.
While phage amino acids adjoining the peptides typically did not
provide a significant contribution to the binding affinity of the
peptide, the peptides according to the presently disclosed subject
matter can comprise, in their amino acid sequence, phage amino
acids adjoining the peptide at the N-terminus (SS) and at the
C-terminus (SR). The peptide sequence shown in SLIDMYGVCHNFDGAYDS
(SEQ ID NO: 130) represents site directed mutagenesis of the first
cysteine residue of CLIDMYGVCHNFDGAYDS (SEQ ID NO: 131) to a serine
residue.
[0158] The phage-derived sequences were further evaluated as
synthetic peptides. Peptides according to the presently disclosed
subject matter can be synthesized using any method known to those
skilled in the art including, but not limited to, solid phase
synthesis, solution phase synthesis, linear synthesis,
recombinantly, and a combination thereof. In this example, peptides
were synthesized using standard solid-phase peptide synthesis
techniques on a peptide synthesizer using standard Fmoc chemistry.
After all residues were coupled, simultaneous cleavage and side
chain deprotection was achieved by treatment with a trifluoroacetic
acid (TFA) cocktail. Crude peptide was precipitated with cold
diethyl ether and purified by high performance liquid
chromatography (HPLC) using a linear gradient of water/acetonitrile
containing 0.1% TFA. Homogeneity of the synthetic peptides was
evaluated by analytical reverse phase-HPLC, and the identity of the
peptides was confirmed with mass spectrometry.
[0159] A typical binding assay for glycopeptide antibiotic was
performed according to the following procedure. Briefly, synthetic
peptides comprising an amino acid sequence to be characterized for
binding were biotinylated to facilitate immobilization on
streptavidin-coated 96-well plates. The microtiter plates were
coated with streptavidin by adding 50 .mu.l of a 10 .mu.g/ml
streptavidin solution in 0.1 M NaHCO.sub.3, and incubating the
plates for at least 3 hours. The plate wells were blocked by adding
150 .mu.l of a 1% BSA solution in 0.1 NaHCO.sub.3 with incubation
for at least 2 hours, and the plates were stored at 4.degree. C.
until needed. Before use, the streptavidin plates were washed
extensively in buffer-T. Added per well was peptide (100 .mu.l 0.1
.mu.M peptide in buffer-T), and then incubated for 30 minutes at
room temperature with shaking. 200 .mu.l of 0.5 mM biotin in
buffer-T was added to block the remaining streptavidin sites, and
plates were incubated for 15 minutes at room temperature. Plates
were then washed with buffer-T to remove the excess biotin and
peptide. Serial dilutions of biotinylated glycopeptide antibiotic
in buffer-T were added (100 .mu.l) to each well, representing a
range of concentrations between 100 .mu.M and 100 .mu.M. Plates
were incubated for 30 minutes at room temperature with shaking
prior to washing several times with buffer-T. Glycopeptide
antibiotic was then detected by adding 100 .mu.l of a diluted
streptavidin-alkaline phosphatase conjugate to each well and
incubated at room temperature for 30 minutes. Excess conjugate was
removed by repeated washes with buffer-T, and the amount of
alkaline phosphatase remaining in the well was detected using a
pNPP (para-nitrophenylphosphate) colorimetric enzymatic assay. The
relative amount of glycopeptide antibiotic captured by the peptides
was determined by measuring the absorbance at 405 nm of the colored
product of the alkaline phosphatase reaction. The EC50 was
determined for each peptide relative to the binding affinity for
the glycopeptide antibiotic used in the assay, as shown in Table 8
(with vancomycin as a representative glycopeptide antibiotic).
TABLE-US-00008 TABLE 8 Peptide sequences isolated by phage
selections using vancomycin SEQ EC50 (.mu.M) ID NO: Amino acid
sequence for vancomycin binding 131 CLIDMYGVCHNFDGAYDS 0.10 132
CLFDIFGVCHSFDGAYDS 0.06 133 PCELIDMFGNDHCP 0.82 134
SCDMLFCENFSGSGNNWFS 10 130 SLIDMYGVCHNFDGAYDS 10
[0160] To identify additional peptides capable of binding
vancomycin, a scanning degenerate codon mutagenesis study was
performed using (SEQ ID NO: 131). To rapidly test variants of the
isolated vancomycin binding peptide, a bacterial expression system
was designed. Under this system, a peptide sequence was placed
under the transcriptional control of a T7 promoter. The peptide was
expressed with an N-terminal OmpA signal peptide, targeting it for
secretion. An HA-tag was engineered downstream of the peptide
sequence for antibody-mediated detection, a rhinovirus protease
cleavage site was engineered for peptide liberation, and the DNA
sequence encoding alkaline phosphatase was engineered for p-NPP
colorimetric detection. Using this expression system, a scanning
mutagenesis study was performed in which new peptide sequences were
generated using mutagenic oligonucleotide primers and tested for
vancomycin binding. The C-terminal His6 tag enabled the
high-throughput peptide purification on Ni.sup.2+ columns or beads
(Qiagen; Cat #30600). After PCR mutagenesis and cloning of a
sequence into the vector, competent cells were transformed and
cultured overnight on 2.times. YT-KAN-BCIP(40 ug/ml) plates at
37.degree. C. Transformed colonies were grown in 2.times. YT-KAN
broth overnight. Peptide-AP fusion-containing supernatants were
harvested and tested for binding to vancomycin. Briefly, the
variant peptides were tested for vancomycin binding as follows. A
streptavidin coated microtiter plate was coated with biotinylated
vancomycin. The concentrations of the alkaline-phosphatase linked
variant peptides were normalized to equal levels based on the
alkaline-phosphatase activity as determined in a kinetic assay with
the alkaline-phosphatase specific chromogenic substrate
p-nitrophenyl phosphate (p-NPP). A streptavidin coated microtiter
plate was coated with biotinylated vancomycin. Normalized amounts
of alkaline-phosphatase linked peptides were allowed to bind to the
immobilized vancomycin and detected by addition of the
alkaline-phosphatase specific chromogenic substrate p-NPP. The
results of the mutagenesis study are shown in Table 9.
TABLE-US-00009 TABLE 9 Scanning degenerate codon mutagenesis (SEQ
ID NO: 131) Substitution Position Acceptable Unacceptable Reduced
Binding C1 C A E G P S V D K S W L2 L M C G P Q T L I3 I M A G P S
L D4 D E H S Y A M5 M I F H K R W V Y6 Y A D E G K N S V G7 G A R S
V L E V8 V R K Q C P G S W D C9 C D E G W R H10 H A E G K L M N P R
T N11 N D M S C G E F12 F E H K L P Q R S Y D13 D L T V C A Y G14 G
R S A F K T V W Y A15 A G C P S Y16 Y M W G C L Y D17 D I L P
[0161] From an alignment of the amino acid sequence of the peptides
identified by phage selections using vancomycin as the illustrative
glycopeptide antibiotic in Table 1, a consensus glycopeptide
antibiotic binding domain sequence was constructed representing all
of SEQ ID NOs: 131-134 and taking into account the results of the
mutagenesis study with SEQ ID NO: 131. The consensus glycopeptide
antibiotic binding domain SEQ ID NO: 135 is as follows:
CXaa.sub.0-3DMFGXaa.sub.0-3C (SEQ ID NO: 135), wherein Xaa
represents any amino acid, the 2 cysteine residues are disulfide
bonded, and the length between the 2 cysteine residues can range
from 4 to 10 amino acids.
[0162] Similarly, from an alignment of the amino acid sequence of
the peptides identified by phage selections using vancomycin as the
illustrative glycopeptide antibiotic in Table 1X, a consensus
glycopeptide antibiotic binding domain sequence was constructed
representing all of SEQ ID NOs: 131-134 and taking into account the
results of the mutagenesis study with SEQ ID NO: 131. The consensus
glycopeptide antibiotic binding domain SEQ ID NO: 136 is as
follows:
Xaa.sub.1Xaa.sub.2X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7Xaa.su-
b.3X.sub.8X.sub.9 (SEQ ID NO: 136), wherein the sequence comprises
at least 2 cysteine residues; wherein Xaa is any amino acid unless
stated otherwise; wherein either Xaa.sub.1 or Xaa.sub.2 is C and
Xaa.sub.2 can be absent if Xaa.sub.1 is C; wherein X.sub.1 s L, M,
I, V or A; wherein X.sub.2 is I, M or F; wherein X.sub.3is D;
wherein X.sub.4 is M or I; wherein X.sub.5is F or Y; wherein
X.sub.6is G; wherein X.sub.7 is any amino acid except C or P;
wherein if X.sub.8 or X.sub.5 is C, Xaa.sub.3 is any amino acid
except C and can be absent; wherein X.sub.8 is C or H unless
Xaa.sub.3 or X.sub.9 is C and then X.sub.8 is not C; and wherein
X.sub.9 is H or C unless Xaa.sub.3 or X.sub.8 is C and then X.sub.9
is not C.
