Compositions for the detection of proteases in biological samples and methods of use thereof
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.
Claims33
A fluorogenic composition for the detection of the activity of a protease, said composition having the formula:
- ##STR13## wherein:
- Y is a composition selected from the group consisting of compounds of formulas:
- ##STR14## C.sup.1.sub.5, C.sup.1.sub.4, C.sup.1.sub.3, P.sub.2, P.sub.1, P'.sub.1, P'.sub.2, C.sup.2.sub.3, and C.sup.2.sub.4 are amino acids;
- P.sub.2 -P.sub.1 -P.sub.1 '-P.sub.2 ' comprises a protease binding site;
- F.sup.1 and F.sup.2 are fluorophores;
- S.sup.1 and S.sup.2 are peptide spacers ranging in length from 1 to about 50 amino acids;
- n and k are independently 0 or 1 and when n is 1, S.sup.1 is joined to C.sup.1.sub.5 by a peptide bond through a terminal alpha amino group of C.sup.1, and when k is 1, S.sup.2 is joined to C.sup.2.sup.3 or to C.sup.2.sub.4 by a peptide bond through a terminal alpha carboxyl group of the respective C.sup.2.sub.3 or C.sup.2.sub.4 ;
- and wherein C.sup.1.sub.5 -C.sup.1.sub.4 -C.sup.1.sub.3 and C.sup.2.sub.3 -C.sup.2.sub.4 or C.sup.2.sub.3 introduce a bend into the composition thereby creating a generally U-shaped configuration of said composition in which said fluorophores are adjacent to each other with a separation of less than about 100 .ANG..
A method of detecting the activity of a protease in a biological sample, said method comprising the steps of:
- contacting said biological sample with a fluorogenic composition having the formula:
- ##STR15## wherein:
- Y is a composition selected from the group consisting of compounds of formulas:
- ##STR16## C.sup.1.sub.5, C.sup.1.sub.4, C.sup.1.sub.3, P.sub.2, P.sub.1, P'.sub.1, P'.sub.2, C.sup.2.sub.3, and C.sup.2.sub.4 are amino acids;
- P.sub.2 -P.sub.1 -P.sub.1 '-P.sub.2 ' comprises a protease binding site;
- F.sup.1 and F.sup.2 are fluorophores;
- S.sup.1 and S.sup.2 are peptide spacers ranging in length from 1 to about 50 amino acids;
- n and k are independently 0 or 1 and when n is 1, S.sup.1 is joined to C.sup.1.sub.5 by a peptide bond through a terminal alpha amino group of C.sup.1, and when k is 1, S.sup.2 is joined to C.sup.2.sub.3 or to C.sup.2.sub.4 by a peptide bond through a terminal alpha carboxyl group of the respective C.sup.2.sub.3 or C.sup.2.sub.4 ;
- and wherein C.sup.1.sub.5 -C.sup.1.sub.4 -C.sup.1.sub.3 and C.sup.2.sub.3 -C.sup.2.sub.4 or C.sup.2.sub.3 introduce a bend into the composition thereby creating a generally U-shaped configuration of said composition in which said fluorophores are adjacent to each other with a separation of less than about 100 .ANG.;
- and
- detecting a change in fluorescence of said fluorogenic composition wherein an increase in fluorescence indicates protease activity.
Dependencies are inferred from the claim text.