Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-01-12
2004-12-07
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S254110, C435S254210, C435S254220, C536S023200
Reexamination Certificate
active
06828092
ABSTRACT:
The present invention relates to a method for identifying compounds which modulate sterol biosynthesis. In particular the invention provides in vivo assays for inhibitors of sterol biosynthesis wherein inhibition leads to a change in the level of expression of a reporter gene. The invention also relates to nucleic acids and recombinant cells for use in the above assays.
Two general approaches may be taken to the identification of inhibitors of a metabolic pathway. In the first approach, one or more individual enzymes from the pathway are selected, and compounds are screened for their ability to inhibit these enzymes in in vitro reactions. This approach can be labour intensive, and, depending on the assay and enzymes used, expensive. An alternative approach is to construct a system which reports on the activity of the entire metabolic pathway, either by directly measuring the levels of the product of the pathway, or by using an indirect measurement of these levels. In both prokaryotes and eukaryotes the products of a metabolic pathway sometimes regulate the expression of the genes which encode the enzymes in the pathway. By coupling the regulatory regions of these genes to reporter genes it is possible to obtain reporter gene assays for inhibitors of metabolic pathways.
In mammalian cells, the product of sterol biosynthesis (cholesterol) regulates the transcription of the genes encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and HMG-CoA reductase. The sequences within the promoter regions of these genes which mediate the regulation of transcription in response to sterols have been determined, and are referred to as sterol response elements (SREs: Brown and Goldstein (1990) Nature 343, 425-430). A transcription factor which binds to these sequences has been characterised, and a mechanism for sterol regulation of the activity of this factor, involving sterol-regulated proteolysis, has been proposed (Wang et al (1994) Cell 77, 53-62). Construction of sterol-responsive reporter genes using SRE sequences has been described. For example, in mammalian cells, additional of exogenous sterols inhibits sterol biosynthesis and will also inhibit the activity of SRE-driven reporter genes (Osborne et al (1985) Cell 42, 203-212).
The regulation of genes encoding enzymes in fungal sterol biosynthesis is less well characterised. No sequence element equivalent to the SRE has been defined. HMG-CoA reductase is subject to feedback regulation by sterol levels in the budding yeast
Saccharomyces cerevisiae
(
S. cerevisiae
), but this regulation occurs at the translational level and is mediated by the 5′ untranslated region of the HMG1 mRNA (Dimster-Denk et al (1994) Mol. Biol. Cell 5, 655-665). Limited studies have been performed which examine the activities of other enzymes in the sterol biosynthetic pathway in the
S. cerevisiae
under conditions which inhibit ergosterol production. Squalene synthase levels are increased 2-3 fold by application of the HMG-CoA reductase inhibitor lovastatin (Robinson et al (1993) Mol. Cell Biol. 13, 2706-2717), and squalene epoxidase activity can increase up to 5 fold in mutant yeast strains containing limited sterol amounts. Messenger RNA levels of lanosterol 14-a-demethylase accumulate during anaerobic growth (Turi and Loper (1992) J. Biol. Chem. 267, 2046-2052); induction of this gene forms the basis for a patent application by Kirsch (EP 0627 491 A1) for a screen for inhibitors of ergosterol biosynthesis. This screen, which also requires additional manipulation of the yeast to reduce flux through the sterol biosynthetic pathway (by deletion of the HMG1 gene), only appears to detect compounds acting on lanosterol 14-a-demethylase or further down the sterol biosynthesis pathway. For example, tolnaftate, an inhibitor of squalene monooxygenase, was not detected in this screen (Kirsch, p. cit.).
We have now devised a assay which is capable of detecting a wide range of inhibitors of sterol biosynthesis. In this assay inhibition of ergosterol synthesis results in the induction of reporter gene activity in a yeast tester strain. The assay is simple, cheap and robust and may be employed in high throughput mode to screen large chemical collections, natural product collections and compound libraries. Sterol biosynthesis inhibitors have a number of applications. As inhibitors of an essential process in eukaryotes they are useful as antifungal drugs and agrochemicals, and may also have applications as biocides and as anti-parasitic agents. In humans, cholesterol may be synthesised de novo or absorbed from the intestine. Inhibition of de novo synthesis reduces lipid levels in the bloodstream, resulting in reduced risk, of atherosclerosis. Sterol biosynthesis provides metabolic intermediates which are used in lipid modification (farnesylation and geranylgeranylation) of proteins. Inhibition of the early stages of sterol biosynthesis may indirectly inhibit lipid modification. This may be of therapeutic application, for example in inhibiting tumour growth driven by activated ras genes.
Therefore in a first aspect of the present invention we provide a method for the identification of agents which modulate sterol biosynthesis which method comprises contacting a test compound with a host cell comprising a DNA sequence which controls expression of a yeast acetoacetyl CoA thiolase (ACoAT) gene operably linked to a reporter system such that modulation of sterol biosythesis in the host cell leads to a detectable change in cell phenotype, and determining whether any such detectable change has occurred.
By “operably linked” we mean linked in such a way as to provide the basic sequence signals necessary for initiation of gene transcription and initiation of gene translation.
By “a DNA sequence which controls expression” we mean a sequence which confers responsiveness of the activity of adjacent genes to change(s) in intracellular metabolic pathways such as sterol biosynthesis. In general such sequences are protein binding sites. Such proteins may regulate events such as transcriptional activation. Their ability to perform this regulation may be influenced by intracellular processes which feedback from sterol biosynthesis.
Acetoacetyl-CoA thiolase (EC 2.3.1.9) catalyses the condensation of two acetyl-CoA molecules to acetoacetyl-CoA. This enzyme precedes HMG-CoA synthase in the sterol biosynthetic pathway. There have been no studies of the regulation of the expression of this gene, and only limited studies on the regulation of the enzyme activity. ACoAT activity can be repressed 12 fold by addition of exogenous sterol (Trocha and Sprinson, Arch. Biochem. Biophys. (1976) 174, 45-51). Servouse and Karst (Biochem. J. (1986) 240, 541-547) provide similar evidence, showing that ergosterol starvation increases ACoAT activity, whereas ergosterol excess reduces ACoAT activity. These studies examined enzyme activities rather than the expression of the ACoAT gene, and thus it is not possible to determine whether the regulation is due to alterations in the enzyme itself, such as covalent modifications, or the binding of a modulator molecule, or whether the level of the protein is regulated through control of gene expression. The ACoAT gene was cloned from the brewing yeast
Saccharomyces uvarum
(Dequin et al (1988) Curr. Genet. 13, 471-478). These authors report that this gene is highly expressed. Furthermore, overexpression of this gene did not lead to an increase in ergosterol production, leading Dequin et al to conclude that ACoAT is not a rate-limiting enzyme in the production of ergosterol. Despite the availability of DNA probes which could be used to measure mRNA levels under different conditions, no studies of the expression of the ACoAT gene in fungi have been conducted under conditions of ergosterol depletion through anaerobic, mutant or inhibitor conditions. We have found that when the promoter region of
S. cerevisiae
acetoacetyl CoA thiolase is linked to a reporter gene the reporter gene may be induced by sterol biosynthesis inhibitors. This assay provides a co
Broad Peter Michael
Dixon Graham Keith
Scanlon David John
Astrazeneca UK Limited
Fish & Richardson P.C.
Prouty Rebecca E.
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