[0163] In another embodiment, a consensus glycopeptide antibiotic
binding domain sequence was constructed (SEQ ID NO: 137)
representing all of SEQ ID NOs: 130-134 and taking into account the
results of the mutagenesis study with SEQ ID NO: 131 shown in Table
X2. The consensus glycopeptide antibiotic binding domain is as
follows:
Xaa.sub.1Xaa.sub.2X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7Xaa.su-
b.3X.sub.8X.sub.9, (SEQ ID NO: 137) wherein the sequence comprises
at least 2 cysteine residues; wherein Xaa is any amino acid unless
stated otherwise; wherein either Xaa.sub.1 or Xaa.sub.2 is C and
Xaa.sub.2 can be absent if Xaa.sub.1 is C; wherein X.sub.1 is not
C, G, P, Q or T; wherein X.sub.2is not A, G, P or S; wherein
X.sub.3 is D or C; wherein X.sub.4 is M or I; wherein X.sub.5 is F
or Y; wherein X.sub.6is not A, R, S or V; wherein X.sub.7 is any
amino acid except C or P; wherein if X.sub.8 or X.sub.9 is C,
Xaa.sub.3 is any amino acid except C and can be absent; wherein
X.sub.8 is C or H unless Xaa.sub.3 or X.sub.9 is C and then X.sub.8
is not C; and wherein X.sub.9 is H or C unless Xaa.sub.3 or X.sub.8
is C and then X.sub.9 is not C.
[0164] Thus, a peptide binding domain sequence motif is provided
having binding affinity for glycopeptide antibiotic. A peptide
according to the glycopeptide antibiotic binding domain of SEQ ID
NOs: 135-137 can further comprise modifications according to the
presently disclosed subject matter including, for example, one or
more of a terminal modification, and a modification to facilitate
linking of the peptide. Thus, such a peptide can have an amino acid
sequence selected from the group consisting of SEQ ID NOs: 131-137.
Preferably, the peptide according to the presently disclosed
subject matter has a binding affinity for glycopeptide antibiotic
of EC50 less than 1 .mu.M.
EXAMPLE 14
Substrate Binding Peptides having Binding Affinity for a Metal
Medical Device
[0165] This Example describes substrate binding peptides having
binding affinity for a metal substrate medical device discovered
according to the methods for utilizing phage display technology
outlined herein previously in Example 1. More specifically, the
following subject matter describing substrate-binding peptides
having binding affinity for a substrate that is a medical device is
taken from PCT International Patent Application Publication No.
WO2007/081942, which is herein incorporated by reference in its
entirety.
[0166] Illustrative substrate binding peptides having binding
affinity for a metal substrate medical device according to the
presently disclosed subject matter were described in Patent
Application Publication No. WO2007/081942 and conform to the
following sequence motif:
X.sub.1--H--X--X--X.sub.2--X.sub.2--X.sub.2--K--X.sub.1--X.sub.1--X--K--X-
.sub.1--X.sub.1--N--K (SEQ ID NO:138); where X is any amino acid;
X.sub.1 is K, N, or S, but preferably either K or N; and X.sub.2 is
K, N, or H. The illustrative peptides are further covalently
coupled to one or both a hydrophobic interaction tag and a charged
interaction tag according to the methods detailed herein at Example
12.
[0167] Similarly, a shortened version of the peptide sequence motif
for metal binding shown above (SEQ ID NO:138) is described herein
above at Example 5 and comprises a 4 amino acid linker sequence at
the C-terminal end: HKKNNPKKKNKTRGSSK (SEQ ID NO:123). SEQ ID
NO:123 is also disclosed herein at Example 5 comprising the
covalently coupled hydrophobic tags of the presently disclosed
subject matter. Peptides useful for binding metal substrates
according to the presently disclosed methods conform to the
following consensus sequence:
X.sub.1--X.sub.1--K--X.sub.2--X.sub.2--X--K--X.sub.2--X.sub.2--N--K
(SEQ ID NO:139), where X is any amino acid; X.sub.1 is K, N, or H,
and X.sub.2 is K, N, or S, but preferably either K or N. Peptides
conforming to the above sequence motifs can also be covalently
coupled to one or both a hydrophobic interaction tag and a charged
interaction tag according to the methods detailed in herein at
Example 12.
EXAMPLE 15
Substrate Binding Peptides having Binding Affinity for a Target
Molecule Cell
[0168] This Example describes substrate binding peptides having
binding affinity for a substrate target molecule that is a cell
discovered according to the methods for utilizing phage display
technology outlined herein previously in Example 1. More
specifically, the following subject matter describing
substrate-binding peptides having binding affinity for a target
molecule that is a cell is taken from PCT International Patent
Application Publication No. WO/2007/081943, which is herein
incorporated by reference in its entirety.
[0169] Illustrative substrate binding peptides according to the
presently disclosed subject matter having binding affinity for a
target molecule that is a cell were described in Patent Application
Publication No. WO2007/081943 and conform to the following sequence
motif:
C--X.sub.1--X--X--X--X.sub.2--X--X.sub.3--P--X--X--X--X.sub.2--X--P--X.su-
b.4--X.sub.1--C (SEQ ID NO:140); where X is any amino acid; X.sub.1
is Asn or Gln; X.sub.2 is Leu or Ile; X.sub.3 is a positively
charged amino acid comprising Lys, Arg, or His; and X.sub.4 is a
negatively charged amino acid comprising Glu or Asp. The
illustrative peptides are further covalently coupled to one or both
a hydrophobic interaction tag and a charged interaction tag
according to the methods detailed herein at Example 12.
EXAMPLE 16
Substrate Binding Peptides having Binding Affinity for Target
Molecule Bone Morphogenic Proteins
[0170] This Example describes substrate binding peptides having
binding affinity for bone morphogenic proteins (BMPs) discovered
according to the methods for utilizing phage display technology
outlined herein previously in Example 1. More specifically, the
following subject matter describing substrate-binding peptides
having binding affinity for a BMP target molecule is taken from PCT
International Patent Application Publication No. WO2006/098744A2,
which is herein incorporated by reference in its entirety.
[0171] Illustrative substrate binding peptides having binding
affinity for a BMP target molecule according to the presently
disclosed subject matter were described in Patent Application
Publication No. WO2006/098744A2 and matter fall into 2 different
"sequence clusters". Each sequence cluster contains a common
sequence motif. For the first sequence cluster of BMP-binding
peptides, the common motif is designated as "Motif 1" and is as
follows: Aromatic-X--X-Phe-X-"Small"-Leu (Aromatic=Trp, Phe, or
Tyr; X=any amino acid; "Small"=Ser, Thr, Ala, or Gly; (SEQ ID
NO:141). The second sequence cluster motif "Motif 2" comprises the
sequence (Leu or Val)-X-Phe-Pro-Leu-(Lys or Arg)-Gly (SEQ ID
NOs:142). The illustrative substrate binding peptides were shown to
bind BMP-2 with an affinity ranging from about 10-100 nM. The
illustrative peptides are further covalently coupled to one or both
a hydrophobic interaction tag and a charged interaction tag
according to the methods detailed herein at Example 12.
EXAMPLE 17
First and Second Substrate Binding Peptides Localizing Growth
Factors to a Suture Medical Device
[0172] This Example describes substrate binding peptides having
binding affinity for growth factors discovered according to the
methods for utilizing phage display technology outlined herein
previously in Example 1. More specifically, the following subject
matter describing substrate-binding peptides having binding
affinity for a growth factor target molecule is taken from PCT
International Patent Application Publication No. WO2009/032943,
which is herein incorporated by reference in its entirety.
[0173] Illustrative substrate binding peptides having binding
affinity for a GDF growth factor target molecule according to the
presently disclosed subject matter were described in Patent
Application Publication No. WO2009/032943 and are shown in Table
10. The compounds are listed as a linear sequence, with "AUD"
representing aminoundecanoic acid, "MYR" representing myristic
acid; "Ahx" represents a fatty acid comprising aminohexanoic acid;
"B" represents biotin; and "NH2" means a modified C-terminal amino
acid that has been amidated. The illustrative peptides in Table x
comprising a hydrophobic interaction tag can be further covalently
coupled to one or both an additional hydrophobic interaction tag
and a charged interaction tag according to the methods detailed in
herein at Example 12. Those peptides in Table 10 that do not
comprise an interaction tag can also be covalently coupled to one
or both a hydrophobic interaction tag and a charged interaction tag
according to the methods detailed in herein at Example 12.
