Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 25 or more amino acid residues in defined sequence
Patent
1990-11-19
1994-12-13
Draper, Garnette D.
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
25 or more amino acid residues in defined sequence
435 692, 4351721, C07K 710, C12N 1500
Patent
active
053730909
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to novel aprotinin analogues and to a process for their production.
BACKGROUND OF THE INVENTION
Throughout the present specification the term naturally occuring amino acid (or amino acid residue) refers to one of the .alpha.-amino acids which are listed below together with the symbols used to designate the individual amino acid residues:
______________________________________ Asp Aspartic acid Ile Isoleucine
Thr Threonine Leu Leucine
Ser Serine Tyr Tyrosine
Glu Glutamine acid
Phe Phenylalanine
Pro Proline His Histidine
Gly Glycine Lys Lysine
Ala Alanine Arg Arginine
Cys Cysteine Trp Tryptophane
Val Valine Gln Glutamine
Met Methionine Asn Asparagine
______________________________________
All amino acids (or amino acid residues) mentioned in the present specification have the L-configuration, except glycine which has no chiral center.
Aprotinin (bovine pancreatic trypsin inhibitor, BPTI) is a polypeptide present in several bovine organs and tissues, such as lymph nodes, pancreas, lung, parotid gland, spleen and liver. It is a single chain polypeptide consisting of 58 amino acid residues cross-linked by three disulphide bridges in the following sequence: ##STR1##
The three disulphide bridges are situated between Cys(5)-Cys(55), Cys(14)-Cys(38) and Cys(30)-Cys(51), respectively.
Aprotinin inhibits various serine proteases, such as trypsin, chymotrypsin, plasmin and kallikrein and is used for the treatment of acute pancreatitis, various states of shock syndroms, hyperfibrinolytic haemorrhage, and myocardial infarction. Administration of aprotinin in high doses significantly reduces blood loss in connection with cardiac surgery.
Aprotinin is extracted from various bovine organs or tissues, such as lung, pancreas and parotid glands. Extraction from animal tissues is a cumbersome process and requires large amounts of the bovine organ or tissue. Aprotinin may also be produced by recombinant DNA-technology by insertion of a gene coding for aprotinin in a suitable microorganism which when cultured in a suitable nutrient medium produces the desired product.
Production of aprotinin analogues in E. coli is described in EP published patent application No. 238,993 and production of aprotinin in yeast is described in Danish patent application No. 4501/87.
Certain aprotinin analogues and derivatives have been described, see for instance Jering H. and Tschesche H., Eur.J.Biochem. 61 (1976), 453-463 describing replacement of Lys(15) with Arg, Phe or Trp or U.S. Pat. No. 4,595,674 describing aprotinin analogues in which the lysine residue in position 15 in the active center of the aprotinin has been replaced by Gly, Ala, Val, Leu, Ile, Met, Arg, L-.alpha.-amino butyric acid, L-norvaline, L-norleucine, dehydroalanine or L-homoserine. Also, the above mentioned EP No. 238,993 describes aprotinin analogues having Lys(15) substituted by Arg, Val, Ile, Leu, Phe, Gly, Ser, Trp, Tyr or Ala and/or Met(52) substituted by Glu, Leu, Val, Thr or Ser.
The known aprotinin analogues are claimed to have modified effects and efficacies towards different proteinases. For instance aprotinin(15 Val) has a relatively high selectivity for granulocyte elastase and an inhibition effect on collagenase, aprotinin(15Ala) has only a weak inhibitory effect on elastase and aprotinin(15Gly) has an outstanding antitrypsin activity and surprisingly inhibits kallikrein.
SUMMARY OF THE INVENTION
It is the purpose of the present invention to provide novel aprotinin analogues having a more specific inhibitory effect towards certain serine proteases, such as elastase, kallikrein, t-PA, urokinase and coagulation factors, such as thrombin.
The present invention is based on the surprising fact that replacement of Arg in position 17 of aprotinin(3-58;42Ser) with Ala or of Arg in position 17 with Ala and of Ile in position 19 of aprotinin (3-58;42Ser) with Glu gives rise to a substantial increase in the inhibition of human plasma kallikrein. This is even more pronounced on replacement
REFERENCES:
patent: 4595674 (1986-06-01), Tschesche et al.
patent: 5118668 (1992-06-01), Auerswald et al.
Schnabel et al., Biol. Chem. Hoppe-Seyler, vol. 367, pp. 1167-1176 (1986).
Fiovett: et al. "Primary Structure and Antiproteolytic Activity of a Kunits-type Inhibitor from Bovine Spleam." JBC, 260 (21) 11451-11455. 1985.
Norris Kjeld
Petersen Lars C.
Agris Cheryl H.
Draper Garnette D.
Novo Nordisk A S
Teng Sally P.
Zelson Steve T.
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