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  Method of screening for protein inhibitors and activatorsUSPTO Application #: 20060019329Title: Method of screening for protein inhibitors and activators Abstract: Chemical agents which are inhibitors or activators of a protein whose expression affects a phenotypic characteristic of the cell, especially a cultural or morphological characteristic, can be identified by their more pronounced effect on cells producing higher, usually non-naturally occurring, levels of the protein, than on cells producing little or none of the protein. Such chemical agents are used to inhibit or activate a protein in a cell. By this method, tamoxifen is used to inhibit PKC activity in a cell. (end of abstract) Agent: Kenyon & Kenyon - New York, NY, US Inventor: Gerard Housey USPTO Applicaton #: 20060019329 - Class: 435029000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Viable Micro-organism The Patent Description & Claims data below is from USPTO Patent Application 20060019329. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application is a continuation-in-part of U.S. Ser. No. 07/154,206, filed Feb. 10, 1988, incorporated by reference herein, the benefit of whose filing date is claimed pursuant to 35 U.S.C. Sec. 120. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] This invention relates to a general screening method for the discovery and identification of both inhibitors and activators of enzymes, receptors, and other proteins. In particular, it is concerned with a method of screening for substances which specifically inhibit or activate a particular protein affecting the cultural or morphological characteristics of the cell expressing the protein, especially in a manner apparent to the naked eye. [0004] 2. Information Disclosure Statement [0005] A number of assay systems are currently in use for the discovery of new modulators of cell growth, and in particular, in the search for new anti-cancer drugs which are specifically toxic to cancer cells but not to normal cells. A variety of changes may be scored for, but the most common ones are reversion of the transformed phenotype, significant changes in cell morphology, or cytotoxicity. The assays include: (1) in vitro cytotoxicity assays; (2) soft agar colony formation assays; (3) in vitro anti-microbial assays; and (4) assays which detect changes in cellular morphology. [0006] In vitro cytotoxicity assays involve the measurement of cellular parameters which are indicative of inhibition of cellular growth or cytotoxicity. These include, for example, the measurement of the inhibition of certain cellular metabolic pathways in response to treatment with cytotoxic agents. The papers by Von Hoff, et al. (1985), and Catino, et al. (1985) describe typical methods which use this technique. These methods are somewhat complex technically, and require the use of radioactive tracers in some cases. Furthermore, the results are non-specific since any agent which alters the growth properties of cells will score positively in these assay systems. [0007] Agents have also been tested for their ability to inhibit transformed (cancerous) cells from growing in soft agar. This method is based upon the finding by Freedman and Shin (1974) that the formation of colonies of cells in soft agar is the in vitro test which shows the highest correlation in predicting whether the cells will be tumorigenic in an experimental animal. This method is relatively simple to perform since colony growth will, after two or more weeks, generally be large enough to be seen with the naked eye. Scoring the final results, therefore, can be performed either by a technician without extensive training in tissue culture, or, as we describe in the current application, by an automated absorbance detection system. In its present form, however, this method is also non-specific for the same reasons as described above. In other words, any agent which inhibits cellular growth in any way will scores positively in this assay system as it is currently used, whether or not it inhibits the protein of interest. [0008] In vitro anti-microbial assays involve the use of bacterial or yeast strains which are used as test organisms for screening for agents with generalized growth inhibitory properties (also described in Catino, et al., 1985). In this method, the bacterial or yeast strain is grown on standard media plates and potential agents are applied to various spots on the plates. If an agent has growth inhibitory properties, a clear zone results at the site of its application on the plate, resulting from the inability of the test strain to grow in the area. This method is rapid and can be performed by a technician without extensive training in tissue culture techniques, but the results are generally non-specific because agents which are effective against bacterial or yeast strains are frequently less effective (or completely ineffective) in modulating the growth of mammalian cells, as shown in the paper by Catino et al. (1985). [0009] Still other screening systems depend upon a morphologic alteration of the test cells by exposure to the potential agents in order to determine the effectiveness of a given agent. This method is currently the most effective one for developing specific agents which interact with a given protein or alter a specific cellular property, as evidenced by the representative paper by Uehara, et. al. (1985). However, these screening systems are the most difficult ones to apply in practice, since the morphologic effect