Enos mutations used for genetic therapy and therapeutic screening

FIELD: medicine, genetics, biochemistry.

SUBSTANCE: invention relates to new NOS-variants or mutants that comprise structural modifications in site Akt-dependent phosphorylation. Modified NOS-proteins or peptides, in particular, human proteins or eNOS-peptides having change of amino acid residue corresponding to S/T in motif of the consensus-sequence RXRXXS/T of NOS-polypeptide of wild type and nucleic acid molecules encoding thereof can be used in genetic therapy and proteins and NOS-peptides can be used in screening methods of agents modulating activity of NOS. The advantage of invention involves the creature of new NOS-variants or mutants that can be used in genetic therapy.

EFFECT: valuable medicinal properties of mutants.

25 cl, 1 tbl, 9 dwg, 3 ex

 

Cross references to related applications

In the present application claims the priority of Provisional application U.S. No. 60/129550 filed April 16, 1999, which in its entirety is introduced into the present description by reference.

Confirmation of government subsidies

Research for the development of the present invention has been partly funded by the Federal government allocated in accordance with the grant NL 57665 and HL 61371.

The scope to which the invention relates.

The present invention relates to new NOS-variants or mutants, which contain a structural modification in the website Akt-dependent phosphorylation. Modified NOS proteins or peptides and encoding molecules are nucleic acids can be used in gene therapy as agents for the treatment of diseases including restenosis after angioplasty, hypertension, atherosclerosis, heart failure, diabetes, and diseases with defective angiogenesis.

Background of invention

Atherosclerosis and vascular thrombosis is the major cause of morbidity and mortality resulting from a lesion of the coronary artery, myocardial infarction and stroke. Atherosclerosis begins with changes in the endothelium lining the blood vessels. Changes in the endothelium may, ultimately, lead to the development of endothelial lesion, caused partly by the absorption of oxidized cholesterol low-density lipoprotein (LDL). The gap in the area of the lesion may lead to thrombosis and occlusion of the blood vessel. In case of defeat of coronary artery rupture in place of the combined lesion may lead to myocardial infarction and, in the case of the carotid artery can lead to stroke.

In ischemic heart disease, accompanied by atherosclerosis, endothelial dysfunction can lead to decreased production of vasodilator substances such as nitric oxide. Myocardial ischemia occurs when the violation of the self-regulating expansion of vessels regardless of whether it is a consequence krovotecheniyah stenosis of the coronary artery or endothelial dysfunction. In both cases, blood flow can no longer be increased in proportion to emerging needs oxygen. In other situations, myocardial ischemia can occur in the case of a constant oxygen demand, but on the background of the primary reduction of coronary blood flow, mediated by spasm of the coronary artery, the rapid development of atherosclerotic plaques formed on the inner wall of the vessel and causing narrowing of the lumen of the coronary blood vessel, and/or periodic formation of traffic jams in capillariasis platelet aggregation.

Balloon angioplasty is mainly used to open the blood vessel narrowed by plaque. Although in most cases, balloon angioplasty is sufficient for the opening of the vessel, but often this process leads to the removal of endothelium and damage to the vessel. Such damage causes migration and proliferation of smooth muscle cells of the blood vessel at the site of injury with formation of pathology, known as hyperplasia neointima or restenosis. This newly formed pathology leads to recurrent symptoms after three to six months after angioplasty in a significant number of patients.

Atherosclerosis, thrombosis and restenosis is also a violation of the normal function of blood vessels with a tendency rather to their contraction than expansion. Excessive narrowing of the vessel, in addition, leads to a reduction of the vessel lumen, thereby reducing blood flow. This can lead to symptoms such as angina (damage arteries in the heart) or transient ischemia of the brain (i.e. "minor heart attack" when the damaged vessel in the brain). This dysfunction of the blood vessels.(excessive vasoconstriction or inadequate vasodilatation) also occurs in other pathological conditions. Hypertension (high blood pressure) is caused by excessive contraction is of Asadov, as well as thickening of the walls of blood vessels, and in particular the walls of small vessels in the circulatory system. This process can affect the blood vessels of the lungs and may also cause pulmonary (lung) hypertension. Other disorders which are known to be associated with excessive vasoconstriction or with inadequate vasodilatation, are atherosclerosis vessel graft, congestive heart failure, toxemia of pregnancy, the phenomenon, Raynaud's disease, Prinzmetal's angina (coronary vasospasm), cerebral vasospasm, hemolytic uremia and impotence.

The substance released by the endothelium, originally called "endothelial relaxing factor" (EDRF), plays an important role in the inhibition of these pathological processes. It is now known that EDRF is a nitric oxide (NO). NO has many functions in human physiology, including relaxation of vascular smooth muscle, inhibition of platelet aggregation, inhibition of mitogenesis, proliferation of vascular smooth muscle and leukocyte adhesion. Since NO is the most powerful endogenous vasodilator and because he is highly responsible for induced physical stress expansion of the arteries of reserveren, increased NO synthesis should also help improve the ability to perenise the Oia physical activity in normal individuals and in individuals with vascular disease.

