US2009137779A1PendingUtilityA1
Peptide inhibitors of thrombin as potent anticoagulants
Est. expiryFeb 27, 2023(expired)· nominal 20-yr term from priority
A61K 38/00C07K 14/815
55
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Claims
Abstract
The tetrapeptide Phe-Asn-Pro-Arg (SEQ ID NO: 3) is a structurally-optimized sequence for binding to the active site of thrombin. By conjugating this tetrapeptide or variants thereof to a C-terminal fragment of hirudin, we were able to generate a series of new multivalent inhibitors of thrombin containing only genetically encodable natural amino acids. We found that synergistic binding to both the active site and an exosite of thrombin can be enhanced through substitutions of amino acid residues at the P 4 , P 3 and P 3 ′ sites of the active-site directed sequence, Xaa (P 4 )-Yaa (P 3 )-Pro (P 2 )-Arg (P 1 )-Pro(P 1 ′)-Gln(P 2 ′)-Zaa(P 3 ′). Complementary to rational design, a phage library was constructed to explore further the residue requirements at the P 4 , P 3 and P 3 ′ sites for multivalent and optimized bridge-binding. Panning of the phage library has led to thrombin-inhibitory peptides possessing strong anti-clotting activities in the low nanomolar range and yet interfering only partially with the catalytic active site of thrombin. In all, the availability of potent and genetically-encodable polypeptide inhibitors of thrombin opens the door for much wider applications of this clinically-successful class of anticoagulants, e.g. through more cost-effective recombinant peptide production, in areas such as gene therapy as well as to improve clinical efficacy/safety through the incorporation of homing peptides for targeted delivery.
Claims
exact text as granted — not AI-modified1 . An isolated or purified polypeptide inhibitor of thrombin consisting of all naturally-occurring amino acids, said inhibitor having general formula (1) of:
SBM-PBM-EBM (1)
wherein
SBM is a sequence moiety comprising P-subsite residues P4-P3-Pro-Arg of the thrombin inhibitor,
PBM is a sequence moiety comprising P′-subsite residues Pro-P2′-P3′-P4′-P5′-P6′-P7′ of the thrombin inhibitor, and
EBM is a peptide sequence moiety that binds to the fibrinogen recognition exosite of thrombin,
or a pharmaceutically acceptable salt thereof,
wherein P4, P3, P2′, P3′, P4′, P5′, P6′ and P7′ are natural amino acid residues, where P3′ is Arg, Leu, Ile or Met, and
wherein the P-subsite and P′-subsite residues represent a division of the peptide sequence at the peptide bond hydrolyzed specifically by thrombin.
2 . The Inhibitor of claim 1 , wherein SBM is Trp-Xaa-Pro-Arg, where Xaa is a natural amino acid residue.
3 . The inhibitor of claim 1 , wherein SBM is selected from the group consisting of:
Trp-Asp-Pro-Arg,
(SEQ ID NO: 5)
Tyr-Asn-Pro-Arg,
(SEQ ID NO: 44)
Tyr-Ser-Pro-Arg,
(SEQ ID NO: 45)
Ile-Gln-Pro-Arg,
(SEQ ID NO: 46)
Gly-Ser-Ile-Gln-Pro-Arg,
(SEQ ID NO: 47)
Ile-Asn-Pro-Arg,
(SEQ ID NO: 48)
Val-Gln-Pro-Arg,
(SEQ ID NO: 49)
Ala-Val-Pro-Arg,
(SEQ ID NO: 50)
Gly-Ser-Ala-Val-Pro-Arg,
(SEQ ID NO: 51)
Ala-Leu-Pro-Arg,
(SEQ ID NO: 52)
and
Ala-Ile-Pro-Arg.
(SEQ ID NO: 53)
4 . The inhibitor of claim 2 , wherein Xaa is Asp or Asn.
5 . The inhibitor of claim 1 , wherein PBM is selected from the group consisting of:
Pro-Gln-Arg-His-Asn-Asp-Gly,
(SEQ ID NO: 55)
Pro-Gln-Arg-Pro-Asn-Asp-Gly,
(SEQ ID NO: 56)
Pro-Gln-Ile-His-Asn-Asp-Gly,
(SEQ ID NO: 58)
Pro-Gln-Leu-His-Asn-Asp-Gly,
(SEQ ID NO: 59)
and
Pro-Gln-Met-His-Asn-Asp-Gly.
(SEQ ID NO: 60)
6 . The inhibitor of claim 1 , wherein EBM is selected from the group consisting of:
(SEQ ID NO: 64)
Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln,
(SEQ ID NO: 65)
Asp-Phe-Glu-Pro-Ile-Pro-Glu-Glu-Tyr-Leu-Gln,
(SEQ ID NO: 66)
Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Tyr-Leu-Gln,
(SEQ ID NO: 67)
Asp-Phe-Glu-Pro-Ile-Pro-Glu-Glu-Tyr-Leu,
and
(SEQ ID NO: 68)
Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Tyr-Leu.
