Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-03-15
2001-06-26
Carlson, Karen Cochrane (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S012200, C530S350000
Reexamination Certificate
active
06251853
ABSTRACT:
SUMMARY AND OBJECTS OF THE INVENTION
(i) Field of the Invention
The present invention relates to novel human G proteins, in particular &bgr;3 subunits of G proteins, a process for their preparation and their use in diagnosis and therapy.
(ii) Description of Related Art
Heterotrimeric guanine nucleotide-binding proteins (G proteins) have an outstanding importance in intracellular signal transduction. They mediate the relaying of extracellular signals after stimulation of hormone receptors and other receptors which undergo a conformational change after receptor activation. This leads to activation of G proteins which may subsequently activate or inhibit intracellular effectors (eg. ion channels, enzymes). Heterotrimeric G proteins consist of three subunits, the &agr;, &bgr; and &ggr; subunits. To date, several different &agr; subunits, 5 &bgr; subunits and about 12 &ggr; subunits have been detected by biochemical and molecular biological methods (Birnbaumer, L. and Birnbaumer, M. Signaltransduction by G proteins: 1994 edition. J.Recept.Res. 15:213-252, 1995; Offermanns, S. and Schultz, G. Complex information processing by the transmembrane signaling system involving G proteins. Naunyn Schmiedebergs Arch.Pharmacol. 350:329-338, 1994; Nürnberg, B., Gudermann, T., and Schultz, G. Receptors and G proteins as primary components of transmembrane signal transduction. Part 2. G proteins: structure and function. J.Mol.Med. 3:123-132, 1995; Neer, E. J. Heterotrimeric G proteins: Organizers of Transmembrane Signals. Cell 80:249-257, 1995; Rens-Domiano, S. and Hamm, H. E. Structural and functional relationships of heterotrimeric G-proteins. FASEB J. 9:1059-1066, 1995).
Receptor-mediated activation of certain &agr; subunits can be inhibited by pretreatment with pertussis toxin (PTX). These include, in particular, the &agr; isoforms &agr;i1, &agr;i2 and &agr;i3, and various &agr;o subunits. G Proteins of these types are also referred to as PTX-sensitive G proteins.
&bgr;&ggr; Subunits carry out essential functions in G protein activation and in the modulation of intracellular reactions. All G protein &bgr; subunits disclosed to date have high degrees of homology at the level of the nucleotide sequence and at the level of the amino-acid sequence. Moreover these similarities are found not only within the human &bgr; subunits (&bgr;1, &bgr;2, &bgr;3) but also in comparison with &bgr; subunits of other species, for example fruit fly or yeast. X-Ray structural analyses have been able to determine those amino acids in &agr;, &bgr; and &ggr; subunits which are in contact with one another and are necessary for ordered formation of the heterotrimer.
All G protein &bgr; subunits disclosed to date belong to the WD repeat proteins. The N terminus of the &bgr; subunit interacts predominantly with &ggr; subunits, and the C terminus is involved in the interaction with receptors.
&bgr; Subunits form what are called propeller structures. The &bgr; propellers of the G&bgr; subunits consist of 7 &bgr; propeller blades, each propeller blade consisting of 4 amino-acid regions in antiparallel arrangement. The seven-fold symmetry of the &bgr; propeller can be detected at the level of the amino-acid sequence, which comprises 7 WD repeats. A WD repeat motif comprises about 40 amino acids and has a number of conserved amino acids, including Trp-Asp dipeptide sequences This WD motif frequently terminates the WD repeat (FIG.
1
).
SUMMARY AND OBJECTS OF THE INVENTION
It has now been found, surprisingly, that G protein &bgr;3 subunits which consist only of 6 instead of the 7 WD repeat motifs otherwise described occur, for example, in hypertensives. The cellular activatability of PTX-sensitive G proteins is increased in these hypertensives compared with normotensives.
Molecular analysis revealed a novel amino-acid sequence for the &bgr;3 subunit in these hypertensives, being shorter by 41 amino acids than the known sequence. The sequence is depicted in SEQ ID NO:2. Formally, it is derived from the known human &bgr;3 subunit by deletion of amino acids 167-207.
The corresponding DNA sequence coding therefor is described in SEQ ID NO:1.
The reason for the occurrence of the shortened G&bgr;3 subunit in hypertensives is presumably an alternative splicing of the relevant gene. At the DNA level, there is an intron exactly in front of the putative splicing site. The intron starts behind nucleotide 497 in the open reading site (numbering as in SEQ ID NO;1).
It was also possible by PCR on genomic DNA to detect an intron starting at about nucleotide 620. The shortened form apparently comes about through deletion of a complete exon. The invention furthermore relates to a process for preparing shortened forms of human G&bgr;3 subunits as mentioned above by expression of a nucleic acid sequence coding therefor in a host organism.
The recombinant expression preferably takes place by preparing a gene construct which, besides the coding nucleic acid sequence, also comprises other signal and regulatory sequences such as promoters, terminators, ribosome binding sites, polyadenylation sites and the like. The general procedure for recombinant expression of a gene is familiar to the skilled worker.
The invention furthermore relates to the use of the nucleic acid sequences according to the invention for producing drugs for gene therapy. Introduction of these nucleic acid sequences in direct form or after preparation of an appropriate gene vector into patients' cells is able to achieve an increased activatability of G proteins therein.
This is desirable in a number of disorders in which there is dysregulation associated with G protein.
Diseases associated with G protein dysregulation mean those disorders in which G protein is involved in signal transduction and does not perform its function in a physiological manner.
These disorders include cardiovascular disorders, metabolic disturbances and immunoloigcal disorders.
Cardiovascular disorders which should be mentioned are: hypertension, pregnancy hypertension (gestosis, hypertension in pregnancy), coronary heart disease, localized and/or generalized atherosclerosis, stenoses of blood vessels, restenosis after revascularizing interventions on vessels (eg. PTCA with and without stent implantation), proneness to stroke, thrombophilia and increased platelet aggregation.
Metabolic disturbances which should be mentioned are: metabolic syndrome, insulin resistance and hyperinsulinemia, type II diabetes mellitus, diabetic complications (eg. nephropathy, neuropathy, retinopathy, etc.), lipid metabolism disturbances, disturbances of central chemoreception (CO
2
tolerance, acidosis tolerance, sudden infant death (SIDS)).
Immunologial disorders which should be mentioned are; impaired strength of the body's immune response (formation of immunoglobulins, aggressiveness of T cells and NK cells), impaired general tendency to proliferation, including wound-healing capacity, tendency to tumor development and proliferation including metastatic potential of malignantly transformed cells, duration of the latency period after HIV infection until the disorder becomes clinically apparent, Kaposi sarcoma, tendency to cirrhosis of the liver, transplant tolerance and transplant rejection.
The invention furthermore relates to the use of the nucleic acid sequences according to the invention for the diagnosis of disorders, in particular including determination of the risk of suffering from a disease associated with G protein dysregulation.
Besides determining the risk of certain disorders, it is also possible to make general physiological data and statements through the use according to the invention, for example on central chemoreception, CO
2
tolerance, acidosis tolerance, risk of sudden infant death (SIDS), fitness for certain types of sport.
The invention furthermore relates to the use of nucleic acid sequences which are complementary to the nucleic acid sequences coding for the shortened form of the G&bgr;3 subunit. Sequences of this type can be used as antisense constructs for the treatment or prevention of
Carlson Karen Cochrane
Nixon & Peabody LLP
Safran David S.
LandOfFree
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