Endothelial synthase nitric oxide (eNOS) represents the synthase isoforms of nitric oxide (NOS)is responsible for maintaining systemic blood pressure, vascular remodelling and angiogenesis (Shesely et al., 1996; Huang et al., 1995; Rudic et al., 1998; Murohara et al., 1998). Because the deficit of NO production in the endothelium is sustainable early sign of atherosclerosis and vascular lesions, it was confirmed that eNOS is an attractive object for vascular gene therapy. Although the mechanism of regulation of the activation of eNOS, basically, is not defined, it is known that eNOS fosfauriliruetsa in response to various forms of stimulation of the cells (Michel et al., 1993; Garcia-Cardena et al., 1996; Corson et al., 1996), however, the role of phosphorylation in the regulation of the production of nitric oxide (NO) and which kinase(s) responsible for this is yet unknown.

Brief description of the invention

The present invention is based in part on the discovery of the fact that serine/treningowy protein kinase, Akt (protein kinase B), can directly fosforilirovanii eNOS on serine residue corresponding to residue 1179 in bovine eNOS or residue in 1177 eNOS person, and to activate the enzyme, which leads to production of NO. Mutant eNOS (S1179A or S1177A) is resistant to phosphorylation and activation of Akt and mutant eNOS (S1179D and S1177D) or (S1179E and S1177E) is the con is titative active. In addition, Akt, activated gene transfer mediated by adenovirus, increases basal release of NO from endothelial cells and Akt deficit activation reduces VEGF-stimulated production of NO. Thus, eNOS represents just described substrate for Akt, mediating signaling through Akt with the release of gaseous secondary transmitter NO. The present invention is also based in part on the discovery of the fact that the mutant eNOS (S1179D) is characterized by an increased rate of production of NO and increased reductase activity.

The present invention relates to NOS, polypeptides or proteins and encoding them isolated nucleic acid molecules, where the specified polypeptide or protein NOS contains the replaced amino acid residue corresponding to residue 1179 bovine eNOS, residue 1177 of human eNOS, residue 1412 rat nNOS, or residue 1415 human nNOS. Preferred substitutions are amino acids with negatively charged R groups, including aspartic acid and glutamic acid.

The present invention also relates to the polypeptides or proteins of NOS and coded them isolated nucleic acid molecules, where the specified polypeptide or protein NOS contains the replaced amino acid residue, sootvetstvujushij OST the weave 1179 bovine eNOS, the residue 1177 of human eNOS, residue 1412 rat nNOS, or residue 1415 human nNOS. Preferred substitutions are amino acids with positively charged R groups, such as alanine.

The present invention relates to methods of stimulating the development of collateral vessels in ischemic disease with deficiency of endogenous angiogenesis, and in particular with peripheral vascular disease and/or myocardial ischemia in a patient, where the method includes delivery of a transgene encoding a NOS polypeptide of the present invention or the polypeptide Akt.

The present invention also relates to a transgenic animal, not a person, which expresses the NOS polypeptide of the present invention.

Finally, the present invention relates to a method of identifying an agent that modulates Akt-regulated the activity of NOS, where these methods include General stages: (a) processing the agent purified NOS, preferably eNOS or nNOS, or cells expressing NOS, and preferably eNOS or nNOS, and Akt; and (b) measurement of Akt-regulated activity of NOS, and preferably eNOS or nNOS.

Brief description of the graphical material

Figa-1B. Akt wild type, but not the inactive kinase Akt, increases the release of NO from cells expressing membrane-associated eNOS. figa COS cells were transferrable plasmids for eNOS in the absence or in the presence or inactive Akt kinase Akt (K179M), and production of NO (analyzed as NO2-) were determined using chemiluminescence. On FIGU COS cells were transferrable different NOS-plasmids described above. On figa and figv values for the production of NO2-subtracted from the levels obtained from cells transfected only with cDNA β-galactosidase. The insert illustrates the expression of proteins in total cell lysates. Data represent the average ± srcvar., n=3-7 experiments; * indicates p<0,05.

