Method for identification of protease activity inhibitors and assaying the presence of protease activity

Abstract

A system for the identification of proteases and protease inhibitors is provided. The system has at least two components. The first component is a reporter construct with at least one binding site, a transcriptional promoter, an inducible promoter region, and at least one reporter gene, all functionally connected for expression of the reporter gene(s) in functional coordination with a transcriptional activation agent. The second component is a transcriptional activation agent comprising a nucleic acid binding domain, at least one protease substrate domain, and at least one transcriptional activation domain for an inducible promoter. The system allows detection and evaluation of agents affecting protease activity directed to the protease substrate domain. The system also allows for the detection of the presence of proteases in environmental samples.

1 . A system for the identification of proteases and protease inhibitors, comprising:
 a reporter construct comprising at least one binding site, an inducible promoter region, and at least one reporter gene, all functionally connected for expression of the reporter gene(s), in functional coordination with a transcriptional activation agent;   the transcriptional activation agent comprising a nucleic acid binding domain, at least one protease substrate domain, and at least one transcriptional activation domain for an inducible promoter.   
     
     
         2 . The system of  claim 1 , further comprising at least one protease or protease candidate, wherein the protease or protease candidate specifically targets the protease substrate domain. 
     
     
         3 . The system of  claim 2 , wherein at least one of the transcriptional activation agent or the protease/protease candidate are provided into the system as products of nucleic acid constructs. 
     
     
         4 . The system of  claim 2 , wherein at least one of the transcriptional activation agent or the protease/protease candidate are provided into the system as a protein. 
     
     
         5 . The system of  claim 1 , further comprising at least one reporter gene selected from the group consisting of a Venus yellow fluorescent protein, yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP); blue fluorescent protein (BFP), red fluorescent protein (RFP), fluorescing mutants thereof, bioluminescent proteins, Gaussia luciferase, renilla luciferase, click beetle luciferase, and firefly luciferase may also be used to quantify the activity of the reporter vector. 
     
     
         6 . The system of  claim 5  comprising at least two reporter genes transcribed from one inducible promoter. 
     
     
         7 . The system of  claim 1 , wherein the reporter gene construct comprises one to eight binding site sequence repeats. 
     
     
         8 . The system of  claim 1 , wherein the reporter gene construct comprises five binding site sequence repeats. 
     
     
         9 . The system of  claim 1 , wherein the binding site is selected from the group consisting of Gal4 and LexA. 
     
     
         10 . The system of  claim 1 , wherein the inducible reporter region comprises a TATA promoter region. 
     
     
         11 . The system of  claim 1 , wherein the protease substrate is selected from the group consisting of BoNT protease substrate, anthrax protease, caspase, alpha virus NSP2 protease, HIV processing proteases, SUMO processing proteases, ubiquitin processing proteases, ISG15 processing proteases, autophagy related ATG4 like processing proteases and Hepatitis virus processing proteases or a combination thereof. 
     
     
         12 . The system of  claim 10 , wherein the protease substrate is selected from the group consisting of SNAP-25, VAMP-2, and Syntaxin1a or a combination thereof. 
     
     
         13 . The system of  claim 10 , wherein the protease substrate comprises SNAP-25 and VAMP-2 protease substrate domains. 
     
     
         14 . The system of  claim 11  wherein the protease is a BoNT protease. 
     
     
         15 . The system of  claim 1 , wherein the protease substrate is located on the transcriptional activator agent between the binding domain and the transcriptional activator domain and the transcriptional activation agent is in a cellular compartment where transcription takes place. 
     
     
         16 . The system of  claim 1 , wherein the protease substrate domain is at one end of the molecule and the binding domain and the transcriptional activator domain are located in functional proximity to each other and at an opposite end of the transcriptional activator agent from the protease substrate domain, such as cleavage of the protease substrate domain releases the binding domain and the transcriptional activator rendering a functional transcriptional activator agent fragment. 
     
     
         17 . The system of  claim 15 , engineered such as the transcriptional activator agent is sequestered outside of a cell nucleus but cleavage with a protease releases a functionally active transcriptional activator agent fragment, comprising the binding domain and the transcriptional activator domain and capable to reach a cellular compartment where transcriptional activity takes place. 
     
     
         18 . The system of  claim 2  in a tTS cell. 
     
     
         19 . A method to identify protease inhibitors, comprising:
 introducing at least one test molecule or compound to a system comprising:
 a reporter gene construct comprising at least one binding site, a transcriptional promoter, an inducible promoter region, and at least one reporter gene, all functionally connected for expression of the reporter gene(s) in functional coordination with a transcriptional activation agent, 
 the transcriptional activation agent comprising a nucleic acid binding domain, at least one protease substrate domain, and at least one transcriptional activation domain for an inducible promoter, and 
 a protease specific for the protease substrate domain; and 
   measuring the effect on the level of expression of the reporter molecule.   
     
     
         20 . A method to identify a protease, comprising:
 introducing at least one test molecule or compound to a system comprising:
 a reporter gene construct comprising at least one binding site, a transcriptional promoter, an inducible promoter region, and at least one reporter gene, all functionally connected for expression of the reporter gene(s) in functional coordination with a transcriptional activation agent, 
 the transcriptional activation agent comprising a nucleic acid binding domain, at least one protease substrate domain, and at least one transcriptional activation domain for an inducible promoter, and 
   measuring the effect on the level of expression of the reporter molecule.