Methods to identify modulators of the mevalonate pathway in sterol synthesis Number:6,803,193 from the United States Patent and Trademark Office (PTO) owispatent An assay for the detection of substances agonistic or antagonistic to the mevalonate pathway are disclosed.<H mevalonate, modulators, Methods, to, iden, 6803193.html, Patent, pto, 6803193

Title: Methods to identify modulators of the mevalonate pathway in sterol synthesis

Abstract: An assay for the detection of substances agonistic or antagonistic to the mevalonate pathway are disclosed.

Patent Number: 6,803,193 Issued on 10/12/2004 to Hopper, et al.

Inventors: Hopper; Anita K. (Hershey, PA); Martin; Nancy C. (Louisville, KY); Benko; Ann (Palmyra, PA); Vaduva; Gabriela (St. Louis, MO)
Assignee: The Penn State Research Foundation (University Park, PA)

Appl. No.: 09/599,662

Filed: June 22, 2000

Current U.S. Class: 435/6 ; 435/254.2; 435/29; 435/34; 435/4

Current International Class: C12Q 1/02 (20060101)

Field of Search: 435/4,6,254.2,29,34

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Primary Examiner: Ketter; James
Assistant Examiner: Lambertson; David
Attorney, Agent or Firm: Medlen & Carroll, LLP

Government Interests

STATEMENT REGARDING FEDERAL SPONSORSHIP

This invention was made in part with government support under grants MCB9506810 and MCB9828216 from the National Science Foundation. The government has certain rights in the invention.

Parent Case Text

PRIORITY

This application for patent under 35 U.S.C. 111(a) claims priority, under 35 U.S.C. .sctn. 119(e), to Provisional Applications Serial No. 60/141,516 (filed on Jun. 23, 1999) and No. 60/199,699 (filed on Apr. 26, 2000); wherein said Provisional Applications were filed under 35 U.S.C. 111(b).

Claims

What is claimed is:

1. A method for screening compounds that are agonistic or antagonistic to the melvalonate pathway in sterol synthesis, comprising: a) providing: i) a test compound, ii) a growth media lacking arginine and containing a canavanine salt, and iii) modified yeast cells derived from wild type yeast cells, wherein said modified yeast cells express reduced cytosolic activity levels of Mod5p as compared to said wild type yeast cells, and wherein said modified yeast cells comprise a CAN1 gene having a nonsense mutation and a gene coding for a nonsense suppressor tRNA; b) mixing said growth media and said modified yeast cells to form an untreated modified yeast cell mixture; c) adding an aliquot of said untreated modified yeast cell mixture in said test compound thereby creating a treated modified yeast cell mixture; and d) measuring the growth of modified yeast cells within said treated modified yeast cell mixture and the growth of modified yeast cells within said untreated yeast cell mixture, wherein a difference in the growth of modified yeast cells within said treated modified yeast cell mixture and the growth of modified yeast cells within said untreated yeast cell mixture indicates the test compound has had an agonistic or antagonistic effect on the melvalonate pathway in sterol synthesis.

2. A method for screening compounds that are agonistic or antagonistic to the melvalonate pathway in sterol synthesis, comprising: a) providing: i) a test compound, ii) a growth media lacking arginine and containing a canavanine salt, and iii) modified yeast cells derived from wild type yeast cells, wherein said modified yeast cells express reduced cytosolic activity levels of Mod5p as compared to said wild type yeast cells, and wherein said modified yeast cells comprise a CAN1 gene having a nonsense mutation and a SUP7 gene coding for a tRNA; b) mixing said growth media and said modified yeast cells to form an untreated modified yeast cell mixture; c) adding an aliquot of said untreated modified yeast cell mixture to said test compound thereby creating a treated modified yeast cell mixture; and d) measuring the growth of modified yeast cells within said treated modified yeast cell mixture and the growth of modified yeast cells within said untreated yeast cell mixture wherein, a difference in the growth of modified yeast cells within said treated modified yeast cell mixture and the growth of modified yeast cells within said untreated yeast cell mixture indicates the test compound has had an agonistic or antagonistic effect on the melvalonate pathway in sterol synthesis.

Description

FIELD OF THE INVENTION

This invention generally relates to a novel assay for the screening of compounds that are agonistic or antagonistic to the mevalonate pathway and sterol and cholesterol synthesis. In selected embodiments this assay incorporates colorimetric, growth, and immunological methods for high throughput screening of compounds.

BACKGROUND

A finely tuned mechanism regulates the biosynthesis of mevalonate, the precursor of isoprenoid groups that are incorporated into more than a dozen classes of end products. These include: sterols, especially cholesterol, involved in membrane structure; haem A and ubiquinone, involved with electron transport; dolichol, required for glycoprotein synthesis; isopenentyladenine, present in some transfer RNAs; and intercellular messengers, such as cytokines in plants, farnesylated mating factors in fungi, juvenile hormones in insects and steroid hormones in animals. Interest in the regulatory importance of mevalonate was heightened by the discovery that growth-regulating p21.sup.ras proteins (encoded by ras proto-oncogenes and oncogenes) and nuclear envelope proteins, are covalently attached to farnesyl residues. These farnesyl residues, in turn, anchor said proteins to cell membranes. Inhibition of mevalonate synthesis prevents farnesylation of these proteins and blocks cell growth. To ensure constant production of the multiple isoprenoid compounds at all stages of growth, cells must precisely regulate mevalonate synthesis while avoiding over accumulation of potentially toxic products such as cholesterol. (Goldstein and Brown, "Regulation of the mevalonate pathway" Nature 343:425-430, 1990).

