Get our Free Patent Expiration Newsletter

Serving leading biopharmaceutical companies globally:

McKesson
McKinsey
Boehringer Ingelheim
AstraZeneca
Dow
Merck

Last Updated: October 19, 2019

DrugPatentWatch Database Preview

Claims for Patent: 6,243,980

See Plans and Pricing

« Back to Dashboard

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)
Application Number:09/035,820
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.

Details for Patent 6,243,980

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

Make Better Decisions: Try a trial or see plans & pricing

Serving leading biopharmaceutical companies globally:

Medtronic
Express Scripts
McKesson
McKinsey
Dow
Johnson and Johnson

Drugs may be covered by multiple patents or regulatory protections. All trademarks and applicant names are the property of their respective owners or licensors. Although great care is taken in the proper and correct provision of this service, thinkBiotech LLC does not accept any responsibility for possible consequences of errors or omissions in the provided data. The data presented herein is for information purposes only. There is no warranty that the data contained herein is error free. thinkBiotech performs no independent verifification of facts as provided by public sources nor are attempts made to provide legal or investing advice. Any reliance on data provided herein is done solely at the discretion of the user. Users of this service are advised to seek professional advice and independent confirmation before considering acting on any of the provided information. thinkBiotech LLC reserves the right to amend, extend or withdraw any part or all of the offered service without notice.