Figa-2D. Phosphorylation of eNOS active kinase Akt in vitro and in vivo. On figa COS cells were transferrable HA-Akt or HA-Akt(K179M), lysates were subjected to and thus were placed in a kinase reaction in vitro with histone 2B (25 mg) or recombinant eNOS (3 mg)used as substrates. The upper panel shows the inclusion of a32P in the substrate, the bottom panel shows the number of the substrate during the application of the gel Kumasi blue. On FIGU32P-labeled wild-type eNOS or double serine mutant of eNOS (eNOS S635/1179) was subjected to affinity purification from transfected COS cells, and then autoradiography (upper panel) or Western blot analysis (bottom panel). Graphical data on FIGU show the relative amount of labeled protein in relation to the number of immunoreactive eNOS in the gel. On figs labeled eNOS hydrolyzed in three of the Shin and the peptides were separated using RP-HPLC. The upper chromatogram shows the pre-labeled trypticase peptide migrates together with its synthetic phosphopeptide standard (lower chromatogram). Insets show the linear nature of the MC labeled peptide (top) and phosphopeptide standard identical mass ions. On fig.2D recombinant wild-type eNOS or eNOS S1179A was purified and equal amounts (2.4 mg) was added to the kinase reaction in vitro with recombinant Akt, as described in "Methods". On the top panel 2D shows the inclusion of a32P eNOS, the bottom panel shows the number of the substrate during the application of the gel Kumasi blue. Graphic data (n=3) show the relative number of labeled samples in relation to the weight of eNOS (Kumasi) in kinase reaction in vitro.

Figure 3. Illustrates that serine 1179 plays a functionally important role in Akt-stimulated release of NO. The COS cells were transferrable plasmids for wild-type eNOS or eNOS mutants in the absence or in the presence of Akt and determined the protein expression and the level of production of NO (analyzed as NO2-). Interestingly, constructs with a mutation S1179 in not activated Akt, and mutation S1179 in D has led to an increase in function. For And the data represent the average ± srcvar., n=4-7 experiments; * indicates significant differences (p<0,05).

FIGA-4C. Akt regulation is the duty to regulate basal and stimulated production of NO in endothelial cells. On figa BLMVEC were infected adenovirus constructs (β-gal as control, mug Akt and AA-Akt) and determined the amount of NO2-produced for 24 hours (n=3). Insets show the expression of eNOS and Akt. On FIGU lysates adenovirus from infected cells BLMVEC were evaluated on the activity of NOS. Equal amounts of protein (50 mg) were incubated with various concentrations of free calcium and determined the activity of NOS (n=3 experiments). On figs BLMVEC were infected with adenoviruses as described above, followed by stimulation of VEGF (40 ng/ml) for 30 minutes and the release of NO2-quantitatively evaluated using chemiluminescence. Data are presented as VEGF-stimulated release of NO2-after subtraction of basal levels. Data represent the average ± srcvar., n=4 experiments; * indicates significant differences (p<0,05).

Figa and 5B. The purity and the ratio of dimer/monomer eNOS wild type and S1179D eNOS. In a and b analysis by electrophoresis in SDS page with LTOs was performed on a 7.5% polyacrylamide gels, colored Kumasi blue. Left shows the molecular mass standards (lane 1) and their size in kDa. eNOS wild-type (lane 2) and S1179D eNOS (lane 3)(1 μg each) were separated, as shown by the arrows. In proteins (2 μg each) were separated by electrophoresis in SDS page with LTOs at 4°C. the molecular mass standards are shown in lane 1. Untreated samples of wild type and S1179D eNOS were divided into tracks 2 and 3, respectively. On track 4 of eNOS-wild-type boiled in buffer with LTOs for a sample.

Figa and 6B. eNOS S1179D has a higher level of NO production (a) and the reductase activity (B)than wild-type eNOS. For a speed of NO produced from eNOS wild-type (•) and S1179D eNOS (O), were determined using an analysis on the capture of hemoglobin depending on the concentration of L-arginine, and the data is represented by two curves inverse relationship. In tests for DCIP and cytochrome C was carried out in the presence or in the absence Itself. Values are presented as mean ± srcvar., n=4-6 definitions. Similar results were obtained using at least three enzyme preparations. Significant differences (p<0,05) between wild-type eNOS and eNOS S1179D are shown by asterisks.

Figa and 7B. NOS activity (a) and the activity of NADPH-dependent reductase () increases when using eNOS S1179D compared with the wild-type enzyme. Tests for capture of hemoglobin (A) and NADPH-dependent recovery of cytochrome C (b) was performed using as eNOS wild type and S1179D eNOS. And the rate of production of NO was determined in the presence of all cofactors NOS (eNOS wild-type (shaded symbols) and S1179D eNOS (where there's no shading characters). The level of cytochrome C reduction was determined from OUTSTA arginine and VN (a) enzyme wild-type (circles) and for S1179D (triangles) in the absence (where there's no shading symbols) or in the presence of 120 nm of calmoduline (shaded symbols). Values are presented as mean ± srcvar., n=3-6 definition, at least for the three enzyme preparations.