The ability to regulate flux through the mevalonate pathway (FIG. 1) is of medical importance because inhibitors of this pathway have been used to treat hypercholesterolemia and, consequently, to diminish the risk of heart attack. (Endo, "The discovery and development of HMG-CoA reductase inhibitors" J Lipid Res 33:1569-1582, 1992). Alteration of the pathway also affects the function of oncogenes (Reviews: Gibbs and Oliff, "The potential of farnesyltransferase inhibitors as cancer chemotherapeutics" Annu Rev Pharmacol Toxicol. 37:143-66, 1997. The mevalonate pathway is an important target for many areas of therapeutic research and application. For example, HMG-CoA reductase catalyzes the rate-limiting step of the mevalonate pathway (Voet and Voet, Biochemistry Wiley, New York, 1990); therefore, inhibitors of this reductase have been developed for administration to patients with hypercholesterolemia in an attempt to lower their blood cholesterol levels (Endo, et al., "Oxygenated cholesterols as ligands for cytosolic-nuclear tumor promoter binding protein: yakkasteroids" Biochem Biophys Res Commun 194:1529-35, 1993). Data reveals that the "statin" class (eg., compactin and lovastatin; see FIG. 1) of reductase inhibitors are reasonably safe and somewhat effective in lowering total cholesterol levels and preventing the progression and reducing the occurrence of coronary disease events (Brown, et al., "Regression of coronary artery disease as a result of intensive lipid-lowering therapy in men with high levels of apolipoprotein B" N Engl J Med 323:1289-98, 1990; Endo, et al., "Beneficial effects of dietary intervention on serum lipid and apolipoprotein levels in obese children" Am J Dis Child 146:303-305, 1992; Nash, et al., "Meeting national cholesterol education goals in clinical practice--a comparison of lovastatin and fluvastatin in primary prevention" Am J Cardiol. 78 (Suppl. 6A):26-31, 1996, but more progress needs to be made in the development of therapies that are more effective.

As an indication of the breadth of potential therapeutic effect regulators of the mevalonate pathway can have, the statins, in addition to regulating cholesterol levels, also stimulate nitric oxide production (Endres et al., "Role of peroxynitrite and neuronal nitric oxide synthase in the activation of poly(ADP-ribose) synthetase in a murine model of cerebral ischemia-reperfusion" Neurosci Lett. 248:41-4, 1998; Laufs et al., "Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors" Circulation 97:1129-35, 1998), have antiproliferative affects on some types of cancer cells (Lee et al., "Inhibition of the 3-Hydroxy-3-methylglutaryl-coenzyme A reductase pathway induces p53-independent transcriptional regulation of p21.sup.waf1/clp1 in human prostate carcinoma cells" J. Biol. Chem. 273: 10618-10623, 1998) and have immunosuppressive affects. Zaragozic acid inhibits the enzyme activity of squalene synthase which is the first step of the pathway committed solely to sterol biosynthesis, but appears not to be currently used in the clinical setting. Zaragozic acid D3 inhibits farnesyl-protein transferase and, therefore, protein prenylation as well (Tanimoto et al., "Inhibitory activity to protein prenylation and antifungal activity of zaragozic acid D3, a potent inhibitor of squalene synthase produced by the fungus, Mollisia sp SANK 10294" J Antibiot (Tokyo) 51:428-431, 1998).

A screen for compounds affecting various steps of the mevalonate pathway, therefore, could identify potential therapeutics for treatment of hypercholesterolemia and other pathological conditions associated with sterol metabolism in addition to compounds which may inhibit oncogene protein prenylation and farnesynelation. Moreover, the availability of a screen for flux through the sterol pathway could be useful for predicting undesirable side effects of drugs designed to treat other illnesses. Furthermore, since the mevalonate pathway is common to most organisms, compounds that regulate the mevalonate pathway may have uses beyond medicine such as agriculture and pest control. Therefore, what is needed is an efficient, flexible, high-throughput assay to screen for agents that are agonistic or antagonistic to mevalonate pathway function.

SUMMARY OF THE INVENTION

The present invention relates to an assay designed to detect flux in the melvonate pathway. In one embodiment the assay is a plate based assay incorporating the yeast strain Saccharomyces cerevisiae. Although it is not intended that the present invention be limited to a specific mechanism, it is believed that the modification of tRNA by Mod5p is in competition with flux through the mevalonate pathway. This competition results from both Mod5p and Erg20p using the same substrate, dimethylallyl-PP (FIG. 1). Mod5p catalyzes the transfer of an isopetenyl moiety to an adenosine generating i.sup.6 A at position 37 of some tRNAs. This modification affects the function of the tRNA in translation and may be measured by monitoring nonsense suppression. As shown in FIGS. 2 & 5F, two yeast strains have been generated that possess limiting cytosolic levels of Mod5p. When there is increased flux through the mevalonate pathway, in one example, by overproduction of Erg20p (FIG. 1), there is less i.sup.6 A modification of tRNA (FIG. 3). This decreases the proliferation of cells which grow on media that will support strains expressing normal levels of Erg20p. See, FIG. 2. Such a highly sensitive assay can differentiate as little as a two-fold difference in the level of i.sup.6 A modification of tRNA. (Benko, et al., "Competition between a sterol biosynthetic enzyme and the tRNA modification in addition to changes in the protein synthesis machinery causes altered nonsense suppression" PNAS 97:61-66, 2000).

Increased cytosolic levels of Mod5p cause a different phenotype easily assayed by growth on media lacking lysine (Zoladek et al., "Mutations altering the mitochondrial-cytoplasmic distribution of Mod5p implicate the actin cytoskeleton and mRNA 3' ends and/or protein synthesis in mitochondrial delivery" Mol Cell Biol. 15:6884-6894, 1995), thereby, providing a means for selecting reagents that decrease flux through the pathway. In one embodiment of the present invention, therefore, yeast growth on particular media can provide an index for both increases and decreases in mevalonate pathway flux. The assays contemplated by the present invention are efficient, inexpensive and provide new methods for screening drugs that alter the clinically significant mevalonate pathway.