7 . The isolated inhibitor of claim 1 , wherein said inhibitor is selected from the group consisting of:
Trp-Asp-Pro-Arg-Pro-Gln-Arg-His-Asn-Asp-Gly-Asp-Phe-Glu-Pro-Ile-Pro-
(SEQ ID NO: 22)
Glu-Glu-Tyr-Leu-Gln,
Trp-Asp-Pro-Arg-Pro-Gln-Arg-His-Asn-Asp-Gly-Asp-Tyr-Glu-Pro-Ile-Pro-
(SEQ ID NO: 23)
Glu-Glu-Tyr-Leu-Gln,
Trp-Asp-Pro-Arg-Pro-Gln-Arg-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 21)
Glu-Glu-Tyr-Leu-Gln,
Trp-Asp-Pro-Arg-Pro-Gln-Leu-His-Asn-Asp-Gly-Asp-Phe-Glu-Pro-Ile-Pro-
(SEQ ID NO: 31)
Glu-Glu-Tyr-Leu-Gln,
Phe-Asn-Pro-Arg-Pro-Gln-Ile-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 26)
Glu-Glu-Tyr-Leu-Gln,
Ile-Gln-Pro-Arg-Pro-Gln-Ile-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-
(SEQ ID NO: 27)
Glu-Tyr-Leu-Gln,
Gly-Ser-Ala-Val-Pro-Arg-Pro-Gln-Ile-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-
(SEQ ID NO: 28)
Pro-Glu-Glu-Tyr-Leu-Gln,
and
Gly-Ser-Ile-Gln-Pro-Arg-Pro-Gln-Ile-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-
(SEQ ID NO: 30)
Pro-Glu-Glu-Tyr-Leu-Gln.
8 . The inhibitor of claim 1 , wherein said inhibitor is selected from the group consisting of:
Tyr-Asn-Pro-Arg-Pro-Gln-Ile-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 72)
Glu-Glu-Tyr-Leu-Gln,
His-Tyr-Pro-Arg-Pro-Gln-Ile-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 73)
Glu-Glu-Tyr-Leu-Gln,
Ile-Leu-Pro-Arg-Pro-Gln-Met-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 76)
Glu-Glu-Tyr-Leu-Gln,
Leu-Thr-Pro-Arg-Pro-Gln-Ile-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 77)
Glu-Glu-Tyr-Leu-Gln,
Gln-Ser-Pro-Arg-Pro-Gln-Leu-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 79)
Glu-Glu-Tyr-Leu-Gln,
His-Val-Pro-Arg-Pro-Gln-Leu-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 80)
Glu-Glu-Tyr-Leu-Gln,
Ile-Asn-Pro-Arg-Pro-Gln-Met-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 83)
Glu-Glu-Tyr-Leu-Gln,
Thr-Asp-Pro-Arg-Pro-Gln-Ile-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 85)
Glu-Glu-Tyr-Leu-Gln,
Pro-Glu-Pro-Arg-Pro-Gln-Leu-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 86)
Glu-Glu-Tyr-Leu-Gln;
Tyr-Ser-Pro-Arg-Pro-Gln-Ile-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 88)
Glu-Glu-Tyr-Leu-Gln,
Ile-Gln-Pro-Arg-Pro-Gln-Met-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 90)
Glu-Glu-Tyr-Leu-Gln,
Ile-Met-Pro-Arg-Pro-Gln-Met-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 91)
Glu-Glu-Tyr-Leu-Gln,
Ile-Ile-Pro-Arg-Pro-Gln-Met-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-
(SEQ ID NO: 92)
Glu-Tyr-Leu-Gln,
Ile-His-Pro-Arg-Pro-Gln-Met-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 93)
Glu-Glu-Tyr-Leu-Gln,
Leu-His-Pro-Arg-Pro-Gln-Leu-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 97)
Glu-Glu-Tyr-Leu-Gln,
Leu-Ile-Pro-Arg-Pro-Gln-Met-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 98)
Glu-Glu-Tyr-Leu-Gln,
Met-Gln-Pro-Arg-Pro-Gln-Met-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 99)
Glu-Glu-Tyr-Leu-Gln,
Met-Met-Pro-Arg-Pro-Gln-Met-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 100)
Glu-Glu-Tyr-Leu-Gln,
Ala-Val-Pro-Arg-Pro-Gln-Ile-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 101)
Glu-Glu-Tyr-Leu-Gln,
Ala-Leu-Pro-Arg-Pro-Gln-Ile-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 102)
Glu-Glu-Tyr-Leu-Gln,
Ala-Gln-Pro-Arg-Pro-Gln-Ile-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 107)
Glu-Glu-Tyr-Leu-Gln,
Ala-Thr-Pro-Arg-Pro-Gln-Leu-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 108)
Glu-Glu-Tyr-Leu-Gln,
Pro-Ile-Pro-Arg-Pro-Gln-Ile-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-
(SEQ ID NO: 110)
Glu-Tyr-Leu-Gln,
Pro-His-Pro-Arg-Pro-Gln-Ile-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 111)
Glu-Glu-Tyr-Leu-Gln,
Pro-Trp-Pro-Arg-Pro-Gln-Leu-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 112)
Glu-Glu-Tyr-Leu-Gln,
Thr-Ile-Pro-Arg-Pro-Gln-Ile-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-
(SEQ ID NO: 114)
Glu-Tyr-Leu-Gln,
and
Asn-Ser-Pro-Arg-Pro-Gln-Leu-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-
(SEQ ID NO: 116)
Glu-Glu-Tyr-Leu-Gln.
9 . The inhibitor of claim 2 , wherein the Trp-Xaa-Pro-Arg is at the carboxy-terminal end of the SBM.
10 . The inhibitor of claim 9 , wherein Xaa is Asp or Asn.
11 . The inhibitor of claim 3 , wherein the SEQ ID NO:5, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53 is at the carboxy-terminal end of SBM.
12 . The inhibitor of claim 6 , wherein the SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68 is at the amino-terminal end of the EBM.Cited by (0)
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