Figa-8D. Calmodulin and Kalnyshevsky activation of NOS and the reductase activity increases slightly for S1179D eNOS. Analysis calmoduline capture of hemoglobin (A) and recovery of cytochrome C (b) was performed using both wild-type eNOS (shaded symbols)and S1179D eNOS (where there's no shading characters). The level of NO production, detektirovanie method of capture of hemoglobin was determined in the presence of all NOS cofactors, whereas the recovery of cytochrome C was determined in the absence of arginine and VN. In C and D are identical experiments were carried out in the presence of increasing concentrations of free calcium. The insets C and D shown Kalnyshevsky cycle of conversion of eNOS wild type and S1179D eNOS as in the analysis on the production of NO, and in the analysis of cytochrome C. the Maximum velocity transformations for eNOS wild type and S1179D was, respectively, And the 22 and 43 min-1; 620 and 1400 min1; From 58 and 100 min1; D 1930 and 3810 min-1. Values are presented as mean ± srcvar., n=3-6 definition, at least for the three enzyme preparations.

Figa and 9B. EGTA-induced inactivation of NOS is reduced eNOS S1179D. Tests for capture of hemoglobin (A) and reductase (B) was carried out as described in the previously with the following modifications. To determine the speed of initiation were monitoring the reaction for 1 minute; and then to the reaction mixture EGTA was added and the reaction rate was monitored for a further 1 minute. The concentration of free calcium in the reaction was 200 μm, and the number of added EGTA gave a final concentration of 0, 200, 400 and 600 μm chelat forming agent. The specific activity was normalized to 100% for eNOS wild type and S1179D. Values are presented as mean ± srcvar., n=3-6 definition, at least for the three enzyme preparations; nd means that the activity for wild-type eNOS was not detected.

Detailed description of the invention And

A. General description

The present invention is based in part on the discovery of the fact that serine/treningowy protein kinase, Akt (protein kinase B), can directly fosforilirovanii eNOS on serine residue (series 1177 in human eNOS) and activate this enzyme, which leads to production of NO, whereas mutant eNOS (S1179A) is resistency to Akt-phosphorylation and activation. In addition, Akt, activated gene transfer mediated by adenovirus, increases basal release of NO from endothelial cells and deficient activation of Akt reduces VEGF-stimulated production of NO. Thus, eNOS is a recently described substrate Akt, TNA is redhouse signaling through Akt with the release of gaseous secondary carrier NO. The present invention is also based in part on the discovery of the fact that the mutant eNOS, for example S1179D, increases the rate of production of NO and increased reductase activity.

The discovery of the fact that the production of NO is regulated by Akt-dependent phosphorylation of eNOS, gives the opportunity to use the new constitutive active eNOS mutants in gene therapy to improve endothelial function in cardiovascular diseases associated with impaired synthesis or biological activity of NO. Such diseases are restenosis after angioplasty, hypertension, atherosclerosis, heart disease, including myocardial infarction, diabetes, and diseases with insufficient angiogenesis. The discovery of this fact also allows you to get a new therapeutic target for the development of the necessary medicines, which can be used to treat diseases associated with impaired synthesis or biological activity of NO.

The present invention also relates to a new constitutive active nNOS-mutants in which the amino acid corresponding to residue 1412 rat nNOS, or 1415 human nNOS, was substituted for the implementation of gene therapy for the treatment of diseases.

C. Specific embodiments of the inventions

Producing the Mut is ntih NOS proteins or polypeptides

The present invention relates to proteins or polypeptides NOS, allelic variants of proteins NOS and conservative substitutions of amino acids NOS proteins, all of which contain a mutation of serine residue present in the website Akt-mediated phosphorylation. So, for example, proteins or polypeptides of the present invention include, but are not limited to (1) human eNOS proteins that contain a mutation at residue 1177 (Janssens et al. (1992) J.Biol.Chem. 267:14519-14522, this work in its entirety is introduced into the present description by reference), as presented in SEQ ID NO: 3 and the corresponding nucleotide sequence represented by SEQ ID NO: 8 (GenBank-room access M), with the substitution of serine for another amino acid such as alanine, and which are resistant to Akt-mediated phosphorylation; (2) bovine eNOS proteins that contain a mutation at residue 1179 (SEQ ID NO:2 of U.S. patent 5498539, which in its entirety is introduced into the present description by reference)as shown in SEQ ID NO:4, with the substitution of serine for another amino acid such as alanine, and which are resistant to Akt-mediated phosphorylation; (3) human nNOS proteins that contain a mutation at residue 1415 with the substitution of serine for another amino acid such as alanine, and which are resistant to Akt-mediated phosphorylation; 4) rat nNOS proteins, which contain a mutation at residue 1412 with the substitution of serine for another amino acid such as alanine, and which are resistant to Akt-mediated phosphorylation; (5) human eNOS proteins that contain a mutation at residue 1177, as presented in SEQ ID NO:5, with the substitution of the serine at amino acid containing negatively charged group R, such as aspartic (as represented in SEQ ID NO:6) or glutamic acid (as represented in SEQ ID NO:7), and which are constitutively active and help to increase the production of NO and the increase in reductase activity; (6) bovine eNOS proteins that contain a mutation at residue 1179 with the replacement of the serine at amino acid containing negatively charged group R, such as aspartic or glutamic acid, and which are constitutively active and help to increase the production of NO and increased reductase activity; (7) human nNOS proteins that contain a mutation at residue 1415 with the replacement of the serine at amino acid containing negatively charged group R, such as aspartic or glutamic acid, and which are constitutively active and help to increase the production of NO and increased reductase activity; (8) rat nNOS proteins that contain a mutation at residue 1412 with the replacement of the serine at amino acid containing OTP is adverse charged group R, such as aspartic or glutamic acid, and which are constitutively active and help to increase the production of NO and increased reductase activity and (9) NOS proteins that are not human, bovine or rat proteins, and modified so that in the position that corresponds serine 1177 of human eNOS, or serine in position 1179 bovine eNOS, serine in position 1412 rat nNOS and serine in position 1415 human nNOS, they contain the amino acid, a non-serine, and which are either resistant to Akt-phosphorylation, or are constitutively active, increase production NO and increased reductase activity. The NOS mutants can be produced by introducing mutations of other amino acids in the motif phosphorylation RXRXXS/T.

