Serine proteases, their activity and their synthetic inhibitors

Details Serine proteases, their activity and their synthetic inhibitors Serine proteases, their activity and their synthetic inhibitors

1997-02-19

2000-07-18

MacMillan, Keith D.

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

514 20, 514 2, 530330, 540130, A61K 3805, C07K 5078

Patent

active

06090786&

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

The present invention lies in the field of serine proteases, in particular serine proteases cleaving a post proline bond, such as the exopeptidase dipeptidyl peptidase IV (DPPIV) and the endopeptidase prolyl oligopeptidase (PO or PEP).
The invention relates to synthetic inhibitors of PEP and dipeptidyl peptidase IV, methods of their preparation and their therapeutic and diagnostic use.
Many biologically important peptide sequences contain proline. It confers unique conformational constraints on the peptide chain because the .alpha.-nitrogen atom of proline is part of the rigid pyrrolidine ring and, at the same time, is covalently bound by means of a secondary amide bond to the N-terminal preceding amino acid. Thus, the side-chain is cyclized back on to the backbone amide position. Inside an .alpha.-helix the possibility of making hydrogen bonds to the preceding turn is thereby lost and a kink will be introduced in the helix. The conformational restrictions imposed by proline motifs in a peptide chain probably implicate important structural or biological functions since a high degree of conservation is bound in many proteins and peptides.
An endo or C-terminal Pro-Pro bond and an endo pre-Pro peptide bond possess a high degree of resistance to any mammalian proteolytic enzyme. Only a limited number of peptidases are known to be able to hydrolyse proline adjacent bonds. Their activity is restricted by the isomeric state and the position of proline in the peptide chain. Dipeptidyl peptidase IV (DPPIV, also commonly referred to as CD26) is one of these peptidases. It is a serine protease which specifically cleaves N-terminal dipeptides from proteins and polypeptides carrying an unsubstituted N-terminus and a penultimate proline (or alanine) residue in trans conformation. DPPIV is a homodimer with subunit molecular weight of 110 kDa. It is a membrane protein expressed on the surface of lymphoid cells, epithelial and endothelial cells. Surface expression is increased significantly by T-cell activation. DPPIV has been reported to play a role in numerous processes such as T-cell costimulation, binding to proteins of the extracellular matrix, attachment of cancer cells to endothelium, binding to plasma adenosine deaminase, cooperation with entry of human immunodeficiency virus (HIV) into lymphoid cells.
The proteolytic activity of DPPIV resides in a stretch of approximately 200 amino acids located at the C-terminal end of the protein. The catalytic residues (Ser-629, Asp-708, His-740) are arranged in a unique order which is different from the classical serine proteases such as chymotrypsin and subtilisin. Proline specific dipeptidyl peptidase activity alters the biological activity of a large number of bioactive proteins and polypeptides comprising, amongst others, the neurotransmitter substance P, human growth hormone-releasing factor, erythropoietin, interleukin 2 and many others. Since many of these peptides have important effector functions, abnormal DPPIV activity--either too low or too high--will be reflected in an abnormal biological effect. Potential DPPIV substrates are listed in tables 1 and 2. The skilled person will easily recognize that interference with the effector function of these polypeptides may result in clinical conditions including inflammation, vascular diseases, auto-immune disease, multiple sclerosis, joint diseases and diseases associated with benign and malign cell transformation.


TABLE 1 ______________________________________ Human cytokines, growth factors, neuro- and vasoactive peptides with a penultimate proline, which are putative substrates for DPP IV Polypeptide N-terminal sequence ______________________________________ Interleukin-1.beta. Ala-Pro-Val-Arg-Ser- Interleukin-2 Ala-Pro-Thr-Ser-Ser- Interleukin-5 Ile-Pro-Thr-Glu-Ile- Interleukin-6 Val-Pro-Pro-Gly-Glu- Interleukin-10 Ser-Pro-Gly-Gln-Gly- Interleukin-13 (recombinant) Ser-Pro-Gly-Pro-Val- Complement C4a Lys-Pro-Arg-Leu-Leu- Granulocyte chemotactic protei

REFERENCES:
patent: 5543396 (1996-08-01), Powers et al.
patent: 5681821 (1997-10-01), Powers et al.
patent: 5686419 (1997-11-01), Powers et al.
Belyaer et al, Tetrahedron Letters, vol. 36, (21), pp. 3755-3758, (1995).
Eltze, Arzheim-Forsch ./Drug Res., 30 (II), No. 7, pp. 1129-1134, (1980).
Harada et al, Biochimica Biophysica Acta, vol. 705, pp. 288-290, (1982).
Uchin, et al, Int. J. Peptide Protein Res., vol. 34, pp. 33-36 (1989).

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