Aggrecanase-1 and -2 peptide substrates and methods
OwnerAnne M. Fourie, Lars Karlsson +1inventors
InventorsAnne M. Fourie, Lars Karlsson, Fawn Coles
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Abstract
The present invention describes synthetic peptide substrates of the metalloproteases, agggrecanase-1 and/or -2 suitable for assays of enzyme activity. The invention also describes methods using these peptides to discover pharmaceutical agents that modulate these proteases.
Claims19
A peptide less than 40 amino acids in length having a cleavage site between a glutamic acid on the N-terminal side of the cleavage site and a non-polar or uncharged residue on the C-terminal side of the cleavage site and wherein the peptide is cleavable by an enzyme having the amino acid sequence of SEQ ID NO:8 and/or SEQ ID NO:9.
A method to identify a compound that inhibits Aggrecanase enzymatic activity comprising the steps of:
- contacting a test compound, an Aggrecanase, and a peptide less than 40 amino acids in length wherein the peptide comprises a cleavage site between a glutamic acid on the N-terminal side of the cleavage site and a non-polar or uncharged amino acid residue on the C-terminal side of the cleavage site and wherein the peptide is cleavable by an enzyme having an amino acid sequence corresponding to SEQ ID NO:8 and/or SEQ ID NO:9;
- and
- detecting cleavage of the peptide wherein inhibition of peptide cleavage in the presence of a test compound indicates compound inhibition of Aggrecanase enzymatic activity.
A method to detect the ability of a compound to inhibit Aggrecanase-1 or -2 enzymatic activity comprising the steps of:
- contacting a test compound, an Aggrecanase secreted by a cell, and a peptide having an amino acid sequence selected from the group consisting of SEQ.ID.NO.:3 or SEQ.ID.NO.:4;
- incubating the compound, enzyme, and peptide to permit enzymatic cleavage of the peptide;
- and
- measuring enzymatic cleavage of the peptide wherein the method is conducted in a single reaction vessel without further manipulation.
A method to identify a compound capable of inhibiting Aggrecanase activity comprising the steps;
- providing a peptide comprising an affinity moiety, an amino acid sequence selected from a group consisting of SEQ.ID.NO.:3 or SEQ.ID.NO.:4 and a detectable label, said affinity moiety and label located on opposite sides of a cleavage site encoded by the amino acid sequence;
- contacting the peptide with an affinity capture coated solid phase support for sufficient time to bind a portion of the peptide;
- washing the support to remove unbound peptide;
- contacting a solution comprising a test compound and functional enzyme with the peptide bound solid phase support for sufficient time to allow enzymatic cleavage of the peptide, thereby releasing the peptide and detectable label into the solution;
- and
- measuring changes in the quantity of the detectable label as a result of compound modulation of expected enzymatic function.
Dependencies are inferred from the claim text.