of each individual agent on the test cells must be studied under the microscope. Hence this method requires extensive observations of the cells by a trained scientist. SUMMARY OF INVENTION [0010] The Method presented in detail in this application combines the rapidity and ease of performance of the soft agar assay with a specificity for detecting an active agent exceeding that of the morphology assay. In brief, the method which we describe herein involves the generation of a cell line purposefully engineered to detect both stimulatory and inhibitory agents which are absolutely specific for any given protein which affects the cultural or morphological characteristics of the cell. [0011] The basis for this invention is my observation that if a protein (the "protein of interest", or POI) which is involved in some manner in cellular growth control is overproduced in cells, then pharmacologic agents which can activate or inhibit the POI can result in altered growth properties of the cells. [0012] The sensitivity of the cells is dependent on their production of the POI, a phenomenon referred to herein as a "graded cellular response" to the pharmacologically active agent. [0013] The present invention provides a rapid, yet powerful screening system for the discovery and identification of both inhibitors and activators of proteins. The method may be applied to virtually any type of protein, including enzymes, receptors, DNA- or RNA-binding proteins, or others which are directly or indirectly involved in regulating cellular growth. [0014] The method involves the insertion of a DNA (or cDNA) sequence encoding the Protein Of Interest (POI) into an appropriate vector and the generation of cell lines which contain either (1) the expression vector alone ("control" cell lines) or (2) the expression vector containing the inserted DNA (or cDNA) sequence encoding the POI ("test" cell lines). Using the appropriate vector system, recipient cell lines, and growth conditions, test cell lines can thus be generated which stably overproduce the corresponding POI. Under the appropriate growth conditions, these cell lines will exhibit a "graded cellular response" to activators or inhibitors of the POI. A screening system can thus be set up whereby the control and test cell lines are propagated in defined growth conditions in tissue culture dishes (or even in experimental animals) and large numbers of compounds (or crude substances which may contain active compounds) can be screened for their effects on the POI. [0015] Substances which inhibit or activate the POI may affect characteristics such as growth rate, tumorigenic potential, anti-tumorigenic potential, anti-metastatic potential, cell morphology, antigen expression, and/or anchorage-independent growth capability. Substances which specifically inhibit or inactivate the POI may be distinguished from substances which affect cell morphology or growth by other mechanisms in that they will have a greater effect on the test lines than on the control lines. [0016] The system has been tested using several cDNA sequences and several recipient cell lines, and can be easily automated. [0017] The appended claims are hereby incorporated by reference as an enumeration of the preferred embodiments. All references cited anywhere in this specification are hereby incorporated by reference to the extent pertinent. BRIEF DESCRIPTION OF THE DRAWINGS [0018] FIG. 1A shows the full-length cDNA sequence, and the deduced amino acid sequence, of one of several forms of PKC which has previously been isolated (cDNA clone RP58), and whose partial sequence has been reported (Housey, et al., 1987). It corresponds to PKCbeta1 according to the nomenclature of Ono, et al. (1987). The deduced amino acid sequence begins with the first in-frame methionine codon at nucleotide position 91 and encodes a 671 amino acid protein with a predicted molecular weight of 76.8 kd. A consensus polyadenylation signal is underlined. [0019] FIG. 1B shows the retrovirus-derived cDNA expression vector, developed in this laboratory, which was used for the present studies. The full-length RP58 cDNA encoding PKCbeta1 (shown in 1A) was cloned into the Eco RI site of plasmid pMV7. The shaded region represents the coding sequence. "E" and "P" designate Eco RI and Pst I restriction sites, respectively. The indicated sizes between restriction sites in the RP58 cDNA are given in kilobases. "LTR" designates the 5' (left) and 3' (right) long terminal repeats of Moloney murine leukemia virus, and "TK-neo" designates the promoter region of the HSV thymidine kinase gene linked to the 5' end of the bacterial neomycin phosphotransferase (neo) gene. [0020] FIG. 1-C outlines in schematic form the overall strategy used to generate cell lines stably overproducing PKC. [0021] FIG. 2. Purification and Autophosphorylation of PKC. PKC activity from each cell line was purified and subjected to reaction conditions favoring autophosphorylation of PKC. Following the autophosphorylation reaction, protein samples were separated by discontinuous polyacrylamide gel electrophoresis. In the lanes bearing odd numbers the reaction mixtures contained 1 mM Ca2+ and phosphatidylserine to activate PKC, and in the lanes bearing even numbers the reaction mixtures contained 1 mM EGTA, 100 ng/ml TPA, and phosphatidylserine. The numbers in the left margin indicate the sizes of molecular weight markers, in kd. Arrows indicate the position of the 75 kd autophosphorylated PKC. Continue reading... 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