It is not intended that the present invention be limited to the identification of compounds for only to a specific therapeutic application. However, in selected embodiments, compounds that regulate the mevalonate pathway may be beneficial in: (1) screening for new drugs to treat hyperlipidemias and other disorders in sterol metabolism such as Addison's disease and Cushing's syndrome; (2) screening for drugs that inhibit the function of farnesylated and prenylated oncogene products; (3) monitoring other drugs for possible side effects in sterol metabolism; (4) identification of yeast mutants with altered sterol metabolism; (5) screening for agents that alter plant physiological processes such as, for example, photosynthesis, cell growth, respiration, architecture and defense against pathogen attack (e.g., antibiotics and antifungals).

The present invention relates to a flexible, high throughput screen for agents that are agonistic or antagonistic to mevalonate pathway function. The present invention is not limited to any particular high throughput assay. Many high throughput assays are contemplated by the present invention. For example, in one embodiment, the present invention contemplates visually scoring plates. In another embodiment, the present invention contemplates culturing cells in microtiter plates, performing the assay, lysing the cells and generating a readout of said cell lysates via a spectrophotometer. In another embodiment the present invention contemplates reading the cells in a flow cytometer to detect changes in cell color and in cell growth. In another embodiment, the present invention contemplates measuring (in one example by scintillation) radiation generated by H.sup.3 -tritium as an index to determine cell growth. In preferred embodiments, the assay may be performed manually or with the use of automation and robotics for any or all steps in the aforementioned procedures.

The present invention relates to yeast strains engineered to provide detectable read-outs for compounds that are agonistic or antagonistic to the mevalonate pathway. In a preferred embodiment, the yeast strains are ALB1 (genotype: MAT.alpha. mod5-M2 SUP7 ade2-1 can1-100 leu2-3, -112 lys1-1 lys2-1 trp1 ura3-1), ALB8 (genotype: MAT.alpha. SUP7 can1-100 ade2-1 leu2-3, -112 lys1-1 lys2-1 trp1 mod5::TRP1 ura3-1::MOD5) and MT8-1D or MD14A with YCfmod5-m2KR6 plasmid for a lysine based assay.

It is not intended that the present invention be limited to the screening of any particular compound or class of compounds. Proteins, lipids, carbohydrates, glycoproteins, lipoproteins, synthetic compounds, compounds contained in combinatorial libraries or compounds and agents already being used as therapeutics may be screened by the present assay. Moreover, the screening of known therapeutics according to the present invention will reveal, heretofore, unknown side effects associated with the modulation of the mevalonate pathway.

It is not intended the present invention be limited to any particular protocol to quantitate or qualitate the assay. In selected embodiments, for example, the assay output may be measured visually, colormetrically, fluorescently, by immunoblot, by radio-immunoassay or by HPLC. In a one embodiment, anti-i.sup.6 A antibody is used to detect i.sup.6 A production in cells treated with a compound. In a preferred embodiment, a Western blot is used to detect the presence of i.sup.6 A. Quantitation is not limited to any particular method. Quantitation may be made by densitometer, chemilumiinescence, and radiography.

The present invention contemplates a composition comprising yeast with the relevant genotype of: SUP7 ade2-1 can1-100 leu2-3 mod5-M2 and designated ALB1. The present invention further contemplates a composition wherein the yeast ALB1 is a strain of Saccharomyces cerevisiae. Still further, the present invention contemplates a composition comprising yeast with the relevant genotype of: SUP7 can1-100 ade2-1 leu2-3 mod5::TRP1 ura3-1::MOD5 and designated ALB8. Even still further, the present invention contemplates a composition wherein the yeast ALB8 is a strain of Saccharomyces cerevisiae. Even further still, the present invention contemplates a composition comprising the yeast strain ALB1 wherein the genotype further comprises: MAT.alpha. mod5-M2 SUP7 ade2-1 can1-100 leu2-3, -112 lys1-1 lys2-1 trp1 ura3-1. Even still further, the present invention contemplates a composition wherein the yeast ALB1 is a strain of Saccharomyces cerevisiae. Even further still, the present invention contemplates a composition comprising yeast with the relevant genotype of:: MAT.alpha. SUP7 can1-100 ade2-1 leu2-3, -112 lys1-1 lys2-1 trp1 mod5::TRP1 ura3-1::MOD5 and designated ALB8. Even further still, the present invention contemplates a composition of claim 7 wherein the yeast is Saccharomyces cerevisiae.

The present invention contemplates a method, comprising: a) providing: i) a test compound, ii) a growth media formulated to allow scoring of nonsense suppression in yeast, and iii) modified yeast cells derived from wild type yeast cells, wherein said modified yeast cells express reduced cytosolic levels of Mod5p, or its homolog, as compared to said wild type yeast cells, and wherein said modified yeast cells comprise a gene with a nonsense mutation and a suppressor tRNA gene coding for a tRNA modified with isopentenyl adenosine by Mod5 or its homolog; b) exposing a portion of said modified yeast cells to said test compound and said growth media to create a treated portion and an untreated portion; and c) measuring for growth of said treated portion.

The present invention also contemplates a measuring of step which comprises examining the color of said yeast cells of said treated portion.

Additionally, the present invention contemplates a measuring of step which comprises comparing said treated portion with said untreated portion, wherein said untreated portion is exposed to said growth media in the absence of said test compound.

The present invention contemplates a method, comprising: a) providing: i) a test compound, ii) a growth media lacking adenine, and iii) modified yeast cells derived from wild type yeast cells, wherein said modified yeast cells express reduced cytosolic levels of Mod5p as compared to said wild type yeast cells, and wherein said modified yeast cells comprise an ADE gene having a nonsense mutation and a gene coding for a nonsense suppressor tRNA; b) exposing a portion of said modified yeast cells to said test compound and said growth media to create a treated portion and an untreated portion; and c) measuring for growth of said treated portion.

