WO1999035494A1 - Method for identifying validated target and assay combinations - Google Patents
Method for identifying validated target and assay combinations Download PDFInfo
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- WO1999035494A1 WO1999035494A1 PCT/US1999/000474 US9900474W WO9935494A1 WO 1999035494 A1 WO1999035494 A1 WO 1999035494A1 US 9900474 W US9900474 W US 9900474W WO 9935494 A1 WO9935494 A1 WO 9935494A1
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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Definitions
- genomics revolution by determining the DNA sequences of great numbers of genes from many different organisms, has considerably broadened the possibilities for drug discovery by identifying large numbers of molecules that are potential targets of drug action.
- These technical advances in genomics have posed an entirely new set of challenges. Specifically, how can one prove that a chosen target molecule is essential to maintaining the disease or disorder to be treated? That is, how does one validate a target? (See “target validation” in Definitions.)
- the invention relates to methods that couple the validation of a target (see Definitions) for drug discovery with the development of an assay to identify compounds that cause a phenotypic effect on the target cell. These procedures can be applied to identifying compounds that bind to and modulate the function of target components of a cell whose function is known or unknown, and cell components that are not amenable to other screening methods.
- the invention relates to procedures for identifying a compound that binds to and modulates (inhibits or enhances) the function of a component of a cell, thereby producing a phenotypic effect in the cell.
- procedures for identifying a biomolecule See Definitions section) that 1) binds to, in vitro, a component of a cell that has been isolated from other constituents of the cell and that 2) causes, in vivo, as seen in an assay upon intracellular expression of the biomolecule, a phenotypic effect (See Definitions section) in the cell which is the usual producer and host of the target cell component.
- intracellular production of the biomolecule can be in cells grown in culture or in cells introduced into an animal.
- One procedure envisioned in the invention is a process for identifying one or more compounds that produce a phenotypic effect on a cell.
- the process is at the same time a method for target validation (See Definitions section).
- the process is characterized by identifying a biomolecule which binds an isolated target cell component, constructing cells comprising the target cell component and further comprising a gene encoding the biomolecular binder which can be expressed to produce the biomolecular binder, testing the constructed cells for their ability to produce, upon expression of the gene encoding the biomolecular binder, a phenotypic effect in the cells (e.g., inhibition of growth), wherein the test of the constructed cells can be a test of the cells in culture or a test of the cells after introducing them into host animals, or both, and further, identifying, for a biomolecular binder that caused the phenotypic effect, one or more compounds that compete with the biomolecular binder for binding to the target cell component.
- a test of the constructed cells after introducing them into host animals is especially well-suited to assessing whether a biomolecular binder can produce a particular phenotype by the expression (regulatable by the researcher) of a gene encoding the biomolecular binder.
- cells are constructed which have a gene encoding the biomolecular binder, and wherein the biomolecular binder can be produced by regulation of expression of the gene.
- the constructed cells are introduced into a set of animals. Expression of the gene encoding the biomolecular binder is regulated in one group of the animals (test animals) such that the biomolecular binder is produced.
- the gene encoding the biomolecular binder is regulated such that the biomolecular binder is not produced (control animals).
- the cells in the two groups of animals are monitored for a phenotypic change (for example, a change in growth rate). If the phenotypic change is observed in cells in the test animals and not in the cells in the control animals, or to a lesser extent in the control animals, then the biomolecular binder has been proven to be effective in binding to its target cell component under in vivo conditions.
- a further embodiment of the invention is a method for determining whether a target cell component of a particular cell type (a "first cell”) is essential to producing a phenotypic effect on the first cell, the method having the steps: isolating the target component of the first cell; identifying a biomolecular binder of the isolated target component of the first cell; constructing a second type of cells (“second cell”) comprising the target component and a regulable, exogenous gene encoding the biomolecular binder; and testing the second cell in culture for an altered phenotypic effect, upon production of the biomolecular binder in the second cell; whereby, if the second cell shows the altered phenotypic effect upon production of the biomolecular binder, then the target component of the first cell is essential to producing the phenotypic effect on the first cell.
- first cell a target cell component of a particular cell type
- the target cell component in this embodiment and in other embodiments not limited to pathogens can be one that is found in mammalian cells, especially cells of a type found to cause or contribute to disease or the symptoms of disease (e.g., cells of tumors or cells of other types of hyperproliferative disorders).
- the invention further relates to methods particularly well suited to a procedure for identifying and/or designing compounds with antimicrobial activity against a pathogen whose target cell component is the subject of studies to identify such compounds.
- a common mechanism of action of an antimicrobial agent is binding to a component of the cells of the pathogen treated with the antimicrobial.
- the procedure includes methods for identifying biomolecules that bind to a chosen target in vitro, methods for identifying biomolecules that also bind to the chosen target and modulate its function during infection of a host mammal in vivo, and methods for identifying compounds that compete with the biomolecules for sites on the target in competitive binding assays.
- Compounds identified by this procedure are candidates for drugs with antimicrobial activity against the pathogen.
- One embodiment of the invention is a method for identifying a biomolecular inhibitor of growth of pathogen cells by using cell culture techniques, comprising contacting one or more types of biomolecules with isolated target cell component of the pathogen, applying a means of detecting bound complexes of biomolecules and target cell component, whereby, if the bound complexes are detected, one or more types of biomolecules have been identified as a biomolecular binder of the target cell component, constructing a pathogen strain having a regulable gene encoding the biomolecular binder, regulating expression of the gene encoding the biomolecular binder to express the gene; and monitoring growth of the pathogen cells in culture relative to suitable control cells, whereby, if growth of the pathogen cells is decreased compared to growth of suitable control cells, then the biomolecule is a biomolecular inhibitor of growth of the pathogen cells.
- a further embodiment of the invention is a method, employing an animal test, for identifying one or more compounds that inhibit infection of a mammal by a pathogen by binding to a target cell component, comprising constructing a pathogen comprising a regulable gene encoding a biomolecule which binds to the target cell component, infecting test animals with the pathogen, regulating expression of the regulable gene to produce the biomolecule, monitoring the test animals and suitable control animals for signs of infection, wherein observing fewer or less severe signs of infection in the test animals than in suitable control animals indicates that the biomolecule is a biomolecular inhibitor of infection, and identifying one or more compounds that compete with the biomolecular inhibitor of growth for binding to the target cell component (as by employing a competitive binding assay), then the compound inhibits infection of a mammal by a pathogen by binding to a target.
- the competitive binding assay to identify binding analogs of biomolecular binders which have been proven to bind to their targets in an intracellular test of binding, can be applied to any target for which a biomolecular binder has been identified, including targets whose function is unknown or targets for which other types of assays are not easily developed and performed. Therefore, the method of the invention offers the advantage of decreasing assay development time when using a gene product of known function as a target cell component and the advantage of bypassing the major hurdle of gene function identification when using a gene product of unknown function as a target cell component.
- cells comprising a biomolecule and a target cell component, wherein the biomolecule is produced by expression of a regulable gene, and wherein the biomolecule modulates function of the target cell component, thereby causing a phenotypic change in the cells.
- cells comprising a biomolecule and a target cell component, wherein the biomolecule is a biomolecular binder of the target cell component, and is encoded by a regulable gene.
- the cells can include mammalian cells or cells of a pathogen, for instance, and the phenotypic change can be a change in growth rate.
- the pathogen can be a species of bacteria, yeast, fungus, or parasite, for example.
- Figure 1 is an illustration showing the steps in cloning of the Pro-3 peptide for regulated expression as a GST (glutathione-S transferase) fusion protein. See Example 1.
- Figure 2 is a photograph of an SDS-polyacrylamide gel showing expression of the Pro-3 peptide in E. coli cells. Production of the Pro-3/GST fusion and production of ⁇ -galactosidase in E. coli cells carrying pC 3 844 or pPROTet-LacZ were analyzed by SDS-PAGE. The concentrations of anhydrotetracycline (aTc) used for induction of gene expression are indicated above the lanes on the gel. See Example 2.
- aTc anhydrotetracycline
- Figure 3 A is a graph showing E. coli growth inhibition by expression of Pro- 3 peptide encoded on the pC 3 844 plasmid.
- the OD 600 of the bacterial cultures in the presence (+aTc) or absence (-aTc) of anhydrotetracychne were monitored at the time points shown. See Example 2.
- Figure 3B is a graph showing results of a control experiment to compare with the results shown in Figure 3A.
- the E. coli cells used in the experiment harbor the pPROTet-GST plasmid.
- Figure 4A is a graph showing functional complementation of growth inhibition by Pro-3 peptide, by expression of a heterologous ProRS gene.
- the growth of E. coli cells DH5 ⁇ PRO/pC 3 844 carrying a Staphylococcus aureus ProRS expression construct was characterized in the presence (Pro3 Expression) or absence (No Pro3 Expression) of 200 ng/ml anhydrotetracychne by monitoring OD 600 at the time points shown.
- Figure 4B is a graph showing the results of a control experiment to compare with the results in Figure 4A.
- the growth of E. coli cells DH5 ⁇ PRO/pC 3 844 carrying pACYC177 was characterized in the presence (Pro3 Expression) or absence (No Pro3 Expression) of 200 ng/ml anhydrotetracychne by monitoring OD 600 at the time points shown.
- Figure 5 is a scanned image of an 18% SDS-polyacrylamide gel stained with Coomassie blue. Inducible expression of chloramphenicol acetyltransferase (CAT) in S. aureus.
- CAT chloramphenicol acetyltransferase
- the whole cell lysates of S aureus RN4220 (Host) or RN4220 harboring pWH353 (C 3 SaE-l) or pWH354 (C 3 SaE-2) with (+) or without (-) tetracycline induction were analyzed by electrophoresis on an 18% SDS- polyacrylamide gel.
- Figure 6 is a graph showing S. aureus MetRS tRNA charging activity (shown as counts per minute of trichloracetic acid precipitable [ 3 H]-methionine) is inhibited by increasing concentrations of JT01.