TABLE-US-00010 TABLE 10 SEQ ID NO: Peptide linear sequence 143
ssGGVGGWALFETLRGKEVsr-(AUD).sub.6-YFRAFRKFVKPFKRA FK-GSSGK-B-NH2
144 YFRAFRKFVKPFKRAFK-(AUD).sub.6-ssGGVGGWALFETLRGKEV
sr-GSSGK-B-NH2 145
(AUD).sub.4-ssGGVGGWALFETLRGKEVsr-(MP).sub.2-YFRAFRKFV
KPFKRAFK-GSSGK-B-NH2 146
MYR-Ahx-ssGGVGGWALFETLRGKEVsr-(MP).sub.2-YFRAFRKF
VKPFKRAFK-GSSGK-B-NH2 147
YFRAFRKFVKPFKRAFK-(MP).sub.2-ssGGVGGWALFETLRGKEV sr-GSSGK-B-NH2 148
ssGGVGGWALFETLRGKEVsr-(MP).sub.2-YFRAFRKFVKPFKRAF K-GSSGK-B-NH2 149
ssGGVGGWALFETLRGKEVsr-P10-YFRAFRKFVKPFKRAFK- GSSGK-B-NH2 150
SWWGFWNGSAAPVWSR-GSSG-ssGGVGGWALFETLRGKEVsr- GSSGK-B-NH2 151
ssGGVGGWALFETLRGKEVsr-GSSG-SWWGFWNGSAAPVWSR- GSSGK-B-NH2 152
ssGGVGGWALFETLRGKEVsr-(MP).sub.2-SWWGFWNGSAAPVWS R-GSSGK-B-NH2 153
ssGGVGGWALFETLRGKEVsr-P10-SWWGFWNGSAAPVWSR- GSSGK-B-NH2 154
ssGGVGGWALFETLRGKEVsr-(AUD).sub.6-SWWGFWNGSAAPVWS R-GSSGK-B-NH2 155
ssGGVGGWALFETLRGKEVsr-GSSG-YFRAFRKFVKPFKRAF K-GSSGK-B-NH2
[0174] The following procedure was performed to test the ability of
the exemplary peptides having binding affinity for a suture medical
device coupled to a peptide having binding affinity for fibrous
connective tissue-inducing growth factor to capture GDF-7 on the
sutures. The peptide compositions described in Table 10 were tested
as follows. ETHIBOND EXCEL 1 sutures (ETHICON) were cut into 0.5 cm
length pieces with razor blade and placed in the wells of a 96-well
polypropylene plate. The plate was blocked with 1% BSA/TBS (high
salt) for 1 hr at RT by shaking. One .mu.M peptide solutions were
prepared in TBST high salt and the peptide solution was added at
1001l/well/suture. Plates were incubated 30 min at RT shaking. The
plates were washed manually with 4.times.250 .mu.l of TBST high
salt. GDF-7 (R&D SYSTEMS) solutions were prepared at a
concentration of 50 nM in TBST high salt and added at serial 1:4
dilutions to the sutures in the 96-well plate at a concentration
range of 0.01 nM-50 nM. The plate was incubated 1 hr at RT shaking.
The plate was washed manually with 4.times.250 .mu.l of TBST high
salt. Detection of GDF-7 was performed using an anti-GDF-7
antibody-secondary antibody-AP conjugate with detection using a
pNPP calorimetric enzymatic assay. A relative EC50 value for GDF-7
capture by the peptide compositions was determined and range from
1-20 nM for peptides SEQ ID NOs:143-146 having a covalently coupled
hydrophobic tag and the remaining peptides (SEQ ID NOs:147-155)
having a relative EC50 value of greater than 20 nm to greater than
100 nM.
EXAMPLE 18
First and Second Substrate Binding Peptides Localizing Vancomycin
to a Metal Medical Device
[0175] In this example, methods are illustrated for coating a metal
substrate with a composition of the presently disclosed subject
matter wherein the target molecule being localized to the metal
substrate is the antibiotic, vancomycin. In one embodiment, the
compositions used in this experiment comprise a first
substrate-binding peptide having binding affinity for a metal bead
representing the medical device or a first substrate-binding
polymer having a positive charge having binding affinity for a
metal bead representing the medical device, a second
substrate-binding peptide having binding affinity for the target
molecule vancomycin, wherein the first and second substrate-binding
peptide/polymer are not covalently linked, and the target molecule
vancomycin. The molecules are combined and coated onto the metal
bead as described herein below and the first and second substrate
binding peptides are shown in Table 11 below. The first substrate
binding polymer having a positive charge is polyethyleneimine of
various molecular weights and is also shown in Table 11. The
combinations of the first and second substrate binding
peptides/polymers used in the experiment and the amount of
vancomycin loaded onto the metal bead is shown in Table 12. Each of
the metal substrate-binding peptides is covalently coupled to at
least one interaction tag selected from the group consisting of a
hydrophobic interaction tag, a positively charged interaction tag,
and a negatively charged interaction tag. The hydrophobic
interaction tags interact with each other and the charged
interaction tags interact with oppositely charged interaction tags
and/or the positively charged polymer. In this manner a
macromolecular network is formed comprising a plurality of
non-covalently coupled first and second substrate-binding
peptides/polymers to load the vancomycin onto the metal bead.
[0176] In another embodiment, the compositions used in this
experiment comprise a first substrate-binding peptide having
binding affinity for a metal bead representing the medical device
covalently linked to a second substrate-binding peptide having
binding affinity for the target molecule vancomycin, and the target
molecule vancomycin. The molecules are combined and coated onto the
metal bead as described herein below. The first and second
covalently linked substrate binding peptides are shown in Table 11
below. The first and second substrate binding peptides used in the
experiment and the resulting amount of vancomycin loaded onto the
metal bead is shown in Table 12. Each of the covalently linked
first and second substrate-binding peptides is covalently coupled
to at least one interaction tag selected from the group consisting
of a hydrophobic interaction tag, a positively charged interaction
tag, and a negatively charged interaction tag. The hydrophobic
interaction tags interact with each other and the charged
interaction tags interact with oppositely charged interaction tags.
In this manner a macromolecular network is formed comprising a
plurality of non-covalently coupled substrate-binding peptides to
load the vancomycin onto the metal bead.
[0177] In another embodiment, the compositions used in this
experiment comprise a first substrate-binding peptide having
binding affinity for a metal bead representing the medical device
covalently linked to a second substrate-binding peptide having
binding affinity for the target molecule vancomycin, another second
substrate-binding peptide, and the target molecule vancomycin. The
molecules are combined and coated onto the metal bead as described
herein below. The first and second covalently linked substrate
binding peptides and the second substrate binding peptides are
shown in Table 11 below. The combinations of the covalently linked
first and second substrate binding peptides with the second
substrate binding peptide used in the experiment and the resulting
amount of vancomycin loaded onto the metal bead is shown in Table
12 below. Each of the covalently linked first and second
substrate-binding peptides and the second substrate binding
peptides are covalently coupled to at least one interaction tag
selected from the group consisting of a hydrophobic interaction
tag, a positively charged interaction tag, and a negatively charged
interaction tag. The hydrophobic interaction tags interact with
each other and the charged interaction tags interact with
oppositely charged interaction tags. In this manner a
macromolecular network is formed comprising a plurality of
non-covalently coupled substrate-binding peptides to load the
vancomycin onto the metal bead.
TABLE-US-00011 TABLE 11 SEQ ID NO: Sequence 156
RRRRRRR-PSSHRTNHKKNNPKKKNKTRGP-RRRRRRR- K(biotin) 157
RRRRRRR-PSSHRTNHKKNNPKKKNKTRGP-(AUD)6- K(Biotin) 158
(Aud)6-PSSHRTNHKKNNPKKKNKTRGP-RRRRRRR- K(biotin) 159
(AUD)6-SSHRTNHKKNNPKKKNKTRGSSG-RRRRRRR- K(biotin) 160
RRRRRRR-miniPeg-SSCLIDIYGVCHNFDAY-miniPeg- miniPeg-HKKNN PKKKN
KTRGSS-K(Biotin) 161 RRRRRRR-miniPeg-SSCLIDIYGVCHNFDAY-(AUD)5-
HKKNNPKKKNKTRGSS-K(Biotin) 162
SSCLIDIYGVCHNFDAY-miniPeg-miniPeg-HKKNNPKKKN
KTRG-miniPeg-RRRRRRR-K(Biotin) 163
SSSCLIDMYGVCHNFDGAYDSSRG-miniPeg-miniPeg- SSHRTNHKKNNPKKKNKTRGSSGK
164 MA-Ahx-SSCLIDIYGVCHNFDAY-miniPeg-miniPeg-
HKKNNPKKKNKTRGSSK(Biotin) 165 MA-AHx-SSCLIDIYGVCHN
FDAY-miniPeg-miniPeg- YFRAFRKFVKPFKRAFKGSSK(Biotin) 166
Pyrene-butyric-SSCLIDIYGVCHNFDAY-miniPeg-
miniPeg-HKKNNPKKKNKTRGSS-K(Biotin) 167
SSCLIDIYGVCHNFDAY-MiniPeg-DDDDDD 168
EEEEEE-MP-SSCLIDIYGVCHNFDAY-amide 169
EEEEEE-miniPeg-SSCLIDIYGVCHNFDAY-miniPeg-EEE EEE 170
DDDDDD-MP-K-dA-dA-acid 171
EEEEEEE-PSSCLIDIYGVCHNFDGAYDSSRGP-EEEEEEE 172
SSCLIDIYGVCHNFDAY-miniPeg-DEDEDE 173
SSCLIDIYGVCHNFDAY-miniPeg-miniPeg-HKKNNPKKKN
KTRG-miniPeg-RRRRRRR-K(Biotin) 174
(AUD)6-SSHRTNHKKNNPKKKNKTR-GSSG-K(RRRRRRR- biotin)
polyethyleneimine, MW = 800,000 (PEI (800K)) polyethyleneimine, MW
= 70,000 (PEI (70K)) polyethyleneimine, MW = 25,000 (PEI (25K))
polyethyleneimine, MW = 10,000 (PEI (10K))
TABLE-US-00012 TABLE 12 Metal Substrate Vancomycin Load Binding
Sequence Vancomycin Binding Sequence (pmol/cm2) SEQ ID NO: SEQ ID
NO: Controls <10 none none <10 156 none Substrate Binding
Peptides with Charged Interaction Tags 8,591 156 167 11,000 156 168
10,050 156 169 32,035 156 170 2,252 156 171 1,079 156 172 Substrate
Binding Peptides with Charged Interaction Tags & Hydrophobic
Interaction Tags 18,898 158 168 8,820 157 167 9,663 157 168 12,690
159 167 2,501 174 167 1st and 2nd Substrate Binding Peptides Linked
+ 2nd Substrate Binding Peptide & Charged Interaction Tag 5,628
160 167 417 161 167 455 173 167 881 162 167 Positively Charged
Polymer + 2nd Substrate Binding Peptide & Charged Interaction
Tag 5,764 PEI (800K) 167 4,760 PEI (70K) 167 6,058 PEI (25K) 167
7,073 PEI (10K) 167 1st and 2nd Substrate Binding Peptides Linked
+/- Hydrophobic Interaction Tags 54 163 278 164 1,700 165 173
166
[0178] The experimental procedure used for the embodiments
described above was as follows. A cleaned and passivated titanium
bead was added to the wells of a 96-well polypropylene plate. 200
ul of the appropriate peptide or polymer (poly(ethyleneimine), MW
ranging from 10,000-700,000, 30% aqueous solution=30 mM
(POLYSCIENCES #17,938)) and vancomycin mixture was added to each
bead set (triplicates). The first substrate binding peptide and
second substrate binding peptide were added at concentrations
ranging from 50-200 uM and the vancomycin was added at
concentrations ranging from 200-600 uM. All three components were
mixed and then applied to the metal bead. The first substrate
binding polymer was added at a concentration ranging from 10-200 uM
to second substrate binding peptide was added at a concentration
ranging from 50-1000 uM and the vancomycin added at a concentration
ranging from 200-1500 uM. All three components were mixed and then
applied to the metal bead. The molecule with the first substrate
binding peptide covalently linked to second substrate binding
peptide was added at a concentration ranging from 50-200 uM and the
vancomycin added at a concentration ranging from 200-600 uM. The
molecule with the first substrate binding peptide covalently linked
to second substrate binding peptide was added at a concentration
ranging from 50-200 uM and the second substrate binding peptide was
added at a concentration ranging from 50-200 uM and the vancomycin
was added at a concentration ranging from 200-600 uM. The mixtures
were applied to the metal beads and incubated for 30 mins at
20.degree. C. with 700 rpm shaking. The beads were washed three
times with 350 ul PBS. Beads were analyzed as follows: 200 ul of
100 mM HCl was added to each bead and incubated for 30 mins at
20.degree. C. with 1,000 rpm shaking. The eluate from each bead was
analyzed by HPLC using Phenomenex Luna 3 um column, 50.times.4.60
mm. Output was compared to a standard curve for vancomycin to
determine the amount of vancomycin retained onto the metal
bead.