The present invention also contemplates a method, comprising: a) providing: i) a test compound, ii) a growth media lacking adenine, and iii) modified yeast cells derived from wild type yeast cells, wherein said modified yeast cells express reduced cytosolic levels of Mod5p as compared to said wild type yeast cells, and wherein said modified yeast cells comprise an ADE gene having a nonsense mutation and a SUP7 gene coding for a tRNA; b) exposing a portion of said modified yeast cells to said test compound and said growth media to create a treated portion and an untreated portion; and c) measuring for growth of said treated portion.

The present invention also contemplates a method, comprising: a) providing: i) a test compound, ii) a growth media lacking arginine and comprising a canavanine salt, and iii) modified yeast cells derived from wild type yeast cells, wherein said modified yeast cells express reduced cytosolic levels of Mod5p as compared to said wild type yeast cells, and wherein said modified yeast cells comprise a CAN1 gene having a nonsense mutation and a gene coding for a nonsense suppressor tRNA; b) exposing a portion of said modified yeast cells to said test compound and said growth media to create a treated portion and an untreated portion; and c) measuring for growth of said treated portion.

The present invention also contemplates a method, comprising: a) providing: i) a test compound, ii) a growth media lacking arginine and comprising a canavanine salt, and iii) modified yeast cells derived from wild type yeast cells, wherein said modified yeast cells express reduced cytosolic levels of Mod5p as compared to said wild type yeast cells, and wherein said modified yeast cells comprise a CAN1 gene having a nonsense mutation and a SUP7 gene coding for a tRNA; b) exposing a portion of said modified yeast cells to said test compound and said growth media to create a treated portion and an untreated portion; and c) measuring for growth of said treated portion.

Additionally, the present invention contemplates a method wherein said gene coding for said nonsense suppressor tRNA is selected from the group consisting of SUP7 and SUP11.

The present invention further contemplates a method comprising: a) providing i) one or more compounds, ii) a first yeast cell line designated ALB1; ii) a second yeast cell line designated ALB8; b) contacting a portion of said cells from i) said first yeast cell line and ii) said second yeast cell line, with said one or more said compounds, so as to create treated portions and untreated portions or cells; and, c) comparing said treated cells with said untreated cells. Even further, the present invention contemplates that the method of comparison of said treated or untreated cells may be by color and cell growth (i.e., the amount of cell division).

The present invention further contemplates a method comprising: a) providing i) one or more compounds and ii) a yeast cell line selected from a group consisting of yeast strains designated ALB1 and ALB8; b) contacting a portion of said cells from said yeast cell line with said one or more said compounds, so as to create treated portions and untreated portions of cells; and, c) i) comparing said treated cells with said untreated cells. The present invention further contemplates the comparison of treated and untreated cells by color and by cell growth.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the mevalonate pathway.

FIG. 2A is an autoradiograph. Low molecular weight RNA was prepared from ALB1 (mod5-M2) with each of the candidate genes or vector alone, ALB8 (MOD5) or MD14A (mod5-1). The RNAs were resolved on polyacrylamide gels, transferred to membranes and probed with anti-isopentenyl adenosine antibody (upper panel) or radiolabeled oligonucleotide complementary to mature tRNA.sup.Tyr (lower panel). This autoradiogaph shows the level of isopentenylated tRNA found in ALB1 over-expressing ERG20 is substantially reduced.

FIG. 2B is a graph. This graph presents data showing the levels of isopentenyl adenosine tRNA found in ALB1 with each of the candidate genes or vector only or in the strain ALB8 or MD14A that were assessed by densitometric analysis of two immunoblots and expressed as a fraction of the level found in the "vector" control. (A) membrane 1 values; (B) membrane 2 values; (C) average values. These data are also consistent with the finding that the level of isopentenylated tRNA found in ALB1 over-expressing ERG20 is substantially reduced.

FIG. 3A presents a model of competition between i.sup.6 A modification of tRNA and sterol biosynthesis.

FIG. 3B presents another model of competition between i.sup.6 A modification of tRNA and sterol biosynthesis.

FIG. 4 presents selected mutations introduced into the MOD5 gene and oligonucleotides. See, Gillman et al., "MOD5 translation initiation sites determine N6-isopentenyladenosine modification of mitochondrial and cytoplasmic tRNA"," Mol Cell Biol 11:2382-2390.

FIG. 5A presents the growth characteristics for ALB1 cells and T8-1D cells (with YCfmod5-M2,KR6) under conditions of normal flux through the sterol biosynthesis pathway.

FIG. 5B presents the growth characteristics for ALB1 cells and T8-1D cells (with YCfmod5-M2,KR6) under conditions such that flux through the sterol biosynthesis pathway is increased.

FIG. 5C presents the growth characteristics for ALB1 cells and T8-1D cells (with YCfmod5-M2,KR6) under conditions such that flux through the sterol biosynthesis pathway is decreased.

FIG. 6 presents a restriction map of YEpMOD5(7.0) and subclones YEpMOD5(1.8) and YEpMOD5(1.9). Numbers in parentheses indicate size of insert. See, Dihanich, et al., "Isolation and characterization of MOD5, a gene required for isopentenylation of cytoplasmic and mitochondrial tRNAs of Saccharomyces cerevisiae" Mol Cell Biol 7:177-184, 1987).

DEFINITIONS

To facilitate understanding of the invention, a number of terms are defined below.

"Genotype" shall be defined as the genetic makeup of an organism as encoded in its DNA.

"Relevant genotype" shall be defined as specific genes that, if expressed, have impact on the investigation under way.