The present invention relates to a constitutive active polypeptides NOS, preferably eNOS or nNOS, characterized by excessive production of NO and increased reductase activity, and contains a mutation at serine residue in the website Akt-mediated phosphorylation. It should also be noted that every person is able to obtain conservative options, such as mutants with substitutions, deletion mutants and insertional mutants of these polypetides NOS, with the ability to increased PR is datirovaniyu NO and to increase the reductase activity. Used herein, the term "conservative variant" means such modifications of amino acid sequences that do not have a negative impact on the ability constitutive active NOS, preferably eNOS or nNOS, to produce NO or reductase activity constitutive active NOS, preferably eNOS or nNOS. It is believed that substitution, insertion or deletion has a negative effect on constitutive active polypeptide NOS, if this modified sequence influences the function of constitutive NOS, with the result that he cannot produce elevated levels of NO and not able to increase the reductase activity compared to wild-type NOS. For example, the total charge, structure or hydrophobic/hydrophilic properties of constitutive NOS can be modified without affecting the activity of constitutive NOS. In accordance with this amino acid sequence of the polypeptide NOS can be modified, for example, so that the polypeptide was more hydrophobic or hydrophilic, but that it had no negative impact on the activity of NOS.

Used herein, the term "constitutive active mutant or variant NOS regardless of whether it is modified or isolated from a natural source, means NOS protein, the site is preferably, eNOS or nNOS, which produces NO at a higher level than native NOS containing serine in his nefosfaurilirovanna form at the position of amino acid residue corresponding to residue 1177 in human NOS or residue 1179 in a bullish NOS. Preferred constitutive active variants contain amino acid with a negatively charged group R, such as asparaginova or glutamic acid, the amino acid residue corresponding to serine in position 1177 human cNOS or serine in position 1179 bullish NOS.

The present invention relates to proteins or polypeptides NOS, allelic variants of proteins NOS and NOS proteins with conservative amino acid substitutions, which contain substituted amino acid residue instead of the corresponding residue 1179 in bovine eNOS, residue 1177 in human eNOS, residue 1412 in rat nNOS, and residue 1415 in human nNOS, where specified substituted amino acid residue containing a non-negative charged group R, such as alanine.

Proteins NOS, preferably proteins eNOS or nNOS, the present invention can be used in an isolated form. They say that used here, the protein is isolated, if for separation of this protein from cellular components normally associated with the protein, use physical, mechanical or chemical methods. Each is a specialist can easily use the standard purification methods to obtain an isolated protein.

The present invention also relates to peptides NOS, including the phosphorylation site corresponding to residue 1179 in bovine eNOS, residue 1177 in human eNOS, residue 1412 in rat nNOS, or residue 1415 human nNOS. The peptides may contain serine site of phosphorylation or preferably they may contain a substitution of serine at the position corresponding to residue 1179 in bovine eNOS, residue 1177 in human eNOS, residue 1412 in rat nNOS, or residue 1415 human nNOS. Such substitutions include, but are not limited to, amino acids with a group R, which simulate the serine in its phosphorylated state, such as aspartic acid or glutamic acid. Such substitutions are amino acids with non-negative charged group R, such as alanine. Peptides, including this site, may have a length of approximately 3, 5, 7, 10, 12, 15, 17, 20, 25, 30, 40, 50 or more amino acids.

Proteins, polypeptides or peptides NOS of the present invention can be obtained by any available means, including recombinant expression of NOS cDNA, which was modified with the replacement or modification of the nucleotide triplet encoding a serine corresponding to serine in position 1177 of human eNOS, at position 1179 bovine eNOS, residue 1412 in rat nNOS, or residue 1415 in human nNOS. To introduce mutations in the nucleotide t is ipled, encoding serine residue, may be used any suitable methods, such as th-Molodizhna recombination, site-directed mutagenesis or PCR mutagenesis (see, Sambrook et al.. Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989). In the case of cDNA can be used in human or bovine NOS cDNA, and cDNA encoding proteins NOS other species, including, but not limited to, rabbit, rat, mouse, pig, sheep, horses and primates, non-human.