- Figure 7 is a graph showing fluorescence polarization plotted with the concentration of compound added, indicating inhibition of Pro3 peptide binding to E. coli ProRS in this assay by known inhibitors: open circles, CB-16914; filled circles, CB-118831 ; open squares. CB-680 negative control.
- Figure 8 is a bar graph showing fluorescence of microtiter wells in the assay described in Example 9. Wells 1-6 contain Met- IF alone. Wells 7 and 8 contain S.
- aureus MetRS methionyl-tRNA synthetase alone
- wells 9-18 and 19-28 contain Met-1 peptide from 640 nM to 1.25 nM
- wells 29-96 contain 1% DMSO (dimethyl sulfoxide).
- Target (also, "target component of a cell,” or “target cell component”) a constituent of a cell which contributes to and is necessary for the production or maintenance of a phenotype of the cell in which it is found.
- a target can be a single type of molecule or can be a complex of molecules.
- a target can be the product of a single gene, but can also be a complex comprising more than one gene product (for example, an enzyme comprising ⁇ and ⁇ subunits, mRNA, tRNA, ribosomal RNA or a ribonucleoprotein particle such as a snRNP).
- Targets can be the product of a characterized gene (gene of known function) or the product of an uncharacterized gene (gene of unknown function).
- Target Validation the process of determining whether a target is essential to the maintenance of a phenotype of the cell type in which the target normally occurs. For example, for pathogenic bacteria, researchers developing antimicrobials want to know if a compound which is potentially an antimicrobial agent not only binds to a target in vitro, but also binds to, and modulates the function of, a target in the bacteria in vivo, and especially under the conditions in which the bacteria are producing an infection ⁇ those conditions under which the antimicrobial agent must work to inhibit bacterial growth in an infected animal or human.
- Phenotypic Effect a change in an observable characteristic of a cell which can include, e.g., growth rate, level or activity of an enzyme produced by the cell, sensitivity to various agents, antigenic characteristics, and level of various metabolites of the cell.
- a phenotypic effect can be a change away from wild type (normal) phenotype, or can be a change towards wild type phenotype, for example.
- a phenotypic effect can be the causing or curing of a disease state, especially where mammalian cells are referred to herein.
- a phenotypic effect can be the slowing of growth rate or cessation of growth.
- Biomolecule a molecule which can be produced as a gene product in cells that have been appropriately constructed to comprise one or more genes encoding the biomolecule. Preferably, production of the biomolecule can be turned on, when desired, by an inducible promoter.
- a biomolecule can be a peptide, polypeptide, or an RNA or RNA oligonucleotide, a DNA or DNA oligonucleotide, but is preferably a peptide.
- the same biomolecules can also be made synthetically. For peptides, see Merrifield, J., J. Am. Chem. Soc. 85: 2140-2154 (1963).
- Biomolecular Binder (of a target): a biomolecule which has been tested for its ability to bind to an isolated target cell component in vitro and has been found to bind to the target.
- Biomolecular Inhibitor of Growth a biomolecule which has been tested for its ability to inhibit the growth of cells constructed to produce the biomolecule in an "in culture” test of the effect of the biomolecule on growth of the cells, and has been found, in fact, to inhibit the growth of the cells in this test in culture.
- Biomolecular Inhibitor of Infection a biomolecule which has been tested for its ability to ameliorate the effects of infection, and has been found to do so. In the test, pathogen cells constructed to regulably express the biomolecule are introduced into one or more animals, the gene encoding the biomolecule is regulated so as to allow production of the biomolecule in the cells, and the effects of production of the biomolecule are observed in the infected animals compared to one or more suitable control animals.
- Isolated term used herein to indicate that the material in question exists in a physical milieu distinct from that in which it occurs in nature.
- an isolated target cell component of the invention may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs.
- the absolute level of purity is not critical, and those skilled in the art can readily determine appropriate levels of purity according to the use to which the material is to be put.
- the isolated material will form part of a composition (for example, a more or less crude extract containing other substances), buffer system or reagent mix.
- the material may be purified to essential homogeneity, for example as determined by PAGE or column chromatography (for example, HPLC).
- Pathogen or Pathgenic Organism an organism which is capable of causing disease, detectable by signs of infection or symptoms characteristic of disease.
- Pathogens can include procaryotes (which include, for example, medically significant Gram- positive bacteria such as Streptococcus pneumoniae, Enterococcus faecalis and Staphylococcus aureus, Gram-negative bacteria such as Escherichia coli, Pseudomonas aeroginosa and Klebsiella pneumoniae, and "acid-fast" bacteria such as Mycobacte ⁇ a, especially M. tuberculosis), eucaryotes such as yeast and fungi (for example, Candida albicans and Aspergillus fumigatus) and parasites.
- procaryotes which include, for example, medically significant Gram- positive bacteria such as Streptococcus pneumoniae, Enterococcus faecalis and Staphylococcus aureus
- Gram-negative bacteria such as Escherichi
- pathogens can include such organisms as soil-dwelling organisms and "normal flora" of the skin, gut and orifices, if such organisms colonize and cause symptoms of infection in a human or other mammal, by abnormal proliferation or by growth at a site from which the organism cannot usually be cultured.
- the present invention relates to methods that couple the validation of a target cell component for drug discovery with the development of a validated assay to identify compounds that cause a phenotypic effect on the target cell (cell harboring the target cell component).
- the target cells are cells of a pathogenic organism, compounds identified by this procedure are candidates for drugs with antimicrobial activity against the pathogen.
- the method utilized for target validation provides a test of how a biomolecule produced intracellularly binds to a specific site on a target cell component and alters the target's function in a cell during an established infection or disease.
- the technology to validate the target identifies a biomolecule specific to the target that can be used in a screening assay to identify drug leads, thereby coupling target validation with drug lead identification.
- the method also validates specific sites on a target molecule for drug discovery, which is especially important for proteins involved in multiple functions.
- Described herein are methods that result in the identification of compounds that cause a phenotypic effect on a cell.
- the general steps described herein to find a compound for drug development can be thought of as these: (1) identifying a biomolecule that can bind to an isolated target cell component in vitro, (2) confirming that the biomolecule, when produced in cells with the target cell component, can cause a desired phenotypic effect and (3) identifying, by an in vitro screening method, for example, compounds that compete with the biomolecule for binding to the target cell component.
- Advantages of the these steps are that it is not necessary to identify the function of the target cell component and it is not necessary to develop an assay tailored to the function (e.g., enzyme activity) of the target cell component.
- a biomolecule is a gene product (e.g., polypeptide, RNA, peptide or RNA oligonucleotide) of an exogenous gene — a gene which has been introduced in the course of construction of the cell. See also Definitions section.
- Biomolecules that bind to and alter the function of a candidate target are identified by various in vitro methods. Upon production of the biomolecule within a cell either in vitro or within an animal model system, the biomolecule binds to a specific site on the target, alters its intracellular function, and hence produces a phenotypic change (e.g. cessation of growth, cell death). When the biomolecule is produced in engineered pathogen cells in an animal model of infection, cessation of growth or death of the engineered pathogen cells leads to the clearing of infection and animal survival, demonstrating the importance of the target in infection and thereby validating the target.
- a phenotypic change e.g. cessation of growth, cell death
- a method for ( 1 ) identifying a biomolecule that produces a phenotypic effect on a cell can comprise steps of introducing into an animal a cell comprising an exogenous regulable gene encoding the biomolecule. regulating expression of the gene to produce the biomolecule in the cell, and monitoring said cell in the animal for a phenotypic effect, compared to a suitable control cell. If the cell of this test manifests a phenotypic effect, this indicates that the biomolecule produced in the cell causes a phenotypic effect on the cell.
- the biomolecule can be termed a "biomolecular inhibitor” or a “biomolecular inhibitor of growth.” It may be desirable to perform another test of intracellular function, using cell culturing techniques, wherein the cell comprising an exogenous regulable gene encoding the biomolecule of interest, and comprising the target cell component, is treated so as to turn on expression of the gene encoding the biomolecule, and one or more phenotypic characteristics of the cells in culture are monitored relative to suitable control cells, where the control cells do not produce the biomolecule. It may be preferable, where both "in culture” and “in animal” intracellular tests are performed, to do an "in culture” test first.
- the purpose of intracellular validation for the combination of a potential target for drug action and molecule for drug development is two-fold.
- the biomolecule in an intracellular test is exposed to a multitude of potential binding partners in the living cell, and interaction with one or more of these binding partners in the cell may be unproductive or result in undesirable effects. These effects are not detectable in an in vitro binding test.
- a biomolecule has been shown previously by in vitro tests to bind to a target cell component (that is, the biomolecule can be called a "biomolecular binder" of the target cell component; See Definitions section)
- intracellular validation provides proof that the target cell component is essential to the maintenance of the original phenotype of the cell. Therefore, the target is validated for drug discovery and the biomolecule can then be utilized in a competitive binding assay to identify compounds that will have an effect on target molecule function. Efficient binding between a biomolecule and a target cell component may be demonstrated in vitro; even binding that inhibits activity of a target enzyme may be demonstrated in vitro. However, in the living cells, there could exist a redundant system that nullifies the effect of the biomolecule binding to the target cell component.
- production of an enzyme having similar activity to that of the target cell component may be induced in the cells.
- the cell could escape any effect the biomolecule might otherwise cause by binding to the target cell component.
- Using an intracellular test to validate biomolecule/target cell component interaction is superior to using only an in vitro test using isolated molecules, because the intracellular test ensures that the target cell component is in its natural conformation and that the biomolecule "sees" the target cell component in that conformation, as that conformation occurs in a disease state. That in an intracellular test a biomolecule finds a site which ultimately causes a phenotypic effect on the cell indicates that the biomolecule is binding to a functionally relevant site on the target cell component (e.g., an active site of an enzyme).