A "nonsense suppressor tRNA" is meant to indicate one of several known suppressor tRNAs. For example, SUP7-1 codes for an altered tRNA that is one of several efficient tyrosine-inserting UAA suppressors. (H. Laten et al., Nucleic Acids Res. 5:4329 (1978)). Indeed, SUP7 (and SUP11 for that matter) can suppress the lys2-1 nonsense allele (as well as other nonsense mutations). The LYS2 gene encodes an enzyme involved in the biosynthesis of lysine. Mutations in this gene prevent growth on media lacking exogenous lysine.

As used herein, the term "scoring of nonsense suppression in yeast" refers to determining the level of suppression due to translation though the nonsense codon in mRNA relative to a control sample wherein said control sample comprises reduced or absent translation through the nonsense codon in mRNA because of differences in suppressor tRNA.

As referred to herein, cells with the mod5-M2 allele (strain ALB1 projected in FIG. 2) contains a mutation at codon 12 preventing the initiation of Mod5p-II translation (and allow only Mod5p-I production) have about 50% of the SUP7 pool modified resulting in intermediate levels of suppression. These cells show intermediate growth on canavanine and on media lacking adenine.

As referred to herein, cells with the mod5-M2KR6 allele (T8-1D with Ycfmod5-M2KR6 as projected in FIG. 5) have a very small cytosolic pool of Mod5p and the cells are unable to grow in the absence of lysine.

Yeast strain "ALB1" is defined as a substantially pure population of yeast with the genotype of: MAT.alpha. mod5-M2 SUP7 ade2-1 can1-100 leu2-3, -112 lys1-1 lys2-1 trp1 ura3-1 and the relevant genotype of: SUP7 ade2-1 can1-100 leu2-3-112 mod5-M2. The yeast may be of the species Saccharomyces cerevisiae.

Yeast strain "ALB8" shall be defined as a substantially pure population of yeast with the genotype of: MAT.alpha. SUP7 can1-100 ade2-1 leu2-3, -112 lys1-1 lys2-1 trp1 mod5::TRP1 ura3-1::MOD5. The yeast may be of the species Saccharomyces cerevisiae.

Yeast strain "T8-1D" shall be defined as a substantially pure population of yeast with the genotype of: MAT.alpha. SUP11 ade2-1 leu2-3, -112 mod5-1 lys2-1 his4-519 ura 3-1. The yeast may be of the species Saccharomyces cerevisiae.

As used herein, CAN1 refers to a sequence encoding an arginine permease that allows the uptake of the arginine analog canavanine. Canavanine interferes with the process of translation and cells cannot grow in its presence. Therefore, cells with wild-type CAN1 are sensitive to canavanine, but cells with the mutant can 1-100, that does not encode a properly functioning permease, are resistant to canavanine and can grow in its presence. See, Bun-Ichiro Ono, et al., "Nonsense Mutations in the can1 Locus of Saccharomyces cervisiae", Journal of Bacteriology, June 1983, pp. 1476-1479.

As used herein, ADE2 refers to a sequence which encodes an enzyme involved in the synthesis of adenine. Cells with ade2-1 turn red in color and fail to grow on defined medium lacking adenine (Ade), whereas cells producing functional Ade2p can grow on such medium (Ade+) and are white. Cells with the ade2-1 allele and sufficient i6A-modified suppressor tRNA can grow in the absence of exogenous adenine and generate white colonies on rich medium, whereas cells with insufficient i6A-modified tRNA are unable to grow in the absence of exogenous adenine and generate red colonies on rich medium. Cells with intermediate levels of i6A modified tRNA have intermediate phenotypes in colony color and intermediate rates of growth in the absence of exogenous adenine.

As used herein a, "substantially deficient adenine growth media" refers to a culture media that, in one embodiment, has less than 20 mg/L of adenine while in a preferred embodiment has less than 5 mg/L of adenine.

"Wild type" shall be defined as the genomic makeup of an organism (the genotype) before modifications have been made. In other words, it is the parent strain of the organism.

The term "binding interaction" when used in relation to RNA shall be defined as the ability of two or more macromolecules to bind to each other (e.g., to produce an aggregate). The present invention makes no limit on the stringency of the binding interaction so long as the interaction can be detected by methods known to those practiced in the art (e.g., by Western blot, coimmunoprecipitation, spectrophotometry, colorimetric assay, etc.).

The term "homology" when used in relation to proteins refers to a degree of similarity. There may be partial homology or complete homology (i.e., identity). A partially similar sequence is one that may partially inhibit a similar sequence from performing its function (e.g., enzymatic, binding, etc) in vivo or in vitro and is referred to using the functional term "substantially homologous." The inhibition function of the substantially similar sequence may be examined using an enzymatic assay, a binding assay or other assay designed to measure the particular function of the substantially similar protein. A substantially homologous proteins may compete for or interfer with a homolog and inhibit its function (e.g., the binding or enzymatic function). This is not to say that conditions of low stringency are such that non-specific interaction is permitted; low stringency conditions require that the interaction be a specific (i.e., selective) interaction.

Low stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42.degree. C. in a solution consisting of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2 PO.sub.4.H.sub.2 O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5.times.Denhardt's reagent [50.times.Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 ug/ml denatured salmon sperm DNA followed by washing in a solution comprising 5.times.SSPE, 0.1% SDS at 42.degree. C. when a probe of about 500 nucleotides in length is employed.

High stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42.degree. C. in a solution consisting of 5.times.SSPE (43.8 g/l NaCl, 6.9 g/l NaH.sub.2 PO.sub.4.H.sub.2 O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5.times.Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1.times.SSPE, 1.0% SDS at 42.degree. C. when a probe of about 500 nucleotides in length is employed.

When used in reference to nucleic acid hybridization the art knows well that numerous equivalent conditions may be employed to comprise either low or high stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of either low or high stringency hybridization different from, but equivalent to, the above listed conditions.

