Odyssey Thera Inc.

Odyssey Pharmaceuticals, Inc.

Stephen William Watson Michnick

Ingrid Remy

Joelle Nina Pelletier

Marnie L MacDonald

Jane Lamerdin

John K Westwick

Helen Yu

Joelle Nina Polletier

Katja M Arndt

Normand Brisson

André Galarneau

Brigitte Keon

Andr&eacute; Galarneau

Marnie Macdonald

We describe a strategy for designing and implementing protein-fragment complementation assays (PCAs) to detect biomolecular interactions in vivo and in vitro. The design, implementation and broad applications of this strategy are illustrated with a large number of enzymes with particular detail provided for the example of murine dihydrofolate reductase (DHFR). Fusion peptides consisting of N- and C-terminal fragments of murine DHFR fused to GCN4 leucine zipper sequences were coexpressed ingrown in minimal medium, where the endogenous DHFR activity was inhibited with trimethoprim. Coexpression of the complementary fusion products restored colony formation. Survival only occurred when both DHFR fragments were present and contained leucine-zipper forming sequences, demonstrating that reconstitution of enzyme activity requires assistance of leucine zipper formation. DHFR fragment-interface point mutants of increasing severity (Ile to Val, Ala and Gly) resulted in a sequential increase indoubling times illustrating the successful DHFR fragment reassembly rather than non-specific interactions between fragments. This assay could be used to study equilibrium and kinetic aspects of molecular interactions including protein-protein, protein-DNA, protein-RNA, protein-carbohydrate and protein-small molecule interactions, for screening cDNA libraries for binding of a target protein with unknown proteins or libraries of small organic molecules for biological activity. The selection and design criteria applied here is developed for numerous examples of clonal selection, colorometric, fluorometric and other assays based on enzymes whose products can be measured. The development of such assay systems is shown to be simple, and provides for a diverse set of protein fragment complementation applications.

Protein fragment complementation assays for the detection of biological or drug interactions

The present invention is directed to Protein-fragment Complementation Assays (PCAs) and assay compositions based on fluorescent proteins. The invention provides methods for fragmenting fluorescent proteins and generating mutant fragments with desired spectral characteristics for PCA. The invention encompasses assays and compositions based on fluorescent proteins from the speciesand. In particular, the invention is directed to fragments of mutant fluorescent proteins having improved spectral properties over the wild-type proteins. The invention encompasses fragments of mutant versions ofgreen fluorescent protein (GFP), in particular yellow fluorescent proteins (EYFP and super-EYFP), 'Venus', cyan, 'citrine', blue, cyan-green, and photoactivatable variants of GFP The invention also encompasses red fluorescent PCAs based on Discosoma red fluorescent protein (RFP PCA) and a kindling fluorescent protein PCA (KFP1 PCA) derived from. Any useful mutation of a fluorescent protein can be engineered into a fragment, generating a wide range of assays useful for drug discovery, target validation, high-throughput screening, high-content screening, pathway mapping, drug mechanism-of-action studies, biosensors, and diagnostics.

Fragments of fluorescent proteins for protein fragment complementation assays

The present invention describes a method for detecting biomolecular interactions said method comprising: (a) selecting an appropriate reporter molecule selected from the group consisting of a protein, a fluorescent protein, a luminescent protein and a phosphorescent protein; (b) effecting fragmentation of said reporter molecule such that said fragmentation results in reversible loss of reporter function; (c) fusing or attaching fragments of said reporter molecule separately to other molecules; followed by (d) reassociation of said reporter fragments through interactions of the molecules that are fused to said fragments; and (e) detecting said biomolecular interactions by reconstitution of activity of the reporter molecule.

The present invention describes an assay method comprising: (A) generating (1) at least a first fragment of a reporter molecule linked to a first interacting domain and at least a second fragment of a reporter molecule linked to a second interacting domain, or (2) nucleic acid molecules that code for (A)(1) and subsequently allowing said nucleic acid molecules to produce their coded products; then, (B) allowing interaction of said domains; and (C) detecting reconstituted reporter molecule activity, where said reporter molecule can react with a penicillin- or a cephalosporin-class substrate.

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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.