[0179] To access the ability of the coated beads to bind, retain
and release a quantity of vancomycin sufficient to kill bacteria,
beads were coated as described above. Coated beads were transferred
to the wells of a polypropylene 96-well plate. 150 ul of human
plasma was added to each well and incubated at 37.degree. C. with
250 rpm shaking. Plasma was removed after 1 hr and assayed for
antibiotic activity. To measure inhibition of bacterial growth, 100
ul of the sample was added to 100 ul of TSB medium and inoculated
with 10 ul of S. aureus (OD600 of 0.1, diluted 14 fold in TSB). The
plate was sealed with an aluminum cover and incubated for 18 h at
37.degree. C. with 250 rpm shaking. Positive and negative controls
(minus bacteria/minus antibiotic; (plus bacteria/minus antibiotic;
plus bacteria/plus antibiotic) were prepared and run in parallel.
100 ul of the solution in each well was transferred to Costar 9017
polystyrene plate and the absorbance read at 600 nm. The level of
vancomycin loading on the beads is shown in Table 12. The coatings
in Table x that delivered>100 pmol/cm2 showed inhibition of S.
aureus growth.
EXAMPLE 19
In vivo Prevention of Bacterial Colonization of a Coated Titanium
Implant
[0180] This Example describes the delivery of vancomycin from a
titanium implant coated with a composition of the presently
disclosed subject matter that prevents implant colonization by
Staphylococcus aureus in vivo. The goal of this experiment was to
assess the ability of the self-assemblying peptides SEQ ID NO:
158/SEQ ID NO:168 to deliver vancomycin from the surface of a
titanium implant and prevent implant colonization in an infected
tibia of a rat. Peptides SEQ ID NO: 158, SEQ ID NO: 168 and
vancomycin were mixed at a final concentration of 100 uM, 100 uM
and 600 uM, respectively in phosphate buffered saline (PBS). 12mm x
0.8mm titanium pins were cleaned by sonication in a succession of
solutions, water, acetone, 10% Contrad, water, 10% Citrisurf, water
for 15-30 min each. After cleaning, the titanium was passivated by
treatment with 20% nitric acid for 30 min followed by multiple
washes with distilled water. Pins were dried and stored under
nitrogen. 15 pins were placed into microfuge tubes and coated with
the peptide/vancomycin mixture for 20 min at room temperature.
[0181] Staphylococcus aureus was grown overnight at 37 C on a Blood
Agar plate. Colonies were picked from the plate and resuspended in
Trypticase Soy Broth (TSB) at an optical density (OD) of 0.2 which
represents 2.times.10.sup.6 CFU per 10 uL. S. aureus was then
diluted to 10.sup.4 CFU per 10 uL in saline. Rats were anesthetized
with isoflurane and their left hind leg was shaved, depilated, and
disinfected. Skin and fascia at the proximal tibial metaphysis was
incised and a hole bored into the top of the tibia to access the
medullary cavity at the proximal metaphysis. After reaming out the
medullary cavity, 10.sup.4 CFU in 10 uL of S. aureus was added
followed by the insertion of either a treated or untreated titanium
pin. The incision was sutured and the rats allowed to recover.
After 48 hr, the rats were euthanized and the titanium pins removed
from the tibia. Pins were sonicated to remove S. aureus that had
colonized the pins and the sonicates were plated onto TSA plates.
After overnight incubation at 37 C, the number of colonies on the
plates were counted and used to determine the number of bacteria
that had colonized the titanium implants. The animal protocol was
repeated to give a total of 24 treated and 24 untreated samples.
The results are shown in table 13.
TABLE-US-00013 TABLE 13 S. aureus colonization of titanium implants
in infected rat tibia Untreated Treated CFU on CFU on Animal # Pin
Animal # Pin 1 46000 3 0 2 0 5 0 4 0 6 0 7 4600 8 0 11 225000 9 0
12 0 10 0 13 6200 15 0 14 17500 16 0 19 350 17 0 20 1950 18 0 21
1200 22 0 24 20000 23 0 25 8050 26 0 30 1200 27 0 33 34500 28 0 35
0 29 0 36 0 31 0 37 0 32 0 38 1250 34 0 40 11000 39 0 42 37500 41 0
44 0 43 0 45 300 46 0 47 150 48 0
Analysis of the colonization of the titanium pins showed 71% of the
untreated pins were colonized with S. aureus while none of the pins
treated with the peptide/vancomycin mixture were colonized.
EXAMPLE 20
Crosslinking of the Compositions of the Presently Disclosed Subject
Matter
[0182] This Example describes how an additional crosslinking step
can be applied to achieve covalent crosslinks between the substrate
binding peptides comprised in the macromolecular network of the
presently disclosed subject matte. The following surface binding
peptides (SBD-1 and SBD-2) were synthesized for crosslinking
strategy for implementation after non-covalent coupling of the
surface binding peptides as described herein above. A first surface
binding peptide is synthesized using standard peptide chemistry
described previously and an additional cysteine residue is
incorporated at the N-terminus (e.g.,
Cys-RRRRRRR--P--SSHRTNHKKNNPKKKNKTRG-P--RRRRRRR--K (Biotin)-amide;
(SEQ ID NO: 175)). The peptide is purified by HPLC under reduced
conditions. A second surface binding peptide, for example:
Maleoyl-propionic acid-SSCLIDIYGVCHNFDAY-DDDDDD-amide (SEQ ID NO:
176) is synthesized using standard peptide chemistry and the
cyclization and purification is carried out using Acetonitrile/TFA
(0.1% TFA) method. A 3-Maleimidopropionic acid N-hydroxysuccinimide
ester (Obiter Research, LLC) (MPA) group is coupled at the
N-terminus of the peptide sequence of SEQ ID NO: X in DMF using
excess TEA as base. The MPA-peptide conjugate is purified by HPLC
and the lyophilized solid is stored at -20 C. Care is taken to
avoid hydrolysis of the MPA group. A crosslink is formed between
the first (SBD-1) and second (SBD-2) substrate binding peptides as
follows. Dissolve SBD-1 and SBD-2 peptide (5 fold excess) is
dissolved in PBS buffer--Adjust pH to 7.5. The cysteine sulfhydryls
in SBD-1 undergo covalent addition across the maleimide group to
form a thioether bridge. This cross-linking reaction can be
facilitated due to the association of the peptides by virtue of
self assembly. The covalent complex formation is confirmed by
LC-MS.
EXAMPLE 21
Substrate Binding Peptides having Binding Affinity for
Demineralized Bone Matrix
[0183] This Example describes substrate binding peptides having
binding affinity for a substrate tissue that is bone discovered
according to the methods for utilizing phage display technology
outlined herein previously in Example 1. More specifically, the
following subject matter describing substrate-binding peptides
having binding affinity for a substrate tissue that is bone taken
from PCT International Patent Application Publication No.
