Fluorogenic peptides for the detection of protease activity
AssigneeOncoimmunin, Inc.
InventorsAkira Komoriya, Beverly S. Packard
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Abstract
The present invention provides for novel reagents whose fluorescence increases in the presence of particular proteases. The reagents comprise a characteristically folded peptide backbone each end of which is conjugated to a fluorophore. When the folded peptide is cleaved, as by digestion with a protease, the fluorophores provide a high intensity fluorescent signal at a visible wavelength. Because of their high fluorescence signal in the visible wavelengths, these protease indicators are particularly well suited for detection of protease activity in biological samples, in particular in frozen tissue sections. Thus this invention also provides for methods of detecting protease activity in situ in frozen sections.
Claims38
A fluorogenic composition for the detection of the activity of a protease, said composition having the formula:
- ##STR19## wherein, P is a peptide comprising a protease binding site for said protease, said binding site consisting of about 2 to about 8 amino acids;
- F.sup.1 and F.sup.2 are fluorophores and F.sup.1 is attached to the amino terminal amino acid and F2 is attached to the carboxyl terminal amino acid;
- S.sup.1 and S.sup.2, when present, are peptide spacers ranging in length from 1 to about 50 amino acids and S.sup.1, when present, is attached to the amino terminal amino acid and S.sup.2, when present, is attached to the carboxyl terminal amino acid;
- i, j, k, 1, m, n, o, p, q, and r are independently 0 or 1;
- aa.sup.1 and aa.sup.10 are independently selected from the group consisting of lysine, ornithine and cysteine;
- aa.sup.2, aa.sup.3, aa.sup.8, and aa.sup.9 are independently selected from the group consisting of Asp, Glu, Lys, Ornithine, Arg, Citulline, homocitrulline, Ser, homoserine, Thr, and Tyr;
- aa.sup.5, aa.sup.4, aa.sup.6, and aa.sup.7 are independently selected from the group consisting of proline, 3,4-dehydroproline, hydroxyproline, alpha aminoisobutyric acid and N-methyl alanine;
- X is selected from the group consisting of Gly, .beta.Ala, .gamma.Abu ,Gly-Gly, Ahx, .beta.Ala-Gly, .beta.Ala-.beta.Ala, .gamma.Abu-Gly, .beta.Ala-.gamma.Abu, Gly-Gly-Gly (SEQ ID NO:37), .gamma.Abu-.gamma.Abu, Ahx-Gly, .beta.Ala-Gly-Gly, Ahx-.beta.Ala, .beta.Ala-.beta.Ala-Gly, Gly-Gly-Gly-Gly, Ahx-.gamma.Abu, .beta.Ala-.beta.Ala-.beta.Ala, .gamma.Abu-.beta.Ala-Gly, .gamma.Abu-.gamma.Abu-Gly, Ahx-Ahx, .gamma.Abu-.gamma.Abu-.beta.Ala, and Ahx-Ahx-Gly;
- Y is selected from the group consisting of Gly, .beta.Ala, .gamma.Abu, Gly-Gly, Ahx, Gly-.beta.Ala, .beta.Ala-.beta.Ala, Gly-.gamma.Abu, .gamma.Abu-.beta.Ala, Gly-Gly-Gly, .gamma.Abu-.gamma.Abu, Gly-Ahx, Gly-Gly-.beta.Ala,.beta.Ala-Ahx, Gly-.beta.Ala-.beta.Ala, Gly-Gly-Gly-Gly (SEQ ID NO:37), .gamma.Abu-Ahx, .beta.Ala-.beta.Ala-.beta.Ala, Gly-.beta.Ala-.gamma.Abu, Gly-.gamma.Abu-.gamma.Abu, Ahx-Ahx, .beta.Ala-.gamma.Abu-.gamma.Abu, and Gly-Ahx-Ahx;
- when i is 1, S.sup.1 is joined to aa.sup.1 by a peptide bond through a terminal alpha amino group of aa.sup.1 ;
- and
- when r is 1, S.sup.2 is joined to aa.sup.10 by a peptide bond through a terminal alpha carboxyl group of aa.sup.10.
Dependencies are inferred from the claim text.