- a functionally relevant site on the target cell component e.g., an active site of an enzyme
- molecules that are found to be structural analogs of the biomolecule and to compete with the biomolecule for a binding site on the target will also interact with the functionally relevant site of the target cell component, as functional analogs.
- a functional analog of the biomolecule can be found through competitive binding assays of the biomolecule against compounds (as in a library of compounds) that are potential binders of the target cell component.
- Structural analogs can also be found by rational drug design once a biomolecular binder is identified, by designing drugs that mimic the structure of the biomolecular binder. These structural analogs can be tested for their binding properties by techniques described herein.
- a further advantage of the intracellular test in which the biomolecule is produced from one or more genes in the cell is that the biomolecule does not have to pass through a cell membrane or rely on inefficient uptake mechanisms of the cell. Intracellular production of the biomolecule ensures that a biomolecule that interacts with a functional site on a target cell component to produce an effect will be detected, even if uptake of the biomolecule into the cell is limited. By the intracellular test, more biomolecules testing as being able to cause the desired phenotypic effect can be detected as candidates for further testing to find functional analogs for drug development. In a test employing extracellular addition of biomolecules.
- biomolecules that bind the target cell component but are taken up by the cell only to a limited extent could be missed as candidates for further testing to find functional analogs for drug development.
- Limited uptake of a biomolecule which has been found to bind to a target in vitro is not necessarily a barrier to further steps towards drug development, as a structural portion of the ultimate compound to be administered as a drug can be selected for its stability, membrane solubility, efficient uptake, etc., and can be chemically combined with a compound whose structure mimics the active binding portion of the biomolecule.
- Intracellular production of the biomolecule in an intracellular test of the effect of a biomolecule, as opposed to uptake from outside the cell, can also minimize degradation of the biomolecules from extracellular and intracellular degradative enzymes (e.g., proteases).
- one or more compounds that can also produce the phenotypic effect caused by the biomolecule can be identified in an in vitro competitive binding assay (which may be adapted for high-throughput screening) as compounds that compete with the biomolecule for a binding site on the target cell component.
- Target cell components can be isolated from the type of cell in which the phenotypic effect is desired (for instance, cells of pathogenic bacteria, yeast or fungi; mammalian cells, such as tumor cells), or from cells engineered to produce the cell component or a derivative of the cell component that would provide (at least some) structurally identical binding sites (e.g., a fusion protein).
- Compounds that produce the phenotypic effect observed with the biomolecule can be found in the competitive binding assay upon screening of libraries of compounds (for example, small molecule compounds or natural products or libraries that can be selected for having as their members compounds that have greater intracellular stability than biomolecules such as peptides or RNA oligonucleotides).
- the invention includes methods for identifying compounds that inhibit the growth of cells having a target cell component.
- the target cell component can first be identified as essential to the growth of the cells in culture and/or under conditions in which it is desired that the growth of the cells be inhibited.
- These methods can be applied, for example, to various types of cells that undergo abnormal or undesirable proliferation, including cells of neoplasms (tumors or growths, either benign or malignant) which, as known in the art, can originate from a variety of different cell types.
- neoplasms tumors or growths, either benign or malignant
- Such cells can be referred to, for example, as being from adenomas, carcinomas, lymphomas or leukemias.
- the method can also be applied to cells that proliferate abnormally in certain other diseases, such as arthritis, psoriasis or autoimmune diseases.
- Described herein are similar methods for identifying inhibitors of target molecules or target cell components of pathogenic organisms. These methods can include a target validation procedure using an animal model for confirming that a cell component of a pathogenic organism is essential, after infection with the organism has been established in a host, and that the inhibitor is effective against the organism after the organism has established the infection.
- a goal of the procedure is to identify compounds and/or gain the knowledge required to design compounds that can be used as antimicrobial agents to treat a human or other mammal having an infection of the organism.
- the invention provides methods for in vitro and in vivo validation of target and assay combinations. Following selection of biomolecular binders to the isolated target cell component of interest, the invention can incorporate steps for (1) regulable (e.g., inducible) intracellular expression of a gene encoding the biomolecular binder and (2) monitoring cell viability in culture (e.g., cell growth in liquid media or agar plates) or in vivo (e.g., growth of introduced cells or pathogen virulence in an animal infection model) or both. If intracellular expression of the biomolecular binder inhibits the function of a target essential for growth (presumably by binding to the target at a biologically relevant site) cells monitored in step (2) will exhibit a slow growth or no growth phenotype.
- regulable e.g., inducible
- Targets found to be essential for growth by these methods are validated starting points for drug discovery, and can be incorporated into assays to identify more stable compounds that bind to the same site on the target as the biomolecule.
- the invention provides a procedure to examine the activity of target (pathogen) cell components in an animal infection model. Controlled expression in cells of biomolecular binders to the target of interest mimics the environment for traditional antimicrobial therapy and validates targets as essential and appropriate for drug discovery.
- the technology facilitates choosing the best antimicrobial targets for drug discovery by facilitating direct observation of the effect (phenotype) produced by target inhibition at a specific target subsite.
- the process is broadly applicable to a variety of targets.
- the process also validates target and biomolecular binder combinations as a direct path to high throughput screening for binding analogs of the biomolecular binder, and is equally facile with targets that are gene products of genes of unknown function or genes of known function.
- Validated target and biomolecular binder assay combinations can be used directly in in vitro or in vivo competitive binding assays for screening chemical compound files. Compounds that compete with the biomolecular binders are identified as potential medicinal chemistry leads.
- a target cell component a gene product of a particular cell type (e.g., a type of pathogenic bacteria), wherein the target cell component is already known as being encoded by a characterized gene, as a potential target for a modulator to be identified.
- the target cell component can be isolated directly from the cell type of interest, assuming suitable culture methods are available to grow a sufficient number of cells, using methods appropriate to the type of cell component to be isolated (e.g., protein purification methods such as differential precipitation, ion exchange chromatography, gel chromatography, affinity chromatography, HPLC).
- the target cell component can be produced recombinantly, which requires that the gene encoding the target cell component be isolated from the cell type of interest.
- This can be done by any number of methods, for example known methods such as PCR, using template DNA isolated from the pathogen or a DNA library produced from the pathogen DNA, and using primers based on known sequences or combinations of known and unknown sequences within or external to the chosen gene. See, for example, methods described in "The Polymerase Chain Reaction," Chapter 15 of Current Protocols in Molecular Biology, (Ausubel, F.M. et al., eds), John Wiley & Sons, New York, 1998.
- Other methods include cloning a gene from a DNA library (e.g., a cDNA library from a eucaryotic pathogen) into a vector (e.g., plasmid, phage, phagemid, virus, etc.) and applying a means of selection or screening to clones resulting from a transformation of vectors (including a population of vectors now having inserted genes) into appropriate host cells.
- the screening method can take advantage of properties given to the host cells by the expression of the inserted chosen gene (e.g., detection of the gene product by antibodies directed against it, detection of an enzymatic activity of the gene product), or can detect the presence of the gene itself (for instance, by methods employing nucleic acid hybridization).
- Target proteins can be expressed with E. coli or other prokaryotic gene expression systems, or in eukaryotic gene expression systems. Since many eukaryotic proteins carry unique modifications that are required for their activities, e.g. glycosylation and methylation, protein expression can in some cases be better carried out in eukaryotic systems, such as yeast, insect, or mammalian cells that can perform these modifications. Examples of these expression systems have been reviewed in the following literature: Methods in Enzymology, Volume 185, eds D.V.
- the gene can be identified and cloned by a method such as that used in Shiba et al., US 5,759,833, Shiba et al, US 5,629,188, Martinis et al, US 5,656,470 and Sassanfar et al, US 5,756.327.
- a method such as that used in Shiba et al., US 5,759,833, Shiba et al, US 5,629,188, Martinis et al, US 5,656,470 and Sassanfar et al, US 5,756.327.
- the teachings of these four patents are incorporated herein by reference in their entirety.
- a segment of DNA containing an open reading frame (ORF; a cDNA can also be used, as appropriate to a eukaryotic cell) which has been isolated from a cell of a type that is to be an object of drug action (e.g., tumor cell, pathogen cell) can be cloned into a vector, and the target gene product of the ORF can be produced in host cells harboring the vector.
- the gene product can be purified and further studied in a manner similar to that of a gene product that has been previously isolated and characterized.
- the open reading frame (in some cases, cDNA) can be isolated from a source of DNA of the cells of interest (genomic DNA or a library, as appropriate), and inserted into a fusion protein or fusion polypeptide construct.
- This construct can be a vector comprising a nucleic acid sequence which provides a control region (e.g., promoter, ribosome binding site) and a region which encodes a peptide or polypeptide portion of the fusion polypeptide wherein the polypeptide encoded by the fusion vector endows the fusion polypeptide with one or more properties that allow for the purification of the fusion polypeptide.
- the vector can be one from the pGEX series of plasmids (Pharmacia) designed to produce fusions with glutathione S-transferase.
- the isolated DNA having an open reading frame, whether encoding a known or an as yet unidentified gene product, when inserted into an expression construct, can be expressed to produce the target cell component in host cells.
- Host cells can be, for example, Gram-negative or Gram-positive bacterial cells such as Escherichia coli or Bacillus subtilis, respectively, or yeast cells such as Saccharomyces cerevisiae, Schizosaccharomyces pombe or Pichia pastoris. It is preferable that the target cell component to be used in target validation studies be produced in a host that is genetically related to the pathogen from which the gene encoding it was isolated. For example, for a Gram-negative bacterial pathogen, an E.
- coli host is preferred over a Pichia pastoris host.
- the target cell component so produced can then be isolated from the host cells.
- Many protein purification methods are known that separate proteins on the basis of, for instance, size, charge, or affinity for a binding partner (e.g., for an enzyme, a binding partner can be a substrate or substrate analog), and these methods can be combined in a sequence of steps by persons of skill in the art to produce an effective purification scheme.
