U.S. patent application number 15/490437 was filed with the patent office on 2017-10-26 for potent compstatin analogs.
This patent application is currently assigned to The Trustees of the University of Pennsylvania. The applicant listed for this patent is THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA. Invention is credited to Madan Katragadda, John D. Lambris.
Application Number | 20170305971 15/490437 |
Document ID | / |
Family ID | 37964706 |
Filed Date | 2017-10-26 |
United States Patent
Application |
20170305971 |
Kind Code |
A1 |
Lambris; John D. ; et
al. |
October 26, 2017 |
POTENT COMPSTATIN ANALOGS
Abstract
Compounds comprising peptides and peptidomimetics capable of
binding C3 protein and inhibiting complement activation are
disclosed. These compounds display greatly improved complement
activation-inhibitory activity as compared with currently available
compounds. Methods of making and using the compounds are also
disclosed.
Inventors: |
Lambris; John D.;
(Philadelphia, PA) ; Katragadda; Madan;
(Ypsilanti, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA |
Philadelphia |
PA |
US |
|
|
Assignee: |
The Trustees of the University of
Pennsylvania
Philadelphia
PA
|
Family ID: |
37964706 |
Appl. No.: |
15/490437 |
Filed: |
April 18, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14850686 |
Sep 10, 2015 |
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15490437 |
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13007196 |
Jan 14, 2011 |
9169307 |
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14850686 |
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11605182 |
Nov 28, 2006 |
7888323 |
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13007196 |
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60740205 |
Nov 28, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 25/28 20180101; C07K 14/472 20130101; A61P 25/00 20180101;
A61P 27/02 20180101; A61K 38/00 20130101; C07K 1/00 20130101; C07K
7/64 20130101; A61P 37/00 20180101; A61P 43/00 20180101; C07K 7/08
20130101; A61P 25/16 20180101; A61P 19/02 20180101; A61P 37/06
20180101 |
International
Class: |
C07K 7/64 20060101
C07K007/64; C07K 1/00 20060101 C07K001/00; C07K 14/47 20060101
C07K014/47; C07K 7/08 20060101 C07K007/08 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under GM
62134 awarded by the National Institutes of Health. The government
has certain rights in the invention.
Claims
1. A compound that inhibits complement activation, comprising a
peptide having a sequence: TABLE-US-00010 (SEQ ID NO: 26)
Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa3-Gly-Xaa4-His- Arg-Cys-Xaa5;
wherein: Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a
dipeptide comprising Gly-Ile; Xaa2 is Trp or an analog of Trp,
wherein the analog of Trp has increased hydrophobic character as
compared with Trp, with the proviso that, if Xaa3 is Trp, Xaa2 is
the analog of Trp; Xaa3 is Trp or an analog of Trp comprising a
chemical modification to its indole ring wherein the chemical
modification increases the hydrogen bond potential of the indole
ring; Xaa4 is His, Ala, Phe or Trp; Xaa5 is L-Thr, D-Thr, Ile, Val,
Gly, a dipeptide comprising Thr-Asn or Thr-Ala, or a tripeptide
comprising Thr-Ala-Asn, wherein a carboxy terminal --OH of any of
the L-Thr, D-Thr, Ile, Val, Gly or Asn optionally is replaced by
--NH.sub.2; and the two Cys residues are joined by a disulfide
bond.
2. The compound of claim 1, wherein: (1) Xaa2 participates in a
nonpolar interaction with C3; (2) Xaa3 participates in a hydrogen
bond with C3; or (c) Xaa2 participates in a nonpolar interaction
with C3, and Xaa3 participates in a hydrogen bond with C3.
3. The compound of claim 1, wherein the analog of Trp of Xaa2
comprises a halogenated tryptophan.
4. The compound of claim 1, wherein the analog of Trp of Xaa2
comprises a lower alkoxy or lower alkyl substituent at the 1 or 5
position of tryptophan, or a lower alkanoyl substituent at the 1
position of tryptophan.
5. The compound of claim 1, wherein the analog of Trp of Xaa3
comprises a halogenated tryptophan.
6. The compound of claim 1, wherein Xaa4 is Ala.
7. The compound of claim 1, wherein Xaa2 comprises a lower alkanoyl
or lower alkyl substituent at the 1 position of tryptophan, Xaa3
optionally comprises a halogenated tryptophan and Xaa4 is Ala.
8. The compound of claim 7, wherein Xaa2 is 1-methyltryptophan or
1-formyltryptophan and Xaa3 optionally comprises
5-fluoro-l-tryptophan.
9. The compound of claim 1, which comprises a peptide produced by
expression of a polynucleotide encoding the peptide.
10. The compound of claim 1, wherein the compound is produced at
least in part by peptide synthesis.
11. The compound of claim 1, wherein the compound is PEGylated.
12. The compound of claim 1, further comprising an additional
peptide component that extends the in vivo retention of the
compound.
13. The compound of claim 22, wherein the additional peptide
component is an albumin binding peptide or albumin binding small
molecule.
14. A method of making a compound that inhibits complement
activation, wherein the compound comprises a peptide having a
sequence: TABLE-US-00011 (SEQ ID NO: 26)
Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa3-Gly-Xaa4-His- Arg-Cys-Xaa5;
wherein: Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a
dipeptide comprising Gly-Ile; Xaa2 is Trp or an analog of Trp,
wherein the analog of Trp has increased hydrophobic character as
compared with Trp, with the proviso that, if Xaa3 is Trp, Xaa2 is
the analog of Trp; Xaa3 is Trp or an analog of Trp comprising a
chemical modification to its indole ring wherein the chemical
modification increases the hydrogen bond potential of the indole
ring; Xaa4 is His, Ala, Phe or Trp; Xaa5 is L-Thr, D-Thr, Ile, Val,
Gly, a dipeptide comprising Thr-Asn or Thr-Ala, or a tripeptide
comprising Thr-Ala-Asn, wherein a carboxy terminal --OH of any of
the L-Thr, D-Thr, Ile, Val, Gly or Asn optionally is replaced by
--NH.sub.2; wherein the method comprises synthesizing the peptide
by condensation of the amino acid residues or analogs thereof, or
expressing a polynucleotide encoding the peptide.
15. The method of claim 14, further comprising cyclizing the
peptide through formation of a disulfide bond between the two Cys
residues.
16. The method of claim 14, further comprising post-synthesis
modification of the compound selected from one or more of (a)
acetylation of Xaa1, (b) replacement of the terminal --OH of Xaa4
with --NH.sub.2, (3) PEGylation of the compound and (4)
synthesizing the compound with an additional peptide component that
extends the in vivo retention of the compound.
17. A method of inhibiting complement activation in or on a medium
in which complement activation is occurring, comprising contacting
the medium with a complement inhibitor, wherein the complement
inhibitor comprises a peptide having a sequence: TABLE-US-00012
(SEQ ID NO: 26) Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa3-Gly-Xaa4-His-
Arg-Cys-Xaa5;
wherein: Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a
dipeptide comprising Gly-Ile; Xaa2 is Trp or an analog of Trp,
wherein the analog of Trp has increased hydrophobic character as
compared with Trp, with the proviso that, if Xaa3 is Trp, Xaa2 is
the analog of Trp; Xaa3 is Trp or an analog of Trp comprising a
chemical modification to its indole ring wherein the chemical
modification increases the hydrogen bond potential of the indole
ring; Xaa4 is His, Ala, Phe or Trp; Xaa5 is L-Thr, D-Thr, Ile, Val,
Gly, a dipeptide comprising Thr-Asn or Thr-Ala, or a tripeptide
comprising Thr-Ala-Asn, wherein a carboxy terminal --OH of any of
the L-Thr, D-Thr, Ile, Val, Gly or Asn optionally is replaced by
--NH.sub.2; and the two Cys residues are joined by a disulfide
bond.
18. The method of claim 17, wherein complement activation is
inhibited in one or more of: (a) blood or serum; (b) artificial
organs or implants; and (c) in physiological fluids during
extracorporeal shunting of the fluids.
19. The method of claim 17, wherein the complement activation is
inhibited as part of a treatment of a disease or condition in which
complement activation contributes to cell damage or tissue
injury.
20. The method of claim 17, adapted to design a peptide analog or
peptidomimetic or to screen a small molecule library to identify
other compounds that inhibit complement activation or compete with
the complement inhibitor for binding to C3 or a C3 fragment.
Description
[0001] Continuation of U.S. application Ser. No. 14/850,686, filed
Sep. 10, 2015, which is a continuation of U.S. application Ser. No.
13/007,196, filed Jan. 14, 2011 and issued Oct. 27, 2015 as U.S.
Pat. No. 9,169,307, which is a continuation of U.S. application
Ser. No. 11/605,182, filed Nov. 28, 2006 and issued Feb. 15, 2011
as U.S. Pat. No. 7,888,323 which claims benefit of U.S. Provisional
Application No. 60/740,205, filed Nov. 28, 2005, the entire
contents of each of which are incorporated by reference herein.
FIELD OF THE INVENTION
[0003] This invention relates to activation of the complement
cascade in the body. In particular, this invention provides
peptides and peptidomimetics capable of binding the C3 protein and
inhibiting complement activation.
BACKGROUND OF THE INVENTION
[0004] Various publications, including patents, published
applications, technical articles and scholarly articles are cited
throughout the specification. Each of these cited publications is
incorporated by reference herein, in its entirety. Full citations
for publications not cited fully within the specification are set
forth at the end of the specification.
[0005] The complement system is the first line of immunological
defense against foreign pathogens. Its activation through the
classical, alternative or lectin pathways leads to the generation
of anaphylatoxic peptides C3a and C5a and formation of the C5b-9
membrane attack complex. Complement component C3 plays a central
role in activation of all three pathways. Activation of C3 by
complement pathway C3 convertases and its subsequent attachment to
target surface leads to assembly of the membrane attack complex and
ultimately to damage or lysis of the target cells. C3 is unique in
that it possesses a rich architecture that provides a multiplicity
of diverse ligand binding sites that are important in immune
surveillance and immune response pathways.
[0006] Inappropriate activation of complement may lead to host cell
damage. Complement is implicated in several disease states,
including various autoimmune diseases, and has been found to
contribute to other clinical conditions such as adult respiratory
syndrome, heart attack, rejection following xenotransplantation and
burn injuries. Complement-mediated tissue injury has also been
found to result from bioincompatibility situations such as those
encountered in patients undergoing dialysis or cardiopulmonary
bypass.
[0007] Complement-mediated tissue injuries are directly mediated by
the membrane attack complex, and indirectly by the generation of
C3a and C5a. These peptides induce damage through their effects on
various cells, including neutrophils and mast cells. In vivo,
regulation of complement at the C3 and C5 activation steps is
provided by both plasma and membrane proteins. The plasma protein
inhibitors are factor H and C4-binding protein, and the regulatory
membrane proteins located on cell surfaces are complement receptors
1 (CR1), decay-accelerating factor (DAF), and membrane cofactor
protein (MCP). These proteins inhibit the C3 and C5 convertases
(multi-subunit proteases), by promoting dissociation of the
multisubunit complexes and/or by inactivating the complexes through
proteolysis (catalyzed by factor I). Several pharmacological agents
that regulate or modulate complement activity have been identified
by in vitro assay, but most have been shown in vivo to be of low
activity or toxic.
[0008] To date, there are no inhibitors of complement activation
approved for use in the clinic, though certain candidates for
clinical use exist, specifically, a recombinant form of complement
receptor 1 known as soluble complement receptor 1 (sCR1) and a
humanized monoclonal anti-05 antibody (5G1.1-scFv). Both of these
substances have been shown to suppress complement activation in in
vivo animal models (Kalli K R et al., 1994; and, Wang et al.,
1996). However, each substance possesses the disadvantage of being
a large molecular weight protein (240 kDa and 26 kDa, respectively)
that is difficult to manufacture and must be administered by
infusion. Accordingly, recent research has emphasized the
development of smaller active agents that are easier to deliver,
more stable and less costly to manufacture.
[0009] U.S. Pat. No. 6,319,897 to Lambris et al. describes the use
of a phage-displayed combinatorial random peptide library to
identify a 27-residue peptide that binds to C3 and inhibits
complement activation. This peptide was truncated to a 13-residue
cyclic segment that maintained complete activity, which is referred
to in the art as compstatin. Compstatin inhibits the cleavage of C3
to C3a and C3b by C3 convertases. Compstatin has been tested in a
series of in vitro, in vivo, ex vivo, and in vivo/ex vivo interface
experiments, and has been demonstrated to: (1) inhibit complement
activation in human serum (Sahu A et al., 1996); (2) inhibit
heparin/protamine-induced complement activation in primates without
significant side effects (Soulika A M et al., 2000); (3) prolong
the lifetime of a porcine-to-human xenograft perfused with human
blood (Fiane A E et al., 1999a; Fiane A E et al., 1999b; and, Fiane
A E et al., 2000); (4) inhibit complement activation in models of
cardio-pulmonary bypass, plasmapheresis, and dialysis
extra-corporeal circuits (Nilsson B et al., 1998); and (5) possess
low toxicity (Furlong S T et al., 2000).
