Claims for Patent: 6,127,139

Summary for Patent: 6,127,139

Title:	Method for assaying proteolytic enzymes using fluorescence-quenched substrates
Abstract:	A method is disclosed for assaying a proteolytic enzyme comprising: (a) incubating an enzyme-containing sample with an immobilized fluorescence-quenched peptide having the formula Que-Sub-Flu-Spa-Car or Flu-Sub-Que-Spa-Car wherein Sub is a peptide chain containing a specific cleavage site for said proteolytic enzyme; Flu is a fluorophore; Que is a quencher capable of absorbing fluorescent radiation emitted by the fluorophore; Spa is a direct bond or a spacing chain; and Car is a water-insoluble and/or macromolecular carrier; (b) optionally separating the liquid from the carrier material; (c) irradiating said carrier material and measuring fluorescence. Also disclosed are immobilized substrates containing specific amino acid sequences for use in such an assay, especially in an assay for aggrecanase, and metalloproteinase-1, -3 and -13 activity.
Inventor(s):	Te Koppele; Johannes Maria (Leiderdorp, NL), Beekman; Bob (Leiden, NL)
Assignee:	Nederlands Organisatle voor Toegepast-Natuurwetenschappelijk Onderzoek (Delft, NL)
Application Number:	09/101,167
Patent Claims:	1. A method for assaying a proteolytic enzyme comprising: (a) incubating an enzyme-containing sample with an immobilized fluorescence-quenched peptide having the formula or wherein Sub is a peptide chain containing a specific cleavage site for said proteolytic enzyme; Flu is a fluorophore; Que is a quencher capable of absorbing fluorescent radiation emitted by the fluorophore; Spa is a direct bond or a spacing chain; and Car is an water-insoluble and/or macromolecular carrier, (b) optionally separating the liquid from the carrier material; (c) irradiating said carrier material and measuring fluorescence. 2. The method according to claim 1, wherein Flu is an aminonaphthalenesulphonic acid group, and Que is an aminophenylazobenzoyl group. 3. The method according to claim 1, wherein Car is a polyacrylate or polyacrylamide. 4. The method according to claim 1, wherein Sub contains the amino acid sequence Xab-Glu-Xad-Xae (SEQ ID NO:31), wherein Xab is Gly, Ala, Abu, Val, Nva, Leu, Glu or Gln, Xad is Gly, Ala, Abu, Val, Nva or Leu, and Xae is Arg, Gly, Ala, Abu, Val, Nva or Leu, for assaying aggrecanase activity. 5. The method according to claim 1, wherein Sub contains the amino acid sequence Xai-Asn-Phe-Xaj (SEQ ID NO:32), wherein Xai is Pro or Hyp, and Xaj is Phe, Tyr, Trp or Cha, for assaying aggrecan degradation by matrix metalloproteinases. 6. The method according to claim 1 wherein Sub contains the amino acid sequence Xad-Arg-Xaf-Xag (SEQ ID NO:33), wherein Xad is Gly, Ala, Abu or Val, Xaf is Gly or Ala, and Xag is any amino acid, Ser, Thr, Ala, Abu or Hse, or the amino acid sequence Xam-Xan-Xao-Glu, (SEQ ID NO:34) wherein Xam is Arg, Lys, Orn, His or Trp, especially Arg, Xan is Gly, Ala, Abu or Val, Xao is Ala, Abu, Val, Nva, Leu, Nle or Ile, especially Leu, for assaying matrix metalloproteinase-13 activity. 7. The method according to claim 1 wherein said immobilized peptide is covalently or non-covalently bound to a solid support, such as a glass plate, for assaying proteinase activity in tissue sections. 