Insect metalloproteinase inhibitors

Patent metadata

Field	Value
Publication number	US-2016159885-A1
Application number	US-201414906887-A
Country	US
Kind code	A1
Filing date	Jul 28, 2014
Priority date	Jul 26, 2013
Publication date	Jun 9, 2016
Grant date	—

Abstract

A polypeptide having at least 70% homology, in particular 80%, 90% or 95% homology to the polypeptide of SEQ ID NO:2 representing the wild-type of the protein insect metalloproteinase inhibitor IMPIα and having at least one mutation at position 35, 36 and/or 39 of the amino acid sequence of IMPIα and the polypeptide having an IC 50 value to thermolysine of less than the IC 50 value of IMPIα wherein
the nonpolar amino acid isoleucine at position 35 of IMPIα is replaced either by a nonpolar amino acid selected from the group consisting of leucine, methionine and phenylalanine or by polar amino acid selected from the group consisting of cysteine, asparagine, glutamine, histidine, lysine and arginine; and/or
the nonpolar amino acid isoleucine at position 36 of IMPIα is replaced either by a nonpolar amino acid selected from the group consisting of valine, phenylalanine and tryptophan or by polar amino acid selected from the group consisting of tyrosine, serine, threonine, asparagine, glutamine, histidine, lysine and arginine; and/or
the polar amino acid position 39 of IMPIα is replaced either by the nonpolar amino acid valine or by the polar amino acids histidine or lysine.

First claim

1 . A polypeptide having at least 70% homology, in particular 80%, 90% or 95% homology to the polypeptide of SEQ ID NO: 2 representing the wild-type of the protein insect metalloproteinase inhibitor IMPIα and having at least one mutation at position 35, 36 and/or 39 of the amino acid sequence of IMPIα and the polypeptide having an IC 50 value to thermolysine of less than the IC 50 value of IMPIα wherein
the nonpolar amino acid isoleucine at position 35 of IMPIα is replaced either by a nonpolar amino acid selected from the group consisting of leucine, methionine and phenylalanine or by polar amino acid selected from the group consisting of cysteine, asparagine, glutamine, histidine, lysine and arginine; and/or
the nonpolar amino acid isoleucine at position 36 of IMPIα is replaced either by a nonpolar amino acid selected from the group consisting of valine, phenylalanine and tryptophan or by polar amino acid selected from the group consisting of tyrosine, serine, threonine, asparagine, glutamine, histidine, lysine and arginine; and/or
the polar amino acid position 39 of IMPIα is replaced either by the nonpolar amino acid valine or by the polar amino acids histidine or lysine.

2 . The polypeptide of claim 1 having the amino acid sequences of SEQ ID NOs: 10, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84.

3 . The polypeptide of claim 1 having chemical modifications in the side chain or at the N and/or C terminal for improving biological or chemical properties such as bio availability, stability, effectivity or comprising a detectable label.

4 . A fusion polypeptide comprising a wtIMPIα, wtIMPIβ and/or a polypeptide of claim 1 fused with at least one polypeptide having a physiological function selected from the group consisting of an antibody or antibody fragment, scaffolds, functional peptides, peptides useful for diagnostic applications, peptide tags enabling immobilization on technical surfaces, or a transferase.

5 . The fusion polypeptide of claim 4 being linked by a peptide of 1 to 100 amino acids in length.

6 . The fusion polypeptide of claim 4 wherein a chemical linking group conjugates the polypeptide of claim 1 and the at least one polypeptide having a physiological function.

7 . A polynucleotide coding for the polypeptide claim 1 .

8 . The polynucleotide of claim 7 operably linked to the heterologous promoter.

9 . A host cell comprising the recombinant nucleic acid of claim 7 .

10 . A method of producing a polypeptide comprising culturing the host cell of claim 9 , expression of said nucleic acid in the host cell and isolation of the polypeptide.

11 . A kit for diagnosis comprising a peptide of claim 3 .

12 . A pharmaceutical composition comprising the
polypeptide having at least 70% homology, in particular 80%, 90% or 95% homology to the polypeptide of SEQ ID NO: 2 representing the wild-type of the protein insect metalloproteinase inhibitor IMPIα, in particular having at least one mutation at position 35, 36 and/or 39 of the amino acid sequence of IMPIα, wherein the nonpolar amino acid isoleucine at position 35 of IMPIα is replaced either by a nonpolar amino acid selected from the group consisting of leucine, methionine and phenylalanine or by polar amino acid selected from the group consisting of cysteine, asparagine, glutamine, histidine, lysine and arginine; and/or the nonpolar amino acid isoleucine at position 36 of IMPIα is replaced either by a nonpolar amino acid selected from the group consisting of valine, phenylalanine and tryptophan or by polar amino acid selected from the group consisting of tyrosine, serine, threonine, asparagine, glutamine, histidine, lysine and arginine; and/or the polar amino acid position 39 of IMPIα is replaced either by the nonpolar amino acid valine or by the polar amino acids histidine or lysine, and/or the fusion polypeptide of claim 4 .

13 . The polypeptide having at least 70% homology, in particular 80%, 90% or 95% homology to the polypeptide of SEQ ID NO: 2 representing the wild-type of the protein insect metalloproteinase inhibitor IMPIα and having at least one mutation at position 35, 36 and/or 39 of the amino acid sequence of IMPIα, wherein
the nonpolar amino acid isoleucine at position 35 of IMPIα is replaced either by a nonpolar amino acid selected from the group consisting of leucine, methionine and phenylalanine or by polar amino acid selected from the group consisting of cysteine, asparagine, glutamine, histidine, lysine and arginine; and/or
the nonpolar amino acid isoleucine at position 36 of IMPIα is replaced either by a nonpolar amino acid selected from the group consisting of valine, phenylalanine and tryptophan or by polar amino acid selected from the group consisting of tyrosine, serine, threonine, asparagine, glutamine, histidine, lysine and arginine; and/or
the polar amino acid position 39 of IMPIα is replaced either by the nonpolar amino acid valine or by the polar amino acids histidine or lysine, or the fusion polypeptide of claim 4 for use in the treatment of an animal or human infected by microorganisms secreting bacterial toxins of the M4 or Metzincin family of metalloproteinases.

14 . A method for detecting the presence or activity of proteases belonging to the M4 family in a sample using the polypeptide of claim 3 .

15 . The fusion polypeptide of claim 4 wherein the scaffold is selected from the group consisting of lipocalin, ankyrin, fibronectin, transferrin, tetranectin, adnectin, albumin, uteroglobin, and protein A.

16 . A polynucleotide coding for the fusion polypeptide of claim 5 .

17 . The pharmaceutical composition of claim 12 wherein the polar amino acid position 39 of IMPIα is replaced by a polynucleotide comprising a section coding for the polypeptide of claim 1 .

18 . The polypeptide of claim 13 wherein the polar amino acid position 39 of IMPIα is replaced by the polynucleotide of claim 7 .

19 . The polypeptide of claim 13 wherein the bacterial toxins of the M4 or Metzincin family of metalloproteinases are selected from the group thermolysine, aureolysin, bacillolysin, pseudolysin, vibriolysin or anthrax npr599.

20 . A method for detection of the presence or activity of proteases belonging to the M4 family in a sample using a fusion polypeptide of claim 4 .

Assignees

• Fraunhofer Ges Forschung

Inventors

• Vilcinskas Andreas
• Salzig Mark
• Fischer Rainer

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C07K14/43563

  from insects · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title