[0010] Compstatin is a peptide comprising the sequence
ICVVQDWGHHRCT-NH.sub.2 (SEQ ID NO:1), where Cys2 and Cys12 form a
disulfide bridge. Its three-dimensional structure was determined
using homonuclear 2D NMR spectroscopy in combination with two
separate experimentally restrained computational methodologies. The
first methodology involved distance geometry, molecular dynamics,
and simulated annealing (Morikis D et al., 1998; WO99/13899) and
the second methodology involved global optimization (Klepeis et
al., J. Computational Chem., 20:1344-1370, 1999). The structure of
compstatin revealed a molecular surface that comprises of a polar
patch and a non-polar patch. The polar part includes a Type I
.beta.-turn and the non-polar patch includes the disulfide bridge.
In addition, a series of analogs with alanine replacements (an
alanine scan) was synthesized and tested for activity, revealing
that the four residues of the .beta.-turn and the disulfide bridge
with the surrounding hydrophobic cluster play important roles in
compstatin's inhibitory activity (Morikis et al., 1998;
WO99/13899).
[0011] Using a complement activity assay comprising measuring
alternative pathway-mediated erythrocyte lysis, the IC.sub.50 of
compstatin has been measured as 12 .mu.M. Certain of the analogs
previously tested have demonstrated activity equivalent to or
greater than that of compstatin. Published International
application No. WO2004/026328 discloses compstatin analogs and
mimetics with variations at the N- and C-termini, and at positions
4 and 9, which imparted improved activity in the aforementioned
assay. Improvements of up to 99-fold over compstatin were reported
for certain analogs (see also, Mallik et al., 2005). The
development of compstatin analogs or mimetics with even greater
activity would constitute a significant advance in the art.
SUMMARY OF THE INVENTION
[0012] The present invention provides analogs and mimetics of the
complement-inhibiting peptide, compstatin
(HOOC-ICVVQDWGHHRCT-NH.sub.2; SEQ ID NO:1), which have improved
complement-inhibiting activity as compared to compstatin.
[0013] In one aspect, the invention features a compound that
inhibits complement activation, which comprises a peptide having a
sequence:
[0014] Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa3-Gly-Xaa4-His-Arg-Cys-Xaa5
(SEQ ID NO:26); wherein:
[0015] Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a dipeptide
comprising Gly-Ile; Xaa2 is Trp or an analog of Trp, wherein the
analog of Trp has increased hydrophobic character as compared with
Trp, with the proviso that, if Xaa3 is Trp, Xaa2 is the analog of
Trp;
[0016] Xaa3 is Trp or an analog of Trp comprising a chemical
modification to its indole ring wherein the chemical modification
increases the hydrogen bond potential of the indole ring;
[0017] Xaa4 is His, Ala, Phe or Trp;
[0018] Xaa5 is L-Thr, D-Thr, Ile, Val, Gly, a dipeptide comprising
Thr-Asn, or a dipeptide comprising Thr-Ala, or a tripeptide
comprising Thr-Ala-Asn, wherein a carboxy terminal --OH of any of
the L-Thr, D-Thr, Ile, Val, Gly or Asn optionally is replaced by
--NH2; and
[0019] the two Cys residues are joined by a disulfide bond.
[0020] In certain embodiments, Xaa2 participates in a nonpolar
interaction with C3. In other embodiments, Xaa3 participates in a
hydrogen bond with C3. In other embodiments, Xaa2 participates in a
nonpolar interaction with C3, and Xaa3 participates in a hydrogen
bond with C3.
[0021] In various embodiments, the analog of Trp of Xaa2 is a
halogenated trpytophan, such as 5-fluoro-1-tryptophan or
6-fluoro-1-tryptophan. In other embodiments, the Trp analog at Xaa2
comprises a lower alkoxy or lower alkyl substituent at the 5
position, e.g., 5-methoxytryptophan or 5-methyltryptophan. In other
embodiments, the Trp analog at Xaa 2 comprises a lower alkyl or a
lower alkanoyl substituent at the 1 position, with exemplary
embodiments comprising 1-methyltryptophan or 1-formyltryptophan. In
other embodiments, the analog of Trp of Xaa3 is a halogenated
tryptophan such as 5-fluoro-1-tryptophan or
6-fluoro-1-tryptophan.
[0022] In certain embodiments, Xaa2 comprises a lower alkanoyl or
lower alkyl substituent at the 1 position of tryptophan, Xaa3
optionally comprises a halogenated tryptophan and Xaa4 comprises
Alanine. In particular embodiments, Xaa2 is 1-methyltryptophan or
1-formyltryptophan and Xaa3 optionally comprises
5-fluoro-1-tryptophan. Some exemplary compounds of the invention
comprise any of SEQ ID NOS: 15-25.
[0023] In some embodiments, the compound comprises a peptide
produced by expression of a polynucleotide encoding the peptide. In
other embodiments, the compound is produced at least in part by
peptide synthesis. A combination of synthetic methods can also be
used.
[0024] In certain embodiments, the compstatin analogs are, wherein
the compound is PEGylated, as exemplified by the compound
comprising SEQ ID NO:36.
[0025] In other embodiments, the compstatin analog further
comprises an additional peptide component that extends the in vivo
retention of the compound. For example, the additional peptide
component can be an albumin binding peptide. One exemplary
compstatin-albumin binding peptide conjugate comprises SEQ ID
NO:39.
[0026] Another aspect of the invention features a compound that
inhibits complement activation, comprising a non-peptide or partial
peptide mimetic of SEQ ID NO:26 or any of the other sequences of
analogs and conjugates described hereinabove. These non-peptide or
partial peptide mimetics are designed to bind C3 and inhibit
complement activation with at least 100-fold greater activity than
does a peptide comprising SEQ ID NO:1 under equivalent assay
conditions.
[0027] The compstatin analogs, conjugates and mimetics of the
invention are of practical utility for any purpose for which
compstatin itself is utilized, as known in the art and described in
greater detail herein. Certain of these uses involve the
formulation of the compounds into pharmaceutical compositions for
administration to a patient. Such formulations may comprise
pharmaceutically acceptable salts of the compounds, as well as one
or more pharmaceutically acceptable diluents, carriers excipients,
and the like, as would be within the purview of the skilled
artisan.
[0028] Various features and advantages of the present invention
will be understood by reference to the detailed description,
drawings and examples that follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1. Activity of expressed compstatin and its analogs.
Plots of percent complement inhibition versus peptide concentration
for Ac-V4W/H9A (SEQ ID NO:5) (squares) and expressed compstatin
with tryptophan (SEQ ID NO:15) (circles), 5-fluoro-tryptophan (SEQ
ID NO:16) (triangles), 6-fluoro-tryptophan (SEQ ID NO:17 (stars),
5-hydroxy-tryptophan (SEQ ID NO:27) (hexagons), 7-aza-tryptophan
(SEQ ID NO: 28) (diamonds).
[0030] FIG. 2. Activity of synthetic compstatin analogs. Plots of
percent complement inhibition versus peptide concentration for
Ac-V4W/H9A (SEQ ID NO:5) (squares) and the compstatin analogs with
5-fluoro-l-tryptophan incorporation at position 4 (SEQ ID NO:18)
(circles), position 7 (SEQ ID NO:19) (triangles), both positions 4
and 7 (SEQ ID NO:20) (diamonds).
[0031] FIG. 3A. Activity of additional synthetic compstatin
analogs. FIG. 3A is a plot of percent complement inhibition vs.
peptide concentration for Ac-V4W/H9A (SEQ ID NO:5) (triangles)
compared to Ac-V4(5f-l-W)/H9A (SEQ ID NO:18) (inverted triangle),
Ac-V4(5-methyl-W)/H9A (SEQ ID NO:22) (circles),
Ac-V4(1-methyl-W)/H9A (SEQ ID NO:23) (diamonds), Ac-V4(2-Nal)/H9A
(SEQ ID NO:7) (squares).
[0032] FIG. 3B. Activity of additional synthetic compstatin
analogs. FIG. 3B is a plot of percent complement inhibition vs.
peptide concentration for Ac-V4W/H9A (SEQ ID NO:5) (triangles)
compared to Ac-V4W/W7(5f-l-W)/H9A (SEQ ID NO:19) (hexagons).
[0033] FIG. 3C. Activity of additional synthetic compstatin
analogs. FIG. 3C is a plot of percent complement inhibition vs.
peptide concentration for wild-type compstatin (SEQ ID NO:1)
(triangles) compared to Ac-V4(1-methyl-W)/W7(5f-l-W)/H9A (SEQ ID
NO:24) (triangles pointing left).
[0034] FIG. 4A. Thermodynamic characterization of the interaction
of additional compstatin analogs with C3. ITC data representing the
binding of Ac-V4W/H9A (SEQ ID NO:5) to C3. The plots were obtained
by fitting the corrected raw data to "one set of sites" model in
Origin 7.0.
[0035] FIG. 4B. Thermodynamic characterization of the interaction
of additional compstatin analogs with C3. ITC data representing the
binding of Ac-V4(5f-l-W)/H9A (SEQ ID NO:18) to C3. The plots were
obtained by fitting the corrected raw data to "one set of sites"
model in Origin 7.0.
[0036] FIG. 4C. Thermodynamic characterization of the interaction
of additional compstatin analogs with C3. ITC data representing the
binding of Ac-V4(5-methyl-W)/H9A (SEQ ID NO:22) to C3. The plots
were obtained by fitting the corrected raw data to "one set of
sites" model in Origin 7.0.
[0037] FIG. 4D. Thermodynamic characterization of the interaction
of additional compstatin analogs with C3. ITC data representing the
binding of Ac-V4(1-methyl-W)/H9A (SEQ ID NO:23) to C3. The plots
were obtained by fitting the corrected raw data to "one set of
sites" model in Origin 7.0.
[0038] FIG. 4E. Thermodynamic characterization of the interaction
of additional compstatin analogs with C3. ITC data representing the
binding of Ac-V4(2-Nal)/H9A (SEQ ID NO:7) to C3. The plots were
obtained by fitting the corrected raw data to "one set of sites"
model in Origin 7.0.
[0039] FIG. 4F. Thermodynamic characterization of the interaction
of additional compstatin analogs with C3. ITC data representing the
binding of Ac-V4W/W7(5f-l-W)/H9A (SEQ ID NO:19) to C3. The plots
were obtained by fitting the corrected raw data to "one set of
sites" model in Origin 7.0.
[0040] FIG. 5A. Plot showing the relation between hydrophobicity of
the analogs denoted by log P and the inhibitory constant.
[0041] FIG. 5B. Plot showing the relation between hydrophobicity of
the analogs denoted by log P and entropy denoted by -T.DELTA.S.
[0042] FIG. 5C. Plot showing the relation between hydrophobicity of
the analogs denoted by log P and the binding constant.
[0043] FIG. 6. Activity of an additional synthetic compstatin
analog. Plots of percent complement inhibition vs. peptide
concentration for Ac-V4(1-methyl-W)/H9A (SEQ ID NO:23) (circles)
and Ac-V4(1-formyl-W)/H9A (SEQ ID NO:25) (squares)
[0044] FIG. 7. Activity of the PEGylated compstatin analog. Plots
of percent complement inhibition vs. peptide concentration for
Ac-V4(1-methyl-W)/H9A (SEQ ID NO:23) (circles) and
Ac-V4(1-methyl-W)/H9A-K-PEG 5000 (SEQ ID NO:36) (squares).
[0045] FIG. 8. Activity of the albumin binding protein-conjugated
compstatin analog. Plots of percent complement inhibition vs.
peptide concentration for Ac-V4(1-methyl-W)/H9A (SEQ ID NO:23)
(circles) and the fusion peptide
(Ac-ICV(1MeW)QDWGAHRCTRLIEDICLPRWGCLWEDD-NH.sub.2) (SEQ ID NO:39)
(squares).
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0046] Various terms relating to the methods and other aspects of
the present invention are used throughout the specification and
claims. Such terms are to be given their ordinary meaning in the
art unless otherwise indicated. Other specifically defined terms
are to be construed in a manner consistent with the definition
provided herein.
Definitions
[0047] The following abbreviations may be used in the specification
and examples: Ac, acetyl group; NH.sub.2, amide; MALDI,
matrix-assisted laser desorption ionization; TOF, time of flight;
ITC, isothermal titration calorimetry; HPLC, high performance
liquid chromatography; NA, not active; dT, D-threonine; 2-Nal,
2-napthylalanine; 1-Nal,1-napthylalanine; 2-Igl, 2-indanylglycine;
Dht, dihydrotryptophan; Bpa, 4-benzoyl-L-phenylalanine; 5f-l-W,
5-fluoro-l-tryptophan; 6f-l-W, 6-fluoro-l-tryptophan; 5-OH--W,
5-hydroxytryptophan; 5-methoxy-W, 5-methoxytryptophan; 5-methyl-W,
5-methyltryptophan; 1-methyl-W, 1-methyltryptophan; amino acid
abbreviations use the standard three- or single-letter
nomenclature, for example Trp or W for tryptophan.
[0048] The term "about" as used herein when referring to a
measurable value such as an amount, a temporal duration, and the
like, is meant to encompass variations of .+-.20% or .+-.10%, in
some embodiments .+-.5%, in some embodiments .+-.1%, and in some
embodiments .+-.0.1% from the specified value, as such variations
are appropriate to make and used the disclosed compounds and
compositions.
