Aprotinin analogues and process for the production thereof

Claim 2

Aprotinin analogues having the formula X₁-Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-X₂-X₃-X₄-X₅-X₆-X₇-­X₈-X₉ Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-Cys-Gln-Thr-­Phe-Val-Tyr-Gly-Gly-X₁₀-Arg-Ala-X₁₁-X₁₂-Asn-Asn-Phe-Lys-­Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala in which X₁ means Arg-Pro, Pro or hydrogen, X₄ and X₁₀ are both Cys or both Ala or both another naturally occuring amino acid residue, X₂ and X₃ are independently any naturally occuring amino acid residue; X₅ is Lys, Arg, Val, Thr, Ile, Leu, Phe, Gly, Ser, Met, Trp, Tyr or Ala with the proviso that when X₅ is different from Lys then at least one of X₂ to X₄ and X₆ to X₉ is different from the amino acid residue at the corresponding position in native aprotinin; X₆ is Ala or Gly; X₇ is any naturally ocurring amino acid residue; X₈ is Ile, Leu, Met, Val or Phe; X₉ is any naturally occuring amino acid residue; X₁₁ is any naturally occuring amino acid residue; X₁₂ is Lys, Arg or Ser at least one of the amino acid residues X₂ to X₉ being different from the corresponding amino acid residue in native aprotinin.

Claim 3

Aprotinin analogues according to Claim 1, wherein X₁ is Arg-Pro, Pro or hydrogen, preferably Arg-Pro or hydrogen, most preferred hydrogen.

Claim 4

Aprotinin analogues according to Claim 1 or 2, wherein X₂ is Gly.

Claim 5

Aprotinin analogues according to any of the preceding claims, wherein X₃ is Pro.

Claim 6

Aprotinin analogues according to any of the preceding claims, wherein X₄ and X₁₀ are Cys.

Claim 7

Aprotinin analogues according to any of the preceding claims wherein X₅ is Lys, Arg, Val, Thr, Ile, Leu, Phe, Gly, Ser, Met, Trp, Tyr or Ala, preferably Lys or Arg with the proviso that when X₅ is different from Lys, then at least one of X₂ to X₄ and X₆ to X₉, preferably X₆ to X₉ is different from the amino acid residue at the corresponding position in native aprotinin.

Claim 8

Aprotinin analogues according to any of the preceding claims, wherein X₆ is Ala or Gly, preferably Ala.

Claim 9

Aprotinin analogues according to any of the preceding claims, wherein X₇ is Ala or Gly, preferably Ala.

Claim 10

Aprotinin analogues according to any of the preceding claims wherein X₈ is Ile, Leu, Met, Val or Phe, preferably Ile.

Claim 11

Aprotinin analogues according to any of the preceding claims wherein X₉ is Ile or Glu, preferably Ile.

Claim 12

Aprotinin analogues according to any of the preceding claims wherein X₁₁ is Lys or Arg, preferably Lys.

Claim 13

Aprotinin analogues according to any of the preceding claims wherein X₁₂ is Lys, Arg o Ser, preferably Arg or Ser.

Claim 14

Aprotinin analogue according to claim 1 having the following structure: Arg-Pro-Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-Cys-­Lys-Ala-Ala-Ile-Glu-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-­Leu-Cys-Gln-Thr-Phe-Val-Tyr-Gly-Gly-Cys-Arg-Ala-Lys-Arg-­Asn-Asn-Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-­Gly-Ala.

Claim 15

Aprotinin analogue according to claim 1 having the following structure: Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-Cys-Lys-Ala-­Ala-Ile-Glu-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-Cys-­Gln-Thr-Phe-Val-Tyr-Gly-Gly-Cys-Arg-Ala-Lys-Arg-Asn-Asn-­Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala.

Claim 16

Aprotinin analogue according to claim 1 having the following structure: Arg-Pro-Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-Cys-­Lys-Ala-Ala-Ile-Glu-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-­Leu-Cys-Gln-Thr-Phe-Val-Tyr-Gly-Gly-Cys-Arg-Ala-Lys-Ser-­Asn-Asn-Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-­Gly-Ala.

Claim 17

Aprotinin analogue according to claim 1 having the following structure: Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-Cys-Lys-Ala-­Ala-Ile-Glu-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-Cys-­Gln-Thr-Phe-Val-Tyr-Gly-Gly-Cys-Arg-Ala-Lys-Ser-Asn-Asn-­Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala.

Claim 18

Aprotinin analogue according to claim 1 having the following structure: Arg-Pro-Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-Cys-­Lys-Ala-Ala-Ile-Ile-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-­Leu-Cys-Gln-Thr-Phe-Val-Tyr-Gly-Gly-Cys-Arg-Ala-Lys-Arg-­Asn-Asn-Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-­Gly-Ala.

Claim 19

Aprotinin analogue according to claim 1 having the following structure: Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-Cys-Lys-Ala-­Ala-Ile-Ile-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-Cys-­Gln-Thr-Phe-Val-Tyr-Gly-Gly-Cys-Arg-Ala-Lys-Arg-Asn-Asn-­Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala.

