Screen assay for selecting protein-protein interaction modulators
AssigneeMyriad Genetics, Inc.
InventorJohn Manfredi
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Abstract
A method for selecting compounds capable of modulating protein-protein interactions is provided, in which two fusion proteins are prepared and allowed to interact with each other in the presence of test compounds. The interaction between the two fusion proteins leads to protein trans-splicing, producing an active reporter. Compounds that disrupt or enhance protein-protein interactions can be selected based on the presence or absence of the active reporter.
Claims26
A method for selecting compounds capable of modulating an interaction between a first test agent and a second test agent, comprising:
- providing a first fusion construct and a second fusion construct, said first fusion construct having an N-intein and said first test agent, said second fusion construct having a C-intein and said second test agent, wherein at least one of the two fusion constructs has an inactive reporter capable of being converted to an active reporter upon trans-splicing through said N-intein and said C-intein;
- allowing said first test agent in said first fusion construct to interact with said second test agent in said second fusion construct in the presence of one or more test compounds;
- and
- detecting said active reporter.
A method for selecting compounds capable of interfering with an interaction between a first test polypeptide and a second test polypeptide comprising:
- introducing into a host cell a first chimeric gene and a second chimeric gene, said first chimeric gene encoding a first fusion protein having said first test polypeptide, an N-intein, and a first inactive reporter polypeptide fused to the N-terminus of an N-intein, said second chimeric gene encoding a second fusion protein having said second test polypeptide, a C-intein, and a second inactive reporter polypeptide fused to the C-terminus of said C-intein, wherein ligation between the C-terminus of said first inactive reporter polypeptide and the N-terminus of said second inactive reporter polypeptide forms an active reporter protein;
- expressing said first fusion protein and said second fusion protein in said host cell in the presence of one or more test compounds;
- and
- determining the production of said active reporter protein, wherein the inhibition of the production of said active reporter protein would indicate that at least one of said one or more test compounds is capable of interfering with the interaction between said first test polypeptide and said second test polypeptide.
A method for selecting compounds capable of interfering with an interaction between a first test polypeptide and a second test polypeptide comprising:
- introducing into a first yeast haploid cell a first chimeric gene encoding a first fusion protein having said first test polypeptide, an N-intein, and a first inactive reporter polypeptide fused to the N-terminus of an N-intein;
- introducing a second chimeric gene into a second yeast haploid cell of a mating type opposite to that of said first yeast haploid cell, said second chimeric gene encoding a second fusion protein having said second test polypeptide, a C-intein, and a second inactive reporter polypeptide fused to the C-terminus of said C-intein, wherein ligation between the C-terminus of said first inactive reporter polypeptide and the N-terminus of said second inactive reporter polypeptide forms an active reporter protein that is counterselectable;
- mating said first and second yeast haploid cells to form a yeast diploid cell and expressing said first fusion protein and said second fusion protein in said yeast diploid cell in the presence of one or more test compounds;
- and
- determining the production of said active reporter protein, wherein the inhibition of the production of said active reporter protein would indicate that at least one of said one or more test compounds is capable of interfering with the interaction between said first test polypeptide and said second test polypeptide.
Dependencies are inferred from the claim text.