[0049] The terms "pharmaceutically active" and "biologically
active" refer to the ability of the compounds of the invention to
bind C3 or fragments thereof and inhibit complement activation.
This biological activity may be measured by one or more of several
art-recognized assays, as described in greater detail herein.
[0050] As used herein, "alkyl" refers to an optionally substituted
saturated straight, branched, or cyclic hydrocarbon having from
about 1 to about 10 carbon atoms (and all combinations and
subcombinations of ranges and specific numbers of carbon atoms
therein), with from about 1 to about 7 carbon atoms being
preferred. Alkyl groups include, but are not limited to, methyl,
ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl,
cyclopentyl, isopentyl, neopentyl, n-hexyl, isohexyl, cyclohexyl,
cyclooctyl, adamantyl, 3-methylpentyl, 2,2-dimethylbutyl, and
2,3-dimethylbutyl. The term "lower alkyl" refers to an optionally
substituted saturated straight, branched, or cyclic hydrocarbon
having from about 1 to about 5 carbon atoms (and all combinations
and subcombinations of ranges and specific numbers of carbon atoms
therein). Lower alkyl groups include, but are not limited to,
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl,
n-pentyl, cyclopentyl, isopentyl and neopentyl.
[0051] As used herein, "halo" refers to F, Cl, Br or I.
[0052] As used herein, "alkanoyl", which may be used
interchangeably with "acyl", refers to an optionally substituted a
straight or branched aliphatic acylic residue having from about 1
to about 10 carbon atoms (and all combinations and subcombinations
of ranges and specific numbers of carbon atoms therein), with from
about 1 to about 7 carbon atoms being preferred. Alkanoyl groups
include, but are not limited to, formyl, acetyl, propionyl,
butyryl, isobutyryl pentanoyl, isopentanoyl, 2-methyl-butyryl,
2,2-dimethylpropionyl, hexanoyl, heptanoyl, octanoyl, and the like.
The term "lower alkanoyl" refers to an optionally substituted
straight or branched aliphatic acylic residue having from about 1
to about 5 carbon atoms (and all combinations and subcombinations
of ranges and specific numbers of carbon atoms therein. Lower
alkanoyl groups include, but are not limited to, formyl, acetyl,
n-propionyl, iso-propionyl, butyryl, iso-butyryl, pentanoyl,
iso-pentanoyl, and the like.
[0053] As used herein, "aryl" refers to an optionally substituted,
mono- or bicyclic aromatic ring system having from about 5 to about
14 carbon atoms (and all combinations and subcombinations of ranges
and specific numbers of carbon atoms therein), with from about 6 to
about 10 carbons being preferred. Non-limiting examples include,
for example, phenyl and naphthyl.
[0054] As used herein, "aralkyl" refers to alkyl radicals bearing
an aryl substituent and have from about 6 to about 20 carbon atoms
(and all combinations and subcombinations of ranges and specific
numbers of carbon atoms therein), with from about 6 to about 12
carbon atoms being preferred. Aralkyl groups can be optionally
substituted. Non-limiting examples include, for example, benzyl,
naphthylmethyl, diphenylmethyl, triphenylmethyl, phenylethyl, and
diphenylethyl.
[0055] As used herein, the terms "alkoxy" and "alkoxyl" refer to an
optionally substituted alkyl-O-- group wherein alkyl is as
previously defined. Exemplary alkoxy and alkoxyl groups include
methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, and heptoxy, among
others.
[0056] As used herein, "carboxy" refers to a --C(.dbd.O)OH
group.
[0057] As used herein, "alkoxycarbonyl" refers to a
--C(.dbd.O)O-alkyl group, where alkyl is as previously defined.
[0058] As used herein, "aroyl" refers to a --C(.dbd.O)-aryl group,
wherein aryl is as previously defined. Exemplary aroyl groups
include benzoyl and naphthoyl.
[0059] Typically, substituted chemical moieties include one or more
substituents that replace hydrogen at selected locations on a
molecule. Exemplary substituents include, for example, halo, alkyl,
cycloalkyl, aralkyl, aryl, sulfhydryl, hydroxyl (--OH), alkoxyl,
cyano (--CN), carboxyl (--COOH), acyl (alkanoyl: --C(.dbd.O)R);
--C(.dbd.O)O-alkyl, aminocarbonyl (--C(.dbd.O)NH.sub.2),
--N-substituted aminocarbonyl (--C(.dbd.O)NHR''), CF.sub.3,
CF.sub.2CF.sub.3, and the like. In relation to the aforementioned
substituents, each moiety R'' can be, independently, any of H,
alkyl, cycloalkyl, aryl, or aralkyl, for example.
[0060] As used herein, "L-amino acid" refers to any of the
naturally occurring levorotatory alpha-amino acids normally present
in proteins or the alkyl esters of those alpha-amino acids. The
term D-amino acid" refers to dextrorotatory alpha-amino acids.
Unless specified otherwise, all amino acids referred to herein are
L-amino acids.
[0061] "Hydrophobic" or "nonpolar" are used synonymously herein,
and refer to any inter- or intra-molecular interaction not
characterized by a dipole.
[0062] As used herein, "pi character" refers to the capacity of
compstatin to participate in a pi bond with C3. Pi bonds result
from the sideways overlap of two parallel p orbitals.
[0063] As used herein, "hydrogen bond potential" refers to the
capacity of compstatin to participate in an electrostatic
attraction with C3 involving electronegative moieties on the
modified tryptophan residues or tryptophan analogs on compstatin
and hydrogen atoms on C3. A non-limiting example of such an
electronegative moiety is a fluorine atom.
[0064] "PEGylation" refers to the reaction in which at least one
polyethylene glycol (PEG) moiety, regardless of size, is chemically
attached to a protein or peptide to form a PEG-peptide conjugate.
"PEGylated means that at least one PEG moiety, regardless of size,
is chemically attached to a peptide or protein. The term PEG is
generally accompanied by a numeric suffix that indicates the
approximate average molecular weight of the PEG polymers; for
example, PEG-8,000 refers to polyethylene glycol having an average
molecular weight of about 8,000.
[0065] As used herein, "pharmaceutically-acceptable salts" refers
to derivatives of the disclosed compounds wherein the parent
compound is modified by making acid or base salts thereof. Examples
of pharmaceutically-acceptable salts include, but are not limited
to, mineral or organic acid salts of basic residues such as amines;
alkali or organic salts of acidic residues such as carboxylic
acids; and the like. Thus, the term "acid addition salt" refers to
the corresponding salt derivative of a parent compound that has
been prepared by the addition of an acid. The
pharmaceutically-acceptable salts include the conventional salts or
the quaternary ammonium salts of the parent compound formed, for
example, from inorganic or organic acids. For example, such
conventional salts include, but are not limited to, those derived
from inorganic acids such as hydrochloric, hydrobromic, sulfuric,
sulfamic, phosphoric, nitric and the like; and the salts prepared
from organic acids such as acetic, propionic, succinic, glycolic,
stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic,
hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,
sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic,
methanesulfonic, ethane disulfonic, oxalic, isethionic, and the
like. Certain acidic or basic compounds of the present invention
may exist as zwitterions. All forms of the compounds, including
free acid, free base, and zwitterions, are contemplated to be
within the scope of the present invention.
Description
[0066] In accordance with the present invention, information about
the biological and physico-chemical characteristics of compstatin
have been employed to design compstatin analogs with significantly
improved activity compared to the parent compstatin peptide. In
some embodiments, the analogs have at least 50-fold greater
activity than does compstatin. In other embodiments, the analogs
have 60-, 65-, 70-, 75-, 80-, 85-, 90-, 95-, 100-, 105-, 110-,
115-, 120-, 125-, or 130-fold or greater activity than does
compstatin. In still other embodiments, the analogs have, 135-,
140-, 145-, 150-, 155-, 160-, 165-, 170-, 175-, 180-, 185-, 190-,
195-, 200-, 205-, 210-, 215-, 220-, 225-, 230-, 235-, 240-, 245-,
250-, 255-, 260-, 265-fold or greater activity than does
compstatin, as compared utilizing the assays described in the
examples.
[0067] Compstatin analogs synthesized in accordance with other
approaches have been shown to possess somewhat improved activity as
compared with the parent peptide, i.e., up to about 99-fold
(Mallik, B. et al, 2005, supra; WO2004/026328). The analogs
produced in accordance with the present invention possess even
greater activity than either the parent peptide or analogs thereof
produced to date, as demonstrated by in vitro assays as shown in
the figures and in the Examples herein.
[0068] Table 1B shows amino acid sequence and complement inhibitory
activities of compstatin and selected analogs with significantly
improved activity. The selected analogs are referred to by specific
modifications of designated positions (1-13) as compared to the
parent peptide, compstatin (SEQ ID NO:1) and to the peptides of SEQ
NOS: 2-14, shown in Table 1A, which were described in
WO2004/026328. The peptides of SEQ ID NOS: 15-24 are representative
of modifications made in accordance with the present invention,
resulting in significantly more potent compstatin analogs. As
described in greater detail below, it will be understood that
certain of the modifications made to tryptophan at position 4 as
set forth in SEQ ID NOS: 2-13 may be combined with a tryptophan
analog substitution at position 7, to form yet additional potent
compstatin analogs.
TABLE-US-00001 {26 TABLE 1 SEQ ID Activity over Peptide Sequence
NO: compstatin A. Compstatin and Previously Described Analogs
Compstatin H-ICVVQDWGHHRCT-CONH2 1 * Ac-compstatin
Ac-ICVVQDWGHHRCT-CONH2 2 3xmore Ac-V4Y/H9A Ac-ICVYQDWGAHRCT-CONH2 3
19xmore Ac-V4W/H9A-OH Ac-ICVWQDWGAHRCT-COOH 4 25xmore Ac-V4W/H9A
Ac-ICVWQDWGAHRCT-CONH2 5 55xmore Ac-V4W/H9A/T13dT-OH
Ac-ICVWQDWGAHRCdT-COOH 6 55xmore Ac-V4(2-Nal)/H9A
Ac-ICV(2-Nal)QDWGAHRCT-CONH2 7 99xmore Ac V4(2-Nal)/H9A-OH
Ac-ICV(2-Nal)QDWGAHRCT-COOH 8 39xmore Ac V4(1-Nal)/H9A-OH
Ac-ICV(1-Nal)QDWGAHRCT-COOH 9 30xmore Ac-V42Ig1/H9A
Ac-ICV(2-IgI)QDWGAHRCT-CONH2 10 39xmore Ac-V42Ig1/H9A-OH
Ac-ICV(2-IgI)QDWGAHRCT-COOH 11 37xmore Ac-V4Dht/H9A-OH
Ac-ICVDhtQDWGAHRCT-COOH 12 5xmore Ac-V4(Bpa)/H9A-OH
Ac-ICV(Bpa)QDWGAHRCT-COOH 13 49xmore +G/V4W/H9A +AN-OH
H-GICVWQDWGAHRCTAN-COOH 14 38xmore B. Exemplary Analogs Described
Herin +G/V4W/H9A +N-OH H-GICVWQDWGAHRCTN-COOH 15 45xmore
+G/V4(5f-l-W)/W7(5f-l-W)/ H-GICV(5f-l-W)QD(5f-l-W)GAHRCTN-COOH 16
112xmore H9A +N-OH +G/V4(6f-l-W)/W7(6f-l-W)/
H-GICV(6f-l-W)QD(6f-l-W)GAHRCTN-COOH 17 126xmore H9A +N-OH
Ac-V4(5f-l-W)/H9A Ac-ICV(5f-l-W)QDWGAHRCT-CONH.sub.2 18 31xmore
Ac-V4W/W7(5f-l-W)/H9A Ac-ICVWQD(5f-l-W)GAHRCT-CONH.sub.2 29
121xmore Ac-V4(5f-l-W)/W7(5f-l-W)/
Ac-ICV(5f-I-W)QD(5f-l-W)GAHRCT-CONH.sub.2 23 161xmore H9A
Ac-V4(5-methoxy-W)/H9A Ac-ICV(1-methoxy-W)QDWGAHRCT-CONH.sub.2 21
76xmore Ac-V4(5-methyl-W)/H9A
Ac-ICV(5-Methyl-W)QDWGAHRCT-CONH.sub.2 22 67xmore
Ac-V4(1-methyl-W)/H9A Ac-ICV(1-Methyl-W)QDWGAHRCT-CONH.sub.2 23
264xmore Ac-V4(1-methyl-W)/W7 Ac-ICV(1-methyl-W)QD(5f-l-W)GAHRCT-
24 264xmore (5f-l-W)/H9A CONH.sub.2 Ac-V4(formyl-W)/H9A
Ac-ICV(1-formyl-W)QDWGAHRCT-CONH.sub.2 25 264xmore Abbreviations
used in this table are as follows: dT = D-threonine 2-Nal =
2-napthylalanine 1-Nal = 1-napthylalanine 2-IgI = 2-indanlglycine
Dht = dihydrotryptophan Bpa = 4-benzoyl-L-phenylalanine 5f-l-W =
5-fluoro-l-tryptophan 6f-l-W = 6-fluoro-l-tryptophan 5-OH-W =
5-hydroxylryptophan 5-methoxy-W = 5-methoxytryptophan 5-methyl-W =
5-methyltryptophan 1-methyl-W = 1-methyltryptophan 1-formyl-W =
1-formyltryptophan
[0069] Modifications at the N-Terminus.
