ABSTRACT
Going against tradition: although most kinase inhibitors are ATP competitive, lariat peptides inhibit Abl kinase activity in an ATP-uncompetitive manner. Further, lariat peptides discriminated Src family kinases, and recognize the allosteric region that lies adjacent to the ATP binding pocket in the Abl kinase catalytic cleft.
Subject(s)
Peptides/metabolism , Peptides/pharmacology , Proto-Oncogene Proteins c-abl/antagonists & inhibitors , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Allosteric Regulation , Amino Acid Sequence , Binding Sites , Catalytic Domain , Cell Line, Tumor , Enzyme Activation/drug effects , Humans , Kinetics , Peptides/chemistry , Protein Binding , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-abl/metabolismABSTRACT
We developed a high throughput yeast two-hybrid (Y2H) assay for screening pools of combinatorial cyclic peptide preys against pools of bait proteins. The assay used the PI (pooling with imaginary tags) deconvolution pooling strategy to generate pools of baits and a random pooling strategy to generate pools of cyclic peptide preys. Haploid yeast, expressing pools of baits or preys, were arrayed and mated to each other to generate diploid arrays, where the yeast express both baits and preys. Diploid arrays were scored for activation of the Y2H reporter genes. We used this Y2H pooling strategy, referred to as 'PI-pool-on-random pool', to screen a cyclic peptide library for interactions against Bcr-Abl domains. Seven Bcr-Abl domain baits and LexA control were pooled using the PI deconvolution pooling strategy. The cyclic peptide library was randomly arrayed into pools of ~1000 members. Cyclic peptides were isolated for six of seven Bcr-Abl domain baits. The PI-pool-on-random pooling Y2H assay using high stringency Y2H reporter genes produced no false positives, while missing 20% of real interactions. The high specificity of the PI-pool-on-random pooling Y2H assay eliminates the need to validate interactions. Pooling of baits and preys allows large prey libraries to be screened against multiple baits and takes advantage of PI-deconvolution to determine protein interactions with high sensitivity and specificity. The scalability of this assay allows the peptide preys to be isolated in a high throughput manner against a large number of baits and provides an avenue for generating affinity agents against entire proteomes in the future.
Subject(s)
Fusion Proteins, bcr-abl/antagonists & inhibitors , Peptide Library , Peptides, Cyclic/genetics , Peptides, Cyclic/pharmacology , Two-Hybrid System Techniques , Amino Acids , Fusion Proteins, bcr-abl/chemistry , Fusion Proteins, bcr-abl/metabolism , Genes, Reporter , High-Throughput Screening Assays/methods , Humans , Protein Binding , Protein Structure, Tertiary , Yeasts/geneticsABSTRACT
Functional genomic analyses provide information that allows hypotheses to be formulated on protein function. These hypotheses, however, need to be validated using reverse genetic approaches, which are difficult to perform on a large scale and in diploid organisms. We developed a genetic screen for isolating "lariat" peptides that function as trans dominant inhibitors of protein function. A lariat consists of a lactone-cyclized peptide with a covalently attached transcription activation domain, which allows combinatorial lariat libraries to be screened for protein interactions using the yeast two-hybrid assay. We isolated lariats against the bacterial repressor protein LexA. LexA regulates bacterial SOS response and LexA mutants that cannot undergo autoproteolysis make bacteria more sensitive to, and inhibit resistance against, cytotoxic reagents. We showed that an anti-LexA lariat blocked LexA autoproteolysis and potentiated the antimicrobial activity of mitomycin C.