Used here is a nucleic acid molecule encoding a protein or polypeptide NOS, and preferably a protein or polypeptid eNOS or nNOS, the present invention is called "isolated"if the specified nucleic acid molecule, mostly separated from the impurity nucleic acid encoding other polypeptides, and isolated from this source of nucleic acids.

The present invention also relates to fragments of the coding molecule of nucleic acid. Used herein, the term "fragment encoding nucleic acid molecule" means a small part of the full protein coding sequence. The size of this fragment is determined in accordance with the purposes of its use. For example, if the fragment is chosen so that it encodes an active part of the protein, then this segment should be large enough he to whom irovel functional part (s) of this protein, including the site Akt-phosphorylation. If this fragment is used as a nucleic acid probe or PCR primer, then the length of the fragment should be selected so that was a relatively small number of false-positive events during the sensing/priming in relation to the area that includes or flanks the site of Akt-phosphorylation NOS.

Fragments of the coding nucleic acid molecules of the present invention (i.e., synthetic oligonucleotides)that are used as probes or specific primers for polymerase chain reaction (PCR), or to synthesize gene sequences encoding proteins of the present invention can easily be synthesized by chemical methods, for example phosphocreatine method described by Matteucci and others 1981, SOC. 103:3185-3191), or automatic methods of synthesis. In addition, larger DNA segments can be easily obtained well-known methods, such as synthesis of a group of oligonucleotides that define the various modular segments of the gene, with subsequent legirovaniem oligonucleotides to construct a full modified gene.

Encoding the nucleic acid molecule of the present invention can be also modified so that they contain detektiruya label for diagnostic whole the th and for sensing. A number of such labels known in the art and these labels can be easily used as described here-coding molecules. Suitable labels include, but are not limited to, Biotin, radioactively labeled nucleotides, etc. Every person is able to use any of these famous labels to get labeled an encoding nucleic acid molecule.

The present invention also relates to recombinant DNA molecules (rDNA), which contain NOS-encoding sequence described above. Used here rDNA molecule is a DNA molecule that has been subjected to molecular manipulation. Methods generation of rDNA molecules are well known in the art and described, for example, Sambrook et al., Molecular Cloning (1989). In the preferred rDNA molecules encoding DNA sequence is functionally attached to regulating the expression of the sequences and/or vector sequences.

The choice of vector and/or regulating the expression of the sequences, which is functionally attached to one of the sequences that encode a family of proteins of the present invention, depends directly, as is well known specialists from the desired functional properties, such as expression of protein from the transformed host cell. Vector, rasmar is applied in the present invention, at least capable of directing the replication or integration into the chromosome of the host, and preferably also to regulate the expression of a structural gene included in the rDNA-molecule.

Regulating the expression of the elements used for regulating the expression of functionally attached protein coding sequence, known in the art, and such elements include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, and other regulatory elements. Preferably, the inducible promoter was easily adjustable, so that he was susceptible to nutritional substance present in the environment of the host cell.

In one of the embodiments of the present invention, the vector containing the encoding nucleic acid molecule includes prokarioticheskie the replicon, i.e., the DNA sequence having the ability to direct Autonomous replication and maintain extrachromosomal recombinant DNA molecule in a prokaryotic cell host, such as a bacterial cell host transformed by this molecule. Such replicons known in the art. In addition, vectors, containing a prokaryotic replicon can also include a gene whose expression is reported to him the detected marker, such as resistantanti drug. Typical bacterial genes of resistance to the drug are the genes that tell the resistance to ampicillin or tetracycline.

Vectors containing a prokaryotic replicon can also include a prokaryotic or bacteriotherapy a promoter capable of directing the expression (transcription or translation) of sequences encoding gene in a bacterial cell host, such as E. coli. The promoter is an element regulating expression and formed by the DNA sequence that makes it possible for the binding of RNA polymerase, resulting in the transcription. Promoter sequences compatible with bacterial hosts are typically echodata in plasmid vectors containing the appropriate restriction sites to embed a segment of DNA of the present invention. Usually, such vector plasmids are pUC8, pUC9, pBR322 and pBR329 supplied by Biorad Laboratories (Richmond, CA), pPL and RCK supplied by Pharmacia Piscataway, NJ.

Expressing the vectors compatible with eukaryotic cells, preferably compatible with vertebrate cells, can also be used to obtain rDNA molecules containing the coding sequence. Expressing the vectors in eukaryotic cells are well known in the art and are supplied NESCO is Kimi commercial firms. Typically, such vectors contain appropriate restriction sites to embed the desired segment of DNA. Typical vectors are pSVL and pKSV-10 (Pharmacia), pBPV-l/pML2d (International Biotechnologies, Inc.), pTDT1 (ATCC #31225) and the like eukaryotic expressing vectors.