- a binding partner e.g., for an enzyme, a binding partner can be a substrate or substrate analog
- An isolated cell component or a fusion protein comprising the cell component can be used in a test to identify one or more biomolecular binders of the isolated product (general step ( 1)).
- a biomolecular binder of a target cell component (See Definitions section) can be identified by in vitro assays that test for the formation of complexes of target and biomolecular binder noncovalently bound to each other.
- the isolated target can be contacted with one or more types of biomolecules under conditions conducive to binding, the unbound biomolecules can be removed from the targets, and a means of detecting bound complexes of biomolecules and targets can be applied.
- the detection of the bound complexes can be facilitated by having either the potential biomolecular binders or the target labeled or tagged with an adduct that allows detection or separation (e.g., radioactive isotope or fluorescent label; streptavidin, avidin or biotin affinity label).
- an adduct that allows detection or separation e.g., radioactive isotope or fluorescent label; streptavidin, avidin or biotin affinity label.
- both the potential biomolecular binders and the target can be differentially labeled.
- WO 98/19162 see, e.g., WO 98/19162.
- the biomolecules to be tested for binding to a target can be from a library of candidate biomolecular binders, (e.g., a peptide or oligonucleotide library).
- a peptide library can be displayed on the coat protein of a phage (see, for examples of the use of genetic packages such as phage display libraries, Koivunen, E. et al., J. Biol. Chem. 265:20205-20210 (1993)).
- the biomolecules can be detected by means of a chemical tag or label attached to or integrated into the biomolecules before they are screened for binding properties.
- the label can be a radioisotope, a biotin tag, or a fluorescent label.
- biomolecular binders Those molecules that are found to bind to the target molecule can be called biomolecular binders.
- An isolated target cell component, an antigenically similar portion thereof, or a suitable fusion protein comprising all of or a portion of or the entire target can be used in a method to select and identify biomolecules which bind specifically to the target.
- the target cell component comprises a protein
- fusion proteins comprising all of, or a portion of, the target linked to a second moiety not occurring in the target as found in nature, can be prepared for use in another embodiment of the method.
- Suitable fusion proteins for this purpose include those in which the second moiety comprises an affinity ligand (e.g., an enzyme, antigen, epitope).
- the fusion proteins can be produced by the insertion of a gene encoding a target or a suitable portion of such gene into a suitable expression vector, which encodes an affinity ligand (e.g., pGEX-4T-2 and pET-15b, encoding glutathione S-transferase and His-Tag affinity ligands, respectively).
- the expression vector can be introduced into a suitable host cell for expression. Host cells are lysed and the lysate, containing fusion protein, can be bound to a suitable affinity matrix by contacting the lysate with an affinity matrix under conditions sufficient for binding of the affinity ligand portion of the fusion protein to the affinity matrix.
- the fusion protein can be immobilized on a suitable affinity matrix under conditions sufficient to bind the affinity ligand portion of the fusion protein to the matrix, and is contacted with one or more candidate biomolecules (e.g., a mixture of peptides) to be tested as biomolecular binders, under conditions suitable for binding of the biomolecules to the target portion of the bound fusion protein.
- candidate biomolecules e.g., a mixture of peptides
- the affinity matrix with bound fusion protein can be washed with a suitable wash buffer to remove unbound biomolecules and non- specifically bound biomolecules. Biomolecules which remain bound can be released by contacting the affinity matrix with fusion protein bound thereto with a suitable elution buffer.
- Wash buffer can be formulated to permit binding of the fusion protein to the affinity matrix, without significantly disrupting binding of specifically bound biomolecules.
- elution buffer can be formulated to permit retention of the fusion protein by the affinity matrix, but can be formulated to interfere with binding of the test biomolecule(s) to the target portion of the fusion protein.
- a change in the ionic strength or pH of the elution buffer can lead to release of biomolecules, or the elution buffer can comprise a release component or components designed to disrupt binding of biomolecules to the target portion of the fusion protein.
- Immobilization can be performed prior to, simultaneous with, or after contacting the fusion protein with biomolecule, as appropriate.
- Various permutations of the method are possible, depending upon factors such as the biomolecules tested, the affinity matrix-ligand pair selected, and elution buffer formulation.
- a suitable elution buffer a matrix elution buffer, such as glutathione for a GST fusion.
- the fusion protein comprises a cleavable linker, such as a thrombin cleavage site
- cleavage from the affinity ligand can release a portion of the fusion with the biomolecules bound thereto.
- Bound biomolecule can then be released from the fusion protein or its cleavage product by an appropriate method, such as extraction.
- One or more candidate biomolecular binders can be tested simultaneously. Where a mixture of biomolecules is tested, the biomolecules selected by the foregoing processes can be separated (as appropriate) and identified by suitable methods (e.g., PCR, sequencing, chromatography). Large libraries of biomolecules (e.g., peptides, RNA oligonucleotides) produced by combinatorial chemical synthesis or other methods can be tested (see e.g., Ohlmeyer, M.H.J. et al., Proc. Natl. Acad. Sci. USA 90:10922-10926 ( 1993) and DeWitt, S.H. et al., Proc. Natl. Acad. Sci.
- RNA molecules which bind to a target can also be screened according to the present method to select RNA molecules which bind to a target.
- biomolecules selected from a combinatorial library by the present method carry unique tags
- identification of individual biomolecules by chromatographic methods is possible.
- biomolecules do not carry tags chromatographic separation, followed by mass spectrometry to ascertain structure, can be used to identify individual biomolecules selected by the method, for example.
- the two-hybrid system or interaction trap is an in vivo system that can can be used to identify polypeptides, peptides or proteins (candidate biomolecular binders) that bind to a target protein.
- both candidate biomolecular binders and target cell component proteins are produced as fusion proteins.
- the two-hybrid system and variations on it have been described (US 5,283,173 and US 5,468,614; Golemis, E.A. et al, pages 20.1.1-20.1.35 In Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., John Wiley and Sons, containing supplements up through Supplement 40, 1997; two-hybrid systems available from Clontech, Palo Alto, CA).
- biomolecular binders of a cell component Once one or more biomolecular binders of a cell component have been identified, further steps can be combined with those taken to identify the biomolecular binder, to identify those biomolecular binders that produce a phenotypic effect on a cell (where "a cell” can mean cells of a cell strain or cell line).
- a method for identifying a biomolecule that produces a phenotypic effect on a first cell can comprise the steps of identifying a biomolecular binder of an isolated target cell component of the first cell; constructing a second cell comprising the target cell component and a regulable exogenous gene encoding the biomolecular binder; and testing the second cell for the phenotypic effect, upon production of the biomolecular binder in the second cell, where the second cell can be maintained in culture or introduced into an experimental animal. If the second cell shows the phenotypic effect upon intracellular production of the biomolecular binder, then a biomolecule that produces a phenotypic effect on the first cell has been identified.
- Host cells also, "second cells” in the terminology used above
- the cell type e.g., species of pathogenic bacteria
- the target was isolated from (or the gene encoding the target was originally isolated from, if the target is produced by recombinant methods)
- the ability to regulate the expression of the biomolecular binder is desirable because constitutive expression of the biomolecular binder could be lethal to the cell.
- inducible or regulated expression gives the researcher the ability to control if and when the biomolecular binder is expressed.
- the gene expressing the biomolecular binder can be present in one or more copies, either on an extrachromosomal structure, such as on a single or multicopy plasmid, or integrated into the host cell genome. Plasmids that provide an inducible gene expression system in pathogenic organisms can be used. For example, plasmids allowing tetracycline-inducible expression of a gene in Staphylococcus aureus have been developed. See Example 6.
- the genes for expression can be carried on plasmid-based or virus-based vectors, or on a linear piece of DNA or RNA.
- expression vectors see Hosfield and Lu, Biotechniques 25:306-309, 1998; Stephens and Cockett, Nucleic Acid Research 77:71 10, 1989; Wohlgemuth et al. Gene Therapy 3:503-512, 1996; Ramirez-Solis et al, Gene
- the genetic material can be introduced into cells using a variety of techniques, including whole cell or protoplast transformation, electroporation, calcium phosphate-DNA precipitation or DEAE- Dextran transfection. liposome mediated DNA or RNA transfer, or transduction with recombinant viral or retroviral vectors. Expression of the gene can be constitutive (e.g., ADH1 promoter for expression in S.
- inducible systems can be utilized, for example, E. coli Lac repressor/operator system and TnlO Tet repressor/operator systems have been engineered to govern regulated expression in organisms from bacterial to mammalian cells. Regulated gene expression can also be achieved by activation.
- gene expression governed by HIV LTR can be activated by HIV or SIV Tat proteins in human cells; GAL4 promoter can be activated by galactose in a nonglucose-containing medium.
- the location of the biomolecule binder genes can be extrachromosomal or chromosomally integrated.
- the chromosome integration can be mediated through homologous or nonhomologous recombinations.
- biomolecule binders For proper localization in the cells, it may be desirable to tag the biomolecule binders with certain peptide signal sequences (for example, nuclear localization signal (NLS) sequences, mitochondria localization sequences).
- NLS nuclear localization signal
- mitochondria localization sequences secretion sequences have been well documented in the art.
- biomolecular binders For presentation of the biomolecular binders in the intracellular system, they can be fused N-terminally, C-terminally, or internally in a carrier protein (if the biomolecular binder is a peptide), and can be fused (5', 3' or internally) in a carrier RNA or DNA molecule (if the biomolecular binder is a nucleic acid).
- the biomolecular binder can be presented with a protein or nucleic acid structural scaffold. Certain linkages (e.g., a 4-glycine linker for a peptide or a stretch of A's for an RNA can be inserted between the biomolecular binder and the carrier proteins or nucleic acids.
- the effect of this biomolecular binder on the phenotype of the cells can be tested, as a manifestation of the binding (implying binding to a functionally relevant site, thus, an activator, or more likely, an inhibitory) effect of the biomolecular binder on the target used in an in vitro binding assay as described above.
