Compositions for the detection of enzyme activity in biological samples and methods of use thereof
AssigneeOnco Immunin, Inc.
InventorsAkira Komoriya, Beverly S. Packard
Life of this patent
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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.
Claims15
A fluorogenic composition for the detection of the activity of a protease, said composition having the formula:
- wherein, P has the sequence Leu-Glu-His-Asp-Gly-Ile-Asn (SEQ ID NO:212);
- 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, l, 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 and aa3 are independently selected from the group consisting of Asp, Glu, Lys, Ornithine, Arg, Citulline, homocitrulline, Ser, Pro, homoserine, Aib, Thr, and Tyr;
- 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, βAla, γAbu, Gly-Gly, Ahx, 8-aminocaprylic acid, βAla-Gly, βAla-βAla, γAbu-Gly, βAla-γAbu, Gly-Gly-Gly, γAbu-γAbu, Ahx-Gly, βAla-Gly-Gly, Ahx-βAla, βAla-βAla-Gly, Gly-Gly-Gly-Gly (SEQ ID NO:223), Ahx-γAbu, βAla-βAla-βAla, γAbu-βAla-Gly, γAbu-γAbu-Gly, Ahx-Ahx, γAbu-γAbu-βAla, and Ahx-Ahx-Gly,
- Y is selected from the group consisting of Gly, βAla, γAbu, Gly-Gly, Ahx, 8-aminocaprylic acid, Gly-βAla, βAla-βAla, Gly-γAbu, γAbu-βAla, Gly-Gly-Gly, γAbu-γAbu, Gly-Ahx, Gly-Gly-βAla, βAla-Ahx, Gly-βAla-βAla, Gly-Gly-Gly-Gly (SEQ ID NO:223), γAbu-Ahx, βAla-βAla-βAla, Gly-βAla-γAbu, Gly-γAbu-γAbu, Ahx-Ahx, βAla-γAbu-γAbu, 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.
Dependencies are inferred from the claim text.