Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1994-05-25
1998-12-29
Saunders, David
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 6, 435 8, C12Q 168, G01N 33566
Patent
active
058540040
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a screening method of determining the modulating effect of substances on a receptor-dependent signal transduction pathway in human or animal cells.
BACKGROUND OF THE INVENTION
Conventional tests for discovering pharmacologically active substances are frequently assays in which a substance is investigated to find out to what extent it is able to displace a (labelled) ligand bound to a receptor (radioligand test). Tests of this kind are only able to identify those substances which influence the binding of known ligand receptor binding sites. Thus, these tests only cover the binding of the substance but not a functional response of the cell and therefore cannot distinguish whether the binding substance has an agonistic or antagonistic activity. For radioligand tests, relatively large amounts of receptors are required and frequently receptor-containing membrane fractions are used which are isolated from animal tissue. These tissues may consist of several types of cells containing different or heterologous receptors. In spite of the great significance of such substrates the heterologous composition or--in the case of investigating drugs for their pharmacological effect in humans--the species difference between humans and animals and the resulting difference in the binding properties of ligands to the human receptor and to animal receptors may give rise to problems in interpreting the results.
Many transmembrane signal transduction systems consist of the following membrane-bound components: a) a cell surface receptor; b) a guanine-nucleotide-binding and GTP-cleaving regulatory protein, which is known as a G-protein and which can be coupled both to the receptor and to its effector; c) a so-called "effector", e.g. an ion channel or adenylate cyclases, guanylate cyclases or phospholipases.
The so-called G-protein-coupled receptors pass on the effects of very different extracellular signals such as light, smells, (peptide) hormones, neurotransmitters, etc.; they have been identified in organisms which are evolutionally as far apart as humans and yeasts (Dohlman et al., 1991). Almost all G-protein-coupled receptors have similarities with one another in their sequence; it is assumed that all are based on a similar topological motif common to them all which consists of seven hydrophobic (possibly .alpha.-helical) sections which penetrate the lipid double layer.
Cell surface receptors recognise the appropriate liqands from a variety of extracellular stimuli. The bonding of the ligand to the receptor activates a signal cascade which begins with the activation of the heterotrimeric G-protein; the activation of the receptor over a lengthy period results in desensitisation which is caused by various modifications of the receptor. The interaction of the G-protein with the activated receptor causes the replacement of guanosindiphosphate (GDP), bound to the .alpha.-subunit, by guanosintriphosphate (GTP), dissociation of the .alpha.-GTP-complex from the .beta.-.gamma.-heterodimer and hydrolysis of GTP into GDP. A single receptor can activate numerous G-protein molecules, thereby intensifying the ligand binding phenomenon. The .alpha.-subunit to which the GTP is bound and the free .beta.-.gamma.-subunit may interact with the effectors, thereby further intensifying the signal by forming so-called "second messengers". Lower molecular second messengers such as CAMP (cyclic AMP), triggered by activation of adenylate cyclase, cGMP (cyclic GMP), triggered by activation of guanylate cyclase, or inositol-1,4,5-triphosphate (IP.sub.3) and diacylglycerols (DAG), triggered by activation of phospholipases, optionally with the participation of hydrolases such as phospholipase C or phospholipase D (Billah et al., 1989), in turn bring about intracellular changes. These include the selective phosphorylation of proteins by activation of protein kinases (e.g. PKC by IP.sub.3 /DAG, PKA by cAMP), influencing the regulation of the transcription of certain genes, reorganisation of the cytosk
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Voraberger
Czernilofsky Armin Peter
Himmler Adolf
Lamche Herbert
Schafer Renate
Stratowa Christian
Boehringer Ingleheim GmbH
Saunders David
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