[0070] Acetylation of the N-terminus typically increases the
complement-inhibiting activity of compstatin and its analogs, as
can be seen specifically by comparing SEQ ID NO: 1 with SEQ ID
NO:2. Accordingly, addition of an acyl group at the amino terminus
of the peptide, including but not limited to N-acetylation, is one
preferred embodiment of the invention, of particular utility when
the peptides are prepared synthetically. However, it is sometimes
of advantage to prepare the peptides by expression of a
peptide-encoding nucleic acid molecule in a prokaryotic or
eukaryotic expression system, or by in vitro transcription and
translation. For these embodiments, the naturally-occurring
N-terminus may be utilized. One example of a compstatin analog
suitable for expression in vitro or in vivo is represented by SEQ
ID NOS:15-17, wherein the acetyl group is replaced by unmodified
glycine at the N-terminus. SEQ ID NOS:15-17, which additionally
comprise modifications within the peptides and at the C-termini as
discussed below, are between about 45- and about 125-fold more
active than compstatin in the complement inhibition assay described
herein.
[0071] Modification within the Peptide.
[0072] Using computational methods that the rank low lying energy
sequences, it was previously determined that Tyr and Val were the
most likely candidates at position 4 to support stability and
activity of the peptide (Klepeis J L et al., 2003). It was
disclosed in WO2004/026328 that Trp at position 4, especially
combined with Ala at position 9, yields many-fold greater activity
than that of the parent peptide (for example, compare activities of
SEQ ID NOS: 4, 5 and 6 with those of SEQ ID NOS: 2 and 3).
WO2004/026326 also disclosed that peptides comprising the
tryptophan analogs 2-napthylalanine (SEQ ID NOS: 7, 8),
1-naphthylalanine (SEQ ID NO: 9), 2-indanylglycine (SEQ ID NOS: 10,
11) or dihydrotryptophan (SEQ ID NO: 12) at position 4 were all
found to possess increased complement-inhibitory activity, ranging
from 5-fold to 99-fold greater than compstatin. In addition, a
peptide comprising the phenylalanine analog, 4-benzoyl-L-alanine,
at position 4 (SEQ ID NO: 13) possessed 49-fold greater activity
that did compstatin.
[0073] In accordance with the present invention, peptides
comprising 5-fluoro-l-tryptophan (SEQ ID NO:19) or either
5-methoxy-, 5-methyl- or 1-methyl-tryptophan, or
1-formyl-tryptophan (SEQ ID NOS: 21, 22, 23 and 25, respectively)
at position 4 possess 31-264-fold greater activity than does
compstatin. Incorporation of 1-methyl- or 1-formyl-tryptophan
increased the activity and the binding affinity the most in
comparison to other analogs. It is believed that an indole
`N`-mediated hydrogen bond is not necessary at position 4 for the
binding and activity of compstatin. The absence of this hydrogen
bond or reduction of the polar character by replacing hydrogen with
lower alkyl, alkanoyl or indole nitrogen at position 4 enhances the
binding and activity of compstatin. Without intending to be limited
to any particular theory or mechanism of action, it is believed
that a hydrophobic interaction or effect at position 4 strengthens
the interaction of compstatin with C3. Accordingly, modifications
of Trp at position 4 (e.g., altering the structure of the side
chain according to methods well known in the art), or substitutions
of Trp analogs that maintain or enhance the aforementioned
hydrophobic interaction are contemplated in the present invention
to produce analogs of compstatin with even greater activity. Such
analogs are well known in the art and include, but are not limited
to the analogs exemplified herein, as well as unsubstituted or
alternatively substituted derivatives thereof. Examples of suitable
analogs may be found by reference to the following publications,
and many others: Beene, et al. (2002) Biochemistry 41: 10262-10269
(describing, inter alia, singly- and multiply-halogenated Trp
analogs); Babitzky & Yanofsky (1995) J. Biol. Chem. 270:
12452-12456 (describing, inter alia, methylated and halogenated Trp
and other Trp and indole analogs); and U.S. Pat. Nos. 6,214,790,
6,169,057, 5,776,970, 4,870,097, 4,576,750 and 4,299,838. Trp
analogs may be introduced into the compstatin peptide by in vitro
or in vivo expression, or by peptide synthesis, as known in the art
and described in greater detail in the examples.
[0074] In certain embodiments, Trp at position 4 of compstatin is
replaced with an analog comprising a 1-alkyl substituent, more
particularly a lower alkyl (e.g., C.sub.1-C.sub.5) substituent as
defined above. These include, but are not limited to, N(.alpha.)
methyl tryptophan and 5-methyltryptophan. In other embodiments, Trp
at position 4 of compstatin is replaced with an analog comprising a
1-alkanoyl substituent, more particularly a lower alkanoyl (e.g.,
C.sub.1-C.sub.5) substituent as defined above. In addition to
exemplified analogs, these include but are not limited to
1-acetyl-L-tryptophan and L-.beta.-homotryptophan.
[0075] Thermodynamic experiments showed that incorporation of
5-fluoro-l-tryptophan at position 7 in compstatin increased
enthalpy of the interaction between compstatin and C3, relative to
wildtype compstatin, whereas incorporation of 5-fluoro-tryptophan
at position 4 in compstatin decreased the enthalpy of this
interaction. Without intending to be bound to any particular
mechanism, the former results indicate that replacement of indole
hydrogens with a fluorine atom on a Trp residue at position 7 of
compstatin can strengthen hydrogen bonding potential of the indole
ring, introduce new hydrogen bonding potential, or mediate an
interaction with C3 through a water molecule at the binding
interface. (Katragadda M et al., 2004). Hence, modifications of Trp
at position 7 (e.g., altering the structure of the side chain
according to methods well known in the art), or substitutions of
Trp analogs that maintain or enhance the aforementioned hydrogen
bonding potential, or mediate an interaction with C3 through a
water molecule at the binding interface, are contemplated in the
present invention to produce analogs with even greater activity. In
certain embodiments, Trp analogs whose indole rings have
modifications that result in increased hydrogen bonding potential
or mediate an interaction with C3 through a water molecule at the
binding interface may be introduced into position 7 of the
compstatin peptide by in vitro or in vivo expression, or by peptide
synthesis. A peptide comprising the tryptophan analog
5-fluoro-tryptophan (SEQ ID NO:19) at position 7 was found to
possess a 121-fold increased activity as compared with
compstatin.
[0076] In another embodiment, Trp analogs are incorporated at both
positions 4 and 7 of the compstatin molecule, and His at position 9
of compstatin is optionally replaced by Ala. Thermodynamic
experiments showed that incorporation of 5-fluoro-tryptophan at
positions 4 and 7 in compstatin increased enthalpy of the
interaction between compstatin and C3, relative to wildtype
compstatin. Accordingly, modifications of Trp at positions 4 and 7
(e.g., altering the structure of the side chain according to
methods well known in the art), or substitutions of Trp analogs
that maintain or enhance the aforementioned hydrophobic interaction
with C3 via position 4 and maintain or enhance the aforementioned
hydrogen bonding potential with C3 via position 7, or interaction
with C3 through a water molecule at the binding interface via
position 7, are contemplated in the present invention to produce
compstatin analogs with even greater activity. Such modified Trp or
Trp analogs may be introduced into the compstatin peptide at
positions 4 and 7 by in vitro or in vivo expression, or by peptide
synthesis. Peptides comprising tryptophan analogs
5-fluoro-tryptophan (SEQ. ID. NO:16) and comprising tryptophan
analogs 6-fluoro-tryptophan (SEQ. ID. NO: 17) at positions 4 and 7
were found to possess significantly increased activity over
compstatin, ranging from a 112- to a 264-fold increase in activity.
In addition, peptides comprising the tryptophan analog
1-methyl-tryptophan at position 4 and 5-fluoro-tryptophan at
position 7 (SEQ ID NO: 24) were found to possess a 264-fold
increase in activity relative to compstatin.
[0077] Modifications at the Carboxy Terminus.
[0078] Peptides produced by synthetic methods are commonly modified
at the carboxy terminus to comprise an amide instead of an acid;
this common modification can be seen in Table 1 in compstatin (SEQ
ID NO:1) and several analogs. Indeed, in some instances, it has
been determined that the terminal amide-containing peptides possess
greater activity than do the terminal acid-containing peptides
(compare, for example, SEQ ID NOS: 5 and 7 with SEQ ID NOS: 4 and
8, respectively). Accordingly, one preferred embodiment of the
invention utilizes the C-terminal amide modification. However, some
circumstances favor the use of an acid at the C-terminus. Such
circumstances include, but are not limited to solubility
considerations and the expression of the peptides in vitro or in
vivo from peptide-encoding nucleic acid molecules.
[0079] The carboxy-terminal residue of compstatin is threonine. In
some embodiments of the present invention, the C-terminal threonine
is replaced by one or more naturally-occurring amino acids or
analogs. For example, the peptide having SEQ ID NO:6 comprises
D-threonine instead of L-threonine, and further possesses a COOH
group at the C-terminus. This peptide shows activity equal to that
of peptide SEQ ID NO:5, comprising L-threonine and CONH.sub.2 at
the C-terminus. Further, Ile has been substituted for Thr at
position 13, to obtain a peptide with 21-fold greater activity than
that of compstatin. In addition, the peptides of SEQ ID NOS: 14-17,
which comprise a C-terminal peptide extension of Asn, or a
dipeptide extension of Ala-Asn, along with a COOH at the C-terminus
and a non-acetylated N-terminus, demonstrate between 38- and
126-fold greater activity than does compstatin. They are also
suitable for production via a prokaryotic or eukaryotic expression
system, as described in greater detail below.
[0080] The compstatin analogs of the present invention may be
prepared by various synthetic methods of peptide synthesis via
condensation of one or more amino acid residues, in accordance with
conventional peptide synthesis methods. For example, peptides are
synthesized according to standard solid-phase methodologies, such
as may be performed on an Applied Biosystems Model 431A peptide
synthesizer (Applied Biosystems, Foster City, Calif.), according to
manufacturer's instructions. Other methods of synthesizing peptides
or peptidomimetics, either by solid phase methodologies or in
liquid phase, are well known to those skilled in the art. During
the course of peptide synthesis, branched chain amino and carboxyl
groups may be protected/deprotected as needed, using commonly-known
protecting groups. An example of a suitable peptide synthetic
method is set forth in Example 3. Modification utilizing
alternative protecting groups for peptides and peptide derivatives
will be apparent to those of skill in the art.
[0081] Alternatively, certain peptides of the invention may be
produced by expression in a suitable prokaryotic or eukaryotic
system. For example, a DNA construct may be inserted into a plasmid
vector adapted for expression in a bacterial cell (such as E. coli)
or a yeast cell (such as Saccharomyces cerevisiae), or into a
baculovirus vector for expression in an insect cell or a viral
vector for expression in a mammalian cell. Such vectors comprise
the regulatory elements necessary for expression of the DNA in the
host cell, positioned in such a manner as to permit expression of
the DNA in the host cell. Such regulatory elements required for
expression include promoter sequences, transcription initiation
sequences and, optionally, enhancer sequences.
[0082] The peptides of SEQ ID NOS:14-17, and others similarly
designed, are suitable for production by expression of a nucleic
acid molecule in vitro or in vivo. A DNA construct encoding a
concatemer of the peptides, the upper limit of the concatemer being
dependent on the expression system utilized, may be introduced into
an in vivo expression system. After the concatemer is produced,
cleavage between the C-terminal Asn and the following N-terminal G
is accomplished by exposure of the polypeptide to hydrazine.
[0083] The peptides produced by gene expression in a recombinant
procaryotic or eucaryotic system may be purified according to
methods known in the art. Examples 1 and 2 set forth methods
suitable for use in the present invention. In one embodiment, a
commercially available expression/secretion system can be used,
whereby the recombinant peptide is expressed and thereafter
secreted from the host cell, to be easily purified from the
surrounding medium.
[0084] A combination of gene expression and synthetic methods may
also be utilized to produce compstatin analogs. For example, an
analog can be produced by gene expression and thereafter subjected
to one or more post-translational synthetic processes, e.g., to
modify the N- or C-terminus or to cyclize the molecule.
[0085] The structure of compstatin is known in the art, and the
structures of the foregoing analogs are determined by similar
means. Once a particular desired conformation of a short peptide
has been ascertained, methods for designing a peptide or
peptidomimetic to fit that conformation are well known in the art.
See, e.g., G. R. Marshall (1993), Tetrahedron, 49: 3547-3558; Hruby
and Nikiforovich (1991), in Molecular Conformation and Biological
Interactions, P. Balaram & S. Ramasehan, eds., Indian Acad. of
Sci., Bangalore, PP. 429-455). Of particular relevance to the
present invention, the design of peptide analogs may be further
refined by considering the contribution of various side chains of
amino acid residues, as discussed above (i.e., for the effect of
functional groups or for steric considerations).
