Peptide and polypeptide inhibitors of complement C1s

• US 20020102256A1
• Filed: 06/18/2001
• Published: 08/01/2002
• Est. Priority Date: 06/21/2000
• Status: Active Grant

• Current Assignee: Zymogenetics Incorporated (Bristol-Myers Squibb Company)
• Original Assignee: Zymogenetics Incorporated (Bristol-Myers Squibb Company)
• Inventors: Sheppard, Paul O., Fox, Brian A., West, Robert R.
• Granted Patent: US 7,071,299 B2
• Time in Patent Office: Days
• US Class Current: 424/131.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: C07K 14/43536 from worms
• CPC Class Codes: C07K 14/815 from leeches, e.g. hirudin, eglin

Abstract

The complement system plays an important role in providing resistance to infections and in the pathogenesis of tissue injury. Yet an inappropriate activation of complement can result in a variety of disorders. The present invention provides C1s catalytic site-directed moieties, C1s exosite binding moieties, and bivalent polypeptide inhibitors comprising such moieties, which can be used to treat conditions characterized by inappropriate complement activation.

Claims

1. A polypeptide that inhibits complement C1s, wherein the polypeptide is characterized by the formula:

“

P-N-[DE](2)-[YX₁X₂X₃]-[DE](2)-[YX₁X₂X₃]-[DE]-[YX₁X₂X₃]-[DE](1,2),”

where amino acid residues in square brackets indicate acceptable amino acids, numbers in parentheses indicate the number of amino acid residues, “

X₁”

represents Phe-(p-CH₂)SO₃H, “

X₂”

represents sulfated tyrosine, and “

X₃”

represents 2-sulfotyrosine (SEQ ID NO;

     127).

2. The polypeptide of claim 1, wherein the polypeptide is characterized by the formula:

“

P-N-[DE](2)-[YX₁X₂X₃]-[DE](2)-[YX₁X₂X₃]-[DE]-[YX₁X₂X₃]-[DE]”

(SEQ ID NO;

129).

3. The polypeptide of claim 2, wherein the polypeptide is characterized by the formula:

“

P-N-E-E-[YX₁X₂X₃]-E-[YX₁X₂X₃]-E-[YX₁X₂X₃]-E”

(SEQ ID NO;

     130).

4. The polypeptide of claim 3, wherein the polypeptide consists of the amino acid sequence:

18. composition, comprising a carrier, and a peptide or a polypeptide of any one of claims 1, 5, or 6.

5. A polypeptide that inhibits complement C1s, wherein the polypeptide comprises an amino acid sequence that is characterized by the formula:

“

[AP]-N-[DE](2)-[X₁X₂X₃]-[DE](2)-[X₁X₂X₃]-[DE]-[X₁X₂X₃]-[DE](1,2)”

where amino acid residues in square brackets indicate acceptable amino acids, numbers in parentheses indicate the number of amino acid residues, “

X₁”

represents Phe-(p-CH₂)SO₃H, “

X₂”

represents sulfated tyrosine, and “

X₃”

represents 2-sulfotyrosine (SEQ ID NO;

     126).

6. A peptide or polypeptide that inhibits complement C1s, wherein the peptide or polypeptide comprises the amino acid sequence “

CRLGC”

(amino acid residues 64 to 68 of SEQ ID NO;

     1), wherein the peptide or polypeptide consists of five to thirty amino acid residues.

7. The peptide or polypeptide of claim 6, wherein the polypeptide consists of the amino acid sequence:

8. The peptide of claim 6, wherein the peptide consists of the amino acid sequence:

“

CRLGC.”

9. A complement C1s inhibitor, wherein the inhibitor consists of:

(a) a C1s catalytic site-directed moiety (CCSDM), which is selected from the group consisting of;

(i) CH₃-Lys(Cbo)-Gly-Arg-pNA-AcOH, where “

Cbo”

represents benzyloxycarbonyl;

(ii) CH₃-Lys(Cbo)-Gly-Arg;

(iii) H-D-Val-Ser-Arg-pNA.HCl;

(iv) H-D-Val-Ser-Arg;

(v) Leu-Xaa-Arg, where “

Xaa”

represents alanine, glutamine, or glycine;

(vi) LQRALEILPN RVTIKANRPF LVFI (SEQ ID NO;

118), (vii) serine protease inhibitor;

(viii) heterocyclic protease inhibitor;

(ix) transition state analogue;

(x) benzamidine;