WO/2008/134329A1, which is herein incorporated by reference in its
entirety.
[0184] Illustrative substrate binding peptides according to the
presently disclosed subject matter having binding affinity for a
substrate tissue that is bone were described in PCT International
Patent Application Publication No. WO/2008/134329A1 and conform to
the following sequence motif 1: ZZXZZXXXXXXXZ (SEQ ID NO:177) and
sequence motif 2: ZXXZZZXXXXXX (SEQ ID NO:178); wherein Z is F
(phenylalanine), W (tryptophan), or Y (tyrosine); and X is any
amino acid. The peptides were shown to have binding affinity for
bone, including demineralized bone matrix, demineralized cortical
bone, and cancellous bone. The illustrative peptides are further
covalently coupled to one or both a hydrophobic interaction tag and
a charged interaction tag according to the methods detailed herein
at Example 12.
[0185] The foregoing description of the specific embodiments of the
present invention has been described in detail for purposes of
illustration. In view of the descriptions and illustrations, others
skilled in the art can, by applying, current knowledge, readily
modify and/or adapt the present invention for various applications
without departing from the basic concept of the present invention;
and thus, such modifications and/or adaptations are intended to be
within the meaning and scope of the appended claims.
Sequence CWU 1
1
178113PRTArtificialsynthesized 1Phe Leu Ser Phe Val Phe Pro Ala Ser
Ala Trp Gly Gly1 5 10213PRTArtificialSynthesized 2Phe Tyr Met Pro
Phe Gly Pro Thr Trp Trp Gln His Val1 5
10313PRTArtificialSynthesized 3Leu Phe Ser Trp Phe Leu Pro Thr Asp
Asn Tyr Pro Val1 5 10413PRTArtificialSynthesized 4Phe Met Asp Ile
Trp Ser Pro Trp His Leu Leu Gly Thr1 5
10513PRTArtificialSynthesized 5Phe Ser Ser Leu Phe Phe Pro His Trp
Pro Ala Gln Leu1 5 10619PRTArtificialSynthesized 6Ser Cys Ala Met
Ala Gln Trp Phe Cys Asp Arg Ala Glu Pro His His1 5 10 15Val Ile
Ser719PRTArtificialArtificial 7Ser Cys Asn Met Ser His Leu Thr Gly
Val Ser Leu Cys Asp Ser Leu1 5 10 15Ala Thr
Ser819PRTArtificialsynthesized 8Ser Cys Val Tyr Ser Phe Ile Asp Gly
Ser Gly Cys Asn Ser His Ser1 5 10 15Leu Gly
Ser919PRTArtificialsynthesized 9Ser Cys Ser Gly Phe His Leu Leu Cys
Glu Ser Arg Ser Met Gln Arg1 5 10 15Glu Leu
Ser1019PRTArtificialsynthesized 10Ser Cys Gly Ile Leu Cys Ser Ala
Phe Pro Phe Asn Asn His Gln Val1 5 10 15Gly Ala
Ser1119PRTArtificialsynthesized 11Ser Cys Cys Ser Met Phe Phe Lys
Asn Val Ser Tyr Val Gly Ala Ser1 5 10 15Asn Pro
Ser1219PRTArtificialsynthesized 12Ser Cys Pro Ile Trp Lys Tyr Cys
Asp Asp Tyr Ser Arg Ser Gly Ser1 5 10 15Ile Phe
Ser1318PRTArtificialsynthesized 13Ser Cys Leu Phe Asn Ser Met Lys
Cys Leu Val Leu Ile Leu Cys Phe1 5 10 15Val
Ser1419PRTArtificialsynthesized 14Ser Cys Tyr Val Asn Gly His Asn
Ser Val Trp Val Val Val Phe Trp1 5 10 15Gly Val
Ser1519PRTArtificialsynthesized 15Ser Cys Asp Phe Val Cys Asn Val
Leu Phe Asn Val Asn His Gly Ser1 5 10 15Asn Met
Ser1619PRTArtificialsynthesized 16Ser Cys Leu Asn Lys Phe Phe Val
Leu Met Ser Val Gly Leu Arg Ser1 5 10 15Tyr Thr
Ser1719PRTArtificialsynthesized 17Ser Cys Cys Asn His Asn Ser Thr
Ser Val Lys Asp Val Gln Phe Pro1 5 10 15Thr Leu
Ser1813PRTArtificialsynthesized 18Phe Phe Pro Ser Ser Trp Tyr Ser
His Leu Gly Val Leu1 5 101913PRTArtificialsynthesized 19Phe Phe Gly
Phe Asp Val Tyr Asp Met Ser Asn Ala Leu1 5
102013PRTArtificialsynthesized 20Leu Ser Phe Ser Asp Phe Tyr Phe
Ser Glu Gly Ser Glu1 5 102113PRTArtificialsynthesized 21Phe Ser Tyr
Ser Val Ser Tyr Ala His Pro Glu Gly Leu1 5
102213PRTArtificialsynthesized 22Leu Pro His Leu Ile Gln Tyr Arg
Val Leu Leu Val Ser1 5 102319PRTArtificialsynthesized 23Ser Cys Tyr
Val Asn Gly His Asn Ser Val Trp Val Val Val Phe Trp1 5 10 15Gly Val
Ser2419PRTArtificialsynthesized 24Ser Cys Asn Ser Phe Met Phe Ile
Asn Gly Ser Phe Lys Glu Thr Gly1 5 10 15Gly Cys
Ser2519PRTArtificialsynthesized 25Ser Cys Phe Gly Asn Leu Gly Asn
Leu Ile Tyr Thr Cys Asp Arg Leu1 5 10 15Met Pro
Ser2619PRTArtificialsynthesized 26Ser Cys Ser Phe Phe Met Pro Trp
Cys Asn Phe Leu Asn Gly Glu Met1 5 10 15Ala Val
Ser2719PRTArtificialsynthesized 27Ser Cys Phe Gly Asn Val Phe Cys
Val Tyr Asn Gln Phe Ala Ala Gly1 5 10 15Leu Phe
Ser2819PRTArtificialsynthesized 28Ser Cys Cys Phe Ile Asn Ser Asn
Phe Ser Val Met Asn His Ser Leu1 5 10 15Phe Lys
Ser2919PRTArtificialsynthesized 29Ser Cys Asp Tyr Phe Ser Phe Leu
Glu Cys Phe Ser Asn Gly Trp Ser1 5 10 15Gly Ala
Ser3019PRTArtificialsynthesized 30Ser Cys Trp Met Gly Leu Phe Glu
Cys Pro Asp Ala Trp Leu His Asp1 5 10 15Trp Asp
Ser3119PRTArtificialsynthesized 31Ser Cys Phe Trp Tyr Ser Trp Leu
Cys Ser Ala Ser Ser Ser Asp Ala1 5 10 15Leu Ile
Ser3219PRTArtificialsynthesized 32Ser Cys Phe Gly Asn Phe Leu Ser
Phe Gly Phe Asn Cys Glu Ser Ala1 5 10 15Leu Gly
Ser3319PRTArtificialsynthesized 33Ser Cys Leu Tyr Cys His Leu Asn
Asn Gln Phe Leu Ser Trp Val Ser1 5 10 15Gly Asn
Ser3419PRTArtificialsynthesized 34Ser Cys Phe Gly Phe Ser Asp Cys
Leu Ser Trp Phe Val Gln Pro Ser1 5 10 15Thr Ala
Ser3519PRTArtificialsynthesized 35Ser Cys Asn His Leu Gly Phe Phe
Ser Ser Phe Cys Asp Arg Leu Val1 5 10 15Glu Asn
Ser3619PRTArtificialsynthesized 36Ser Cys Gly Tyr Phe Cys Ser Phe
Tyr Asn Tyr Leu Asp Ile Gly Thr1 5 10 15Ala Ser
Ser3719PRTArtificialsynthesized 37Ser Cys Asn Ser Ser Ser Tyr Ser
Trp Tyr Cys Trp Phe Gly Gly Ser1 5 10 15Ser Pro
Ser3813PRTArtificialsynthesized 38Phe Gly His Gly Trp Leu Asn Thr
Leu Asn Leu Gly Trp1 5 103913PRTArtificialsynthesized 39Phe Ser Pro
Phe Ser Ala Asn Leu Trp Tyr Asp Met Phe1 5
104013PRTArtificialsynthesized 40Val Phe Val Pro Phe Gly Asn Trp
Leu Ser Thr Ser Val1 5 104113PRTArtificialsynthesized 41Phe Trp Asn
Val Asn Tyr Asn Pro Trp Gly Trp Asn Tyr1 5
