Multiprotein assemblies

Abstract

The present invention provides compositions and methods of use in investigations of the formation of mulitprotein assemblies implicated in disease. Also provided are assays for screening candidate compounds of potential utility in preventing and/or treating such diseases by preventing the assembly of or disrupting the function of multiprotein assemblies.

1 . A method for assaying a candidate compound for its ability to interfere with the function of a multiprotein assembly implicated in a disease, wherein said multiprotein assembly participates in folding of a second protein, formation of multiprotein structures comprising said second protein or a combination thereof said method comprising:
 (a) in a cell-free translation system comprising a ribosome, translating an m-RNA sequence encoding a protein substrate of said multiprotein assembly, wherein said m-RNA sequence is a truncated m-RNA sequence lacking a stop codon at its 3′-terminus such that, upon completion of translation of said m-RNA sequence, said protein substrate is complexed to said ribosome at a first site accessible to an A site, and said protein substrate is not complexed to a t-RNA;   (b) expressing said second protein in said cell-free translation system;   (c) contacting said cell-free system with said candidate compound; and   (c) determining whether said multiprotein assembly is bound to said protein substrate, wherein failure of said substrate to bind to said multiprotein assembly confirms said ability to interfere with the function of said multiprotein assembly of said candidate compound.   
     
     
         2 . The method according to  claim 1 , wherein the candidate compound interferes with a single protein binding site, an interface between two proteins or an interface between three proteins of said multiprotein assembly. 
     
     
         3 . The method according to  claim 1 , wherein the candidate compound interferes by binding to a protein active site of a single protein, protein active sites of two proteins or protein acitve sites of three proteins of said multiprotein assembly. 
     
     
         4 . The method according to  claim 1 , wherein the candidate compound interferes by binding to an allosteric site of a single protein, an allosteric site of each of two proteins or an allosteric site of each of three proteins of said multiprotein assembly. 
     
     
         5 . The method of  claim 1 , wherein said disease is a viral infection. 
     
     
         6 . The method according to  claim 5 , wherein said infection is by one or more member of the group Flaviviridae, Togaviridae, Bunyaviridae, Arenaviridae, Filoviridae, Poxviridae, Orthomyxoviridae, Rhabdoviridae, Herpesviridae, Coronaviridae, Paramyxoviridae, Hepadnaviridae, Bornaviridae, Picornaviridae, Retroviridae, Reoviridae, Papillomaviridae, Adenoviridae, Astroviridae, and Polyomaviridae. 
     
     
         7 . The method according to  claim 1 , wherein said disease results in formation of amyloid fibrils. 
     
     
         8 . The method according to  claim 7 , wherein said disease is a neurological disease. 
     
     
         9 . The method according to  claim 8 , wherein said disease is a member selected from Alzheimer's and Creutzfeldt-Jakob disease. 
     
     
         10 . The method according to  claim 1 , wherein said cell-free translation system is a wheat germ system. 
     
     
         11 . The method according to  claim 10 , wherein wheat germ extract is present in said system in an amount of not more than about 5%. 
     
     
         12 . The method according to  claim 1 , wherein said second protein comprises a detectable label. 
     
     
         13 . The method according to  claim 12 , wherein said detectable label is  35 S-methionine. 
     
     
         14 . The method according to  claim 1 , wherein said determining comprising confirming whether a viral capsid was assembled. 
     
     
         15 . The method according to  claim 1 , wherein following a member selected from step (a), step (b) and a combination thereof, said m-RNA remains complexed to said ribosome. 
     
     
         16 . The method according to  claim 1 , further comprising, contacting said cell-free translation system with puromycin, releasing said protein substrate. 
     
     
         17 . A method of verifying that a target for a candidate compound which interferes with the function of a multiprotein assembly implicated in a disease is a host target, wherein said multiprotein assembly participates in folding of a second protein, formation of a multiprotein structure comprising said second protein or a combination thereof said method comprising:
 (a) contacting an initial medium for a cell-free translation system comprising one or more host protein with an affinity chromatography device comprising said candidate compound immobilized thereon, directly or through a linker, under conditions appropriate to bind at least one member of said multiprotein assembly to said candidate compound;   (b) washing the device with a first eluent, removing species not bound to said candidate compound in a flow through fraction;   (c) washing the device with a second eluent, removing the at least one member of said multiprotein assembly bound to said device in an eluent fraction;   (d) combining said candidate compound with said flow through fraction and using the resulting first mixture for cell-free translation of an m-RNA sequence encoding a protein substrate of said multiprotein assembly, wherein said m-RNA sequence is a truncated m-RNA sequence lacking a stop codon at its 3′-terminus such that, upon completion of translation of said m-RNA sequence, said protein substrate is complexed to said ribosome at a first site accessible to an A site, and said protein substrate is not complexed to a t-RNA;   (e) expressing said second protein in said cell-free translation system;   (f) determining whether said second protein was folded, said multiprotein structure comprising said second protein was formed or both; and   optionally,
 (i) combining said candidate compound, said flow through fraction and said eluent fraction and using the resulting second mixture for cell-free translation of said m-RNA sequence encoding said protein substrate of said multiprotein assembly, wherein said m-RNA sequence is said truncated m-RNA sequence lacking a stop codon at its 3′-terminus such that, upon completion of translation of said m-RNA sequence, said protein substrate is complexed to said ribosome at a first site accessible to an A site, and said protein substrate is not complexed to a t-RNA; 
 (ii) expressing said second protein in said second mixture; 
 (ii) determining whether said second protein was folded, said multiprotein structure comprising said second protein was formed or both, 
   wherein lack of said folding of said protein or formation of said multiprotein structure comprising said second protein confirms that said initial medium does not include a host target for said candidate compound or the host target for the candidate compound is removed by said contacting with said immobilized candidate compound, and confirmation of said folding of said protein or formation of said multiprotein structure comprising said second protein confirms that said host target for said candidate compound is removed by said contacting with said immobilized candidate compound.   
     
     
         18 . The method according to  claim 17 , wherein said multiprotein structure is a viral capsid. 
     
     
         19 . An isolated ribosomal complex comprising:
 a ribosome having a first site accessible to an A site, wherein said first site is complexed to a protein synthesized from a m-RNA lacking a stop codon, wherein said protein is not complexed to a t-RNA.   
     
     
         20 . The isolated ribosomal complex according to  claim 19 , wherein said truncated m-RNA is complexed to said ribosome. 
     
     
         21 . The isolated ribosomal complex according to  claim 19 , wherein said complex is bound to a candidate compound immobilized directly or through a linker on an affinity chromatography device. 
     
     
         22 . A mixture of the isolated ribosomal complex according to  claim 19  and a candidate compound. 
     
     
         22 . The isolated ribosomal complex according to  claim 19  in an assay format comprising, at least one candidate compound and at least one additional component appropriate for assaying the effect of said at least one candidate compound on said isolated ribosomal complex. 
     
     
         23 . The isolated ribosomal complex according to  claim 19 , wherein said isolated ribosomal complex is isolated from a cell-free translation system in which it was assembled and is free of essentially all components of said cell-free translation system other than those components forming said complex.