"Stringency" when used in reference to nucleic acid hybridization typically occurs in a range from about T.sub.m -5.degree. C. (5.degree. C. below the T.sub.m of the probe) to about 20.degree. C. to 25.degree. C. below T.sub.m. As will be understood by those of skill in the art, a stringent hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences. Under "stringent conditions" a nucleic acid sequence of interest will hybridize to its exact complement and closely related sequences.

As used herein, the term "purified" or "to purify" refers to the removal of contaminants from a sample. The present invention contemplates purified compositions (e.g. the ALB1 and ALB8 stains of S. cerevisiae).

As used herein, the term "substantially purified" refers to the removal of a significant portion of the contaminants of a sample to the extent that the substance of interest is recognizable by techniques known to those skilled in the art as the most abundant substance in the mixture.

As used herein the term "portion" when in reference to a protein (as in "a portion of a given protein") refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid. In one embodiment, the present invention contemplates "functional portions" of a protein. Such portions are "functional" if they contain a binding region (i.e., a region having affinity for another molecule) and such binding can take place (i.e., the binding region functions, albeit with perhaps lower affinity than that observed for the full-length protein). Such "functional portions" of the gene product are typically greater than 50 amino acids in length, and more typically greater than 100 amino acids in length. "Functional portions" may also be "conserved portions" of the protein. The alignment of the yeast and human gene products (described herein) permit one skilled in the art to select conserved portions of the protein (i.e., those portions in common between yeast and man) as well as unconserved portions (i.e., those portions unique to either yeast or man). The present invention contemplates conserved portions 20 amino acids in length or greater, and more typically greater than 50 amino acids in length.

As used herein the term "portion" when in reference to an oligonucleotide sequence (as in "a portion of a given sequence") refers to fragments of that sequence. The fragments may range in size from four base residues to the entire oligonucleotide sequence minus one base. More typically, such portions are 15 nucleotides in length or greater. Again, such portions may be conserved portions (see FIG. 8). On the other hand, such portions may be unique portions of the gene.

"Compound" refers to any chemical entity, pharmaceutical, drug, and the like that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function. Compounds comprise both known and potential therapeutic compounds. A compound can be determined to have therapeutic potential by screening, e.g., using the screening methods of the present invention. A "known therapeutic compound" refers to a therapeutic compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention.

"Antibody" shall be defined as a glycoprotein produced by B cells that binds with high specificity to the agent (usually, but not always, a peptide), or a structurally similar agent (e.g., i.sup.6 A), that generated its production (e.g., the i.sup.6 A antibody). Antibodies may be produced by any of the known methodologies [Current Protocols in Immunology (1998) John Wiley and Sons, Inc., N.Y.] and may be either polyclonal or monoclonal.

"Antigen" shall be defined as a protein, glycoprotein, lipoprotein, lipid or other substance that is reactive with an antibody specific for a portion of the molecule.

The terms "immunoprecipitate", "immunoprecipitated", "immunoprecipitation" refer to the use of antibody to take an antigen out of solution by precipitation.

The term "affinity purification" refers to the use of an antibody to separate its antigen or a portion thereof from a mixture of other molecules because of affinity for the antigen.

"Immunofluorescence" is a staining technique used to identify, mark, label, visualize or make readily apparent by procedures known to those practiced in the art, where a ligand (usually an antibody) is bound to a receptor (usually an antigen) and such ligand, if an antibody, is conjugated to a fluorescent molecule, or the ligand is then bound by an antibody specific for the ligand, and said antibody is conjugated to a fluorescent molecule, where said fluorescent molecule can be visualized with the appropriate instrument (e.g., a fluorescent microscope).

"Staining" shall be defined as any number of processes known to those in the field that are used to better visualize, distinguish or identify a specific component(s) and/or feature(s) of a cell or cells.

"RIA" or "radioimmunoassay" shall be defined as an assay wherein antibody is used to detect antigen and thereafter quantitated with radioisotopes.

"Immunoabsorbant" and "immunoaffinity" shall be defined as binding antigen with antibody reactive to said antigen. The antibody or the antigen may be bound to a solid or semi-solid substrate.

"Western blot" shall be defined as an assay comprising antigen that is detected by antibodies reactive to the antigen after the antigen has been at least partially separated from contaminants by, for example, electrophoresis.

"In operable combination", "in operable order" and "operably linked" as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.

"Heterologous DNA" sequence refers to a nucleotide sequence which is not endogenous to the cell into which it is introduced. Heterologous DNA includes a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Heterologous DNA also includes a nucleotide sequence which is naturally found in the cell into which it is introduced and which contains some modification relative to the naturally-occurring sequence.

"Morphology" shall be defined as the visual appearance of a cell or organism when viewed with the eye, a light microscope, a confocal microscope or an electronmicroscope, as appropriate. Morphology shall include, but is not limited to, cell shape, cell appearance and cell color.

"Patient" shall be defined as a human or other animal, such as a guinea pig or mouse and the like.

GENERAL DESCRIPTION OF THE INVENTION

This invention generally relates to a novel assay for the screening of compounds that are agonistic or antagonistic to the mevalonate pathway and sterol and cholesterol synthesis. In selected embodiments this assay incorporates colorimetric, growth, biochemical (e.g., HPLC) and immunological methods for high through-put screening of compounds.

Baker's yeast Saccharomyces cerevisiae are single celled eukaryotes used extensively as model organisms. This model system provides for the facile manipulations of yeast genes, availability of the complete genomic sequence of baker's yeast, and identification of numerous yeast genes which directly correlate with known human disease genes. One fundamental discovery that has become clear from the use of this model system is that many physiologically significant regulatory pathways and proteins are highly conserved among all eukaryotes. Importantly, mammalian homologues of many yeast proteins are known to be important in regulating cell physiology, including the mevalonate pathway. Therefore, these model systems have been effectively utilized for the rapid identification and functional characterization of compounds that may be of use as therapeutics in higher eukaryotes. Thus yeast is well suited as a model system not only for the elucidation of basic cell biology but also for large-scale screening of compounds which specifically target cell growth.