104213PRTArtificialsynthesized 42Phe Tyr Trp Asp Arg Leu Asn Val
Gly Trp Gly Leu Leu1 5 104313PRTArtificialsynthesized 43Leu Tyr Ser
Thr Met Tyr Pro Gly Met Ser Trp Leu Val1 5
104419PRTArtificialsynthesized 44Ser Cys Phe Tyr Gln Asn Val Ile
Ser Ser Ser Phe Ala Gly Asn Pro1 5 10 15Trp Glu
Cys4519PRTArtificialsynthesized 45Ser Cys Asn Met Leu Leu Asn Ser
Leu Pro Leu Pro Ser Glu Asp Trp1 5 10 15Ser Ala
Cys4619PRTArtificialsynthesized 46Ser Cys Pro Phe Thr His Ser Leu
Ala Leu Asn Thr Asp Arg Ala Ser1 5 10 15Pro Gly
Cys4719PRTArtificialsynthesized 47Ser Cys Phe Glu Ser Asp Phe Pro
Asn Val Arg His His Val Leu Lys1 5 10 15Gln Ser
Cys4819PRTArtificialsynthesized 48Ser Cys Val Phe Asp Ser Lys His
Phe Ser Pro Thr His Ser Pro His1 5 10 15Asp Val
Cys4919PRTArtificialsynthesized 49Ser Cys Gly Asp His Met Thr Asp
Lys Asn Met Pro Asn Ser Gly Ile1 5 10 15Ser Gly
Cys5019PRTArtificialsynthesized 50Ser Cys Asp Phe Phe Asn Arg His
Gly Tyr Asn Ser Gly Cys Glu His1 5 10 15Ser Val
Cys5119PRTArtificialsynthesized 51Ser Cys Gly Asp His Met Thr Asp
Lys Asn Met Pro Asn Ser Gly Ile1 5 10 15Ser Gly
Cys5219PRTArtificialsynthesized 52Ser Cys Tyr Tyr Asn Gly Leu Val
Val His His Ser Asn Ser Gly His1 5 10 15Lys Asp
Cys5318PRTArtificialsynthesized 53Cys Trp Ser Arg Phe Arg Leu Phe
Met Leu Phe Cys Met Phe Tyr Leu1 5 10 15Val
Ser5418PRTArtificialsynthesized 54Cys Ile Lys Tyr Pro Phe Leu Tyr
Cys Cys Leu Leu Ser Leu Phe Leu1 5 10 15Phe
Ser5519PRTArtificialsynthesized 55Ser Cys Phe Trp Phe Leu Arg Trp
Ser Leu Phe Ile Val Leu Phe Thr1 5 10 15Cys Cys
Ser5619PRTArtificialsynthesized 56Ser Cys Glu Ser Val Asp Cys Phe
Ala Asp Ser Arg Met Ala Lys Val1 5 10 15Ser Met
Ser5719PRTArtificialsynthesized 57Ser Cys Val Gly Phe Phe Cys Ile
Thr Gly Ser Asp Val Ala Ser Val1 5 10 15Asn Ser
Ser5819PRTArtificialsynthesized 58Ser Cys Ser Asp Cys Leu Lys Ser
Val Asp Phe Ile Pro Ser Ser Leu1 5 10 15Ala Ser
Ser5919PRTArtificialsynthesized 59Ser Cys Ala Phe Asp Cys Pro Ser
Ser Val Ala Arg Ser Pro Gly Glu1 5 10 15Trp Ser
Ser6018PRTArtificialsynthesized 60Ser Cys Val Asp Val Met His Ala
Asp Ser Pro Gly Pro Asp Gly Leu1 5 10 15Asn
Ser6119PRTArtificialsynthesized 61Ser Cys Ser Ser Phe Glu Val Ser
Glu Met Phe Thr Cys Ala Val Ser1 5 10 15Ser Tyr
Ser6219PRTArtificialsynthesized 62Ser Cys Gly Leu Asn Phe Pro Leu
Cys Ser Phe Val Asp Phe Ala Gln1 5 10 15Asp Ala
Ser6319PRTArtificialsynthesized 63Ser Cys Met Leu Phe Ser Ser Val
Phe Asp Cys Gly Met Leu Ile Ser1 5 10 15Asp Leu
Ser6419PRTArtificialsynthesized 64Ser Cys Val Asp Tyr Val Met His
Ala Asp Ser Pro Gly Pro Asp Gly1 5 10 15Leu Asn
Ser6519PRTArtificialsynthesized 65Ser Cys Ser Glu Asn Phe Met Phe
Asn Met Tyr Gly Thr Gly Val Cys1 5 10 15Thr Glu
Ser6613PRTArtificialsynthesized 66His Lys His Pro Val Thr Pro Arg
Phe Phe Val Val Glu1 5 106718PRTArtificialsynthesized 67Cys Asn Cys
Tyr Val Thr Pro Asn Leu Leu Lys His Lys Cys Tyr Lys1 5 10 15Ile
Cys6818PRTArtificialsynthesized 68Cys Ser His Asn His His Lys Leu
Thr Ala Lys His Gln Val Ala His1 5 10 15Lys
Cys6918PRTArtificialsynthesized 69Cys Asp Gln Asn Asp Ile Phe Tyr
Thr Ser Lys Lys Ser His Lys Ser1 5 10 15His
Cys7018PRTArtificialsynthesized 70Ser Ser Asp Val Tyr Leu Val Ser
His Lys His His Leu Thr Arg His1 5 10 15Asn
Ser7118PRTArtificialsynthesized 71Ser Asp Lys Cys His Lys His Trp
Tyr Cys Tyr Glu Ser Lys Tyr Gly1 5 10 15Gly
Ser7218PRTArtificialsynthesized 72Ser Asp Lys Ser His Lys His Trp
Tyr Ser Tyr Glu Ser Lys Tyr Gly1 5 10 15Gly
Ser7314PRTArtificialsynthesized 73His His Lys Leu Lys His Gln Met
Leu His Leu Asn Gly Gly1 5 107414PRTArtificialsynthesized 74Gly His
His His Lys Lys Asp Gln Leu Pro Gln Leu Gly Gly1 5
107517PRTArtificialsynthesized 75Ser Ser His Lys His Pro Val Thr
Pro Arg Phe Phe Val Val Glu Ser1 5 10
15Arg7622PRTArtificialsynthesized 76Ser Ser Cys Asn Cys Tyr Val Thr
Pro Asn Leu Leu Lys His Lys Cys1 5 10 15Tyr Lys Ile Cys Ser
Arg207722PRTArtificialsynthesized 77Ser Ser Cys Ser His Asn His His
Lys Leu Thr Ala Lys His Gln Val1 5 10 15Ala His Lys Cys Ser Arg
207822PRTArtificialsynthesized 78Ser Ser Cys Asp Gln Asn Asp Ile
Phe Tyr Thr Ser Lys Lys Ser His1 5 10 15Lys Ser His Cys Ser Arg
207922PRTArtificialsynthesized 79Ser Ser Ser Ser Asp Val Tyr Leu
Val Ser His Lys His His Leu Thr1 5 10 15Arg His Asn Ser Ser Arg
208022PRTArtificialsynthesized 80Ser Ser Ser Asp Lys Cys His Lys
His Trp Tyr Cys Tyr Glu Ser Lys1 5 10 15Tyr Gly Gly Ser Ser Arg
208114PRTArtificialsynthesized 81His His Lys Leu Lys His Gln Met
Leu His Leu Asn Gly Gly1 5 108214PRTArtificialsynthesized 82Gly His
His His Lys Lys Asp Gln Leu Pro Gln Leu Gly Gly1 5
108313PRTArtificialsynthesized 83Cys Phe Val Leu Asn Cys His Leu
Val Leu Asp Arg Pro1 5 108419PRTArtificialsynthesized 84Ser Cys Phe
Gly Asn Phe Leu Ser Phe Gly Phe Asn Cys Glu Tyr Ala1 5 10 15Leu Gly
Ser8513PRTArtificialsynthesized 85Asp Gly Phe Phe Ile Leu Tyr Lys
Asn Pro Asp Val Leu1 5 10867PRTArtificialsynthesized 86Asn His Gln
Asn Gln Thr Asn1 5877PRTArtificialsynthesized 87Ala Thr His Met Val
Gly Ser1 5887PRTArtificialsynthesized 88Gly Ile Asn Pro Asn Phe
Ile1 5897PRTArtificialsynthesized 89Thr Ala Ile Ser Gly His Phe1
59013PRTArtificialsynthesized 90Leu Tyr Gly Thr Pro Glu Tyr Ala Val
Gln Pro Leu Arg1 5 109113PRTArtificialsynthesized 91Cys Phe Leu Thr
Gln Asp Tyr Cys Val Leu Ala Gly Lys1 5
109213PRTArtificialsynthesized 92Val Leu His Leu Asp Ser Tyr Gly
Pro Ser Val Pro Leu1 5 109313PRTArtificialsynthesized 93Val Val Asp
Ser Thr Gly Tyr Leu Arg Pro Val Ser Thr1 5
109413PRTArtificialsynthesized 94Val Leu Gln Asn Ala Thr Asn Val
Ala Pro Phe Val Thr1 5 109513PRTArtificialsynthesized 95Trp Trp Ser
Ser Met Pro Tyr Val Gly Asp Tyr Thr Ser1 5
109619PRTArtificialsynthesized 96Ser Ser Tyr Phe Asn Leu Gly Leu
Val Lys His Asn His Val Arg His1 5 10 15His Asp