8. An immobilized substrate for assaying a proteolytic enzyme, containing a fluorescence-quenched peptide having the formula or wherein Sub is a peptide chain containing a specific cleavage site for said proteolytic enzyme; Flu is a fluorophore; Que is a quencher capable of absorbing fluorescent radiation emitted by the fluorophore; Spa is a direct bond or a spacing chain; and Car is an insoluble and/or macromolecular carrier selected from a glass or polymer bead, a glass plate, a microtiter plate, a paper or synthetic polymer strip, an acrylate or acrylamide gel and a protein. 9. A labelled oligopeptide as a substrate for aggrecanase activity comprising one of the following sequences: Gly-Glu-Ala-Arg (SEQ ID NO:35), Leu-Glu-Gly-Arg (SEQ ID NO:36), Glu-Glu-Gly-Leu (SEQ ID NO:37), Gln-Glu-Ala-Gly (SEQ ID NO:38) and Gln-Glu-Leu-Gly (SEQ ID NO:39). 10. A labelled oligopeptide as a substrate for matrix metalloproteinase-13 activity, comprising the amino acid sequence Xad-Arg-Xaf-Leu (SEQ ID NO:40) wherein Xad is Pro or Hyp and Xaf is Gly, Ala, Abu or Val. 11. A labelled oligopeptide as a substrate for aggrecanase activity comprising the amino acid sequence Xab-Glu-Xad-Xae (SEQ ID NO:31), wherein Xab is Gly, Ala, Abu, Val, Nva, Leu, Glu or Gln, Xad is Gly, Ala, Abu, Val, Nva or Leu, and Xae is Arg, Gly, Ala, Abu, Val, Nva or Leu, to which a fluorescent label and preferably also a quenching group are bound. 12. The oligopeptide according to claim 11, comprising the amino acid sequence Xaa-Xab-Glu-Xad-Xae-Xaf (SEQ ID NO:41), wherein Xaa is Glu, Gln, Ala, Ser or Thr, and/or Xaf is Gly, Glu or Gln, and Xab, Xad and Xae are as defined in claim 11. 13. A labelled oligopeptide as a substrate for aggrecan degrading activity by matrix metalloproteinases comprising the amino acid sequence Xah-Xai-Asn-Phe-Xaj-Xak (SEQ ID NO:42), wherein Xah is Ala, Abu, Val, Nva, Leu, Nle or Ile, Xai is Pro or Hyp, Xaj is Phe, Tyr, Trp or Cha, and Xak is Ala, Abu, Val, Nva, Ile, Leu or Gly, to which a fluorescent label are bound. 14. A labelled oligopeptide as a substrate for matrix metalloproteinase-13 activity, comprising the amino acid sequence Xad-Arg-Xaf-Xag (SEQ ID NO:33), wherein Xad is Gly, Ala, Abu or Val, Xaf is Gly or Ala, and Xag is an aliphatic amino acid, to which a fluorescent label are bound. 15. A labelled oligopeptide as a substrate for matrix metalloproteinase-13 activity, comprising the amino acid sequence Xal-Xam-Xan-Xao-Xap-Xaq (SEQ ID NO:34), wherein Xal is Pro or Hyp, Xam is Arg, Lys, Orn, His or Trp, Xan is Gly, Ala, Abu or Val, Xao is a direct bond or Gly, Ala, Abu, Val, Nva, Leu, Nle or Ile, Xap is an amino acid capable of coupling to an amino group, and Xaq is Gly, Ala, Abu, Val, Nva, Leu, Nle or Ile, to which a fluorescent label are bound. 16. The oligopeptide according to claim 9, having a fluorescent label and a quenching group. 17. A labelled oligopeptide as a substrate for matrix metalloproteinase-1 (interstitial collagenase) activity, comprising the amino acid sequence Cha-Abu-Smc-His (SEQ ID No. 29) to which a fluorescent label and a quenching group are bound. 18. A labelled oligopeptide as a substrate for matrix metalloproteinase-3 (stromelysin) activity, comprising the amino acid sequence Val-Glu-Nva-Trp (SEQ ID No. 30), to which Edans as a fluorescent label and Dabcyl as a quenching group are bound. 19. The oligopeptide according to claim 9, bound to a water-insoluble carrier or to a protein.

Details for Patent 6,127,139

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2016-01-04