公开(公告)号：US07456152B2

公开(公告)日：2008-11-25

申请号：US10546390

申请日：2004-02-27

申请人： Feng Ni ,  Dmitri Tolkatchev ,  Anna Natapova ,  Anatol Koutychenko

发明人： Feng Ni ,  Dmitri Tolkatchev ,  Anna Natapova ,  Anatol Koutychenko

IPC分类号： A61K38/16 ,  A61K38/00

CPC分类号： C07K14/815 ,  A61K38/00

摘要： 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 P4, P3 and P3′ sites of the active-site directed sequence, Xaa (P4)-Yaa (P3)-Pro (P2)-Arg (P1)-Pro(P1′)-Gln(P2′)-Zaa(P3′). Complementary to rational design, a phage library was constructed to explore further the residue requirements at the P4, P3 and P3′ sites for multivalent and optimized bridge-binding. Panning of the phage library has led to thrombin-inhibitory peptides possessing strong anti-cloning 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 polypepticle 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.

摘要翻译： 四肽Phe-Asn-Pro-Arg（SEQ ID NO：3）是结合到凝血酶活性位点的结构优化的序列。 通过将这种四肽或其变体与水蛭素的C末端片段缀合，我们能够产生一系列含有仅基因可编码天然氨基酸的凝血酶的新型多价抑制剂。 我们发现通过在P 3，P 3和P 3上的氨基酸残基的取代可以增强与凝血酶的活性位点和外部位点的协同结合， 活性位点定向序列的3个位点，Xaa（P 3） - Yaa（P 3-3）-Pro（P （P ） - Arg（P 1） - Pro（P 1） > 3 '）。 与理性设计相辅相成，构建了噬菌体文库，进一步探索了P＆lt; 4＆gt;，＆lt; 3＆gt;和＆lt; 3＆gt; 多重和优化的桥接。 噬菌体文库的淘选已导致凝血酶抑制肽在低纳摩尔范围内具有强抗克隆活性，但仅部分地与凝血酶的催化活性位点相互干扰。 总之，有效的和可遗传编码的凝血酶多肽抑制剂的可用性为这种临床上成功的一类抗凝剂的应用开辟了广泛的应用。 通过在基因治疗等领域通过更具成本效益的重组肽生产，以及通过并入用于靶向递送的归巢肽来提高临床疗效/安全性。

公开(公告)号：US20070042946A1

公开(公告)日：2007-02-22

申请号：US10546390

申请日：2004-02-27

申请人： Feng Ni ,  Dmitri Tolkatchev ,  Anna Natapova ,  Anatol Koutychenko

发明人： Feng Ni ,  Dmitri Tolkatchev ,  Anna Natapova ,  Anatol Koutychenko

IPC分类号： A61K38/54 ,  C12N9/99

CPC分类号： C07K14/815 ,  A61K38/00

摘要： The tetrapeptide Phe-Asn-Pro-Arg 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 P4, P3 and P3′ sites of the active-site directed sequence, Xaa(P4)-Yaa(P3)-Pro(P2)-Arg(P1)-Pro(P1′)-Gln(P2′)-Zaa(P3′). Complementary to rational design, a phage library was constructed to explore further the residue requirements at the P4, P3 and P3′ 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.

摘要翻译： 四肽Phe-Asn-Pro-Arg是用于结合凝血酶活性位点的结构优化的序列。 通过将这种四肽或其变体与水蛭素的C末端片段缀合，我们能够产生一系列含有仅基因可编码天然氨基酸的凝血酶的新型多价抑制剂。 我们发现通过在P 3，P 3和P 3上的氨基酸残基的取代可以增强与凝血酶的活性位点和外部位点的协同结合， 3个位点的活性位点定向序列Xaa（P 3） - Yaa（P 3-3）-Pro（P （P 1 SUB）-Gln（P 2） - Zaa（P＆lt; SUB＆gt; > 3'）。 与合理设计相辅相成，构建了噬菌体文库，进一步探索了P＆lt; 4＆gt;，P＆lt; 3＆gt;和＆lt; 3＆gt; 3＆gt; 多重和优化的桥接。 噬菌体文库的淘选导致凝血酶抑制肽在低纳摩尔范围内具有强烈的抗凝血活性，但仅部分地与凝血酶的催化活性位点相互干扰。 总之，有效的和可遗传编码的凝血酶多肽抑制剂的可用性为这种临床上成功的一类抗凝剂的应用开辟了广泛的应用。 通过在基因治疗等领域通过更具成本效益的重组肽生产，以及通过并入用于靶向递送的归巢肽来提高临床疗效/安全性。

公开(公告)号：US20090137779A1

公开(公告)日：2009-05-28

申请号：US12219031

申请日：2008-07-15

申请人： Feng Ni ,  Dmitri Tolkatchev ,  Anna Natapova ,  Anatol Koutychenko

发明人： Feng Ni ,  Dmitri Tolkatchev ,  Anna Natapova ,  Anatol Koutychenko

IPC分类号： C07K14/00

CPC分类号： C07K14/815 ,  A61K38/00

摘要： 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 P4, P3 and P3′ sites of the active-site directed sequence, Xaa (P4)-Yaa (P3)-Pro (P2)-Arg (P1)-Pro(P1′)-Gln(P2′)-Zaa(P3′). Complementary to rational design, a phage library was constructed to explore further the residue requirements at the P4, P3 and P3′ 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.

摘要翻译： 四肽Phe-Asn-Pro-Arg（SEQ ID NO：3）是结合到凝血酶活性位点的结构优化的序列。 通过将这种四肽或其变体与水蛭素的C末端片段缀合，我们能够产生一系列含有仅基因可编码天然氨基酸的凝血酶的新型多价抑制剂。 我们发现通过在活性位点定向序列Xaa（P4）-Yaa（P3）的P4，P3和P3'位点处的氨基酸残基的取代可以增强与凝血酶的活性位点和外部位点的协同结合， -Pro（P2）-Arg（P1）-Pro（P1'）-G1n（P2'）-Zaa（P3'）。 与理性设计相辅相成，构建了噬菌体文库，进一步探索P4，P3和P3位点的残留要求，进行多价和优化的桥接。 噬菌体文库的淘选导致凝血酶抑制肽在低纳摩尔范围内具有强烈的抗凝血活性，但仅部分地与凝血酶的催化活性位点相互干扰。 总之，有效的和可遗传编码的凝血酶多肽抑制剂的可用性为这种临床上成功的一类抗凝剂的应用开辟了广泛的应用。 通过在基因治疗等领域通过更具成本效益的重组肽生产，以及通过并入用于靶向递送的归巢肽来提高临床疗效/安全性。