- An intracellular test can not only determine which biomolecular binders have a phenotypic effect on the cells, but at the same time can assess whether the target in the cells is essential for maintaining the normal phenotype of the cells. For example, a culture of the engineered cells expressing a biomolecular binder can be divided into two aliquots.
- the first aliquot (“test” cells) can be treated in a suitable manner to regulate (e.g., induce or release repression of, as appropriate) the gene encoding the biomolecular binder, such that the biomolecular binder is produced in the cells.
- the second aliquot (“control” cells) can be left untreated so that the biomolecular binder is not produced in the cells.
- a different strain of cells not having a gene that can express the biomolecular binder, can be used as control cells.
- the phenotype of the cells in each culture can then be monitored by a suitable means (e.g., enzymatic activity, monitoring a product of a biosynthetic pathway, antibody to test for presence of cell surface antigen, etc.).
- a suitable means e.g., enzymatic activity, monitoring a product of a biosynthetic pathway, antibody to test for presence of cell surface antigen, etc.
- the growth of the cells in each culture (“test” and “control” cells grown under the same conditions, other than the expression of the biomolecular binder)
- a suitable means e.g., turbidity of liquid cultures, cell count, etc. If the extent of growth or rate of growth of the test cells is less than the extent of growth or rate of growth of the control cells, then the biomolecular binder can be concluded to be an inhibitor of the growth of the cells, or a biomolecular inhibitor.
- biomolecular binder can be concluded to be one that causes a phenotypic effect.
- isolated target cell component having a known function e.g., an enzyme activity
- positive results in these tests should encourage the investigator to continue in the drug discovery process with efforts to find a more stable compound (than a peptide, polypeptide or RNA biomolecule) that mimics the binding properties of the biomolecular binder on the tested target cell component.
- a further test can, again, employ an engineered strain of cells that comprise both the target cell component and one or more genes encoding a biomolecule tested to be a biomolecular binder of the target cell component.
- the cells of the cell strain can be tested in animals to see if regulable expression of the biomolecular binder in the engineered cells produces an observable or testable change in phenotype of the cells.
- Both the "in culture” test for the effect of intracellular expression of the biomolecular binder and the “in animal” test (described below) for the effect of intracellular expression of the biomolecular binder can be applied not only towards drug discovery in the categories of antimicrobials and anticancer agents, but also towards the discovery of therapeutic agents to treat inflammatory diseases, cardiovascular diseases, diseases associated with metabolic pathways, and diseases associated with the central nervous system, for example.
- the object of the test is to see whether production of the biomolecular binder in the engineered strain inhibits growth of these cells after their introduction into an animal by the engineered pathogen.
- Such a test can not only determine which biomolecular binders are inhibitors of growth of the cells, but at the same time can assess whether the target in the cells is essential for maintaining growth of the cells (infection, for a pathogenic organism) in a host mammal.
- Suitable animals for such an experiment are, for example, mammals such as mice, rats, rabbits, guinea pigs, dogs, pigs, and the like. Small mammals are preferred for reasons of convenience.
- the engineered cells are introduced into one or more animals ("test” animals) and into one or more animals in a separate group (“control” animals) by a route appropriate to cause symptoms of systemic or local growth of the engineered cells.
- the route of introduction may be, for example, by oral feeding, by inhalation, by subdermal, intramuscular, intravenous, or intraperitoneal injection as appropriate to the desired result.
- expression of the gene encoding the biomolecular binder is regulated to allow production of the biomolecular binder in the engineered pathogen cells. This can be achieved, for instance, by administering to the test animals a treatment appropriate to the regulation system built into the cells, to cause the gene encoding the biomolecular binder to be expressed. The same treatment is not administered to the control animals, but the conditions under which they are maintained are otherwise identical to those of the test animals.
- the treatment to express the gene encoding the biomolecular binder can be the administration of an inducer substance (where expression of the biomolecular binder or gene is under the control of an inducible promoter) or the functional removal of a repressor substance (where expression of the biomolecular binder gene is under the control of a repressible promoter).
- the test and control animals can be monitored for a phenotypic effect in the introduced cells.
- the introduced cells are constructed pathogen cells
- the animals can be monitored for signs of infection (as the simplest endpoint, death of the animal, but also e.g., lethargy, lack of grooming behavior, hunched posture, not eating, diarrhea or other discharges; bacterial titer in samples of blood or other cultured fluids or tissues).
- the test and control animals can be monitored for the development of tumors or for other indicators of the proliferation of the introduced engineered cells.
- the biomolecule can be also called a biomolecular inhibitor of growth, or biomolecular inhibitor of infection, as appropriate, as it can be concluded that the expression in vivo of the biomolecular inhibitor is the cause of the relative reduction in growth of the introduced cells in the test animals.
- Further steps of the procedure involve in vitro assays to identify one or more compounds that have binding and activating or inhibitory properties that are similar to those of the biomolecules which have been found to have a phenotypic effect, such as inhibition of growth. That is, compounds that compete for binding to a target cell component with the biomolecule would then be structural analogs of the biomolecules. Assays to identify such compounds can take advantage of known methods to identify competing molecules in a binding assay. These steps comprise general step (3) of the method.
- a biomolecular inhibitor (or activator) can be contacted with the isolated target cell component to allow binding, one or more compounds can be added to the milieu comprising the biomolecular inhibitor and the cell component under conditions that allow interaction and binding between the cell component and the biomolecular inhibitor, and any biomolecular inhibitor that is released from the cell component can be detected.
- One suitable system that allows the detection of released biomolecular inhibitor (or activator) is one in which fluorescence polarization of molecules in the milieu can be measured.
- the biomolecular inhibitor can have bound to it a fluorescent tag or label such as fluorescein or fluorescein attached to a linker.
- Assays for inhibition of the binding of the biomolecular inhibitor to the cell component can be done in microtiter plates to conveniently test a set of compounds at the same time.
- a majority of the fluorescently labeled biomolecular inhibitor must bind to the protein in the absence of competitor compound to allow for the detection of small changes in the bound versus free probe population when a compound which is a competitor with a biomolecular inhibitor is added (B. A. Lynch, et al. Analytical Biochemistry 247:11-82 (1997)). If a compound competes with the biomolecular inhibitor for a binding site on the target cell component, then fluorescently labeled biomolecular inhibitor is released from the target cell component, lowering the polarization measured in the milieu.
- the target cell component can be attached to a solid support, contacted with one or more compounds, and contacted with the biomolecular inhibitor.
- One or more washing steps can be employed to remove biomolecular inhibitor and compound not bound to the cell component. Either the biomolecular inhibitor bound to the target cell component or the compound bound to the target cell component can be measured.
- Detection of biomolecular inhibitor or compound bound to the cell compound can be facilitated by the use of a label on either molecule type, wherein the label can be, for instance, a radioactive isotope either incorporated into the molecule itself or attached as an adduct, streptavidin or biotin, a fluorescent label or a substrate for an enzyme that can produce from the substrate a colored or fluorescent product.
- a scintillation counter can be used to measure radioactivity.
- Radiolabeled streptavidin or biotin can be allowed to bind to biotin or streptavidin, respectively, and the resulting complexes detected in a scintillation counter.
- Alkaline phosphatase conjugated to streptavidin can be added to a biotin-labeled biomolecular inhibitor or compound.
- Detection and quantitation of a biotin-labeled complex can then be by addition of pNPP substrate of alkaline phosphatase and detection by spectrophotometry, of a product which absorbs UV light at a wavelength of 405 nm.
- a fluorescent label can also be used, in which case detection of fluorescent complexes can be by a fluorometer. Models are available that can read multiple samples, as in a microtiter plate.
- the method for identifying compounds comprises attaching the target cell component to a solid support, contacting the biomolecular inhibitor with the target cell component under conditions suitable for binding of the biomolecular inhibitor to the cell component, removing unbound biomolecular inhibitor from the solid support, contacting one or more compounds (e.g., a mixture of compounds) with the cell component under conditions suitable for binding of the biomolecular inhibitor to the cell component, and testing for unbound biomolecular inhibitor released from the cell component, whereby if unbound biomolecular inhibitor is detected, one or more compounds that displace or compete with the biomolecular inhibitor for a particular site on the target cell component have been identified.
- one or more compounds e.g., a mixture of compounds
- Those compounds that bind competitively to the target cell component can be considered to be drug candidates. Further appropriate testing can confirm that those compounds which bind competitively with biomolecular inhibitors (or activators) possess the same activity as seen in an intracellular test of the effect of the biomolecular inhibitor or activator upon the phenotype of cells. Derivatives of these compounds having modifications to confer improved solubility, stability, etc., can also be tested for a desired phenotypic effect.
- steps for testing the phenotypic effects of a biomolecule as can be produced in an intracellular test, with steps for identifying compounds that compete with the biomolecule for sites on a target cell component, yields a method for identifying a compound which is a functional analog of a biomolecule which produces a phenotypic effect on a cell.
- steps for identifying a compound which is a functional analog of a biomolecule which produces a phenotypic effect on a cell can be to test, for the phenotypic effect, either in culture or in an animal model, or in both, a cell which produces a biomolecule by regulable expression of an exogenous gene in the cell, and to identify, if the biomolecule caused the phenotypic effect, one or more compounds that compete with the biomolecule for binding to a target cell component.
- the compound is a functional analog of a biomolecule which produces a phenotypic effect on the cell.
- a functional analog can cause qualitatively a similar effect on the cell, but to a similar degree, lesser degree or greater degree than the biomolecule.
- a further embodiment of the invention combining general steps (1) and (2) is a method for determining whether a target component of a cell is essential to producing a phenotypic effect on the cell, comprising isolating the target component from the cell, identifying a biomolecular binder of the isolated target component of the cell, constructing a second cell comprising the target component and a regulable, exogenous gene encoding the biomolecular binder, and testing the second cell in culture for an altered phenotypic effect, upon production of the biomolecular binder in the second cell, whereby, if the second cell shows the altered phenotypic effect upon production of the bimolecular binder, then the target component of the first cell is essential to producing the phenotypic effect on the first cell.
