Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-07-30
2003-07-01
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S174000, C435S334000, C435S810000
Reexamination Certificate
active
06586190
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to a high throughput method and kit for the discovery of small molecule interactors of target proteins, in particular to a high throughput method and kit for parallel analysis of small molecule interactions with a multitude of target proteins of unknown function.
BACKGROUND OF THE INVENTION
Single target High Throughput Screens (HTS) are the method of choice to discover small molecule inhibitors, ligands, agonists or antagonists of specific target proteins in both the Agricultural and Pharmaceutical Research communities. These screens are performed on robotic platforms that can test 100,000 to over a million compounds against one target protein. Several detection methods, ranging from radiolabeled tracers to elaborate fluorescence markers, are currently used. For each target protein to be screened, investigators must know its particular enzymatic or regulatory function in order to identify and prepare a proper substrate or ligand, and to develop a proper workable detection method. Gene products that are known to have a useful physiological role, but whose enzymatic or regulatory function is unknown, are not used as targets in HTS. Thus, this approach requires a large investment in assay development and implementation, allowing only a limited number of target protein HTS to be performed in a given time period.
Whole genome sequencing projects are forcing a shift in the traditional approach to the HTS research paradigm. One important outcome of these sequencing efforts is the identification of large collections of validated targets. However, because the current approaches to small molecule discovery are based on the single target HTS model, scientists are unable to efficiently use and exploit the vast amount of genomics information being generated. Thus, only highly validated targets warrant the development of unique screens, i.e. targets of known function. In addition, the function of a large portion of the newly discovered validated targets is unknown, making the development of a single target HTS impossible in these cases. Clearly, current single target HTS approaches have severe economic, feasibility, and logistic limitations Thus, a HTS capable of analyzing more than just a few targets without development of unique screens for each target would provide a significant advantage in the field of genomic analysis. In addition, an HTS capable of analyzing a large number of targets of unknown function would also provide a significant advantage in the field of genomic analysis.
SUMMARY
The present invention brings together gene cloning and expression, protein purification and modification, ligand identification and synthesis, and assay platform technologies into a novel screen and method that allows for the parallel, massively multiplexed screening of targets on a genomic scale. The present invention further provides a screen and method capable of analyzing in a HTS manner a multitude of targets of unknown function. A protein of unknown function is any protein for which one cannot associate enzymatic, regulatory, structural or receptor activity.
In the preferred embodiment, the method and kit of the present invention use a collection or plurality of gene products, otherwise referred to herein as “target proteins,” based on genomics information about their essentiality to a physiological process. The method and kit of the present invention do not require, however, that the enzymatic or regulatory function of the target proteins be known, nor, for that matter, their essentiality.
In accordance with the present invention, the gene coding for each desired target protein is prepared, transferred into an expression vector and moved into an appropriate host organism, for example
E. coli,
baculovirus, mammalian cells, or yeast. The gene products or target proteins are expressed and then purified.
The purified target proteins are biotinylated and used to pan for phages displaying foreign peptides on their surfaces. Phages containing peptide sequences that bind selectively to the purified target proteins are separately amplified and the DNA encoding the peptides sequenced. The peptide sequences encoded by the DNA are synthesized and used as the surrogate ligands in the method and kit of the present invention.
A target module is prepared by binding selectively a target protein, modified to allow its detection, with a surrogate ligand that is linked to an individually detectable bead. In one embodiment of the present invention, 100 target modules are constructed for 100 target proteins of interest. The 100 target modules are mixed together in each chamber of a multi-chamber container. A compound or collection of compounds to be tested is added to each chamber, and the interaction of a compound with each target module is observed. In the preferred embodiment, analysis of the interaction is implemented by flow cytometry. A compound that is specific for a particular target protein will displace that protein from its target module. The identity of the target module so disrupted is determined by identifying the particular bead to which the surrogate ligand is attached.
In more detail, the method of the present invention includes the steps of obtaining a plurality of target proteins; obtaining a first set of surrogate ligands, wherein each surrogate ligand in the set of surrogate ligands binds selectively to a first target protein; binding the first set of surrogate ligands to first detectable beads to form a first set of surrogate ligand-bead complexes, wherein the first detectable beads can all be detected by the optical characteristics of the first detectable beads; combining the first set of surrogate ligand-bead complexes with the target protein labeled for detection to form a first target module; repeating the above steps, either concurrently or subsequently with a different set of surrogate ligands and detectable beads, and with either the first target protein or a different target protein, to form sets of target modules; adding the sets of target modules to each chamber of a multi-chamber container; adding a test compound, or a collection of test compounds, to each chamber of the multi-chamber container; detecting displacement of a target protein with a test compound; and determining the identity of each target protein that is displaced with a test compound.
The method of the invention further includes obtaining each said set of surrogate ligands by obtaining a phage library, wherein each phage of the library displays foreign peptides; mixing the phage library with each target protein of the plurality of target proteins; isolating phages displaying the foreign peptides that bind selectively to each target protein; isolating DNA encoding the foreign peptides that bind selectively to each target protein; sequencing the DNA; and synthesizing the set of surrogate ligands based on the sequencing.
The method of the invention further includes obtaining the plurality of target proteins by selecting target genes from a genome; expressing each of the target genes to produce the plurality of target proteins; and purifying the target proteins.
The method includes biotinylating the target proteins and linking the target proteins with avidin-phycoerythrin.
The invention further provides target proteins by selecting target genes from a genome, expressing each of the target genes to produce the set of target proteins, and purifying the target proteins.
The invention further provides using a surrogate ligand selected from the group including a peptide, RNA aptamer, and &bgr;-peptide. Surrogate ligands may also be selected from small molecules derived from combinatorial chemistry or from natural compound collections.
The invention also provides screening target proteins having unknown function.
The invention further provides labeling each individual bead with a defined combination of two dyes.
The invention further provides labeling each individual bead with a defined combination of three dyes.
The invention further provides labeling each individual
Bernasconi Paul
Carpenter Ray Donald
Hunt Jennifer Audrey
Petrovich Robert Michael
Powell Gary Keith
Park Hankyel T.
Syngenta Participations (AG)
Vrana Bruce
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