Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-06-22
2004-10-12
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S004000, C435S029000, C435S034000, C435S254200
Reexamination Certificate
active
06803193
ABSTRACT:
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
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
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
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
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
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 contemplat
Benko Ann
Hopper Anita K.
Martin Nancy C.
Vaduva Gabriela
Ketter James
Lambertson David
The Penn State Research Foundation
LandOfFree
Methods to identify modulators of the mevalonate pathway in... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods to identify modulators of the mevalonate pathway in..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods to identify modulators of the mevalonate pathway in... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3327597