- the methods described herein are well suited to the identification of compounds that can inhibit the proliferation of the cells of infectious agents such as bacteria, fungi and the like.
- a procedure such as the one outlined below can be used in the identification of compounds to inhibit the proliferation of cancer cells.
- the two procedures described below further illustrate the use of the methods described herein and would provide proof of principle of these methods with a known target for anticancer therapy.
- DHFR Mammalian dihydrofolate reductase
- Methotrexate (MTX) is one of many existing drugs that inhibit DHFR. It is widely used for anticancer chemotherapy.
- NIH 3T3 is a mouse fibroblast cell line that is able to develop spontaneous transformed cells when cultured in low concentration (2%) of calf serum in molecular, cellular and developmental biology medium 402 (MCDB) (M. Chow and H. Rubin, Proc. Natl. Acad. Sci. USA 95(8):4550-4555 ( 1998)).
- the transformed cells which can be selectively inhibited by MTX (Chow and Rubin), are isolated.
- Both the normal and transformed N1H3T3 cells are transfected with pTet-On plasmid (Clontech; Palo Alto, CA).
- Stable cell lines that express high levels of reverse tetracycline-controlled activator (rtTA) are isolated and characterized for their normal or transformed phenotype (Chow and Rubin).
- the DHFR gene (Genbank Accession # L26316) from the NIH 3T3 cell line is amplified by reverse transcription-PCR (RT-PCR) using poly A * RNA isolated from NIH 3T3 cells (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989). Active DHFR is expressed using the BacPAK Baculovirus Expression System (Clontech) or other appropriate systems. The expressed DHFR is purified and biotinylated and subjected to peptide binder identification as exemplified for bacterial proteins. The identified peptides are biochemically characterized for in vitro inhibition of DHFR activity. Peptides that inhibit DHFR are identified.
- a nucleic acid encoding each peptide can be cloned into a vector such as pGEX-4T2 (Pharmacia) to yield a vector which encodes a fusion polypeptide having the peptide fused to the N-terminus of GST. This can also be done by PCR amplification as exemplified herein for the peptide Pro-3.
- the fusion genes are cloned into plasmid pTRE (Clontech) for regulated expression.
- the constructed plasmid or the vector is cotransfected with pTK-Hyg into the stable NIH 3T3 cell line that expresses rtTA.
- 3T3N-VITA normal 3T3 cells that express rtTA and the DHFR inhibitory peptides
- 3T3T-VITA transformed 3T3 cells that express rtTA and the DHFR inhibitory peptides
- 3T3T-VITA control transformed 3T3 cells that express rtTA and GST
- 10 2 -10 3 of 3T3TNITA or 3T3T-VITA control cells are mixed with 10 5 3T3 ⁇ -VITA and are grown in MCDB 402 medium with 10% calf serum at 37°C for three days. Tetracycline is added to the medium to a final concentration of 0 to 1 ⁇ g/ml. In a control, 200 nM of MTX is added. The cultures are incubated for an additional eight days, and the number of foci formed are counted as described by M. Chow and H. Rubin, Proc. Natl. Acad. Sci. USA 95(8):4550-4555 (1998). Peptides that specifically inhibit foci formation of 3T3 transformed cells are identified.
- a murine model of fibroblastoma (Kogerman, P. et al, Oncogene 75(12):1407-1416 (1997)) is used for evaluting the DHFR/peptide combination for identification of compounds for cancer therapy.
- Various amounts of 3T3T-VITA or 3T3T-VITA control cells (10 3 , 10 4 , 10 ⁇ 10 6 cells) are injected subcutaneously into 5 groups (10 in each group) of athymic nude mice (4-6 weeks old, 18-22 g) to determine the minimal dose needed for development of fibroblastomas in all of the tested animals.
- mice Upon determination of the minimal tumorigenic dose, 6 groups of athymic nude mice (10 each) are injected subcutaneously (s.c.) with the minimal tumorigenic dose for 3T3T-VITA or 3T3T-VITA control cells to develop fibroblastoma.
- group 1 mice start receiving MTX s.c. at 2 mg/kg/day as positive control
- group 2 to 5 start receiving 1, 2, 5, or 10 mg/kg/day of tetracycline
- group 6 start receiving saline (vehicle) as control.
- saline vehicle
- All of the mice are sacrificed and tumors are removed from them. Tumor mass is measured and compared among the groups.
- An effective peptide identified by these in vivo experiments can be used for screening libraries of compounds to identify those compounds that competitively bind to DHFR.
- Transgenic mice that overexpress human Ha-ras have been produced. Such transgenic mice develop salivary and/or mammary adenocarcinomas (Nielsen, L.L. et al, In Vivo 5(5): 1331-1343 (1994)). Secondary transgenic mice that express rtTA can be generated using the pTet-On plasmid from Clontech.
- Human Ha-ras open reading frame cDNA (Genbank Accession #G00277) is amplified by RT-PCR using polyA ' RNA isolated from human mammary gland or other tissues. Active Ha-ras is expressed using the BacPAK Baculovirus Expression System (Clontech) or other appropriate systems. The expressed Ha-ras is purified and biotinylated and subjected to peptide binder identification as exemplified herein for bacterial proteins as target cell components. The identified peptides are biochemically characterized for in vitro inhibition of Ha-ras GTPase activity.
- Peptides that inhibit Ha-ras are cloned into plasmid pTRE (Clontech) for regulated expression as an N-terminal fusion of GST. Such constructs are used to generate tertiary transgenic mice using the secondary transgenic mice. Transgenic mice that are able to overexpress peptide genes are identified by Northern and Western analysis. Control mice that express GST are also identified.
- tetracycline Various doses of tetracycline are administered to the tertiary transgenic mice by s.c. or i.p. injection before or after tumor onset. Prevention or regression of tumors resulting from expression of the peptide genes are analyzed as described above for murine fibroblastoma. Peptides found to be effective in in vivo experiments will be used to screen compounds that inhibit human Ha-ras activity for cancer therapy.
- the method of the invention can be applied more generally to mammalian diseases caused by: (1) loss or gain of protein function, (2) over-expression or loss of regulation of protein activity.
- the starting point is the identification of a putative protein target or metabolic pathway involved in the disease.
- the protocol can sometimes vary with the disease indication, depending on the availability of cell culture and animal model systems to study the disease. In all cases the process can deliver a validated target and assay combination to support the initiation of drug discovery.
- Appropriate disease indications include, but are not limited to, Alzheimer's, arthritis, cancer, cardiovascular diseases, central nervous system disorders, diabetes, depression, hypertension, inflammation, obesity and pain.
- Appropriate protein targets putatively linked to disease indications include, but are not limited to (1) the leptin protein, putatively linked to obesity and diabetes; (2) a mitogen-activated protein kinase putatively linked to arthritis, osteoporosis and atherosclerosis; (3) the interleukin-1 beta converting protein putatively linked to arthritis, asthma and inflammation; (4) the caspase proteins putatively linked to neurodegenerative diseases such as Alzheimer's, Parkinson's and stroke, and (5) the tumor necrosis factor protein putatively linked to obesity and diabetes.
- Appropriate protein targets include also, but are not limited to.
- the arachidonic acid pathway constitutes one of the main mechanisms for the production of pain and inflammation.
- the pathway produces different classes of end products, including the prostaglandins, thromboxane and leukotrienes.
- Prostaglandins an end product of cyclooxygenase metabolism, modulate immune function, mediate vascular phases of inflammation and are potent vasodilators.
- COX cyclooxygenase
- Anti- inflammatory potencies of different NSAIDs have been shown to be proportional to their action as COX inhibitors. It has also been shown that COX inhibition produces toxic side effects such as erosive gastritis and renal toxicity. The knowledge base regarding the toxic side effects of COX inhibitors has been gained through years of monitoring human therapies and human suffering. Two kinds of COX enzymes are now known to exist, with inhibition of COX 1 related to toxicity, and inhibition of COX2 related to reduction of inflammation. Thus, selective COX2 inhibition is a desirable characteristic of new anti-inflammatory drugs.
- the method of the invention can provide a route from identification of potential drug targets to validating these targets (for example, COX1 and COX2) as playing a role in disease (pain and inflammation) to an examination of the phenotype for the inhibition of one or both target isozymes without human suffering. Importantly, this information can be collected in vivo.
- the method of the invention can be used to define the phenotype of "genes of unknown function" obtained from various human genome sequencing projects or to assess the phenotype resulting from inhibition of one isozyme subtype or one member of a family of related protein targets.
- the present invention is more specifically illustrated in the following examples, which are not intended to be limiting in any way.
- Example 1 Isolating a peptide that binds to and inhibits E. coli prolyl-tRNA synthetase Because of its well established genetic and expression systems, Escherichia coli was chosen for initial tests of the methods. Prolyl-tRNA synthetase (ProRS) catalyzes the attachment of proline to its cognate tRNA for protein biosynthesis. It is an enzyme of essential cellular function and is a good candidate for target validation.
- ProRS Prolyl-tRNA synthetase
- the E. coli ProRS gene (Genbank accession number: M97858) was PCR amplified and cloned into the p ⁇ T- 20b vector (Novagen) using standard molecular cloning protocols (J. Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory Press, 1989). The overexpressed ProRS was purified sequentially on Q- and SP-Sepharose columns.
- the purified ProRS was biotinylated using ⁇ Z-linkTM Sulfo-NHS-LC-Biotin from Pierce according to the instructions packaged with the biotinylation compound, captured onto streptavidin-agarose beads and used to select specific binding peptides from a peptide library displayed on coat protein III of M13 phages using a standard protocol (J. K. Scott and G. P. Smith, Science 249:386-390 (1990)). Thirteen clones from phages that were identified as having a high affinity to ProRS were sequenced. These clones share 4 different sequences, 3 of which are closely related (Table 1). See Example 4 of WO98/19162, published 7 May 1998.