[0086] It will be appreciated by those of skill in the art that a
peptide mimic may serve equally well as a peptide for the purpose
of providing the specific backbone conformation and side chain
functionalities required for binding to C3 and inhibiting
complement activation. Accordingly, it is contemplated as being
within the scope of the present invention to produce C3-binding,
complement-inhibiting compounds through the use of either
naturally-occurring amino acids, amino acid derivatives, analogs or
non-amino acid molecules capable of being joined to form the
appropriate backbone conformation. A non-peptide analog, or an
analog comprising peptide and non-peptide components, is sometimes
referred to herein as a "peptidomimetic" or "isosteric mimetic," to
designate substitutions or derivations of the peptides of the
invention, which possess the same backbone conformational features
and/or other functionalities, so as to be sufficiently similar to
the exemplified peptides to inhibit complement activation.
[0087] The use of peptidomimetics for the development of
high-affinity peptide analogs is well known in the art (see, e.g.,
Zhao B et al., 1995; Beeley, N. 1994; and, Hruby, V J 1993)
Assuming rotational constraints similar to those of amino acid
residues within a peptide, analogs comprising non-amino acid
moieties may be analyzed, and their conformational motifs verified,
by means of the Ramachandran plot (Hruby & Nikiforovich 1991),
among other known techniques.
[0088] The compstatin analogs of the present invention can be
modified by the addition of polyethylene glycol (PEG) components to
the peptide. As is well known in the art, PEGylation can increase
the half-life of therapeutic peptides and proteins in vivo. In one
embodiment, the PEG has an average molecular weight of about 1,000
to about 50,000. In another embodiment, the PEG has an average
molecular weight of about 1,000 to about 20,000. In another
embodiment, the PEG has an average molecular weight of about 1,000
to about 10,000. In an exemplary embodiment, the PEG has an average
molecular weight of about 5,000. The polyethylene glycol may be a
branched or straight chain, and preferably is a straight chain.
[0089] The compstatin analogs of the present invention can be
covalently bonded to PEG via a linking group. Such methods are well
known in the art. (Reviewed in Kozlowski A. et al. 2001; see also,
Harris J M and Zalipsky S, eds. Poly(ethylene glycol), Chemistry
and Biological Applications, ACS Symposium Series 680 (1997)).
Non-limiting examples of acceptable linking groups include an ester
group, an amide group, an imide group, a carbamate group, a
carboxyl group, a hydroxyl group, a carbohydrate, a succinimide
group (including without limitation, succinimidyl succinate (SS),
succinimidyl propionate (SPA), succinimidyl carboxymethylate (SCM),
succinimidyl succinamide (SSA) and N-hydroxy succinimide (NHS)), an
epoxide group, an oxycarbonylimidazole group (including without
limitation, carbonyldimidazole (CDI)), a nitro phenyl group
(including without limitation, nitrophenyl carbonate (NPC) or
trichlorophenyl carbonate (TPC)), a trysylate group, an aldehyde
group, an isocyanate group, a vinylsulfone group, a tyrosine group,
a cysteine group, a histidine group or a primary amine. In certain
embodiments, the linking group is a succinimide group. In one
embodiment, the linking group is NHS.
[0090] The compstatin analogs of the present invention can
alternatively be coupled directly to PEG (i.e., without a linking
group) through an amino group, a sulfhydral group, a hydroxyl group
or a carboxyl group. In one embodiment, PEG is coupled to a lysine
residue added to the C-terminus of compstatin.
[0091] PEGylation is one way to increase in vivo retention of
thereaputic peptides and proteins. The in vivo clearance of
peptides can also be reduced by linking the peptides to certain
other peptides. For instance, certain albumin binding peptides
display an unusually long half-life of 2.3 h when injected by
intravenous bolus into rabbits (Dennis et al., 2002). A peptide of
this type, fused to the anti-tissue factor Fab of D3H44 enabled the
Fab to bind albumin while retaining the ability of the Fab to bind
tissue factor (Nguyen et al., 2006). This interaction with albumin
resulted in significantly reduced in vivo clearance and extended
half-life in mice and rabbits, when compared with the wild-type
D3H44 Fab, comparable with those seen for PEGylated Fab molecules,
immunoadhesins, and albumin fusions. As described in Example 11
herein, the inventors have synthesized a compstatin analog fused
with an albumin-binding peptide and demonstrated that the fusion
protein is active in inhibiting complement activation.
[0092] The complement activation-inhibiting activity of compstatin
analogs, peptidomimetics and conjugates may be tested by a variety
of assays known in the art. In a preferred embodiment, the assay
described in Example 4 is utilized. A non-exhaustive list of other
assays is set forth in U.S. Pat. No. 6,319,897, including, but not
limited to, (1) peptide binding to C3 and C3 fragments; (2) various
hemolytic assays; (3) measurement of C3 convertase-mediated
cleavage of C3; and (4) measurement of Factor B cleavage by Factor
D.
[0093] The peptides and peptidomimetics described herein are of
practical utility for any purpose for which compstatin itself is
utilized, as known in the art. Such uses include, but are not
limited to: (1) inhibiting complement activation in the serum,
tissues or organs of a patient (human or animal), which can
facilitate treatment of certain diseases or conditions, including
but not limited to but not limited to, age-related macular
degeneration, rheumatoid arthritis, spinal cord injury, Parkinson's
disease, and Alzheimer's disease; (2) inhibiting complement
activation that occurs during use of artificial organs or implants
(e.g., by coating or otherwise treating the artificial organ or
implant with a peptide of the invention); (3) inhibiting complement
activation that occurs during extracorporeal shunting of
physiological fluids (blood, urine) (e.g., by coating the tubing
through which the fluids are shunted with a peptide of the
invention); and (4) in screening of small molecule libraries to
identify other inhibitors of compstatin activation (e.g., liquid-
or solid-phase high-throughput assays designed to measure the
ability of a test compound to compete with a compstatin analog for
binding with C3 or a C3 fragment).
[0094] The following examples are provided to describe the
invention in greater detail. They are intended to illustrate, not
to limit, the invention. The materials and methods set forth in
Examples 1-5 were utilized to generate the results described in
Examples 6-11.
Example 1
Bacterial Expression of Compstatin
[0095] A compstatin analog with the following sequence,
NH.sub.2-GICVWQDWGAHRCTN-OH ("G(-1)/V4W/H9A/N14") (SEQ ID NO:15)
was expressed in fusion with chitin binding domain and the DnaB
intein (New England Biolabs, Beverly, Mass.). Guided by the peptide
sequence and the codon usage for E. coli the following genetic code
was used to generate a synthetic gene for this peptide with the
following sequence:
TABLE-US-00002 (SEQ ID NO: 29)
.sup.5'ATTTGCGTTTGGCAGGATTGGGGTGCGCACCGTTGCACCAATTAA.sup.3'
[0096] To clone the synthetic gene into the pGEM-T vector, a 5'
flanking region containing a SapI site and 3' flanking region
containing a PstI site were designed. To construct the synthetic
gene, the four overlapping oligonucleotides shown below were
designed using DnaWorks software and synthesized at Invitrogen Inc.
(Carlsbad, Calif.):
TABLE-US-00003 (SEQ ID NO: 30)
.sup.5'GGTGGTGCTCTTCCAACGGTATTTGCGTTTGGCAGGA.sup.3' (SEQ ID NO: 31)
.sup.5'TTGGGGTGCGCACCGTTGCACCAATTAACTGCAGG.sup.3' (SEQ ID NO: 32)
.sup.3'CAACGTGGTTAATTGACGTCCGC.sup.5' (SEQ ID NO: 33)
.sup.3'CATAAACGCAAACCGTCCTAACCCCACGCGTGG.sup.5'
[0097] The overlapping DNA fragments were assembled by PCR as
described by Stemmer et al., 1995. The resulting gene was amplified
using the following primers:
TABLE-US-00004 (SEQ ID NO: 34) .sup.5'CGCCTGCAGTTAATTGGT.sup.3'
(SEQ ID NO: 35) .sup.5'GGTGGTGCTCTTCCAACG.sup.3'
[0098] The PCR-amplified fragments of compstatin were then cloned
into the pGEM-T vector, and the resulting clone was digested with
PstI and SapI. The Pst1-SapI fragment encoding the compstatin
analog was further subcloned into the expression vector pTWIN1,
which had been predigested with PstI and SapI; the sequence of the
clone was verified by DNA sequencing.
[0099] To express the compstatin analog, ER2566 E. coli cells
transformed with the compstatin clone were grown in SOB medium (20
g/L tryptone, 5 g/L yeast extract, 0.5 g/L NaCl, 2.5 mM KCl, 10 mM
MgCl.sub.2) at 37.degree. C. When an OD.sub.600 0.7 was reached,
expression was induced by the addition of IPTG to a final
concentration of 0.3 mM, followed by an additional incubation at
37.degree. C. for 4 hr. Cells were collected by centrifugation and
lysed by sonication in buffer B1 (20 mM phosphate buffer, pH 8.5,
with 500 mM NaCl and 1 mM EDTA) supplemented with 0.2% Tween-20.
The cell extract was centrifuged, and the soluble fraction was
applied to a chitin binding column (New England Biolabs, Beverly,
Mass.) pre-equilibrated with buffer B1. The column was washed with
100 ml of buffer B1, followed by a quick wash with 3 column volumes
of buffer B2 (50 mM ammonium acetate, pH 7.0). The column was
incubated at room temperature for 20 hr, and the peptide was eluted
with Buffer B2, lyophilized and further purified on a C18 HPLC
column. The purified peptide was identified using MALDI-TOF mass
spectrometry.
Example 2
Expression of Tryptophan Analogs of Compstatin in E. coli
[0100] To express compstatin analogs containing tryptophan
derivatives, the pTWIN1-compstatin clone was transformed into the
ER2566 Trp 82 auxotroph. Expression was carried out in M9 minimal
medium supplemented with 1 mM L-tryptophan as described above.
Cells were grown to an OD.sub.600 0.8-1.0, then collected by
centrifugation and resuspended in fresh minimal medium containing 2
mM of the desired tryptophan analog(s): 5-fluoro-tryptophan,
6-fluoro-tryptophan, 7-aza-tryptophan or 5-hydroxy-tryptophan. The
expressed compstatin analogs were further purified as described in
Example 1.
Example 3
Peptide Synthesis
[0101] Peptide synthesis and purification was performed as
described by Sahu et al., 1996; Sahu et al., 2000; and Mallik et
al., 2005. Briefly, peptides were synthesized in an Applied
Biosystem peptide synthesizer (model 431A) using Fmoc amide resin
and standard side chain protecting groups. Peptides were cleaved
from the resin by incubation for 3 hours at 22.degree. C. with a
solvent mixture containing 5% phenol, 5% thioanisole, 5% water,
2.5% ethanedithiol, and 82.5% trifluoroacetic acid (TFA). The
reaction mixture was filtered through a fritted funnel,
precipitated with cold ether, dissolved in 50% acetonitrile
containing 0.1% TFA, and lyophilized.
[0102] The crude peptides obtained after cleavage were dissolved in
10% acetonitrile containing 0.1% TFA and purified using a reverse
phase C-18 column (Waters, Milford, Mass.). Disulfide oxidation was
achieved by an on-resin cyclization method using the reagent
Thallium (III) trifluoroacetate. This method eliminates the dilute
solution oxidation steps and subsequent time-consuming
concentration through lyophilization steps prior to reverse-phase
HPLC. Using this method, the multimer formation was nonexistent and
a high level (.about.90%) of fully deprotected, oxidized or
cyclized material was obtained. The identity and purity of all
peptides were confirmed by laser desorption mass spectroscopy and
HPLC.
[0103] For the synthesis of the 5-fluoro-tryptophan,
1-methyl-tryptophan, and 5-methyl-tryptophan analogs,
Fmoc-dl-derivatives were used. Separation of the enantiomeric
peptides was performed as described by Meyers et al. 1978. The dl
mixture of each peptide was separated into d and l isomeric
peptides on a C18 reversed-phase HPLC column using 10% acetonitrile
in 0.01M ammonium acetate, pH 4.1. The isomeric identity of the
eluted peptides was determined by treating the peptides with V8
protease, followed by analysis using MALDI-TOF mass spectrometry
(MicroMass TOFspec2E).
Example 4
Complement Inhibition Assays
[0104] Inhibitory activity of compstatin and its analogs on the
complement system was determined by measuring their effect on the
activation of the complement system by immunocomplexes. Complement
activation inhibition was assessed by measuring the inhibition of
C3 fixation to ovalbumin-anti-ovalbumin complexes in normal human
plasma. Microtiter wells were coated with 50 .mu.l of ovalbumin (10
mg/ml) for 2 hr at 25.degree. C. (overnight at 4.degree. C.). The
wells were saturated with 200 .mu.l of 10 mg/ml BSA for 1 hr at
25.degree. C. and then a rabbit anti-ovalbumin antibody was added
to form an immunocomplex by which complement can be activated.
Thirty microliters of peptides at various concentrations were added
directly to each well followed by 30 .mu.l of a 1:80 dilution of
human plasma. After 30 min incubation, bound C3b/iC3b was detected
using a goat anti-human C3 HRP-conjugated antibody. Color was
developed by adding ABTS peroxidase substrate and optical density
measured at 405 nm.
