(12) Patent Application Publication (10) Pub. No.: US 2003/0082511 A1 Brown Et Al
Total Page：16
File Type：pdf, Size：1020Kb
US 20030082511A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0082511 A1 Brown et al. (43) Pub. Date: May 1, 2003 (54) IDENTIFICATION OF MODULATORY Publication Classification MOLECULES USING INDUCIBLE PROMOTERS (51) Int. Cl." ............................... C12O 1/00; C12O 1/68 (52) U.S. Cl. ..................................................... 435/4; 435/6 (76) Inventors: Steven J. Brown, San Diego, CA (US); Damien J. Dunnington, San Diego, CA (US); Imran Clark, San Diego, CA (57) ABSTRACT (US) Correspondence Address: Methods for identifying an ion channel modulator, a target David B. Waller & Associates membrane receptor modulator molecule, and other modula 5677 Oberlin Drive tory molecules are disclosed, as well as cells and vectors for Suit 214 use in those methods. A polynucleotide encoding target is San Diego, CA 92121 (US) provided in a cell under control of an inducible promoter, and candidate modulatory molecules are contacted with the (21) Appl. No.: 09/965,201 cell after induction of the promoter to ascertain whether a change in a measurable physiological parameter occurs as a (22) Filed: Sep. 25, 2001 result of the candidate modulatory molecule. Patent Application Publication May 1, 2003 Sheet 1 of 8 US 2003/0082511 A1 KCNC1 cDNA F.G. 1 Patent Application Publication May 1, 2003 Sheet 2 of 8 US 2003/0082511 A1 49 - -9 G C EH H EH N t R M h so as se W M M MP N FIG.2 Patent Application Publication May 1, 2003 Sheet 3 of 8 US 2003/0082511 A1 FG. 3 Patent Application Publication May 1, 2003 Sheet 4 of 8 US 2003/0082511 A1 KCNC1 ITREXCHO KC 150 mM KC 2000000 so 100 mM induced Uninduced Steady state O 100 200 300 400 500 600 700 Time (seconds) FIG. 4 Patent Application Publication May 1, 2003 Sheet 5 of 8 US 2003/0082511 A1 4-Aminopyridine 1500OOO 100 mMKC induced A 4-AP v Uninduced Pre-incubated for >30' in 900 M4-aminopyridine. 100 200 300 400 Time(sec) F.G. 5 Patent Application Publication May 1, 2003 Sheet 6 of 8 US 2003/0082511 A1 BaC 150000 O induced A BaC D 1000000- . w Uninducedt 500000 Pre-incubated for >30' in 30 mMBaC. 100 200 300 400 Time(sec) F.G. 6 Patent Application Publication May 1, 2003 Sheet 7 of 8 US 2003/0082511 A1 FIG. 7 Patent Application Publication May 1, 2003 Sheet 8 of 8 US 2003/0082511 A1 100M 25nM 1 OOOOOO KC Pirozide induced A Uninduced 9 750000 C () 9. 500000 O 2 " 250000 O O 100 200 300 400 Time(sec) FIG. 8 US 2003/0O82511 A1 May 1, 2003 IDENTIFICATION OF MODULATORY requires the target protein be properly expressed, correctly MOLECULES USING INDUCIBLE PROMOTERS localized within the cell, functionally coupled to a Signaling mechanism, and expressed Stably throughout the duration of TECHNICAL FIELD the testing process. However, when the function of the target 0001. The present invention relates generally to the tech is unknown, these requirements can not be tested. nical fields of molecular biology and drug discovery. More 0006 When the target is a membrane protein such as a Specifically, the invention relates to the method of identify G-protein coupled receptor (“GPCR”), it may be mutated ing a drug target modulator using an inducible vector. Such that the protein is expressed in its activated form. Since BACKGROUND OF THE INVENTION ligand binding of the mutated protein frequently causes a drop in activity, an assay that detects a drop in activation 0002 Advances in molecular biology have increased the Suggests the compound binds the target. However, Since this efficiency of gene isolation and Sequencing. Additionally, technique identifies compounds which bind to a mutated the availability of known Sequences and Sequence alignment protein, the compounds may not possess the Same affinity or programs allow comparisons to be made leading to the avidity for a native protein. In addition, this technique is not identification of motifs that are conserved between members available when information regarding the design of an of the same family or Similar classes. This allows genes to activated receptor is unavailable, Such as the active form of be assigned to particular target families, Such as G-protein ion channels. coupled receptorS or ion channels. However, in the case of receptors, Sequence information of the target does not pro 0007 Another frequently used technique to identify vide the identity of the receptor's native ligand or that modulators is to perform competitive binding assayS. How ligand’s biological function. For example, Single transmem ever, competitive binding assays require a native ligand to brane membrane receptors contain a cysteine rich domain, assay the compound, and as previously discussed they are followed by an alpha helix motif, followed by a tyrosine frequently unknown. kinase domain. This may Suggest that the Sequence is a 0008 Lastly, assays that directly measure binding inter receptor, whereby the cysteine rich domain is involved in actions using purified proteins allow the measurement of