Activatable photodynamic therapy agents

1. A conjugate comprising a substrate that comprises a recognition sequence cleavable by proteolytic enzyme, a photoactivatable killing agent, and a quencher, wherein said photoactivatable killing agent is operably linked to one end of said substrate and said quencher is operably linked to another end of said substrate, wherein said proteolytic enzyme is capable of recognizing said recognition sequence in said substrate, wherein said conjugate is capable of providing an intact form and a cleaved form,

wherein in said conjugate in said intact form, the distance between said photoactivatable killing agent and said quencher is sufficient for said quencher to quench photoactivation of said photoactivatable killing agent; and

wherein in said conjugate in said cleaved form, said photoactivatable killing agent is separated from said quencher as a result of said enzyme contacting said substrate for a period of time and conditions sufficient to proteolytically cleave said recognition sequence, thereby said photoactivatable killing agent is not quenched by said quencher.

2. A conjugate comprising:

(a) a cell death protease recognition sequence;

(b) a photoactivatable killing agent comprising a fluorophore;

(c) a fluorescence quencher; and

(d) a targeting ligand;

wherein the photoactivatable killing agent, the fluorescence quencher, and the targeting ligand are covalently linked to the cell death protease recognition sequence, and wherein said cell death protease recognition sequence brings said photoactivatable killing agent and said fluorescence quencher sufficiently close to each other to provide in the conjugate fluorescence quenching of the fluorophore.

3. The conjugate of claim 1 , wherein said enzyme is a protease.

4. The conjugate of claim 1 , wherein said photoactivatable killing agent is a free base or metal complex of a compound selected from the group consisting of a pyropheophorbide, a purpurin, a porphyrin, a chlorin, a bacteriochlorin, a phthalocyanine, a naphthalocyanine, a hypericin, a porphyrin isomer, an expanded porphyrin, a cationic dye, a psoralen, and a merocyanine 540.

5. The conjugate of claim 1 , wherein said quencher is a carotenoid, a metal complex dye, a cyanine dye, a stilbene quinone dye, an azomethine dye, an amine, a phenol, a sulfide, a bilirubin, a biliverdin, a nitroso compound, a nitrone compound and a N-oxy compound.

6. The conjugate of claim 1 , wherein said recognition sequence is a cell death protease recognition sequence.

7. The conjugate of claim 6 , wherein said cell death protease recognition sequence is cleavable by a caspase.

8. The conjugate of claim 6 , wherein said caspase is selected from the group consisting of caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, and caspase-10.

9. The conjugate of claim 6 , wherein said cell death protease recognition sequence comprises a sequence selected from the group consisting of Asp-Glu-Val-Ile(SEQ ID NO:1), Asp-Glu-Thr-Asp(SEQ ID NO:2), Leu-Glu-His-Asp(SEQ ID NO:3), Asp-Glu-His-Asp(SEQ ID NO:4), Trp-Glu-His-Asp(SEQ ID NO:5), Leu-Glu-Thr-Asp(SEQ ID NO:6), Asp-Glu-Val-Asp(SEQ ID NO:7), Val-Glu-His-Asp(SEQ ID NO:8), Ile-Glu-Ala-Asp(SEQ ID NO:9); Gly-Asp-Glu-Val-Asp-Gly-Ser-Gly-Lys (SEQ ID NO:10); and Lys-Gly-Asp-Glu-Val-Asp-Gly-Ser-Gly-Lys (SEQ ID NO:11).

10. The conjugate of claim 9 , wherein said cell death protease recognition sequence comprises a sequence selected from the group consisting of X-Asp-Glu-Val-Ile(SEQ ID NO: 23)-Y, X-Asp-Glu-Thr-Asp(SEQ ID NO: 24)-Y, X-Leu-Glu-His-Asp(SEQ ID NO: 25)-Y, X-Asp-Glu-His-Asp(SEQ ID NO: 26)-Y, X-Trp-Glu-His-Asp(SEQ ID NO: 27)-Y, X-Leu-Glu-Thr-Asp(SEQ ID NO: 28)-Y, X-Asp-Glu-Val-Asp(SEQ ID NO: 29)-Y, X-Val-Glu-His-Asp(SEQ ID NO: 30)-Y, and X-Ile-Glu-Ala-Asp(SEQ ID NO: 31)-Y, wherein X and Y are each independently a polypeptide comprising from one to about 15 amino acids and the N-terminal amino acid of X is covalently linked to said substrate.