Expressing the vectors of eukaryotic cells used to construct rDNA molecules of the present invention may, additionally, include breeding marker that is effective in a eukaryotic cell, preferably a marker for selection for resistance to the drug. The preferred marker of resistance to the drug is a gene the expression of which results in resistance to neomycin, i.e. gene neomycin-phosphotransferase (neo) (Southern et al. (1982), J.Mol.Anal.Genet.1:327-341). Alternatively, this breeding marker may be present on a separate plasmid, and these two vectors can be introduced by cotransfection kletki host with subsequent selection on this selective marker by cultivation in medium with the appropriate drug.

The present invention also relates to the cell host transformed or transfected with a nucleic acid molecule that encodes a protein NOS, and protein preferably eNOS or nNOS of the present invention. A host cell can be the ü or prokaryotic, or eukaryotic. Eukaryotic cells used for expression of the protein of the present invention have no particular restrictions, provided that this cell line is compatible with the methods of cultivation and breeding expressing vector and expression of the gene product. Preferred eukaryotic cell hosts include, but are not limited to, yeast cells, insect cells and mammalian cells, preferably vertebrate, such as a mouse, rat, monkey or human cell lines. Preferred eukaryotic cells host cells are Chinese hamster ovary (Cho), deposited in ADS as CCL61, cell mouse embryos NIH Swiss, NIH/3T3, deposited in ADS as CCL 1658, the kidney cells baby hamster (KSS) and cell lines like eukaryotic tissue culture. For expression of the rDNA molecule that encodes a protein of the present invention may be used in any prokaryotic host. Preferred prokaryotic host, especially for constitutively active NOS mutants is E.coli.

Transformation or transfection of appropriate host cells rDNA molecule of the present invention perform well known methods that typically depend on the type of vector and used system the s-master. With regard to transformation of prokaryotic host cells, the commonly used methods of electroporation and salt processing, see, for example, Cohen et al., (1972) Proc. Natl. Acad. Sci. USA 69:2110; and Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). In the case of transformation of vertebrate cells with vectors containing rDNA, are typically used electroporation techniques and processing methods of cationic lipids and salt, see, for example, Graham et al. (1983) Virol. 52:456; Wigler et al., (1979) Proc. Natl. Acad. Sci. USA 76:1373-76. Successfully transformed or transfected cells, i.e. cells that contain the rDNA molecule of the present invention, can be identified by well known techniques, including the selection of breeding marker. For example, cells obtained by introducing rDNA of the present invention can be cloned to obtain single colonies. Cells from those colonies can be harvested, lysed and their DNA content assessed for the presence of rDNA methods, opisannymi Southern et al. (1975), J.Mol.Biol. 98:503 or Berent et al., (1985) Biotech. 3:208, or the proteins produced from these cells can be analyzed immunological method.

The present invention also relates to methods of producing protein NOS, protein preferably eNOS or nNOS protein of the present invention using the nucleic acid molecules described in the present is awke. In General, the production of recombinant forms of the protein typically involves the following stages. First get a nucleic acid molecule that encodes a protein of the present invention. If the coding sequence is not interrupted by introns, it is suitable for expression in any host. Then, the nucleic acid molecule preferably functionally attached to suitable regulatory sequences, as described above, with the formation of expressing an element containing an open reading frame for a given protein. This expressing the element used for the transformation or transfection of a suitable host and the transformed or transfected host is subjected to cultivation under conditions suitable for producing the recombinant protein. This recombinant protein can be, but not necessarily isolated from the medium or from the cells; and, in some cases, where the valid presence of some impurities, isolation and purification of the protein is optional.

Each of the above stages can be implemented in different ways. For example, the desired coding sequences can be obtained from genomic fragments and used directly in suitable hosts. The construct expressing the vector is in, which operate in different hosts, is performed with the use of appropriate replicons and regulatory sequences mentioned above. Specific regulatory sequence for expressing the vectors and methods of transformation or transfection, depending on the type of host cell used for expression of this gene, and discussed in detail above. Suitable restriction sites, if they are not initially present in the sequence, can be added to the ends of the coding sequence with getting cut out of the gene to be embedded in the data vectors. Each specialist can easily adapt any known system owner/expressing vector for manipulation of the nucleic acid molecules of the present invention for producing the recombinant protein.