I. Assay Development

The Saccharomyces cerevisiae protein Mod5p catalyzes the addition of an isopentenyl group to adenosine (i.sup.6 A) at position 37 of the anticodon loop of some tRNAs (Kline, et al, "N6-(delta-2-isopentenyl)adenosine. Biosynthesis in transfer ribonucleic acid in vitro" Biochemistry 8:4361-4371, 1969; Bartz, et al., "N6-(Delta 2-isopentenyl)adenosine: biosynthesis in vitro in transfer RNA by an enzyme purified from Escherichia coli" Biochem Biophys Res Commun 40:1481-1487, 1970; Rosenbaum and Gefter, "Delta 2-isopentenylpyrophosphate: transfer ribonucleic acid 2-isopentenyltransferase from Escherichia coli. Purification and properties of the enzyme" J Biol Chem 247:5675-5680, 1972; McCloskey and Nishimura, Acc. Chem. Res. 10:403-410, 1977; Dihanich, et al., "Isolation and characterization of MOD5, a gene required for isopentenylation of cytoplasmic and mitochondrial tRNAs of Saccharomyces cerevisiae" Mol Cell Biol 7:177-184, 1987). There are two isoforms of Mod5p, Mod5p-I and Mod5p-II, which differ in the site of their translation initiation codon and in their distribution in the cell. Mod5p-I is translated starting at codon one of the MOD5 ORF and is localized to mitochondria and the cytoplasm. Mod5p-II, translated from codon twelve of the MOD5 ORF, is located in the nucleus and the cytoplasm (Gillman, et al., "MOD5 translation initiation sites determine N6-isopentenyladenosine modification of mitochondrial and cytoplasmic tRNA" Mol Cell Biol 11:2382-2390, 1991; Boguta, et al., "Subcellular locations of MOD5 proteins: mapping of sequences sufficient for targeting to mitochondria and demonstration that mitochondrial and nuclear isoforms commingle in the cytosol" Mol Cell Biol 14:2298-2306, 1994).

Over-expression of Erg20p causes a decrease in i.sup.6 A tRNA-mediated suppression in yeast and an approximately 70% decrease in i.sup.6 A modification of tRNA. These effects are most likely due to the loss of Mod5p substrate to Erg20p. The data demonstrate the dependence of tRNA processing upon changes in components of the sterol pathway. The data also indicate that, at least in yeast, Erg20p may also catalyze a rate-limiting step in sterol biosynthesis. Hence, the tRNA and the sterol biosynthetic pathways are in apparent competition and a delicate balance in protein levels is required to maintain proper functioning of translation and mevalonate metabolism.

The i.sup.6 A modification promotes the efficiency of SUP7 or SUP11 tRNA in cytosolic suppression of UAA nonsense mutations by the insertion of tyrosine (Laten, et al., "Isopentenyladenosine deficient tRNA from an antisuppressor mutant of Saccharomyces cerevisiae" Nucleic Acids Res 5:4329-4342, 1978). Cells possessing only Mod5p-I have limiting cytosolic amounts of isozyme and changes in the subcellular distribution and/or the activity of this isozyme alter nonsense suppression. Hence genetic screens/selections based on nonsense suppression can identify cells with altered cytosolic Mod5p-I activity (Zoladek, et al., "Mutations altering the mitochondrial-cytoplasmic distribution of Mod5p implicate the actin cytoskeleton and mRNA 3' ends and/or protein synthesis in mitochondrial delivery" Mol Cell Biol 15:6884-6894, 1995).

Applicants employed the genetic strategy of using over expression to perturb a pathway (Rine, "Gene overexpression in studies of Saccharomyces cerevisiae" Methods Enzymol 194:239-251, 1991) and developed a protocol for the selection of cells with lower than normal levels of cytosolic Mod5p-I activity. Using this strategy Applicants were able to sample the entire yeast genome and identify genes that, when overexpressed, lead to lower than normal, levels of cytosolic Mod5p-I activity.

As a result of that screen, Applicants have identified two categories of genes. The first category includes genes that affect nonsense suppression via alteration of the protein synthetic machinery. Applicants' studies suggest that the yeast gene product encoded by YDL219w may function in protein synthesis and that the translation elongation factor EF1-.gamma. may function in translational proofreading. SAL6 and ARC1, with previously established effects on protein synthesis, were also recovered.

The second category includes ERG20, involved in sterol biosynthesis. The mevalonate pathway generates sterols, prenylated proteins, heme A, dolichol, ubiquinone and the substrate required for isopentenylation of tRNAs (FIG. 1). Both Mod5p and Erg20p use the same substrate, dimethylallyl pyrophosphate (DMAPP). Recovery of ERG20 coupled with subsequent biochemical assays demonstrating a reduction of i.sup.6 A on tRNA is most easily explained by a model in which Mod5p and Erg20p compete for a limited pool of DMAPP. When more DMAPP is used to make sterols, less is available for modification of tRNA and a reduction in the efficiency of nonsense suppression results. Thus, Applicants have demonstrated that the tRNA biosynthetic pathway and the sterol biosynthetic pathway are in apparent competition for substrate and that Erg20p and Mod5p must be balanced to optimally maintain the protein synthetic machinery. A practical consequence is that it should be possible to adapt the selection the applicants developed to assess the effect of mutations and/or drugs that change the distribution of DMAPP between the sterol pathway and the tRNA biosynthetic pathway.