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

The present invention describes rapid methods to screen for biomolecular interactions in vivo based on protein fragment complementation assays (PCA). We have demonstrated an in vivo library-versus-library screening strategy that has numerous applications in the identification of novel protein-protein interactions and in directed evolution. Also we demonstrate the detection of protein-protein interactions starting with defined (full-length) cDNAs, and the concomitant generation of functional assays that provide initial validation of the cDNA products as being biologically relevant. Also, we screened a large cDNA collection using automated PCA, combined with quantitative detection of protein-protein complexes. The invention enables bait-vs.-library, library-vs.-library and defined gene screening in any type of cell or cellular context, and using a wide range of reporters and detection methods. The invention allows for identifying and validating genes involved in any cellular process and also provide assays to study effects of potential drugs, or gene knockouts on specific pathways.

In vivo screening of protein-protein interactions with protein-fragment complementation assays

The present invention describes a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. It allows for the screening of a protein library against a second protein library and therefore finds numerous applications in the study of protein-protein interactions. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into. Interaction between the library polypeptides was required for reconstitution of the enzymatic activity of mDHFR, allowing bacterial growth. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We applied these selection processes to a library-versus-library sample of 2.0&#xd7;10combinations, and selected a novel leucine zipper pair which may be appropriate for use in further in vivo heterodimerization strategies.

The instant invention describes a method for detecting protein-protein interactions in living organisms and/or cells, said method comprising: (a) synthesizing probe protein fragments from a protein which enables fluorescent or luminescent detection by dissecting the gene coding for the fluorescent or luminescent protein into a least two fragments; (2) constructing fusion proteins consisting of the probe protein fragments linked to protein domains that are to be tested for interactions; (3) coexpressing the fusion proteins; and (d) detecting reconstitution of the fluorescence or luminescence signal.

Protein fragment complementation assays in whole animals applications to drug efficacy, ADME, cancer biology, immunology, infectious disease and gene therapy

The present invention is directed to Protein-fragment Complementation Assays (PCAs) and assay compositions based on fluorescent proteins. The invention provides methods for fragmenting fluorescent proteins and generating mutant fragments with desired spectral characteristics for PCA. The invention encompasses assays and compositions based on fluorescent proteins from the species Aequorea, Anemonia and Anthozoa. In particular, the invention is directed to fragments of mutant fluorescent proteins having improved spectral properties over the wild-type proteins. The invention encompasses fragments of mutant versions ofgreen fluorescent protein (GFP), in particular yellow fluorescent proteins (EYFP and super-EYFP), 'Venus', cyan, 'citrine', blue, cyan-green, and photoactivatable variants of GFP.

The present invention provides protein single-color and multi-color 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 such as monomeric enzymes and fluorescent proteins, the assays may be run in high-throughput or high-content mode and may be used in automated screening of libraries of compounds. 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. The development of such assays provides for a broad, flexible and biologically relevant platform for drug discovery.

The present invention describes a method for detecting biomolecular interactions said method comprising: (a) selecting an appropriate reporter molecule selected from the group consisting of a protein, a fluorescent protein, a luminescent protein and a phosphorescent protein; (b) effecting fragmentation of said reporter molecule such that said fragmentation results in reversible loss of reporter function; (c) fusing or attaching fragments of said reporter molecule separately to other molecules; followed by (d) reassociation of said reporter fragments through interactions of the molecules that are fused to said fragments; and (e) detecting said biomolecular interactions by reconstitution of activity of the reporter molecule with the proviso that said protein is not ubiquitin.

The present invention describes a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. It allows for the screening of a protein library against a second protein library, rather than against a single bait protein, and thus has numerous applications in the study of protein-protein interactions. Additionally, it allows for the application of different selection stringencies. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into. Interaction between the library polypeptides was required for reconstitution of the enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates. We applied these selection processes to a library-versus-library sample of 2.0&#xd7;10combinations, and selected a novel leucine zipper pair which may be appropriate for use in further in vivo heterodimerization strategies.

Vivo library-versus-library selection of optimized protein-protein interactions

Protein Fragment Complementation Assays (PCA) are done in plant material using enzyme fragment constructs, for example, dihydrofolate reductase (DHFR) fragment constructs. Plant material is transformed with at least two different constructs that form different products capable of interacting to reconstitute enzymatic activity in the plant material. Detection of the activity can be done using a substrate for the enzyme in the culture medium which, when reacted with the enzyme, is converted to a detectable product. One embodiment uses a substrate that can be enzymatically converted to a detectable fluorescent product. An inducer such as rapamycin or salicylic acid can be added to the culture medium to increase the level of detectable product.

Mapping molecular interactions in plants with protein fragments complementation assays

Protein fragment complementation assay (PCA) for the detection of protein-protein, protein-small molecule and protein nucleic acid interactions based on the E. coli TEM-1 &bgr;-Lactamase

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Westmount, Canada

Stephen William Watson Michnick