[0105] The absorbance data obtained at 405 nm were translated into
% inhibition based on the absorbance corresponding to 100%
complement activation. The % inhibition was plotted against the
peptide concentration, and the resulting data set was fit to the
logistic dose-response function using Origin 7.0 software. The
concentration of the peptide causing 50% inhibition of C3b/iC3b
deposition was taken as the IC.sub.50 and used to compare the
activities of various peptides. IC.sub.50 values were obtained from
the fitted parameters that achieved the lowest chi-square
value.
Example 5
Isothermal Titration Calorimetry Analysis of the Interaction C3
with Compstatin and its Analogs
[0106] Isothermal titration calorimetry experiments were performed
using the Microcal VP-ITC calorimeter (Microcal Inc, Northampton,
Mass.). Protein concentrations of 3.5-5 .mu.M and peptide
concentrations of 80-200 .mu.M were used for these experiments. All
titrations were performed in PBS (10 mM phosphate buffer with 150
mM NaCl, pH 7.4). In each experiment, the target protein, C3, was
loaded into the cell, and peptide was loaded into the syringe. All
experiments were performed at 25.degree. C. and for each
experiment, 2-.mu.l peptide injections were made into the cell
containing the protein. In each experiment, the raw isotherms were
corrected for the heats of dilution by subtracting the isotherms
representing peptide injections into the buffer. The resulting
isotherms were fit to various models within the Origin 7.0
software, and the model that achieved the lowest chi square value
was deemed to be appropriate for the respective dataset. Binding
affinity and entropy values were plotted against log P values.
Example 6
Role of Tryptophan in C3-Compstatin Interaction as Assessed by
Bacterially Expressed Compstatin Analogs
[0107] Four different tryptophan analogs that differ in the
chemical nature of the indole ring were incorporated into
compstatin using an intein-mediated protein expression system.
Following expression, the peptides were purified in a single step
with a final yield of 2 mg/L of culture. The tryptophan analogs
5-fluoro-tryptophan, 6-fluoro-tryptophan, 7-aza-tryptophan and
5-hydroxy-tryptophan were also expressed using the ER2566/Trp 82
auxotroph as indicated by the MALDI profiles, and the resulting
peptides were purified to homogeneity. Native compstatin and
analogs were cyclized in vivo through a disulfide bond, as
evidenced by their inability to react with PHMB. All peptides were
further purified on a reverse-phase C18 HPLC column.
[0108] The activity of the expressed compstatin analog
G(-1)/V4W/H9A/N14 (SEQ ID NO:15) exhibited an IC.sub.50 of 1.2
.mu.M, which is similar to the activity observed for the Ac-V4W/H9A
analog (SEQ ID NO:5). This finding indicates that the glycine
located at the N-terminus of the expressed peptide plays a role
similar to that of the acetyl group located at the N-terminus of
the Ac-V4W/H9A analog.
[0109] All the expressed compstatin analogs except the 7-aza
tryptophan analog were found to be active at the concentrations
tested. However, the peptide showed different levels of activity
relative to the analog, Ac-V4W/H9A (FIG. 1; Table 2). Compstatin
containing 6-fluoro-tryptophan and 5-fluoro-tryptophan as well as
alanine at position 9 exhibited a 2.8 and 2.5-fold higher activity,
respectively, than that of the Ac-V4W/H9A analog.
TABLE-US-00005 TABLE 2 Complement inhibitory activity of the
expressed peptides SEQ ID Relative Expressed peptide NO: IC.sub.50
(.mu.M) activity* Ac-V4W/H9A.sup.b 5 1.2 45 G(-1)/V4W/H9A/N14 15
1.2 45 G(-1)/V4(5fW)/W7(5fW)/H9A/N14 16 0.48 112
G(-1)/V4(6fW)/W7(6fW)/H9A/N14 17 0.43 126
G(-1)/V4(5-OH.sup.\a-W)/W7(5-OH- 27 33 1.6 W)/H9A/N14
G(-1)/V4(7-aza-W)/W7(7-aza- 28 122 0.44 W)/H9A/N14 *relative to the
activity of the peptide H-I(CVVQDWGHHRC)T-NH.sub.2 (compstatin, SEQ
ID NO: 1) .sup.crepresents hydroxy .sup.bsynthetic peptide
[0110] Without being limited to any particular mechanism, it is
believed that adding fluorine atom increases the activity of the
peptide by increasing the hydrophobicity of the indole ring. The
incorporation of less hydrophobic tryptophan analogs 5-hydroxy
tryptophan and 7-aza-tryptophan was also investigated. In contrast
to the results with the 5-fluoro and 6-fluoro analogs, compstatin
analogs containing 5-hydroxy-tryptophan showed 27.5-fold loss in
the activity compared to the Ac-V4W/H9A analog (SEQ ID NO:5), and
the peptide containing 7-aza-tryptophan showed no activity at all
at the concentrations tested. 7-aza-tryptophan resembles tryptophan
in molecular structure except that it has a nitrogen atom at
position 7 of the indole ring as opposed to a carbon atom. The loss
in activity observed upon substitution of 7-aza-tryptophan shows
the relative importance of this carbon atom.
Example 7
Role of Individual Tryptophans in C3-Compstatin Interaction
[0111] Solid-phase peptide synthesis was used to generate
compstatin analogs with 5-fluoro-tryprophan incorporated
selectively at position 4, position 7, or both positions 4 and 7,
with alanine at position 9. Synthesis was undertaken using
Fmoc-5-fluoro-dl-tryptophan. This reaction yielded an enantiomeric
mixture of the peptides bearing 5-fluoro-d-tryptophan and
5-fluoro-l-tryptophan. Three different peptides were synthesized:
two peptides with single substitution independently at position 4
or 7 and one peptide with substitutions at both positions 4 and 7.
While a mixture of 5-fluoro-l-tryptophan and 5-fluoro-d-tryptophan
analogs could occur in the case of the single substitutions, a
mixture of four enantiomeric combinations was possible in the case
of the double substitution. Each of the peptide mixtures was
further subjected to reversed-phase HPLC to separate the peptide
enantiomers. Identification of the enatiomers was carried out by
digesting the peptides with V8 protease and subsequently analyzing
the digested product using MALDI. V8 protease cleaves at the
C-terminal side of Asp residues only when followed by an l-amino
acid. Identification of cleavage products in the mass spectra
indicated that the l-enantiomeric peptide eluted first followed by
the d-form, where no cleavage fragments were detected.
[0112] All the peptides, containing either 5-fluoro-l-tryptophan or
5-fluoro-d-tryptophan or both, were tested for their complement
inhibitory activity. The synthetic peptide substituted with
5-fluoro-l-tryptophan in both the positions showed a 2.5-fold
higher activity than that of Ac-V4W/H9A (SEQ ID NO:5) (Table
3).
TABLE-US-00006 TABLE 3 Complement inhibitory activity of the
synthetic compstatin analogs containing 5-fluoro-l-tryptophan
Peptide SEQ ID NO: IC.sub.50 (.mu.M) Relative activity* Ac-V4W/H9A
5 1.20 45 Ac-V4(5f-l-W)/H9A 18 1.74 31 Ac-V4W/W7(5f-l-W)/H9A 19
0.446 121 Ac-V4(5f-l-W)/W7(5f-l- 20 0.482 112 W)/H9A *relative to
the activity of the peptide H-I(CVVQDWGHHRC)T-NH.sub.2 (compstatin,
SEQ ID NO: 1)
[0113] Complement inhibition assays (FIG. 2; Table 3) indicated
that (a) substitution of 5-fluoro-l-tryptophan at position 4 alone
rendered the peptide at least 1.5 times less active than Ac-V4W/H9A
(SEQ ID NO:5). Substitution of 5-fluoro-l-tryptophan at position 7
alone increased the activity 2.7-fold when compared to Ac-V4W/H9A.
Substitution of 5-fluoro-l-tryptophan simultaneously at positions 4
and 7 also yielded a 2.5-fold increase in the activity relative to
Ac-V4W/H9A (SEQ ID NO:5). Substitution of 5-fluoro-d-tryptophan at
either position 4 or 7, or both, rendered the peptide inactive.
Example 8
Thermodynamic Basis for the Tryptophan-Mediated Recognition of
Compstatin by C3
[0114] Isothermal titration calorimetry was used to examine the
binding of the peptides to C3 and investigate the thermodynamic
basis for their activities. The calorimetric data obtained for the
interaction of all the peptides with C3 fit to a one set of sites
model with stoichiometry close to 1. It is believed that the
binding of these peptides to C3 occurs in a 1:1 ratio. The
thermodynamic parameters resulting from these fits are shown in
Table 4. As evident from the K.sub.d values, the peptide with a
single substitution of 5-fluoro-l-tryptophan at position 7 and a
double substitution at positions 4 and 7 exhibited tighter binding
than the Ac-V4W/H9A (SEQ ID NO:5) and the Ac-V4(5f-l-W)/H9A (SEQ ID
NO:18) analogs. This finding is in agreement with the relative
activities observed in the complement inhibition assay (Table 3),
indicating that a binding-activity correlation exists.
[0115] All peptides bound to C3 with a negative enthalpy and
positive entropy. Such binding is a characteristic of the
interaction of compstatin with C3. Among all the peptides examined,
the position 7-substituted Ac-V4W/W7(5f-l-W)/H9A analog (SEQ ID
NO:19) exhibited a higher binding enthalpy (.DELTA.H=-21.83,
.DELTA..DELTA.H=-3.69) than did its wild-type counterpart. The
position 4-substituted Ac-V4(5f-l-W)/H9A analog (SEQ ID NO:18)
bound to C3 with an enthalpy of -16.69 kcal/mole, 1.45 kcal/mole
lower than that exhibited by its wild-type counterpart.
[0116] Incorporation of 5-fluoro-tryptophan at position 4 led to a
loss in enthalpy of 1.45 kcal/mole relative to that of tryptophan
at this position (Table 4). Since the only difference between
tryptophan and 5-fluoro-tryptophan is the fluorine atom at C5 of
the indole, this loss in enthalpy can be attributed to the
replacement of hydrogen with fluorine.
TABLE-US-00007 TABLE 4 Thermodynamic parameters for the interaction
of synthetic compstatin analogs containing 5-fluoro-l-tryptophan
and C3 SEQ ID K.sub.d (kcal/mole) peptide NO: (.mu.M) .DELTA.H
.DELTA..DELTA.H -T.DELTA.S -T.DELTA..DELTA.S .DELTA.G
.DELTA..DELTA.G Ac-V4W/H9A 5 0.14 -18.14 0 8.79 0 -9.4 0
Ac-V4(5f-l-W)/H9A 18 0.15 -16.69 1.45 7.39 -1.4 -9.4 0
Ac-V4W/W7(5f-l- 19 0.035 -21.83 -3.69 11.56 2.77 -10.25 -1 W)/H9A
Ac-V4(5f-l-W)/W7(5f-l- 20 0.017 -17.33 0.81 6.73 -2.06 -10.6 -1.2
W)/H9A
[0117] Incorporation of 5-fluoro-tryptophan at position 7 increased
the enthalpy by 3.69 kcal/mole relative to wild-type (Table 4).
Without being limited to any particular mechanism, it is believed
that tryptophan at position 7 is participating in an enthalpically
favorable interaction such as hydrogen bonding. Replacing one of
the indole hydrogens with a fluorine atom might strengthen the
hydrogen bonding character of the indole NH due to the drop in
pK.sub.a. Alternatively, the fluorine forms a hydrogen bond as a
result of its electron-donating nature, as has been demonstrated in
the structure of the tetradeca (3-fluorotyrosyl)glutathione
transferase.
[0118] Another explanation for the observed increase in enthalpy is
that a water molecule is bridging the interaction between the
fluorine atom and a hydrogen acceptor on C3, in which case two
hydrogen bonds (equivalent to about 4 kcal/mole energy) need to be
formed. Support for this theory comes from the decrease in entropy
observed for the interaction of the position 7-substituted
Ac-V4W/W7(5fW)/H9A analog (SEQ ID NO:19) relative to the wild-type
analog (Table 4), a decrease that could be produced by the binding
of an additional water molecule at the interface. Water-mediated
interactions between fluorine atoms and other hydrogen bond
acceptors have been observed in other systems.
[0119] Binding of the double-substituted analog to C3 yielded an
enthalpy change of -19.85 kcal/mole, an entropy change of -9.35
kcal/mole and a free energy change of -10.5 kcal/mole. It is
believed that incorporation of 5-fluoro-tryptophan simultaneously
at both positions abrogates the effects of the single
substitutions.
Example 9
Additional Compstatin Analogs
[0120] Incorporation of Tryptophan Analogs at Position 4.
[0121] It was shown in Examples 5 and 6 that substitution of valine
with tryptophan at position 4 of compstatin increased its activity
45-fold. To further investigate the nature of interaction mediated
by residue at position 4 during the course of the binding of
compstatin to C3, the tryptophan at position 4 was replaced with
tryptophan analogs and 2-napthylalanine.