Ser9718PRTArtificialsynthesized 97Cys His Asp His Ser Asn Lys Tyr
Leu Lys Ser Trp Lys His Gln Gln1 5 10 15Asn
Cys9819PRTArtificialsynthesized 98Ser Cys Lys His Asp Ser Glu Phe
Ile Lys Lys His Val His Ala Val1 5 10 15Lys Lys
Cys9919PRTArtificialsynthesized 99Ser Cys His His Leu Lys His Asn
Thr His Lys Glu Ser Lys Met His1 5 10 15His Glu
Cys10011PRTArtificialsynthesized 100Val Asn Lys Met Asn Arg Leu Trp
Glu Pro Leu1 5 1010119PRTArtificialsynthesized 101Ser Ser His Arg
Thr Asn His Lys Lys Asn Asn Pro Lys Lys Lys Asn1 5 10 15Lys Thr
Arg10219PRTArtificialsynthesized 102Asn His Thr Ile Ser Lys Asn His
Lys Lys Lys Asn Lys Asn Ser Asn1 5 10 15Lys Thr
Arg10314PRTArtificialsynthesized 103Ser Lys Lys His Gly Gly Lys Lys
His Gly Ser Ser Gly Lys1 5 1010414PRTArtificialsynthesized 104Ser
Lys His Lys Gly Gly Lys His Lys Gly Ser Ser Gly Lys1 5
1010519PRTArtificialsynthesized 105Ser His Lys His Gly Gly His Lys
His Gly Gly His Lys His Gly Ser1 5 10 15Ser Gly
Lys10614PRTArtificialsynthesized 106Ser Lys His Lys Gly Gly His Lys
His Gly Ser Ser Gly Lys1 5 1010714PRTArtificialsynthesized 107Ser
His Lys His Gly Gly Lys His Lys Gly Ser Ser Gly Lys1 5
1010816PRTArtificialsynthesized 108Ser Lys His Lys Gly Gly Gly Gly
Lys His Lys Gly Ser Ser Gly Lys1 5 10
1510916PRTArtificialsynthesized 109Ser His Lys His Gly Gly Gly Gly
His Lys His Gly Ser Ser Gly Lys1 5 10
1511014PRTArtificialsynthesized 110Ser His Lys His Gly Gly His Lys
His Gly Ser Ser Gly Lys1 5 1011114PRTArtificialsynthesized 111Ser
His His Lys Gly Gly His His Lys Gly Ser Ser Gly Lys1 5
1011219PRTArtificialsynthesized 112Ser Lys His Lys Gly Gly Lys His
Lys Gly Gly Lys His Lys Gly Ser1 5 10 15Ser Gly
Lys11317PRTArtificialsynthesized 113Gly Gly Ala Leu Gly Phe Pro Leu
Lys Gly Glu Val Val Glu Gly Trp1 5 10
15Ala11413PRTArtificialsynthesized 114Phe Asp Ile Asp Trp Ser Gly
Met Arg Ser Trp Trp Gly1 5 101157PRTArtificialsynthesized 115Leu
Ser Pro Ser Arg Met Lys1 511621PRTArtificialsynthesized 116Ser Arg
Lys Ser Ser Gln Lys Asn Pro His His Pro Lys Pro Pro Lys1 5 10 15Lys
Pro Thr Ala Arg 2011712PRTArtificialsynthesized 117Ala Leu Pro Ser
Thr Ser Ser Gln Met Pro Gln Leu1 5 1011823PRTArtificialsynthesized
118Ser Ser Ser Cys Gln His Val Ser Leu
Leu Arg Pro Ser Ala Ala Leu1 5 10 15Gly Pro Asp Asn Cys Ser Arg
2011912PRTArtificialsynthesized 119His Thr Pro His Pro Asp Ala Ser
Ile Gln Gly Val1 5 101204PRTArtificialsynthesized 120Val Met Asn
Val11214PRTArtificialsynthesized 121Ala Glu Asp
Gly112224PRTArtificialsynthesized 122Ser Ser His Arg Thr Asn His
Lys Lys Asn Asn Pro Lys Lys Lys Asn1 5 10 15Lys Thr Arg Gly Ser Ser
Gly Lys 2012317PRTArtificialsynthesized 123His Lys Lys Asn Asn Pro
Lys Lys Lys Asn Lys Thr Arg Gly Ser Ser1 5 10
15Lys12424PRTArtificialsynthesized 124Ser Ser Ser Cys Leu Ile Asp
Met Tyr Gly Val Cys His Asn Phe Asp1 5 10 15Gly Ala Tyr Asp Ser Ser
Arg Gly 2012517PRTArtificialsynthesized 125Ser Ser Cys Leu Ile Asp
Ile Tyr Gly Val Cys His Asn Phe Asp Ala1 5 10
15Tyr12616PRTArtificialsynthesized 126Ser Ser Cys Leu Ile Asp Ile
Tyr Gly Lys Cys His Asn Pro Leu Arg1 5 10
1512716PRTArtificialsynthesized 127Lys Trp Lys Leu Phe Lys Lys Ile
Gly Ala Val Leu Lys Val Leu Lys1 5 10
151288PRTArtificialsynthesized 128Arg Ala Asp Ala Arg Ala Asp Ala1
51298PRTArtificialsynthesized 129Tyr Trp Ala Trp Ala Tyr Ala Trp1
513018PRTArtificialsynthesized 130Ser Leu Ile Asp Met Tyr Gly Val
Cys His Asn Phe Asp Gly Ala Tyr1 5 10 15Asp
Ser13118PRTArtificialsynthesized 131Cys Leu Ile Asp Met Tyr Gly Val
Cys His Asn Phe Asp Gly Ala Tyr1 5 10 15Asp
Ser13218PRTArtificialsynthesized 132Cys Leu Phe Asp Ile Phe Gly Val
Cys His Ser Phe Asp Gly Ala Tyr1 5 10 15Asp
Ser13314PRTArtificialsynthesized 133Pro Cys Glu Leu Ile Asp Met Phe
Gly Asn Asp His Cys Pro1 5 1013419PRTArtificialsynthesized 134Ser
Cys Asp Met Leu Phe Cys Glu Asn Phe Ser Gly Ser Gly Asn Asn1 5 10
15Trp Phe Ser13512PRTArtificialsynthesized 135Cys Xaa Xaa Xaa Asp
Met Phe Gly Xaa Xaa Xaa Cys1 5 1013612PRTArtificialsynthesized
136Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa1 5
1013712PRTArtificialsynthesized 137Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa1 5 1013816PRTArtificialsynthesized 138Xaa His Xaa
Xaa Xaa Xaa Xaa Lys Xaa Xaa Xaa Lys Xaa Xaa Asn Lys1 5 10
1513911PRTArtificialsynthesized 139Xaa Xaa Lys Xaa Xaa Xaa Lys Xaa
Xaa Asn Lys1 5 1014018PRTArtificialsynthesized 140Cys Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Pro Xaa1 5 10 15Xaa
Cys1417PRTArtificialsynthesized 141Xaa Xaa Xaa Phe Xaa Xaa Leu1
51427PRTArtificialsynthesized 142Xaa Xaa Phe Pro Leu Xaa Gly1
514343PRTArtificialsynthesized 143Ser Ser Gly Gly Val Gly Gly Trp
Ala Leu Phe Glu Thr Leu Arg Gly1 5 10 15Lys Glu Val Ser Arg Tyr Phe
Arg Ala Phe Arg Lys Phe Val Lys Pro 20 25 30Phe Lys Arg Ala Phe Lys
Gly Ser Ser Gly Lys 35 4014443PRTArtificialsynthesized 144Tyr Phe
Arg Ala Phe Arg Lys Phe Val Lys Pro Phe Lys Arg Ala Phe1 5 10 15Lys
Ser Ser Gly Gly Val Gly Gly Trp Ala Leu Phe Glu Thr Leu Arg 20 25
30Gly Lys Glu Val Ser Arg Gly Ser Ser Gly Lys 35
4014543PRTArtificialsynthesized 145Ser Ser Gly Gly Val Gly Gly Trp
Ala Leu Phe Glu Thr Leu Arg Gly1 5 10 15Lys Glu Val Ser Arg Tyr Phe
Arg Ala Phe Arg Lys Phe Val Lys Pro 20 25 30Phe Lys Arg Ala Phe Lys
Gly Ser Ser Gly Lys 35 4014643PRTArtificialsynthesized 146Ser Ser
Gly Gly Val Gly Gly Trp Ala Leu Phe Glu Thr Leu Arg Gly1 5 10 15Lys
Glu Val Ser Arg Tyr Phe Arg Ala Phe Arg Lys Phe Val Lys Pro 20 25
30Phe Lys Arg Ala Phe Lys Gly Ser Ser Gly Lys 35
4014743PRTArtificialsynthesized 147Tyr Phe Arg Ala Phe Arg Lys Phe
Val Lys Pro Phe Lys Arg Ala Phe1 5 10 15Lys Ser Ser Gly Gly Val Gly
Gly Trp Ala Leu Phe Glu Thr Leu Arg 20 25 30Gly Lys Glu Val Ser Arg
Gly Ser Ser Gly Lys 35 4014843PRTArtificialsynthesized 148Ser Ser
Gly Gly Val Gly Gly Trp Ala Leu Phe Glu Thr Leu Arg Gly1 5 10 15Lys