Applicants interest in the distribution of sorting isozymes led to the design of a screen for gene products that function in the subcellular distribution of Mod5p or affect its ability to modify cytosolic tRNAs. Applicants anticipated the discovery of genes affecting transcription of MOD5 and/or tRNAs, nuclear export of tRNA, and the translation process, (in addition to those involved in protein distribution). Three gene products, Sal6p, Tef4p and YDL219w, may affect nonsense suppression via function in protein translation and Arc1p could play a role in nuclear export of tRNA. No gene products affecting MOD5 or tRNA transcription or Mod5p-I subcellular distribution were uncovered. Rather, ERG20, important to sterol biosynthesis, was discovered.

Sal6p/Ppq1p is a serine-threonine protein phosphatase very similar to mammalian phosphatase PP1 (Vincent, et al., "The yeast translational allosuppressor, SAL6: a new member of the PP1-like phosphatase family with a long serine-rich N-terminal extension" Genetics 138:597-607, 1994; Chen, et al., "PPQ, a novel protein phosphatase containing a Ser.sup.+ Asn-rich amino-terminal domain, is involved in the regulation of protein synthesis" Eur J Biochem 218:689-699, 1993). Multiple copies of SAL6 cause antisuppression of nonsense mutations (Vincent, et al., "The yeast translational allosuppressor, SAL6: a new member of the PP1-like phosphatase family with a long serine-rich N-terminal extension" Genetics 138:597-607, 1994) and the sal6-1 allele acts as an allosuppressor (Vincent, et al., "The yeast translational allosuppressor, SAL6: a new member of the PP1-like phosphatase family with a long serine-rich N-terminal extension" Genetics 138:597-607, 1994; Stansfield and Tuite, "Polypeptide chain termination in Saccharomyces cerevisiae" Curr Genet 25:385-395, 1994). Yeast cells containing a disrupted PPQ1 gene exhibit a slowed translation rate and hypersensitivity to inhibitors of protein synthesis (Chen, et al., "PPQ, a novel protein phosphatase containing a Ser+ Asn-rich amino-terminal domain, is involved in the regulation of protein synthesis" Eur J Biochem 218:689-699, 1993). These findings suggest a role for Sal6p in the regulation of the fidelity of translation (Vincent, et al., "The yeast translational allosuppressor, SAL6: a new member of the PP1-like phosphatase family with a long serine-rich N-terminal extension" Genetics 138:597-607, 1994; Chen, et al., "PPQ, a novel protein phosphatase containing a Ser+ Asn-rich amino-terminal domain, is involved in the regulation of protein synthesis" Eur J Biochem 218:689-699, 1993).

Tef4p is the gamma subunit of elongation factor 1 (EF-1). Eukaryotic EF-1 functions in delivering incoming tRNAs to the ribosome and is composed of at least three subunits. EF-1.alpha. binds GTP and then the proper aminoacyl-tRNA and positions the aminoacyl tRNA in the ribosomal A site. Subsequently, GTP is hydrolyzed to GDP and EF-1.beta. catalyzes the exchange of GDP for GTP to restore EF-1.alpha. to its initial state (Kinzy, et al., "Multiple genes encode the translation elongation factor EF-1 gamma in Saccharomyces cerevisiae" Nucleic Acids Res 22:2703-2707, 1994; Hinnebusch and Liebman, in "The molecular and cellular biology of the yeast Saccharomyces: Genomic dynamics, protein synthesis and energetics" eds. Broach, et al. [Cold Spring Harbor Lab Press, Plainview, N.Y.] Vol. 1, pp. 627-735, 1991). The exact function of EF-1.delta. has not been determined; however, in Artemia salina this subunit was observed to enhance the function of EF-1.beta. catalysis. In the same study EF1-.gamma. was found to interact with cell membranes and tubulin suggesting that it might mediate the association of the translational machinery with the cell framework (Janssen and Moller, "Elongation factor 1 beta gamma from Artemia. Purification and properties of its subunits" Eur J Biochem 171:119-129, 1988).

In bacteria the EF-1 counterpart functions in translational proofreading. Incorrectly inserted aminoacyl tRNAs can be removed prior to GTP hydrolysis and prior to the departure of EF-1.alpha. from the ribosome (Hinnebusch and Liebman, in "The molecular and cellular biology of the yeast Saccharomyces: Genomic dynamics, protein synthesis and energetics" eds. Broach, et al. [Cold Spring Harbor Lab Press, Plainview, N.Y.] Vol. 1, pp. 627-735, 1991). If the yeast EF-1 serves a similar function as the . bacterial counterpart, it is unlikely that the a subunit participates in this function as extra copies of the genes encoding EF-1.alpha. decrease fidelity (i.e., they enhance suppression of nonsense mutations; Song, et al., "Elongation factor EF-1 alpha gene dosage alters translational fidelity in Saccharomyces cerevisiae" Mol Cell Biol 9:4571-4575, 1989). Applicants show that over-expression of Tef4p reduces nonsense suppression and therefore it is possible that it is the EF1-.gamma. subunit that functions in EF-1 proofreading.

ORF YDL219w is predicted to code for a 150 amino acid protein with no significant homology to any characterized protein. However two lines of evidence indicate that this protein may function in the translation process. First, the gene possesses an intron [(Saccharomyces Genome Database, http://genome-www.stanford.edu/cgi-bin/dbrun)]. As introns are rare in yeast other than for approximately half of the genes encoding ribosomal proteins (Woolford and Warner, in "The molecular and cellular biology of the yeast Saccharomyces: Genomic dynamics, protein synthesis and energetics" eds. Broach, et al. [Cold Spring Harbor Lab Press, Plainview, N.Y.] Vol. 1, pp. 587-626, 1991), the presence of the intron is suggestive of a role in translation. Second, Applicants show that over expression of YDL219w affects tRNA-mediated nonsense suppression.

Arc1p, or G4p1, was originally discovered as a quadruplex nucleic acid binding protein (Frantz and Gilbert, "A novel y