Fluorogenic peptides for the detection of protease activity
First Claim
1. 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;
F1 and F2 are fluorophores and F1 is attached to the amino terminal amino acid and F2 is attached to the carboxyl terminal amino acid;
S1 and S2, when present, are peptide spacers ranging in length from 1 to about 50 amino acids and S1, when present, is attached to the amino terminal amino acid and S2, 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;
aa1 and aa10 are independently selected from the group consisting of lysine, ornithine and cysteine;
aa2, aa3, aa8, and aa9 are independently selected from the group consisting of Asp, Glu, Lys, Ornithine, Arg, Citulline, homocitrulline, Ser, homoserine, Thr, and Tyr;
aa5, aa4, aa6, and aa7 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, β
Ala, γ
Abu ,Gly-Gly, Ahx, β
Ala-Gly, β
Ala-β
Ala, γ
Abu-Gly, β
Ala-γ
Abu, Gly-Gly-Gly (SEQ ID NO;
37), γ
Abu-γ
Abu, Ahx-Gly, β
Ala-Gly-Gly, Ahx-β
Ala, β
Ala-β
Ala-Gly, Gly-Gly-Gly-Gly, Ahx-γ
Abu, β
Ala-β
Ala-β
Ala, γ
Abu-β
Ala-Gly, γ
Abu-γ
Abu-Gly, Ahx-Ahx, γ
Abu-γ
Abu-β
Ala, and Ahx-Ahx-Gly;
Y is selected from the group consisting of Gly, β
Ala, γ
Abu, Gly-Gly, Ahx, Gly-β
Ala, β
Ala-β
Ala, Gly-γ
Abu, γ
Abu-β
Ala, Gly-Gly-Gly, γ
Abu-γ
Abu, Gly-Ahx, Gly-Gly-β
Ala,β
Ala-Ahx, Gly-β
Ala-β
Ala, Gly-Gly-Gly-Gly (SEQ ID NO;
37), γ
Abu-Ahx, β
Ala-β
Ala-β
Ala, Gly-β
Ala-γ
Abu, Gly-γ
Abu-γ
Abu, Ahx-Ahx, β
Ala-γ
Abu-γ
Abu, and Gly-Ahx-Ahx;
when i is 1, S1 is joined to aa1 by a peptide bond through a terminal alpha amino group of aa1 ; and
when r is 1, S2 is joined to aa10 by a peptide bond through a terminal alpha carboxyl group of aa10.
1 Assignment
0 Petitions
Accused Products
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.
133 Citations
38 Claims
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1. 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;
F1 and F2 are fluorophores and F1 is attached to the amino terminal amino acid and F2 is attached to the carboxyl terminal amino acid; S1 and S2, when present, are peptide spacers ranging in length from 1 to about 50 amino acids and S1, when present, is attached to the amino terminal amino acid and S2, 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; aa1 and aa10 are independently selected from the group consisting of lysine, ornithine and cysteine; aa2, aa3, aa8, and aa9 are independently selected from the group consisting of Asp, Glu, Lys, Ornithine, Arg, Citulline, homocitrulline, Ser, homoserine, Thr, and Tyr; aa5, aa4, aa6, and aa7 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, β
Ala, γ
Abu ,Gly-Gly, Ahx, β
Ala-Gly, β
Ala-β
Ala, γ
Abu-Gly, β
Ala-γ
Abu, Gly-Gly-Gly (SEQ ID NO;
37), γ
Abu-γ
Abu, Ahx-Gly, β
Ala-Gly-Gly, Ahx-β
Ala, β
Ala-β
Ala-Gly, Gly-Gly-Gly-Gly, Ahx-γ
Abu, β
Ala-β
Ala-β
Ala, γ
Abu-β
Ala-Gly, γ
Abu-γ
Abu-Gly, Ahx-Ahx, γ
Abu-γ
Abu-β
Ala, and Ahx-Ahx-Gly;Y is selected from the group consisting of Gly, β
Ala, γ
Abu, Gly-Gly, Ahx, Gly-β
Ala, β
Ala-β
Ala, Gly-γ
Abu, γ
Abu-β
Ala, Gly-Gly-Gly, γ
Abu-γ
Abu, Gly-Ahx, Gly-Gly-β
Ala,β
Ala-Ahx, Gly-β
Ala-β
Ala, Gly-Gly-Gly-Gly (SEQ ID NO;
37), γ
Abu-Ahx, β
Ala-β
Ala-β
Ala, Gly-β
Ala-γ
Abu, Gly-γ
Abu-γ
Abu, Ahx-Ahx, β
Ala-γ
Abu-γ
Abu, and Gly-Ahx-Ahx;when i is 1, S1 is joined to aa1 by a peptide bond through a terminal alpha amino group of aa1 ; and when r is 1, S2 is joined to aa10 by a peptide bond through a terminal alpha carboxyl group of aa10. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38)
- ##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;
Specification