Protein fragment complementation assays for high-throughput and high-content screening

Abstract

The present invention provides protein fragment complementation assays for drug discovery, in particular to identify compounds that activate or inhibit cellular pathways. Based on the selection of an interacting protein pair combined with an appropriate PCA reporter, the assays may be run in high-throughput or high-content mode and may be used in automated screening of libraries of compounds. The interacting pair may be selected by cDNA library screening; by gene-by-gene interaction mapping; or by prior knowledge of a pathway. Fluorescent and luminescent assays can be constructed using the methods provided herein. The selection of suitable PCA reporters for high-throughput or high-content (high-context) assay formats is described for a diversity of reporters, with particular detail provided for examples of monomeric enzymes and fluorescent proteins. Methods are described for constructing such assays for one or more steps in a biochemical pathway; testing the effects of compounds from combinatorial, natural product, peptide, antibody, nucleic acid or other diverse libraries on the protein or pathway(s) of interest; and using the results of the screening to identify specific compounds that activate or inhibit the protein or pathway(s) of interest. Single-color and multi-color assays are disclosed. Further disclosed are universal expression vectors with cassettes that allow the rapid construction of assays for a large and diverse number of gene/reporter combinations. The development of such assays is shown to be straightforward, providing for a broad, flexible and biologically relevant platform for drug discovery.

1 - 16 . (canceled) 
     
     
         17 . Protein fragment complementation assays for drug discovery comprising a reassembly of separate fragments of a reporter molecule wherein reassembly of the reporter fragments generates an optically detectable signal. 
     
     
         18 - 25 . (canceled) 
     
     
         26 . An assay composition for drug discovery comprising complementary fragments of a first reporter molecule, said complementary fragments exhibiting a detectable activity when associated, wherein each fragment is fused to a separate molecule. 
     
     
         27 - 34 . (canceled) 
     
     
         35 . An assay composition according to  claim 26  wherein one or more reporter fragment(s) are derived from the group consisting of a fluorescent protein, a bioluminescent protein, a chemiluminescent protein, an enzyme, a monomeric protein, an antibody and an antigen. 
     
     
         36 - 39 . (canceled)