- the peptide having sequence number 3 was synthesized and tested for inhibition of tRNA charging activity of E. coli ProRS. This peptide inhibits E. coli ProRS aminoacylation activity, demonstrates competitive inhibition with both proline and ATP, and exhibits a K, of 300 nM. This peptide is called Pro-3 (also, Pro3).
- Pro-3 peptide in E. coli was achieved by fusing an oligonucleotide encoding the peptide to the 5' end of a gene encoding a glutathione S-transferase (GST) protein.
- GST glutathione S-transferase
- the Pro-3/GST fusion gene was PCR amplified using a combination of the Pro3, Pro3/GST, and GST/R primers as illustrated in Figure 1.
- Primers Pro3 and Pro3/GST encode the Pro-3 peptide sequence; the latter anneals to the 5' end of the GST gene on plasmid pGEX-4T2 (Pharmacia).
- a 4-amino acid linker was introduced between the Pro-3 peptide and GST for flexibility.
- the PCR product was further amplified with primers Pro3(Kpn) and GST/R(Bam), digested with Kpnl and BamHI restriction endonucleases, and ligated to the Kpnl/BamHI sites of the expression vector pPROTet (Clontech, Palo Alto, CA) using standard cloning protocols.
- pPROTet uses the P L promoter of phage lambda combined with the Tet operator of the TnlO tetracycline resistance operon to direct the regulated expression of the cloned gene (Clontech, PROTM Bacterial Expression System User Manual, PT3161-1, Version PR7Y629).
- DH5 PRO (Clontech)
- E. coli strain expressing the Tet repressor.
- Clone pC 3 844 was sequenced and identified as containing the Pro-3/GST fusion gene. The linker between the Pro3 peptide and GST is Glu-Gly-Gly-Gly.
- pC 3 844 was also transformed into the E. coli strain JM109/pSC, which is JM109 harboring a plasmid expressing Tet repressor that was isolated from BL21PRO (Clontech). The resulting E. coli strain is called JM109/pSC/pC 3 844.
- Anhydrotetracychne is a derivative of tetracycline that acts as a potent inducer of the TetO/R system and is less toxic to E. coli than tetracycline (T. Lederer, et al, Biochemistry, 35:7439-7446 (1996); B. Oliva, et al., Antimicrob. Agents Chemother. 36:913-919 (1992).
- the expressed Pro-3/GST fusion protein was purified on a glutathione- agarose affinity column according to a procedure provided by Pharmacia (see, e.g., procedures manual from Pharmacia P-L Biochemicals, Inc.: GST Gene Fusion System, regarding use of pGEX expression vectors and glutathione-S-transferase fusion proteins, 1993).
- the N-terminal sequence of the purified protein was determined by Edman cycles and confirmed to have the expected Pro-3 peptide sequence.
- the purified Pro-3/GST was also confirmed to inhibit E. coli ProRS activity, with a K s of 180 nM, similar to that of the Pro-3 peptide.
- Example 3 In vivo expression of Pro-3 peptide to cure a lethal infection
- Pro-3 Intracellularly expressed Pro-3 was expected to bind to and inhibit a specific essential cellular target in a manner similar to that of an antimicrobial drug. Pro-3 was expected to cure the infection when its expression was induced in an animal model of bacterial infection. An established animal infection model was used to test this concept (CO. Onyeji et al, Antimicrob. Agents Chemother. 35.T 112-1117 (1994)).
- mice Eight groups of CD-I female mice (5 mice per group, Charles River Laboratories; Wilmington, MA) weighing 20-24 g were used in this experiment.
- the inoculum was prepared from E. coli JM109/pSC/pC 844 which was cultured at 37°C for 17 hr in Luria-Bertani broth containing spectinomycin and chloramphenicol, and then 100 ⁇ l of the culture was diluted to 1 ml with medium for reading OD at 600 nm (0.2403, the medium as blank).
- the turbidity of a 0.5 McFarland standard is equivalent to OD 600 0.1, or 10 8 cfu/ml.
- coli JM109/pSC/pC 844 (1.25 ml) from the overnight culture were diluted to 15 ml with 0.01 M phosphate buffered saline (Sigma P-0261) containing 8% hog gastric mucin (Sigma M-2378), 100 ⁇ g/ml spectinomycin and 68 ⁇ g/ml chloramphenicol.
- Each mouse of groups 1 through 4 was injected with 0.5 ml of the inoculum intraperitoneally (i.p.), equivalent to lxlO 8 cfu/mouse (lethal dose).
- Groups 5 through 8 served as vector control.
- the control inoculum was prepared from E.
- E. coli pPROTet-GST whose vector carries a gene encoding glutathione S-transferase, but no Pro-3 peptide.
- E. coli pPROTet-GST was cultured at 37 °C for 17 hr in Luria-Bertani broth containing spectinomycin and chloramphenicol, and then 100 ⁇ l of the culture were diluted to 1 ml with the medium for reading OD at 600 nm (0.2858, the medium as blank). Then 3xl0 9 cfu (1.05 ml) from the overnight culture were diluted to 15 ml with 0.01 M phosphate buffered saline containing 8% hog gastic mucin. Each mouse of groups 5 through 8 was injected (i.p.) with E. con pPROTet-GST inocula equivalent to lxlO 8 cfu/mouse (lethal dose).
- groups 1 and 5 animals received a saline injection i.p. at 10 ml/kg; groups 2, 3 and 4 animals received i.p. injections of anhydrotetracychne at 2, 1 and 0.5 mg/kg (diluted in saline), respectively; groups 6, 7 and 8 received i.p. injections of anhydrotetracychne at 2, 1 and 0.5 mg/kg, respectively.
- the injection volume for all the animals was 10 ml/kg.
- Example 4 Specific inhibition of ProRS: examination of intracellular tRNA charging levels.
- E. coli growth inhibition was caused by inhibition of ProRS activity.
- 400 ml of fresh LB broth containing 34 ⁇ g/ml chloramphenicol and 50 ⁇ g/ml spectinomycin were inoculated with 4 ml JM109/pSC/pC 844 overnight culture and grown at 37°C to an OD 600 of 0.3.
- the bacterial culture was split into two 200 ml subcultures. To one of them anhydrotetracychne was added to final concentration of 500 ng/ml.
- Example 5 Functional complementation of Pro-3 peptide inhibition by expression of a heterologous ProRS gene
- ProRS is the primary intracellular target of the Pro-3 peptide
- E. coli growth inhibition by Pro-3 peptide should be relieved by functional complementation with a heterologous ProRS. It was found that Pro-3 does not inhibit S. aureus ProRS enzyme activity, which efficiently charges E. coli tRNA Pro . The S.
- aureus ProRS gene (WO 97/26343; EP 785272) was amplified with oligonucleotides S.PRS/XhoI-5' (5'AAT CCG CTC GAG GAT TAT TGC TAT TGG TGC C) (SEQ ID NO: 10) and S.PRS/Hind-3' (5'AAT CGT AAG CTT TTA TTT TAA GTT ATC ATA TTT) (SEQ ID NO: 1 1 ), digested with Xho I / Hind III restriction endonucleases, and cloned into Xho I / Hind III sites in pACYC177. The cloned S.
- aureus ProRS gene carries its own promoter and ribosome binding site, and is in the same orientation as the disrupted kanamycin resistance gene in the vector for efficient expression.
- Either the S. aureus ProRS expression construct or the pACYC177 vector alone was transformed into DH5 ⁇ PRO /pC 844.
- the growth of the resulting E. coli strains was followed in the presence or absence of 200 ng ml anhydrotetracychne.
- Figure 4A and Figure 4B with the S. aureus ProRS expression construct, E. coli cell growth was no longer inhibited by expression of the Pro-3 peptide.
- E. coli/Bacillus shuttle expression vectors pWH353 and pWH354 were obtained from Professor Wolfgang Hillen (Mikrobielle Genetik, Universitat Tubingen, Tubingen, Germany; see M. Geissend ⁇ rfer and W. Hillen, Appl. Microbiol. Biotechnol, 33:657-663 (1990); DE 3934454). These expression vectors carry a Tnl O tet repressor gene. They also contain synthetic promoters with one or two Tet repressor binding sites that are optimized for inducible expression in 73. subtilis. These inducible promoters direct the expression of CAT (chloramphenicol acetyltransferase).
- CAT chloramphenicol acetyltransferase
- pWH353 and pWH354 were transformed into S. aureus RN4220 cells by electroporation (S. Schenk and R. A. Laddaga, CMS Microbiology Letters, 94: 133- 138 (1992)).
- the transformants were tested for inducible expression by growing in LB broth containing 30 ⁇ g/ml kanamycin. After the OD 600 reached 0.5, the cultures were split and tetracycline was added to one set of the cultures to a final concentration of 0.5 ⁇ g/ml. The cultures were maintained with aeration for 3 hours at 37 °C. The S.
- aureus cells were then pelleted and resuspended in 80 mM Tris- HC1, pH 7.4 containing 200 ⁇ g/ml lysostaphin, incubated at 37°C for 5 minutes, frozen and thawed twice on dry ice/ethanol and 37°C water bath. The samples were then sonicated twice and centrifuged at 14.000g for 10 minutes. The supematants were collected and subjected to electrophoretic analysis on an 18% SDS- polyacrylamide gel stained with Coomassie blue ( Figure 5). The CAT activities in these samples were determined (Frankel, A.D. et al., Proc. Natl. Acad. Sci. USA 56:7397-7401 ( 1989)) and summarized in Table 5.
- ND Not detectable.
- Example 7 Identification of small peptides that specifically bind to and inhibit S aureus methionyl-tRNA synthetase (MetRS)
- the S. aureus MetRS gene has been cloned (EP 785269; WO 97/26350).