Glu Val Ser Arg Tyr Phe Arg Ala Phe Arg Lys Phe Val Lys Pro 20 25
30Phe Lys Arg Ala Phe Lys Gly Ser Ser Gly Lys 35
4014943PRTArtificialsynthesized 149Ser Ser Gly Gly Val Gly Gly Trp
Ala Leu Phe Glu Thr Leu Arg Gly1 5 10 15Lys Glu Val Ser Arg Tyr Phe
Arg Ala Phe Arg Lys Phe Val Lys Pro 20 25 30Phe Lys Arg Ala Phe Lys
Gly Ser Ser Gly Lys 35 4015046PRTArtificialsynthesized 150Ser Trp
Trp Gly Phe Trp Asn Gly Ser Ala Ala Pro Val Trp Ser Arg1 5 10 15Gly
Ser Ser Gly Ser Ser Gly Gly Val Gly Gly Trp Ala Leu Phe Glu 20 25
30Thr Leu Arg Gly Lys Glu Val Ser Arg Gly Ser Ser Gly Lys 35 40
4515141PRTArtificialsynthesized 151Ser Ser Gly Gly Val Gly Gly Trp
Ala Leu Phe Glu Thr Leu Arg Gly1 5 10 15Lys Glu Val Ser Arg Gly Ser
Ser Gly Ser Trp Trp Gly Phe Trp Asn 20 25 30Gly Ser Ala Ala Pro Val
Trp Ser Arg 35 4015242PRTArtificialsynthesized 152Ser Ser Gly Gly
Val Gly Gly Trp Ala Leu Phe Glu Thr Leu Arg Gly1 5 10 15Lys Glu Val
Ser Arg Ser Trp Trp Gly Phe Trp Asn Gly Ser Ala Ala 20 25 30Pro Val
Trp Ser Arg Gly Ser Ser Gly Lys 35 4015342PRTArtificialsynthesized
153Ser Ser Gly Gly Val Gly Gly Trp Ala Leu Phe Glu Thr Leu Arg Gly1
5 10 15Lys Glu Val Ser Arg Ser Trp Trp Gly Phe Trp Asn Gly Ser Ala
Ala 20 25 30Pro Val Trp Ser Arg Gly Ser Ser Gly Lys 35
4015442PRTArtificialsynthesized 154Ser Ser Gly Gly Val Gly Gly Trp
Ala Leu Phe Glu Thr Leu Arg Gly1 5 10 15Lys Glu Val Ser Arg Ser Trp
Trp Gly Phe Trp Asn Gly Ser Ala Ala 20 25 30Pro Val Trp Ser Arg Gly
Ser Ser Gly Lys 35 4015547PRTArtificialsynthesized 155Ser Ser Gly
Gly Val Gly Gly Trp Ala Leu Phe Glu Thr Leu Arg Gly1 5 10 15Lys Glu
Val Ser Arg Gly Ser Ser Gly Tyr Phe Arg Ala Phe Arg Lys 20 25 30Phe
Val Lys Pro Phe Lys Arg Ala Phe Lys Gly Ser Ser Gly Lys 35 40
4515637PRTArtificialsynthesized 156Arg Arg Arg Arg Arg Arg Arg Pro
Ser Ser His Arg Thr Asn His Lys1 5 10 15Lys Asn Asn Pro Lys Lys Lys
Asn Lys Thr Arg Gly Pro Arg Arg Arg 20 25 30Arg Arg Arg Arg Lys
3515730PRTArtificialsynthesized 157Arg Arg Arg Arg Arg Arg Arg Pro
Ser Ser His Arg Thr Asn His Lys1 5 10 15Lys Asn Asn Pro Lys Lys Lys
Asn Lys Thr Arg Gly Pro Lys 20 25 3015830PRTArtificialsynthesized
158Pro Ser Ser His Arg Thr Asn His Lys Lys Asn Asn Pro Lys Lys Lys1
5 10 15Asn Lys Thr Arg Gly Pro Arg Arg Arg Arg Arg Arg Arg Lys 20
25 3015931PRTArtificialsynthesized 159Ser Ser His Arg Thr Asn His
Lys Lys Asn Asn Pro Lys Lys Lys Asn1 5 10 15Lys Thr Arg Gly Ser Ser
Gly Arg Arg Arg Arg Arg Arg Arg Lys 20 25
3016041PRTArtificialsynthesized 160Arg Arg Arg Arg Arg Arg Arg Ser
Ser Cys Leu Ile Asp Ile Tyr Gly1 5 10 15Val Cys His Asn Phe Asp Ala
Tyr His Lys Lys Asn Asn Pro Lys Lys 20 25 30 Lys Asn Lys Thr Arg
Gly Ser Ser Lys 35 4016141PRTArtificialsynthesized 161Arg Arg Arg
Arg Arg Arg Arg Ser Ser Cys Leu Ile Asp Ile Tyr Gly1 5 10 15Val Cys
His Asn Phe Asp Ala Tyr His Lys Lys Asn Asn Pro Lys Lys 20 25 30Lys
Asn Lys Thr Arg Gly Ser Ser Lys 35 4016239PRTArtificialsynthesized
162Ser Ser Cys Leu Ile Asp Ile Tyr Gly Val Cys His Asn Phe Asp Ala1
5 10 15Tyr His Lys Lys Asn Asn Pro Lys Lys Lys Asn Lys Thr Arg Gly
Arg 20 25 30Arg Arg Arg Arg Arg Arg Lys
3516348PRTArtificialsynthesized 163Ser Ser Ser Cys Leu Ile Asp Met
Tyr Gly Val Cys His Asn Phe Asp1 5 10 15Gly Ala Tyr Asp Ser Ser Arg
Gly Ser Ser His Arg Thr Asn His Lys 20 25 30Lys Asn Asn Pro Lys Lys
Lys Asn Lys Thr Arg Gly Ser Ser Gly Lys 35 40
4516434PRTArtificialsynthesized 164Ser Ser Cys Leu Ile Asp Ile Tyr
Gly Val Cys His Asn Phe Asp Ala1 5 10 15Tyr His Lys Lys Asn Asn Pro
Lys Lys Lys Asn Lys Thr Arg Gly Ser 20 25 30Ser
Lys16538PRTArtificialsynthesized 165Ser Ser Cys Leu Ile Asp Ile Tyr
Gly Val Cys His Asn Phe Asp Ala1 5 10 15Tyr Tyr Phe Arg Ala Phe Arg
Lys Phe Val Lys Pro Phe Lys Arg Ala 20 25 30Phe Lys Gly Ser Ser Lys
3516634PRTArtificialsynthesized 166Ser Ser Cys Leu Ile Asp Ile Tyr
Gly Val Cys His Asn Phe Asp Ala1 5 10 15Tyr His Lys Lys Asn Asn Pro
Lys Lys Lys Asn Lys Thr Arg Gly Ser 20 25 30Ser
Lys16723PRTArtificialsynthesized 167Ser Ser Cys Leu Ile Asp Ile Tyr
Gly Val Cys His Asn Phe Asp Ala1 5 10 15Tyr Asp Asp Asp Asp Asp Asp
2016823PRTArtificialsynthesized 168Glu Glu Glu Glu Glu Glu Ser Ser
Cys Leu Ile Asp Ile Tyr Gly Val1 5 10 15Cys His Asn Phe Asp Ala Tyr
2016929PRTArtificialsynthesized 169Glu Glu Glu Glu Glu Glu Ser Ser
Cys Leu Ile Asp Ile Tyr Gly Val1 5 10 15Cys His Asn Phe Asp Ala Tyr
Glu Glu Glu Glu Glu Glu 20 251709PRTArtificialsynthesized 170Asp
Asp Asp Asp Asp Asp Lys Ala Ala1 517139PRTArtificialsynthesized
171Glu Glu Glu Glu Glu Glu Glu Pro Ser Ser Cys Leu Ile Asp Ile Tyr1
5 10 15Gly Val Cys His Asn Phe Asp Gly Ala Tyr Asp Ser Ser Arg Gly
Pro 20 25 30Glu Glu Glu Glu Glu Glu Glu
3517223PRTArtificialsynthesized 172Ser Ser Cys Leu Ile Asp Ile Tyr
Gly Val Cys His Asn Phe Asp Ala1 5 10 15Tyr Asp Glu Asp Glu Asp Glu
2017339PRTArtificialsynthesized 173Ser Ser Cys Leu Ile Asp Ile Tyr
Gly Val Cys His Asn Phe Asp Ala1 5 10 15Tyr His Lys Lys Asn Asn Pro
Lys Lys Lys Asn Lys Thr Arg Gly Arg 20 25 30Arg Arg Arg Arg Arg Arg
Lys 3517431PRTArtificialSynthesized 174Ser Ser His Arg Thr Asn His
Lys Lys Asn Asn Pro Lys Lys Lys Asn1 5 10 15Lys Thr Arg Gly Ser Ser
Gly Lys Arg Arg Arg Arg Arg Arg Arg20 25
3017538PRTArtificialSynthesized 175Cys Arg Arg Arg Arg Arg Arg Arg
Pro Ser Ser His Arg Thr Asn His1 5 10 15Lys Lys Asn Asn Pro Lys Lys
Lys Asn Lys Thr Arg Gly Pro Arg Arg 20 25 30Arg Arg Arg Arg Arg Lys
3517623PRTArtificialSynthesized 176Ser Ser Cys Leu Ile Asp Ile Tyr
Gly Val Cys His Asn Phe Asp Ala1 5 10 15Tyr Asp Asp Asp Asp Asp Asp
2017713PRTArtificialSynthesized 177Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa1 5 1017812PRTArtificialSynthesized 178Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10
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