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Summary for Patent: 6,243,980
|Title:||Protease inhibitor assay|
|Abstract:||Heterogenous and homogenous assays are provided for the detection of protease inhibitory activity in a sample or target compound, taking advantage of the chemiluminescent characteristics of 1,2-dioxetanes. In the heterogenous assay, a peptide bearing a cleavage site for the protease of interest is provided with a first member of a first ligand binding pair at one end, and a first member of a second ligand binding pair at the other end. The other member of the first ligand binding pair is attached to a surface, which binds the peptide, or protease substrate, to the surface. The peptide substrate is combined with the protease and target compound or sample. Substrate cleavage, if not inhibited, is allowed to occur, and any unbound cleaved fragments are removed. An enzyme complexed with the second member of the second ligand binding pair is added, and allowed to bind to any of the (uncleaved) first member of the second ligand binding pair remaining. Unbound complex is removed, and a 1,2-dioxetane substrate for the enzyme is added. If any peptide substrate has not been cleaved, the dioxetane will chemiluminesce, indicating inhibitory activity. In a homogenous assay, the same substrate bears at one end a fluorescent energy accepting moiety, and at the other end a 1,2-dioxetane or precursor. If the substrate is cleaved by the protease, the dioxetane and the fluorescent moiety are not in close physical relationship, and no energy transfer occurs when the dioxetane is caused to decompose. If cleavage has not occurred, indicating inhibition, when the dioxetane is caused to decompose, energy is transferred to the fluorescing entity, which releases light of a wavelength recognizably distinct from that of the dioxetane.|
|Inventor(s):||Bronstein; Irena (Newton, MA), Voyta; John (Sudbury, MA), Palmer; Michelle (Arlington, MA), Tillotson; Bonnie (Belmont, MA)|
|Assignee:||Tropix, Inc. (Bedford, MA)|
|Patent Claims:||1. A method for conducting an assay to determine whether a target compound exhibits activity as a protease inhibitor, comprising:
(a) combining a protease and said target compound in an environment further comprising a construct, wherein said environment is such that said protease cleaves said construct in the absence of inhibition of said protease,
said construct comprising an amino acid sequence capable of being cleaved by said protease terminating (1) at a first end with a first member of a first ligand binding pair and (2) at a second end with a first member of a second ligand binding pair, wherein
said first member of said first ligand binding pair binds to a second member of said first ligand binding pair, said second member of said first ligand binding pair is bound to a surface,
(b) removing any fragments of said construct cleaved by said protease,
(c) after step (a), adding a second member of said second ligand binding pair complexed with an enzyme and allowing said second member of said second ligand binding pair to bind to said first member of said second ligand binding pair,
(d) removing any unbound second member of the second ligand binding pair complexed with the enzyme, and
(e) adding a 1,2-dioxetane which is a substrate for said enzyme and observing chemiluminescence released thereby, wherein emission of said chemiluminescence is indicative of protease inhibition activity by said target compound.
2. The method of claim 1, wherein said chemiluminescence permits determination of a degree of inhibitory activity exhibited by said compound.
3. The method of claim 1, wherein said assay is a transfer free single plate endpoint assay.
4. The method of claim 1, wherein said surface is at least a portion of at least one well of a test plate.
5. The method of claim 1, wherein said second member of said second ligand binding pair is an antibody.
6. The method of claim 1, wherein said first ligand binding pair is biotin and a compound which binds to biotin at least as tightly as avidin.
7. The method of claim 1, wherein said second ligand binding pair is biotin and a compound which binds to biotin at least as tightly as avidin.
8. The method of claim 6, wherein said compound which binds to biotin at least as tightly as avidin is avidin or strepavidin.
9. The method of claim 7, wherein said compound which binds as tightly as avidin is avidin or strepavidin.
10. The method of claim 1, wherein said first member of said first ligand binding pair is biotin and said first member of said second ligand binding pair is fluorescein.
11. The method of claim 1, wherein said second ligand binding pair is an eight amino acid peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lvs) known as FLAG and an antibody therefor.
12. The method of claim 1, wherein said protease is selected from the group consisting of a serine protease, a cysteine protease, an aspartic protease and a metallo proteinase.
13. The method of claim 1, wherein said protease is selected from the group consisting of human immunodeficiency virus 1 (HIV-1 protease), caspases, cathhepsins, hydrolase, L-proteinase, calpain, interleukin converting proteases, urokinase, trypsin, thrombin, HIV-2 protease, Yapsin I, Yapsin 3, Plasmepsin I, Plasmepsin II, collagenase, gelatinases, stromelysin, amino peptidase and elastase.
14. The method of claim 13, wherein said protease is HIV-1 protease.
|Applicant||Tradename||Biologic Ingredient||Dosage Form||BLA||Number||Approval Date||Patent No.||Assignee||Estimated Patent Expiration||Status||Orphan||Source|
|Advance Biofactures||SANTYL||collagenase||VIAL||101995||001||1965-06-04||Start Trial||Tropix, Inc. (Bedford, MA)||2036-04-30||RX||search|
|>Applicant||>Tradename||>Biologic Ingredient||>Dosage Form||>BLA||>Number||>Approval Date||>Patent No.||>Assignee||>Estimated Patent Expiration||>Status||>Orphan||>Source|
|Country||Patent Number||Publication Date|
|World Intellectual Property Organization (WIPO)||9839471||Sep 11, 1998|
|United States of America||2001031478||Oct 18, 2001|
|Japan||2001514506||Sep 11, 2001|
|European Patent Office||0972071||Apr 21, 2004|
|European Patent Office||0972071||Jan 19, 2000|
|>Country||>Patent Number||>Publication Date|
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