- the gene was PCR amplified and cloned into pGEX-4T2 (Pharmacia) for expression as a GST fusion using standard molecular cloning protocols.
- the expressed GST-S. aureus MetRS was purified on a glutathione agarose column and the GST moiety was removed by thrombin cleavage.
- the resulting nonfusion S aureus MetRS was then biotinylated using EZ-LinkTM Sulfo-NHS-LC-Biotin from Pierce according to the instructions enclosed with the biotinylation compound.
- the biotinylated S. aureus MetRS was used for selecting binding peptides from a 12-mer peptide library displayed on Ml 3 phages (New England Biolabs). After 4 rounds of selection, 12 phage clones were isolated and sequenced. Out of thse 12 clones, 1 1 yielded 4 different sequences as summarized in Table 6.
- Peptides of sequence JT01 and JT02 were synthesized. JT01 was tested for inhibition of S. aureus MetRS activity.
- the activity of S. aureus MetRS was monitored by the aminoacylation reaction.
- the enzyme was diluted in 50 mM HEPES, pH 7.5, 5 mM DTT and
- a typical 50 ⁇ L reaction mixture contained 30 mM HEPES, pH 7.5,
- the results as depicted in Figure 6 indicate that peptide JTOl is a competitive inhibitor of S. aureus MetRS.
- the K,'s for JTOl are 138 nM (methionine) and 13 nM (ATP).
- the K,'s for JT02 are 1.7 ⁇ M (methionine) and 0.5 ⁇ M (ATP) and 13 nM (ATP).
- Example 8 Development of Assay for E.coli ProRS and Pro3 peptide
- FP Fluorescent Polarization
- the E. coli ProRS FP binding assay involves the incubation of fluorescently labeled Pro3 peptide (Pro3-F) with purified E. coli ProRS. In the bound state. Pro3- F is bound to E. coli ProRS and an increase in ploarization signal is detected using a FP detection system.
- Pro3-F is not bound to E. coli ProRS and there is a decrease in polarization signal. See Example 1 for cloning and purification of E. coli ProRS. See Example 1 (SEQ ID NO:3) for description of Pro3 peptide. Fluorescently labeled Pro3 peptide (Pro3-F) was synthesized and purified by SynPep Corp, Dublin, CA. Its sequence is shown below.
- a 96-well plate (Costar cat #3915) was blocked for 1 hour at room temperature with 150 ⁇ L/well 2 mg/ml BSA (FisherBiotech cat #BP 1600- 100) in 0.1M NaHCO 3 The plate was then washed manually three times with 150 ⁇ L/well of TBST (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% Tween 20). To each well, 10 uL of 10% DMSO/TBST or compound (0.0001 - l OO ⁇ M) in 10% DMSO/TBST was incubated for 20 minutes at room temperature with 50 ⁇ L of 2.36 ⁇ M E.
- coli ProRS in TBST (10 mM Tris-HCL pH 8.0, 150 mM NaCl, 0.05% Tween 20). Following the pre-incubation, 40 ⁇ L of 0.391 ⁇ M Pro3-F in TBST was added to each well, mixed and incubated at room temperature for 60 minutes. The plate was then read in an LJL Analyst (LJL Biosystems, Sunnyvale, CA) in fluorescent polarization mode.
- CB-16914 and CB-1 18831 are known inhibitors of E. coli ProRS (nM and uM inhibitors, respectively) and CB- 680 was used as a negative control. As shown in Figure 7, both CB-16914 and CB- 1 18831 were shown to inhibit Pro3-F binding whereas CB-680 had no effect.
- Figure 7 shows fluorescence polarization binding assay inhibition curves for CB-16914 (0.0001-10 ⁇ M), CB-1 18831 (0.046-100 ⁇ M) and CB-680 (0.0001-10 ⁇ M) with 1.18 ⁇ M E. coli ProRS and 0.625 ⁇ M Pro3-F.
- FP Fluorescence polarization
- MetRS Staphylococcus aureus MetRS
- Metl peptide Metl peptide
- the Sa MetRS FP binding assay involves the incubation of fluorescently labeled Metl peptide (Metl-F) with purified Sa MetRS. In the bound state. Metl-F is bound to Sa MetRS and an increase in polarization signal is detected using a FP detection system. In the free state, Metl-F is not bound to Sa MetRS and there is a decrease in polarization signal.
- a 96-well plate (Costar cat# 3915) was blocked for 1 hour at room temperature with 150 ⁇ L/well 2 mg/ml bovine serum albumin (BSA) (FisherBiotech cat# BP 1600- 100) in 0.1 M NaHC0 3 . The plate was then washed manually three times with 150 ⁇ L/well of TBS (10 mM Tris-HCl pH 8.0, 150 mM NaCl).
- BSA bovine serum albumin
- the seven compounds were tested as well as the unlabeled Met-1 peptide.
- the seven compounds are known inhibitors (IC 50 's in nM to ⁇ M range) of Sa MetRS functional charging assay.
- IC 50 's in nM to ⁇ M range As shown in Table 7, six of the seven inhibitors showed similar IC ⁇ 0 's (within 3-4x) in the FP binding assay as compared to the functional assay.
- the FP assay was unable to detect CB-125552 which may be due to (1) poor compound solubility or (2) compound binding at a site on Sa MetRS different from the site bound by the Met-1 peptide.
- the FP assay for Sa MetRS inhibition has been evaluated to see if it is amenable for high throughput screening (HTS) for drug discovery.
- the FP assay for Sa MetRS has been reduced to 96-well format and has been automated which is necessary for HTS.
- Figure 8 shows results of an experiment on a control plate that was run to assess the performance of the FP HTS assay.
- S a MetRS was at 20 nM final concentration; Metl-F was at 5 nM final concentration.
- Wells 1-6 contain Met- IF alone, wells 7-8 contain Sa MetRS alone, wells 9-18 and 19-28 contain Metl-F from 640 nMto 1.25 ⁇ M, wells 29-96 contain 1% DMSO.
- the mP range between free Metl-F and Metl-F bound by Sa MetRS was 141 mP.
- the IC 50 for Metl was tested on the plate in duplicate and the results were consistent with previously determined values.
- the signal to noise for the plate assay (S/N) was 7: 1 which is acceptable for HTS.
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CA002317653A CA2317653A1 (en) | 1998-01-09 | 1999-01-08 | Method for identifying validated target and assay combinations |
EP99902132A EP1046034A1 (en) | 1998-01-09 | 1999-01-08 | Method for identifying validated target and assay combinations |
AU22181/99A AU2218199A (en) | 1998-01-09 | 1999-01-08 | Method for identifying validated target and assay combinations |
NO20003515A NO20003515L (en) | 1998-01-09 | 2000-07-07 | Procedure for identifying validated measurement and analysis combinations |
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US10021198P | 1998-09-14 | 1998-09-14 | |
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WO2000022112A1 (en) * | 1998-10-13 | 2000-04-20 | The University Of Georgia Research Foundation, Inc. | Stabilized bioactive peptides and methods of identification, synthesis and use |
EP1047453A1 (en) * | 1998-01-21 | 2000-11-02 | Smithkline Beecham Corporation | Method for determining gene essentiality in a pathogen |
WO2001019981A2 (en) * | 1999-09-14 | 2001-03-22 | Sangamo Biosciences, Inc. | Functional genomics using zinc finger proteins |
US6228579B1 (en) | 1997-11-14 | 2001-05-08 | San Diego State University Foundation | Method for identifying microbial proliferation genes |
WO2001040798A2 (en) * | 1999-12-06 | 2001-06-07 | Sangamo Biosciences, Inc. | Methods of using randomized libraries of zinc finger proteins for the identification of gene function |
WO2002016940A2 (en) * | 2000-08-23 | 2002-02-28 | Genome Therapeutics Corporation | Genomics-assisted rapid identification of targets |
US6589738B1 (en) | 1999-11-09 | 2003-07-08 | Elitra Pharmaceuticals, Inc. | Genes essential for microbial proliferation and antisense thereto |
US6780590B2 (en) | 1999-09-14 | 2004-08-24 | Sangamo Biosciences, Inc. | Gene identification |
GB2385124B (en) * | 2001-12-20 | 2006-07-26 | Hoffmann La Roche | Assay and screening method for identification of inhibitors of beta-secretases |
US7365162B2 (en) | 1998-10-13 | 2008-04-29 | University Of Georgia Research Foundation, Inc. | Stabilized bioactive peptides and methods of identification, synthesis, and use |
WO2011083147A1 (en) | 2010-01-08 | 2011-07-14 | Cemm-Forschungsinstitut Für Molekulare Medizin Gmbh | Wave1 inhibition in the medical intervention of inflammatory diseases and/or infections caused by a pathogen |
WO2011131626A1 (en) | 2010-04-19 | 2011-10-27 | Medizinische Universität Innsbruck | Tmem195 encodes for tetrahydrobiopterin-dependent alkylglycerol monooxygenase activity |
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1991017260A1 (en) * | 1990-05-02 | 1991-11-14 | The Upjohn Company | Method of identifying compounds useful as antiparasite drugs |
GB2303209A (en) * | 1995-07-12 | 1997-02-12 | Zeneca Ltd | Screening method for identification of sterol biosynthesis inhibitors |
-
1999
- 1999-01-08 WO PCT/US1999/000474 patent/WO1999035494A1/en not_active Application Discontinuation
- 1999-01-08 AU AU22181/99A patent/AU2218199A/en not_active Abandoned
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- 1999-01-08 EP EP99902132A patent/EP1046034A1/en not_active Withdrawn
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1991017260A1 (en) * | 1990-05-02 | 1991-11-14 | The Upjohn Company | Method of identifying compounds useful as antiparasite drugs |
GB2303209A (en) * | 1995-07-12 | 1997-02-12 | Zeneca Ltd | Screening method for identification of sterol biosynthesis inhibitors |
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AU2218199A (en) | 1999-07-26 |
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