Claim 20

Aprotinin analogue according to claim 1 having the following structure: Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-Cys-Lys-Ala-­Ala-Ile-Ile-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-Cys-­Gln-Thr-Phe-Val-Tyr-Gly-Gly-Cys-Arg-Ala-Lys-Ser-Asn-Asn-­Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala.

Claim 21

Aprotinin analogue according to claim 1 having the following structure: Arg-Pro-Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-Cys-­Lys-Ala-Ala-Ile-Ile-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-­Leu-Cys-Gln-Thr-Phe-Val-Tyr-Gly-Gly-Cys-Arg-Ala-Lys-Ser-­Asn-Asn-Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-­Gly-Ala.

Claim 22

Aprotinin analogue according to claim 1 having the following structure: Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-Cys-Arg-Ala-­Ala-Ile-Ile-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-Cys-­Gln-Thr-Phe-Val-Tyr-Gly-Gly-Cys-Arg-Ala-Lys-Ser-Asn-Asn-­Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala.

Claim 23

Aprotinin analogue according to claim 1 having the following structure: Arg-Pro-Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-Gly-Pro-Cys-­Arg-Ala-Ala-Ile-Ile-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-­Leu-Cys-Gln-Thr-Phe-Val-Tyr-Gly-Gly-Cys-Arg-Ala-Lys-Ser-­Asn-Asn-Phe-Lys-Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-­Gly-Ala.

Claim 24

A process for producing aprotinin analogues according to the general formula (I): X₁-Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-X₂-X₃-X₄-X₅-X₆-X₇-­X₈-X₉-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-Cys-Gln-Thr-­Phe-Val-Tyr-Gly-Gly-X₁₀-Arg-Ala-X₁₁-X₁₂-Asn-Asn-Phe-Lys-­Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala      (I) wherein X₁ is Arg-Pro, Pro or hydrogen; X₂ is any naturally occuring amino acid residue, preferably Gly; X₃ is any naturally occuring amino acid residue, preferably Pro; X₄ and X₁₀ are any naturally occuring amino acid residues, preferably they are both Ala or both Cys; X₅ is Lys, Arg, Val, Thr, Ile, Leu, Phe, Gly, Ser, Met, Trp, Tyr or Ala, preferably Lys or Arg, with the proviso, that when X₅ is different from Lys, then at least one of X₂ to X₄ and X₆ to X₉ is different from the amino acid residue at the corresponding position in native aprotinin; X₆ is Ala or Gly, preferably Ala; X₇ is any naturally occuring amino acid residue, preferably Ala or Gly; X₈ is Ile, Leu, Met, Val or Phe, preferably Ile; X₉ is any naturally occuring amino acid residue, preferably Ile or Glu; X₁₁ is any naturally occuring amino acid residue, preferably Lys or Arg and X₁₂ is Lys, Arg or Ser, preferably Arg or Ser, at least one of the amino acid residues X₂ to X₉, preferably X₅ to X₉, more preferred X₆ to X₉ being different from the corresponding amino acid residue in native protein in yeast comprising culturing a yeast strain containing a replicable expression vector, the vector comprising a gene coding for the aprotinin analogue and DNA sequences that allow for the expression of the aprotinin analogue in a suitable nutrient medium and recovering the expressed aprotinin analogues.

Claim 25

A process for producing aprotinin analogues according to the general formula (I): X₁-Asp-Phe-Cys-Leu-Glu-Pro-Pro-Tyr-Thr-X₂-X₃-X₄-X₅-X₆-X₇-­X₈-X₉-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-Cys-Gln-Thr-­Phe-Val-Tyr-Gly-Gly-X₁₀-Arg-Ala-X₁₁-X₁₂-Asn-Asn-Phe-Lys-­Ser-Ala-Glu-Asp-Cys-Met-Arg-Thr-Cys-Gly-Gly-Ala      (I) wherein X₁ is Arg-Pro, Pro or hydrogen; X₂ is any naturally occuring amino acid residue, preferably Gly; X₃ is any naturally occuring amino acid residue, preferably Pro; X₄ and X₁₀ are any naturally occuring amino acid residues, preferably they are both Ala or both Cys; X₅ is Lys, Arg, Val, Thr, Ile, Leu, Phe, Gly, Ser, Met, Trp, Tyr or Ala, preferably Lys or Arg, with the proviso, that when X₅ is different from Lys, then at least one of X₂ to X₄ and X₆ to X₉ is different from the amino acid residue at the corresponding position in native aprotinin; X₆ is Ala or Gly, preferably Ala; X₇ is any naturally occuring amino acid residue, preferably Ala or Gly; X₈ is Ile, Leu, Met, Val or Phe, preferably Ile; X₉ is any naturally occuring amino acid residue, preferably Ile or Glu; X₁₁ is any naturally occuring amino acid residue, preferably Lys or Arg and X₁₂ is Lys, Arg or Ser, preferably Arg or Ser, at least one of the amino acid residues X₂ to X₉, preferably X₅ to X₉, more preferred X₆ to X₉ being different from the corresponding amino acid residue in native protein in yeast comprising culturing a yeast strain containing a replicable expression vector, the vector comprising a gene coding for the aprotinin analogue and DNA sequences that allow for the expression of the aprotinin analogue in a suitable nutrient medium and recovering the expressed aprotinin analogues.