Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1995-06-05
1998-07-07
Elliott, George C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
43525421, 4353201, 435325, 530350, C12Q 168, C12N 119, C12N 1579, C07K 1400
Patent
active
057766754
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of PCT/GB93/02543, filed Dec. 14, 1993.
The present invention relates to a method for identifying compounds which modulate protein/cell membrane association. The method provides an in vivo assay for inhibitors of protein/cell membrane association wherein modulation of the association leads to a detectable change in cell phenotype. The invention also relates to heterologous proteins, nucleic acid sequences encoding these, corresponding DNA constructs, and recombinant cells, all for use in the above method.
Numerous proteins are modified by the covalent addition of lipids (1). The hydrophobic side-chains the proteins in selected cellular membranes. We refer to this process as membrane association. Inhibition of membrane association of certain lipid-modified proteins is a potential target for therapeutic intervention, since membrane association is usually critical for full biological activity of these proteins. By way of example, inhibition of the membrane association of ras proteins has been proposed as a method for the selective inhibition of oncogenic forms ras (2). Additionally the activity of oncogenic src variants is dependent upon their membrane association through N-myristoylation (3).
Current procedures for identifying inhibitors of lipid modification and membrane association involve screening compounds, such as peptides, against purified samples of individual enzymes involved in the lipid modification process (8). This is time-consuming and expensive since it involves the purification and perhaps cloning of these enzymes. Furthermore, not all steps involved in the membrane association process may be amenable to study in this way. For example, the proposed interaction of lipid-modified proteins with membrane components (9) may be difficult to reproduce in vitro. An alternative approach was reported by Finegold et al (10). Activation of the pheromone response pathway of Saccharomyces cerevisiae (S. cerevisiae) leads to growth arrest. The activity of the pheromone response pathway is dependent upon the integrity of the g subunit of the pheromone receptor-associated G protein. This subunit is isoprenylated. Thus, inhibition of isoprenylation reverses growth arrest in a S. cerevisiae strain in which the pheromone response pathway is active. This provides a growth
o growth assay or inhibitors of isoprenylation. However, this assay is not specific to isoprenylation, since compounds inhibiting the pheromone response pathway at any point will be active.
It has been reported elsewhere that amino acid sequences around the site of lipid modification in some proteins act as membrane association signals (5,6). Hancock et al (5) have demonstrated that fusion of ras membrane association signals to protein A relocates protein A to the inner surface of the plasmamembrane. Similarily Pellman et al (6) showed that fusion of .beta.-globin to a src membrane association signal relocated that fusion to the plasmamembrane. In both cases neither of the fusions conferred a readily measurable phenotype on the cell and the location of the fusion protein could only be determined by time-consuming an labour intensive immunological methods.
We now provide an in vivo assay or inhibitors of protein/cell membrane association wherein modulation of association leads to a detectable change in cell phenotype.
In a first aspect of the present invention we provide a method for identifying compounds which modulate protein/cell membrane association which method comprises contacting a test compound with a cell, having (i) a cell membrane, (ii) a heterologous protein comprising a reporter sequence and a recognition sequence for cell membrane association, (iii) a reporter system which is acted upon by the reporter sequence such that there is a measurable change in cell phenotype upon modulation of protein/cell membrane association by the test compound, and detecting any change in cell phenotype.
The method may be used to identify inhibitors or enhancers of any biological process which leads directly or indirectly to m
REFERENCES:
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Ravanello, et al: "An NH2-terminal peptide from the vaccinia virus L1R protein directs the myristylation and virion envelope localization of a heterologous fusion protein", Journal of Biological Chemistry, vol. 268, No. 10, Apr. 5, 1993, pp. 7585-7593, see the whole document.
Pellman, et al: "An N-terminal peptide from p60src can direct myristylation and plasma membrane localization when fused to heterologous proteins", Nature, vol. 314, Mar. 1985, pp. 374-377, see the whole document.
Resh, et al: "Identification of a 32K plasma membrane protein that binds to the myristylated amino-terminal sequence of p60v-scr", Nature, vol. 346, Jul. 5, 1990, pp. 84-86, see the whole document.
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Elliott George C.
Schwartzman Robert
Zeneca Limited
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