ligand binding, the alpha helix traverses the membrane, and interactions between compounds and targets. Examples of the tyrosine kinase domain is involved in cellular Signaling. direct binding assays are Surface plasmon resonance Spec Unfortunately, Sequencing an unknown receptor's ligand troScopy, thermal denaturation profiling, and multipole cou binding domain does not provide Sufficient information that pling spectroScopy. However, these techniques only detect would easily lead to the identity of the ligand. Similar binding and are not functional assays. They do not distin problems occur when Searching for the function of ion guish between agonists, antagonists, or non-functional inter channels, enzymes, transporters, transcription factors, actions. Moreover, when the targets are membrane proteins nuclear receptors, chaperone proteins and other regulatory in their native form, purification is not always possible. molecules within the cell. Consequently, experiments must When a purified form is unavailable, interaction among be designed and performed to identify the Sequence's func other molecules in the preparation may lead to false posi tion and modulatory compounds. tives or false negatives in the assay. 0.003 Controlled expression of the target sequence is necessary to identify modulatory compounds because con 0009. Therefore there is a need for methods to assay the Stitutive expression often leads to over expression of the effects of compounds on the function of biological targets. protein. This is frequently toxic to the cell or can cause Specifically, there is a need for an assay that allows control down-regulation of the target by Stimulation of internaliza of the expression of the target Sequence, identifies target tion and degradation processes. However gene expression is expressing cells, expresses the target in its native form, can difficult to control in terms of both the level and time course distinguish between agonists, antagonists, and nonfunctional of target expression. Current expression vectors are usually interactions and may be performed within the cellular envi designed to maximize expression levels, and therefore yield rOnment. cells that continuously express the target. Alternatively, techniques Such as transient transfection reduce the target's BRIEF DESCRIPTION OF THE FIGURES duration of expression, but these techniqueS often lead to 0010 FIG. 1 is an illustration of the inducible expression heterogeneous expression among replicate Samples, are vector comprising a tetracycline inducible promoter, a labor-intensive, and may damage the cells or alter their pcDNA4/TO vector construct and a murine KCNC1 potas function due to the need to penetrate the membrane to deliver exogenous genes, making data difficult to collect and sium ion channel gene. analyze. 0011 FIG. 2 is a photograph of a 1.5% agarose gel 0004. The activity of a compound against a target of demonstrating KCNC1 mRNA production of clones 7, 13 interest is determined by a variety of techniques. Some and 22 under non-induced (“(-)Tet”) and induced (“(+)Tet”) examples include randomly Screening the compound against conditions. cells transfected with the target, testing compounds in cells 0012 FIG. 3 is a photograph of immuno-staining of where the target has been mutated to express the protein in KCNC1 produced by clone 22 under non-induced (“(-)Tet’) its active State, and binding Studies between a compound and and induced (“(+)Tet') conditions. an isolated form of the target. However each has problems 0013 FIG. 4 is a graph demonstrating hyperpolarization asSociated with the technique. of an induced population of cells compared to a non-induced 0005 Random screening of transfected cells requires a population of cells and their responses to 50 mM, 100 mM number of assumptions that often may not be tested. It and 150 mM KC1. US 2003/0O82511 A1 May 1, 2003 0.014 FIG. 5 is a graph demonstrating that cells induced 0022. In some embodiments of the invention, the target to overexpress KCNC1 when pre-incubated with 4-ami protein affects ion channel activity of the cell. In one nopyridine, Show characteristics more Similar to uninduced particular embodiment, the target protein is an ion channel cells. protein. 0.015 FIG. 6 is a graph demonstrating that cells induced 0023. In other embodiments of the invention, the target to overexpress KCNC1 when pre-incubated with BaCl, protein is a cell Surface receptor, Such as a G-protein coupled show characteristics more Similar to uninduced cells. receptor. In Still other embodiments, the target protein is 0016 FIG. 7 is an illustration of an inducible expression another type of Signaling molecule or transport molecule. vector comprising a tetracycline inducible promoter, a 0024. One aspect of the present invention includes iden pcDNA4/TO vector construct and a HERG potassium ion tification of the type of Signal being produced by a candidate channel gene. molecule, or more particularly, the method by which the Signal is being produced or by which the modulation occurs.