11. The conjugate of claim 9 , wherein said cell death protease recognition sequence comprises a sequence selected from the group consisting of X-Asp-Glu-Val-Ile(SEQ ID NO: 23)-Y, X-Asp-Glu-Thr-Asp(SEQ ID NO: 24)-Y, X-Leu-Glu-His-Asp(SEQ ID NO: 25)-Y, X-Asp-Glu-His-Asp(SEQ ID NO: 26)-Y, X-Trp-Glu-His-Asp(SEQ ID NO: 27)-Y, X-Leu-Glu-Thr-Asp(SEQ ID NO: 28)-Y, X-Asp-Glu-Val-Asp(SEQ ID NO: 29)-Y, X-Val-Glu-His-Asp(SEQ ID NO: 30)-Y, and X-Ile-Glu-Ala-Asp(SEQ ID NO: 31)-Y, wherein X and Y are each independently a polypeptide comprising from one to about 15 amino acids and the C-terminal amino acid of Y is covalently linked to said substrate.

12. The conjugate of claim 2 , wherein the conjugate has the chemical structure

13. A pharmaceutical composition comprising the conjugate of claim 1 , and a pharmaceutically acceptable carrier.

14. A conjugate comprising a substrate that comprises

(a) a recognition sequence cleavable by a proteolytic enzyme recognizing said recognition sequence, wherein said recognition sequence is selected from the group consisting of Asp-Glu-Val-Ile(SEQ ID NO:1), Asp-Glu-Thr-Asp(SEQ ID NO:2), Leu-Glu-His-Asp(SEQ ID NO:3), Asp-Glu-His-Asp(SEQ ID NO:4), Trp-Glu-His-Asp(SEQ ID NO:5), Leu-Glu-Thr-Asp(SEQ ID NO:6), Asp-Glu-Val-Asp(SEQ ID NO:7), Val-Glu-His-Asp(SEQ ID NO:8), Ile-Glu-Ala-Asp(SEQ ID NO:9); Gly-Asp-Glu-Val-Asp-Gly-Ser-Gly-Lys (SEQ ID NO:10); and Lys-Gly-Asp-Glu-Val-Asp-Gly-Ser-Gly-Lys (SEQ ID NO:11);

(b) a photoactivatable killing agent, wherein said photoactivatable killing agent is selected from the group consisting of a pyropheophorbide, a porphyrin or isomer thereof, an expanded porphyrin, a chlorin, a bacteriochlorin, a phthalocyanine, a naphthalocyanine, a hypericin, a cationic dye, a purpurin, a psoralen, and a merocyanine 540; and

(c) a quencher, wherein said quencher is selected from the group consisting of a carotenoid, an amine, a phenol, a sulfide, a bilirubin, a biliverdin, a nitroso compound, a nitrone compound, an N-oxy compound and a dye selected from the group consisting of a metal complex dye, a cyanine dye, a stilbene quinone dye, and an azomethine dye, wherein the dye is other than the photoactivatable killing agent cationic dye;

wherein said photoactivatable killing agent is operably linked to one end of said substrate and said quencher is operably linked to another end of said substrate, wherein said proteolytic enzyme is capable of recognizing said recognition sequence in said substrate, wherein said conjugate is capable of providing an intact form and a cleaved form,

wherein in said conjugate in said intact form, the distance between said photoactivatable killing agent and said quencher is sufficient for said quencher to quench photoactivation of said photoactivatable killing agent; and

wherein in said conjugate in said cleaved form, said photoactivatable killing agent is separated from said quencher as a result of said enzyme contacting said substrate for a period of time and conditions sufficient to proteolytically cleave said recognition sequence, thereby said photoactivatable killing agent is not quenched by said quencher.

15. A method of treating a cancerous target tissue comprising the steps of:

(a) administering to the target tissue of patient in need thereof a conjugate of claim 4 ; and

(b) irradiating the photoactivatable killing agent of the conjugate, thereby killing said cancerous target tissue,

wherein the killing of the cancerous tissue provides proteolytic cleaving of the conjugate cell death protease recognition sequence, wherein said cleaving removes the quencher from the conjugate, thereby allowing fluorescence detection of the photoactivatable killing agent fluorophore.