[0122] ELISA-based assays were used to test the activity of all the
peptide analogs bearing tryptophan analogs at position 4 and
alanine at position 9. While substitution with 1-methyl-tryptophan
(Ac-V4(1-methyl-W)/H9A) (SEQ ID NO:23) and 2-naphthylalanine
(Ac-V4(2-Nal)/H9A) (SEQ ID NO:7) increased the activity over
compstatin 264 and 99-fold, respectively, substitution of
5-fluoro-tryptophan (Ac-V4(5f-l-W)/W7/H9A) (SEQ ID NO:18 and
5-methyl tryptophan (Ac-V4(5-methyl-W)/H9A) (SEQ ID NO:22) resulted
in a lower activity; to 31 and 67-fold greater than the activity
exhibited by the wild-type peptide (Table 5). FIG. 3 shows the
inhibitory curves depicting the activity and Table 5 shows the
IC.sub.50 values calculated from the curves and the relative
activities of the peptides in comparison to the activity of
original compstatin. FIG. 5 shows inhibitory constants (IC.sub.50)
plotted against log P values of tryptophan analogs and
2-napthylalanine.
TABLE-US-00008 TABLE 5 Complement inhibitory activity of the
compstatin analogs SEQ Relative Peptide ID NO: IC.sub.50 (.mu.M)
activity* Ac-V4W/H9A 5 1.20 45 Ac-V4(5f-l-W)/7W/H9A 18 1.74 31
Ac-V4W/W7(5f-l-W)/H9A 19 0.446 121 Ac-V4(5f-l-W)/W7(5f-l- 20 0.482
112 W)/H9A Ac-V4W/7(5-methoxy 29 0.46 0.5 W)/H9A Ac-V4(5-methoxy 21
0.71 76 W)/7W/H9A Ac-V4(5-methyl 22 0.81 67 W)/7W/H9A
Ac-V4(1-methyl 23 0.205 264 W)/7W/H9A Ac-V4(2-Nal)/W7/H9A 7 0.545
99 Ac-V4(1-methyl W)/W7(5f- 24 0.205 264 l-W)/H9A *Relative to the
activity of H-I(CVVQDWGHHRC)T-NH.sub.2 (compstatin, SEQ ID NO:
1).
[0123] The binding of compstatin peptides was also investigated
using isothermal titration calorimetry. The calorimetric data
obtained for the interaction of all the peptides with C3 fit to a
one set of sites model with stoichiometry close to 1 (FIG. 4). This
result suggests that the binding of these peptides to C3 occurs in
a 1:1 ratio. The thermodynamic parameters resulted from these fits
are shown in Table 6. As evident from the Kd values,
Ac-V4(1-methyl-W)/H9A exhibited higher binding affinity
(K.sub.d=0.015 .mu.M) compared to all other peptides having a
single substitution at position 4. Plotting these values against
the log P values of analogs indicates that a correlation exists
between binding affinity and hydrophobic nature of the tryptophan
analogs and 2-napthylalanine. As per the correlation, binding
affinity increases with an increase in the hydrophobicity of the
analog incorporated at position 4. This observation is consistent
with the correlation shown between log P and the inhibitory
constants.
TABLE-US-00009 TABLE 6 Thermodynamic parameters for the interaction
of synthetic compstatin analogs containing 5-fluoro-l-tryptophan
and C3 SEQ ID K.sub.d (kcal/mole) Peptide NO. (.mu.M) .DELTA.H
.DELTA..DELTA.H -T.DELTA.S -T.DELTA..DELTA.S .DELTA.G
.DELTA..DELTA.G Wild-type 1 0.14 -18.14 0 8.79 0 -9.4 0
Ac-V4(5f-l-W/H9A 18 0.15 -16.69 1.45 7.39 -1.4 -9.4 0
Ac-V4(5-methyl- 22 0.12 -17.75 0.34 8.2 -0.54 -9.55 -0.15 W)/H9A
Ac-V4(1-methyl- 23 0.015 -17.59 0.81 6.94 -1.85 -10.65 -1.1 W)/H9A
Ac-V4(2-Nal)/H9A 7 0.11 -14.27 3.87 4.8 -3.99 -9.5 -0.1
Ac-V4W/W7(5f-l- 19 0.035 -21.83 -3.69 11.56 2.77 -10.25 -0.8 W)/H9A
Ac-V4(1-methyl- 24 0.017 -17.33 0.81 6.73 -2.06 -10.6 -1.2
W)/W7(5f-l-W)/H9A
[0124] All the peptides bound to C3 with a negative enthalpy and
positive entropy, suggesting that the binding is enthalpy-driven.
Such binding is a characteristic of the interaction of compstatin
with C3. However, the binding of these peptides is characterized by
an enthalpy change lower than the wild-type, and entropy change
shifted towards favorable end. FIG. 5B shows a plot of log P vs.
-T.DELTA.S, which indicates that with an increase in the
hydrophobicity of the analogs incorporated at position 4, the
entropy is more favored thus making a positive impact on the free
energy change.
[0125] Incorporation of Tryptophan Analogs at Position 7.
[0126] It was proposed in Example 7 that tryptophan at position 7
makes a hydrogen bond with a residue on C3. To examine this
possibility further, tryptophan at position 7 was replaced with
tryptophan analogs similar to the replacements at position 4 to
elucidate the nature of interaction made by tryptophan at this
position. Substitution with 5-fluoro-tryptophan
(Ac-V4W/W7(5f-l-W)/H9A) (Seq ID NO:19), yielded a 121-fold more
active peptide. (FIG. 3, Table 5). Substitutions of tryptophan 7
with the analog 5-methyl trp or 1-methyl trp rendered compstatin
inactive (data not shown). Thus, no correlation between the
activity and hydrophobicity of tryptophan analogs was observed.
[0127] The thermodynamic properties of the different Trp 7-analogs
was investigated in parallel by calorimetry. (Table 6). Since no
binding was detected for peptides containing either the 5-methyl
trp or 1-methyl trp at position 7, the binding parameters do not
exist. Only the peptide Ac-V4W/W7(5f-l-W)/H9A (SEQ ID NO:19) bound
to C3. The binding affinity was 0.035 .mu.M, which is greater that
that observed for all the peptides having a Trp analog at position
4, except for the peptide Ac-V4(1-methyl-W)/H9A (SEQ ID NO:23). In
contrast to the peptides having a Trp analog at position 4,
Ac-V4W/W7(5f-l-W)/H9A (SEQ ID NO:19) bound to C3 with high
favorable binding enthalpy change (.DELTA.H=-21.83,
.DELTA..DELTA.H=-3.69) and unfavorable entropy change
(-T.DELTA.S=11.56, -T.DELTA..DELTA.S=2.77), suggesting additional
favorable non-covalent interactions of polar nature.
[0128] The results show that incorporation of 5-fluoro-tryptophan
at position 7 results in an increase in the activity of compstatin,
whereas incorporation of analogs 5-methyl-tryptophan and
1-methyl-tryptophan renders compstatin inactive. The loss of
activity of compstatin upon incorporation of 1-methyl-tryptophan
supports the conclusion that the hydrogen bond mediated by N--H of
Trp 7 is important for the interaction of compstatin with C3. In
addition, the complete loss of activity of compstatin upon
incorporation of 5-methyl-tryptophan suggests that a hydrophobic
amino acid is not well tolerated at position 7.
[0129] Incorporation of Tryptophan Analogs at Both Positions 4 and
7.
[0130] Since the substitution of tryptophans at position 4 with
1-methyl-tryptophan and position 7 with 5-fluoro-tryptophan yielded
compstatin analogs that showed a drastic increase in the activity,
a compstatin analog containing substitutions at positions 4 and 7
was generated. The resulting peptide
(Ac-V4(1-methyl-W)/W7(5f-l-W)/H9A) (SEQ ID NO:24) generated an
inhibition curve similar to that of the single substitution with
1-methyl-tryptophan (Ac-V4(1-methyl-W)/H9A) (SEQ ID NO:23), (FIG.
3, Table 5). The binding affinity (K.sub.d=0.017) observed for this
peptide in the calorimeter is also similar to that of
Ac-V4(1-methyl-W)/H9A (SEQ ID NO:23). These observations suggest
that 5-fluoro-tryptophan has no effect at position 7 in the
presence of 1-methyl-tryptophan at position 4 under these
experimental conditions.
[0131] Incorporation of Another Tryptophan Analog at Position
4.
[0132] To further investigate the nature of interaction mediated by
residue at position 4 during the course of the binding of
compstatin to C3, the tryptophan at position 4 was replaced with
the tryptophan analog 1-formyl-tryptophan.
[0133] FIG. 6 shows a comparison of percent complement inhibition
vs. peptide concentration for Ac-V4(1-methyl-W)/H9A (SEQ ID NO:23)
(circles) and Ac-V4(1-formyl-W)/H9A (SEQ ID NO:25). As can be seen,
the 1-formyl-W analog was essentially identical to the 1-methyl-W
analog in its complement inhibition activity.
Example 10
PEGylation of Compstatin Analog
[0134] A prolonged half-life of compstatin is advantageous for its
use in chronic treatments. Extending the half-life of tested
therapeutic peptides has been achieved in several instances through
PEGylation (see Veronese et al., 2001), as PEG has the ability to
delay the elimination of biomolecules from the circulation through
a variety of mechanisms, including decreasing renal clearance,
proteolysis and immunogenicity. PEGylation involves covalent
attachment of PEG polymers to macromolecules, preferably to the
primary amine of lysines.
[0135] This example describes the preparation of a PEGylated
compstatin analog, Ac-V4(1-methyl-W)/H9A-K-PEG 5000 (SEQ ID NO:36)
and evaluation of the compound for its ability to inhibit
complement activation.
[0136] Fmoc-NH-NHS-5000 PEG was purchased from Nektar transforming
therapeutics, 490 discovery Dr, Huntsville, Ala. 35806.
[0137] The compound Ac-V4(1-methyl-W)/H9A-K-PEG 5000 (SEQ ID NO:36)
was synthesized chemically by Fmoc solid-phase peptide chemistry
according to a modified standard protocol. Briefly, PEG was
dissolved in 3 ml of dichloromethane, 1 ml of 2M DIEA was added
manually, and the PEG was mixed for 5 minutes.
[0138] Then the PEG was transferred to the vessel, and left to
couple overnight. The PEG was then deprotected with 20% piperidine
for 20 min.
[0139] Then the synthesis proceeded according to the standard
protocol, with a lysine incorporated at the C-terminus of the
molecule for the purpose of linking the PEG to its side chain.
[0140] Final cleavages of the peptides was achieved with Reagent D
(TFA:H2O:TIS:Phenol, 87.5:5:2.5:5) (4 mL) at 25 C for 90 min, to
provide the desired product. The peptide was then purified on a C18
reversed-phase HPLC column, lyophilized and characterized by
MALDI-TOF.
[0141] The PEGylated compstatin analog was tested for
complement-inhibiting activity using the in vitro assay described
in Example 4. As shown in FIG. 7, the PEGylated analog was active
in inhibiting complement activation, however, seven-fold more
conjugate was required to achieve the same amount of inhibition as
the non-PEGylated analog, Ac-V4(1-methyl-W)/H9A (SEQ ID NO:23).
Example 11
Albumin Binding Protein Conjugate of Compstatin Analog
[0142] Dennis et al. (2002) identified a series of peptides having
the core sequence DICLPRWGCLW (SEQ ID NO:37) that specifically
bound serum albumin from multiple species with high affinity. These
peptides bound to albumin with 1:1 stoichiometry at a site distinct
from known small molecule binding sites. The peptide SA21
(AcRLIEDICLPRWGCLWEDDNH2; SEQ ID NO:38) has an unusually long
half-life of 2.3 h when injected by intravenous bolus into rabbits.
As mentioned in the Detailed Description, a related sequence, fused
to the anti-tissue factor Fab of D3H44 enabled the Fab to bind
albumin with similar affinity to that of SA21 while retaining the
ability of the Fab to bind tissue factor1 (Nguyen et al. 2006).
This interaction with albumin resulted in reduced in vivo clearance
of 25- and 58-fold in mice and rabbits, respectively, when compared
with the wild-type D3H44 Fab. The half-life was extended 37-fold to
32.4 h in rabbits and 26-fold to 10.4 h in mice, achieving 25-43%
of the albumin half-life in these animals. These half-lives exceed
those of a Fab 2 and are comparable with those seen for PEGylated
Fab molecules, immunoadhesins, and albumin fusions.
[0143] This example describes the synthesis of a Compstatin analog
fused with an albumin-binding peptide and its activity in in vitro
assays for complement inhibition.
[0144] The compound 4(1MeW)-ABP was synthesized chemically by Fmoc
solid-phase peptide chemistry according to standard protocols. The
N- and C-termini of the peptide waere protected with acetyl and
amide groups. The peptide was further purified on a C18
reversed-phase HPLC column, lyophilized, and characterized by MALDI
mass spectrometry.
