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 occurrs 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.

1 . A method for conducting an assay to determine whether a target compound exhibits activity as a protease inhibitor, comprising: 
 combining said 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 terminating, at a first end, with a first member of a first ligand binding pair and 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 which second member is bound to a surface,    removing any fragments of said construct cleaved by said protease,    adding a complex of a second member of said second ligand binding pair complexed with an enzyme and allowing said complex to bind to any said first member of said second ligand binding pair present,    removing any unbound complex,    adding a 1,2-dioxetane which is a substrate for said enzyme and observing any 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 observed is measured, and wherein the amount of chemiluminescence observed is correlated with the 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 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 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.  
     
     
         15 . A protease substrate for conducting an assay to determine protease inhibitory activity in a target compound, said substrate comprising: 
 a polypeptide, said polypeptide including a cleavage site specific for said protease, said polypeptide bearing at a first end a first member of a first ligand binding pair and at another end a first member of a second ligand binding pair, wherein said first member of said first ligand binding pair and said first member of said second ligand binding pair do not bind to each other.    
     
     
         16 . The substrate of    claim 15   , wherein said first and second ligand binding pair is comprised, independently, of an antigen and an antibody therefore, or biotin and a compound which binds as tightly to biotin as avidin.  
     
     
         17 . The substrate of    claim 15   , wherein said substrate has the sequence Fluorescein-Spacer-Ser-Glu-Asu-Tyr-Pro-Ile-Val-Glu-Spacer-Biotin, or FLAG-Ser-Nle-Ala-Glu-Phe-Leu-Val-Arg-Ala-Hys-His-Spacer-Biotin.  
     
     
         18 . A homogenous assay for detection of protease inhibitory activity in a target compound, comprising: 
 combining said 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 a polypeptide of 2-10 amino acids, said polypeptide terminating, at a first end, and a moiety which is a 1,2-dioxetane or precursor thereof which can be oxygenated in said environment to provide a 1,2-dioxetane moiety and, at a second end, with an energy accepting fluorescent moiety,    oxygenating said precursor if present to form a 1,2-dioxetane moiety,    causing said 1,2-dioxetane moiety to decompose, and observing the wavelength of light emitted from said environment, wherein said wavelength, if the wavelength of said fluorescent emitter, is indicative of inhibitory activity, and said wavelength, if the wavelength of said dioxetane, is indicative of absence of inhibitory activity.    
     
     
         19 . A construct for conducting a homogenous assay to determine protease inhibitory activity in a target compound, comprising: 
 a polypeptide of 2-10 amino acid residues, wherein said polypeptide includes a cleavage site specific for said protease, said polypeptide bearing, at one end, a fluorescent energy accepting moiety or a chemiluminescece quenching moiety and at another end, a 1,2-dioxetane or 1,2-dioxetane precursor moiety that can be oxygenated to form a 1,2-dioxetane moiety in the course of said assay.    
     
     
         20 . A kit for conducting an assay to determine whether a protease inhibitor is present in a sample, comprising in one or more containers: 
 (a) a peptide which is a substrate for said protease, wherein said peptide substrate is labeled with (i) a first member of a first ligand binding pair, and (ii) a first member of a second ligand binding pair;    (b) a chemiluminescent 1,2-dioxetane substrate containing an enzymatically cleavable group, which substrate is capable of producing light in the presence of an enzyme which cleaves said enzymatically cleavable group from said substrate.    
     
     
         21 . The kit of    claim 20   , wherein said protease is HIV-1.  
     
     
         22 . The kit of    claim 21   , wherein said peptide substrate comprises the amino acid sequence: -Ser-Gln-Asn-Try-Pro-Ile-Val-Gln-.  
     
     
         23 . The kit of    claim 20   , wherein said first member of said second ligand binding pair is fluorescein.  
     
     
         24 . The kit of    claim 20   , wherein said first member of said first ligand binding pair is biotin.  
     
     
         25 . The kit of    claim 20   , additionally comprising a polymeric quaternary onium salt enhancement agent for said 1,2-dioxetane.  
     
     
         26 . The kit of    claim 25   , wherein said enhancement agent comprises polyvinylbenzyltributyl ammonium chloride.  
     
     
         27 . The kit of    claim 20   , further comprising a enzyme complexed with a second member of said second ligand binding pair.  
     
     
         28 . The kit of    claim 27   , wherein said enzyme is alkaline phosphatase.