(xi) X-C1-C2-A-Y, where C1 is a derivative of Arg, Lys, or Om, characterized by a reduced carboxylate moiety or a carboxylate moiety that is displaced from the α

-carbon by a chemical structure characterized by a backbone chain of from 1 to 10 atoms, C2 is a non-cleavable bond, “

X”

is hydrogen or a continuation of the peptide backbone, “

A”

is a backbone chain, and “

Y”

is a bond;

(xii) CDGFK CRLGC TYGFK TDKKG CEAFC TCNT (SEQ ID NO;

121); and

(xiii) X-C-X(8-12)-L-Q-R, where “

X”

represents glycine, serine, or threonine, and numbers in parentheses indicate the number of amino acid residues (SEQ ID NO;

140);

(b) a linker moiety that is either characterized by a backbone chain having a calculated length of between 14 Å and

20 Å

, or that is a polypeptide, which has the amino acid sequence of

KETAC VNIWC TDPYK CNPES GRCED (SEQ ID NO;

123);

 

and

(c) a C1s exosite binding moiety (CEBM), which is selected from the group consisting of;

(i) a polypeptide characterized by the formula;

“

[AP]-N-[DE](2)-[YX₁X₂X₃]-[DE](2)-[YX₁X₂X₃]-[DE]-[YX₁X₂X₃]-[DE](1,2),”

where amino acid residues in square brackets indicate acceptable amino acids, numbers in parentheses indicate the number of amino acid residues, “

X₁”

represents Phe-(p-CH2)SO₃H, “

X₂”

represents sulfated tyrosine, and “

X₃”

represents 2-sulfotyrosine (SEQ ID NO;

126); and

(ii) NEDYEDYEYD (SEQ ID NO;

119);

wherein the C1s catalytic site-directed moiety is bound to the linker moiety, the linker moiety is bound to the C1s exosite binding moiety.

10. The inhibitor of claim 9, wherein the serine protease inhibitor is selected from the group consisting of phenylmethylsulfonylfluoride, diisopropylflouorophosphate, tosylprolylchloromethylketone, and tosyllysl chloromethylketone.

11. The inhibitor of claim 9, wherein the heterocyclic protease inhibitor is an isocoumarin.

12. The inhibitor of claim 9, wherein the transition state analogue is difluoroketomethylene.

13. The inhibitor of claim 9, wherein a moiety having the formula “

X-C1-C2-A-Y”

includes a C1 component selected from the group consisting of β

-homoarginine, an arginine containing a reduced carboxylate moiety, and β

-homoornithine.

14. The inhibitor of claim 13, wherein the arginine containing a reduced carboxylate moiety is Argψ

[CH₂NH].

15. The inhibitor of claim 13, wherein the linker is selected from the group consisting of:

20. A method of inhibiting complement C1s inhibitor, comprising administering the composition of claim 18 to complement C1 s.

21. A method of inhibiting complement C1s inhibitor, comprising administering the composition of claim 19 to complement C1s.

22. The method of claim 20, wherein the composition is administered to a mammalian subject.

23. The method of claim 21, wherein the composition is administered to a mammalian subject.

16. A complement C1s inhibitor, wherein the inhibitor consists of:

(a) a C1s catalytic site-directed moiety (CCSDM), which is selected

(i) GCDGFKCRLG CTYGFKTDKK GCEAFCTCNT (SEQ ID NO;

53);

 

from the group consisting of; and

(ii) CRLGC (amino acid residues 64 to 68 of SEQ ID NO;

1);

(b) a linker moiety characterized by a backbone chain having a calculated length of between 14 Å and

20 Å

; and

(c) a C1s exosite binding moiety (CEBM), which is a polypeptide characterized by the formula;

“

A-N-[DE](2)-[YX₁X₂X₃]-[DE](2)-[YX₁X₂X₃]-[DE]-[YX₁X₂X₃]-[DE](1,2),”

where amino acid residues in square brackets indicate acceptable amino acids, numbers in parentheses indicate the number of amino acid residues, “

X₁”

represents Phe-(p-CH2)SO₃H, “

X₂”

represents sulfated tyrosine, “

X₃”

represents 2-sulfotyrosine (SEQ ID NO;

     128);

wherein the C1s catalytic site-directed moiety is bound to the linker moiety, the linker moiety is bound to the C1s exosite binding moiety.

17. The complement C1s inhibitor of any one of claims 9 or 16, wherein the inhibitor is characterized by the formula:

“

CCSDM-Linker-CEBM.”

19. A composition, comprising a carrier, and the complement C1s inhibitor of any one of claims 9 or 16.