[0145] For cyclization, the peptide-resin (0.10 mmol/g loading
based on amino acid analysis) was swollen in dichloromethane (DCM)
(2 mL) for 5 min, filtered and treated with 94:1:5 DCM/TFA/TIS (5
mL) at 25.degree. C. 3 times.times.2 min each to selectively
deprotect the S-Mmt protecting groups, removing the solvent N2
pressure. These bis(thiol), bis(Acm)-peptide-resin intermediates
were washed with CH.sub.2Cl.sub.2, DMF and NMP (each 5
times.times.2 min, 2 mL), swollen further in NMP (2 mL) for 5 min
and then treated with Et.sub.3N (2 eq.) in NMP at 25.degree. C. for
4 h. The peptide-resin was then washed with DMF and
CH.sub.2Cl.sub.2 (each 5 times.times.2 min, 2 mL). Following
resin-bound formations of the first loop, the peptide-resin was
again washed with DMF (5 times.times.2 min, 2 mL) and swollen in
DMF (2 mL) for 5 min, filtered and treated with Tl(tfa)3 (1.5 eq.)
in DMF-anisole (4 mL) to cyclize the second disulfide loops. After
gentle agitation at 25.degree. C. for 4 h, the thallium reagents
were removed with DMF (8 times.times.2 min, 2 mL) and the
peptide-resins were washed further with CH.sub.2Cl.sub.2 (5
times.times.2 min, 2 mL). Final cleavages of the bicyclic peptide
was achieved with Reagent D (TFA:H.sub.2O:TIS:Phenol, 87.5:5:2.5:5)
(4 mL) at 25.degree. C. for 90 min, to provide the desired
product.
[0146] The resultant conjugated peptide (SEQ ID NO:39) is shown
below.
##STR00001##
[0147] The Albumin-binding peptide-compstatin was tested for
complement-inhibiting activity using the in vitro assay described
in Example 4. As shown in FIG. 8, the conjugate was active in
inhibiting complement activation, however, seven-fold more
conjugate was required to achieve the same amount of inhibition as
the unconjugated analog, Ac-V4(1-methyl-W)/H9A (SEQ ID NO:23).
REFERENCES
[0148] Babitzke P, and Yanofsky C. (1995) Structural features of
L-tryptophan required for activation of TRAP, the trp RNA-binding
attenuation protein of Bacillus subtilis. J. Biol. Chem.
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(5-HT3A) and nicotinic acetylcholine receptors: the anomalous
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D, Damico L A. (2002) Albumin binding as a general strategy for
improving the pharmacokinetics of proteins. J Biol Chem.
277:35035-43 [0152] Fiane A E, Mollnes T E, Videm V, Hovig T,
Hogasen K, Mellbye O J, Spruce L, Moore W T, Sahu A, and Lambris J
D. (1999a) Prolongation of ex vivo-perfused pig xenograft survival
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O J, Spruce L, Moore W T, Sahu A, and Lambris J D. (1999b)
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perfused pig xenografts. Xenotransplantation. 6:52-65. [0154] Fiane
A E, Videm V, Lambris J D, Geiran O R, Svennevig J L, and Mollnes T
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hyperacute rejection in a porcine-to-human xenograft model.
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M M, Gormley J J, Hubbs S J, Lloyd D, Mauger R C, Strimpler A M,
Sylvester M A, Scott C W, and Edwards P D. (2000) C3 activation is
inhibited by analogs of compstatin but not by serine protease
inhibitors or peptidyl alpha-ketoheterocycles. Immunopharmacology.
48:199-212. [0156] Hruby V J. (1993) Conformational and
topographical considerations in the design of biologically active
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and D T. (1994) Therapeutic uses of recombinant complement protein
inhibitors. Springer Semin. Immunopathol. 15:417-31. [0158]
Katragadda M, Morikis D, and Lambris J D. (2004) Thermodynamic
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its inhibitor, compstatin. J. Biol. Chem. 279:54987-95. [0159]
Klepeis J L, Floudas C A, Morikis D, Tsokos C G, Argyropoulos E,
Spruce L, and Lambris J D. (2003) Integrated computational and
experimental approach for lead optimization and design of
compstatin variants with improved activity. J. Am. Chem. Soc.
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Spruce L A, Carafides C, Tsokos C G, Morikis D, and Lambris J D
(2005) Design and NMR Characterization of Active Analogs of
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(1998) Solution structure of Compstatin, a potent complement
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Zhang M, McDonald P, Wong W L, Damico L A, Dennis M S. (2006) The
pharmacokinetics of an albumin-binding Fab (AB.Fab) can be
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Sel. 19:291-7. [0164] Nilsson B, Larsson R, Hong J, Elgue G, Ekdahl
K N, Sahu A, and Lambris J D. (1998) Compstatin inhibits complement
and cellular activation in whole blood in two models of
extracorporeal circulation. Blood. 92:1661-7. [0165] Sahu A, Kay B
K, and Lambris J D. (1996) Inhibition of human complement by a
C3-binding peptide isolated from a phage-displayed random peptide
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Hattori T, Bowen F W, Richardson B A, Hack C E, Sahu A, Edmunds L H
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Mastellos, G. Sfyroera, and J. D. Lambris (2002) Chemical synthesis
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A, Chodera A, and Matis L A. (1996) Amelioration of lupus-like
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[0171] The present invention is not limited to the embodiments
described and exemplified above, but is capable of variation and
modification within the scope of the appended claims.
Sequence CWU 1
1
39113PRTArtificial sequencesynthetic
sequenceDISULFID(2)..(12)MOD_RES(13)..(13)AMIDATION 1Ile Cys Val
Val Gln Asp Trp Gly His His Arg Cys Thr 1 5 10 213PRTArtificial
sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(13)..(13)amida-
tion 2Ile Cys Val Val Gln Asp Trp Gly His His Arg Cys Thr 1 5 10
313PRTArtificial sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(13)..(13)amida-
tion 3Ile Cys Val Tyr Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10
413PRTArtificial sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATION,DISULFID(2)..(12) 4Ile Cys Val
Trp Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10 513PRTArtificial
sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(13)..(13)amida-
tion 5Ile Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10
613PRTArtificial sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(13)..(13)D-thr-
eonine 6Ile Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10
713PRTArtificial sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(4)..(4)Xaa
is a tryptophan analog 2-naphthylalanineMOD_RES(13)..(13)amidation
7Ile Cys Val Xaa Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10
813PRTArtificial sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(4)..(4)Xaa
is a tryptophan analog 2-naphthylalanine 8Ile Cys Val Xaa Gln Asp
Trp Gly Ala His Arg Cys Thr 1 5 10 913PRTArtificial
sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(4)..(4)Xaa
is a tryptophan analog 1-naphthylalanine 9Ile Cys Val Xaa Gln Asp
Trp Gly Ala His Arg Cys Thr 1 5 10 1013PRTArtificial
sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(4)..(4)Xaa
is a tryptophan analog 2-indanylglycine carboxylic
acidMOD_RES(13)..(13)amidation 10Ile Cys Val Xaa Gln Asp Trp Gly
Ala His Arg Cys Thr 1 5 10 1113PRTArtificial sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(4)..(4)Xaa
is a tryptophan analog 2-indanylglycine carboxylic acid 11Ile Cys
Val Xaa Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10
1213PRTArtificial sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(4)..(4)Xaa
is a tryptophan analog dihydrotryptophan 12Ile Cys Val Xaa Gln Asp
Trp Gly Ala His Arg Cys Thr 1 5 10 1313PRTArtificial
sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(4)..(4)Xaa
is a tryptophan analog benzoylphenylalanine 13Ile Cys Val Xaa Gln
Asp Trp Gly Ala His Arg Cys Thr 1 5 10 1416PRTArtificial
sequencesynthetic sequenceDISULFID(3)..(13) 14Gly Ile Cys Val Trp
Gln Asp Trp Gly Ala His Arg Cys Thr Ala Asn 1 5 10 15
1515PRTArtificial sequenceSynthetic sequenceDISULFID(3)..(13) 15Gly
Ile Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys Thr Asn 1 5 10 15
1615PRTArtificial sequencesynthetic
sequenceDISULFID(3)..(13)MOD_RES(5)..(5)5-fluoro-l-tryptophanMOD_RES(8)..-
(8)5-fluoro-l-tryptophan 16Gly Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr Asn 1 5 10 15 1715PRTArtificial sequencesynthetic
sequenceDISULFID(3)..(13)MOD_RES(5)..(5)6-fluoro-l-tryptophanMOD_RES(8)..-
(8)6-fluoro-l-tryptophan 17Gly Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr Asn 1 5 10 15 1813PRTArtificial sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(4)..(4)5-fluor-
o-l-tryptophanMOD_RES(13)..(13)amidation 18Ile Cys Val Trp Gln Asp
Trp Gly Ala His Arg Cys Thr 1 5 10 1913PRTArtificial
sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(7)..(7)5-fluor-
o-l-tryptophanMOD_RES(13)..(13)amidation 19Ile Cys Val Trp Gln Asp
Trp Gly Ala His Arg Cys Thr 1 5 10 2013PRTArtificial
sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(4)..(4)5-fluor-
o-l-tryptophanMOD_RES(7)..(7)5-fluoro-l-tryptophanMOD_RES(13)..(13)amidati-
on 20Ile Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10
2113PRTArtificial sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(4)..(4)5-metho-
xytryptophanMOD_RES(13)..(13)amidation 21Ile Cys Val Trp Gln Asp
Trp Gly Ala His Arg Cys Thr 1 5 10 2213PRTArtificial
sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(4)..(4)5-methy-
ltryptophanMOD_RES(13)..(13)amidation 22Ile Cys Val Trp Gln Asp Trp
Gly Ala His Arg Cys Thr 1 5 10 2313PRTArtificial sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(4)..(4)1-methy-
ltryptophanMOD_RES(13)..(13)amidation 23Ile Cys Val Trp Gln Asp Trp
Gly Ala His Arg Cys Thr 1 5 10 2413PRTArtificial sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(4)..(4)1-methy-
ltryptophanMOD_RES(7)..(7)5-fluoro-l-typtophanMOD_RES(13)..(13)amidation
24Ile Cys Val Trp Gln Asp Trp Gly Ala His Arg Cys Thr 1 5 10
2513PRTArtificial sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(4)..(4)1-formy-
ltryptophanMOD_RES(13)..(13)amidation 25Ile Cys Val Trp Gln Asp Trp
Gly Ala His Arg Cys Thr 1 5 10 2616PRTArtificial sequencesynthetic
sequenceVARIANT(1)..(1)Xaa is Gly, or is missingVARIANT(2)..(2)Xaa
is Ile, Val, Leu, Ac-Ile, Ac-Val or
Ac-LeuDISULFID(3)..(13)VARIANT(5)..(5)Xaa is Trp or an analog of
TrpVARIANT(8)..(8)Xaa is Trp or an analog of
TrpVARIANT(10)..(10)Xaa is His, Ala, Phe or TrpVARIANT(14)..(14)Xaa
is L-Thr, D-Thr, Ile, Val or GlyVARIANT(15)..(15)Xaa is Asn or Ala,
or is missingVARIANT(16)..(16)Xaa is Asn, or is missing 26Xaa Xaa
Cys Val Xaa Gln Asp Xaa Gly Xaa His Arg Cys Xaa Xaa Xaa1 5 10 15
2715PRTArtificial sequencesynthetic
sequenceDISULFID(3)..(13)MOD_RES(5)..(5)5-hydroxytryptophanMOD_RES(8)..(8-
)5-hydroxytryptophan 27Gly Ile Cys Val Trp Gln Asp Trp Gly Ala His
Arg Cys Thr Asn 1 5 10 15 2815PRTArtificial sequencesynthetic
sequenceDISULFID(3)..(13)MOD_RES(5)..(5)7-aza-tryptophanMOD_RES(8)..(8)7--
aza-tryptophan 28Gly Ile Cys Val Trp Gln Asp Trp Gly Ala His Arg
Cys Thr Asn 1 5 10 15 2945DNAArtificial sequencesynthetic sequence
29atttgcgttt ggcaggattg gggtgcgcac cgttgcacca attaa
453037DNAArtificial sequencesynthetic sequence 30ggtggtgctc
ttccaacggt atttgcgttt ggcagga 373135DNAArtificial sequencesynthetic
sequence 31ttggggtgcg caccgttgca ccaattaact gcagg
353223DNAArtificial sequencesynthetic sequence 32caacgtggtt
aattgacgtc cgc 233333DNAArtificial sequencesynthetic sequence
33cataaacgca aaccgtccta accccacgcg tgg 333418DNAArtificial
sequencesynthetic sequence 34cgcctgcagt taattggt
183518DNAArtificial sequencesynthetic sequence 35ggtggtgctc
ttccaacg 183614PRTArtificial sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(4)..(4)1-methy-
ltryptophanMOD_RES(14)..(14)pegylated 36Ile Cys Val Trp Gln Asp Trp
Gly Ala His Arg Cys Thr Lys 1 5 10 3711PRTArtificial
sequencesynthetic sequence 37Asp Ile Cys Leu Pro Arg Trp Gly Cys
Leu Trp 1 5 10 3818PRTArtificial sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATION 38Arg Leu Ile Glu Asp Ile Cys
Leu Pro Arg Trp Gly Cys Leu Trp Glu 1 5 10 15 Asp Asp
3931PRTArtificial sequencesynthetic
sequenceMOD_RES(1)..(1)ACETYLATIONDISULFID(2)..(12)MOD_RES(4)..(4)1-methy-
ltryptophanDISULFID(20)..(26) 39Ile Cys Val Trp Gln Asp Trp Gly Ala
His Arg Cys Thr Arg Leu Ile 1 5 10 15 Glu Asp Ile Cys Leu Pro Arg
Trp Gly Cys Leu Trp Glu Asp Asp 20 25 30
* * * * *