Gene therapy

The present invention encompasses any suitable delivery system genes, combined with a suitable expression system gene, using the most appropriate methods of delivery. For example, the mutant NOS genes or variants, preferably a variant or mutant genes eNOS or nNOS present invention or Akt genes, can be travelrobe into the cells of the heart or skeletal muscles, including the heart myocytes and myocytes of skeletal muscle in vitro or in vivo for direct selling is tiravanija of the encoded protein. Particularly effective are the genes Akt person and NOS mutants, preferably eNOS person containing amino acid with a negatively charged group R, such as aspartic or glutamic acid at the position corresponding serine in 1177 eNOS person. Introduction methods mutant or variant NOS genes include, but are not limited to, intravascular, intramuscular, intraperitoneal, intradermal or intra-arterial injection.

The delivery system gene using adenovirus has several advantages: adenovirus (i) allows you to enter a relatively large insert DNA; (ii) can grow to high titer; (iii) capable of infecting mammalian cells with a wide range; and (iv) can be used with a large number of available vectors containing different promoters. In addition, because adenoviruses are stable in the bloodstream, they can be entered by intravenous injection. A preferred vector for delivery is the human adenovirus 5 defective in helper-independent replication, although there are and can be used and other ways of delivery, including direct delivery of nucleic acids directly into the desired cell (see Sawa et al. (1998) Gene Ther. 5 (11): 1472-80; Labhasetwar et al. (1998) J. Pharm. Sci. 87 (11): 1347-50; Lin et al. (1997) Hypertension 30:307-313; Chen et al. (1997) Circ.Res. 80(3):327-335; Channon et al. (1996) Cardiovasc. Res. 32:962-972; HarvHeart. Lett. (1999)9(8):5-6; and Nabel et al.(1999) Nat.Med. 5(2):141-2.

Using system adenovirus 5 frequency transfection of more than 60% have been demonstrated in myocardial cells in vivo by a single intracoronary injection (Giordano & Hammond (1994) Clin. Res. 42:123A). Dereplication recombi-Nannie adenoviral vectors are particularly effective at transfection coronary endothelium and cardiac myocytes and provide a highly efficient transfection after intracoronary injection. Dereplication recombinant adenoviral vectors are also effective for transfection of the desired cells of peripheral vascular system (see U.S. patent 5792453, which in its entirety are introduced in the present description by reference).

Adenoviral vectors used in the present invention, can be konstruirovanie method “rescue” recombination described by Graham et al. (1988) Virology 163:614-617. Briefly, the transgene eNOS clone in a Shuttle vector that contains a promoter, polylinker and partial flanking adenoviral sequences that were delegated genes EA/EV. Examples Shuttle vector may be a plasmid pAC1 (Virology 163:614-617, 1988) (or its equivalent), which encodes fragments of the left end of the genome of a human adenovirus 5 (Virology 163:614-617, 1988) without early protein coding sequence is th EA and EB, which play an important role in virus replication, and plasmid ACCMVPLPA (J.Bio.Chem. 267:25129-25134, 1992), which contains polylinker, the CMV promoter and SV40 polyadenylation signal, flanked incomplete adenoviral sequences from which they were delegated genes EA/EV. The use of plasmid PAC1 or ACCMVPLPA facilitates the cloning process. Then, a Shuttle vector cotransfected with a plasmid containing the entire genome of a human adenovirus 5, which is too large for inkapsulirovanie in 293 cells. Cotransfected can be carried out by precipitation of calcium phosphate or lipofectin (Biotechniques, 15:868-872, 1993). Plasmid JM17 encodes the complete genome of a human adenovirus 5 plus fragments of the vector pBR322, including the gene for resistance to ampicillin (4,3 TPN). Although JM17 adenovirus encodes all the proteins necessary for obtaining Mature viral particles, it is too large for inkapsulirovanie (40 TPN compared with 36 TPN for wild type). In a small sub-cotransfection cells, rescue recombination between transgenderism Shuttle vector, such as plasmid RAS, and the plasmid having the complete genome of adenovirus 5, such as a plasmid pJM17, yields a recombinant genome, which is scarce on sequences EA/EV and which contains and representing the interest of the transgene, but again in the recombination process, he loses an additional sequence such as the sequence of pBR322, and therefore it becomes small enough to be encapsulated. Regulated by the CMV promoter and encoding beta-galactosidase adenovirus HCMVSP1 lacZ (Clin.Res. 42:123F, 1994) can be used to assess the efficiency of gene transfer by processing X-gal.

In another embodiment of the invention the gene encoding NOS, preferably eNOS or nNOS, can be introduced in vivo by attenuating or defective DNA virus, non-limiting examples of which are herpes simplex virus (HSV), papillomavirus, Epstein-Barr (EBV), adenovirus and adeno-associated virus (AAV). Preferred are defective viruses, which entirely or almost entirely lack viral genes. Defective virus after its introduction into the cell is non-communicable. The use of defective viral vectors allows to introduce them into cells in a specific local area, not fearing that this vector can infect other cells. Thus, this vector can be specifically targeted to a specific area, for example in the brain or spine. In a specific embodiment, the invention can be used defective herpes virus 1 (HSV1) (Kaplitt et al., (1991) Molec.Cel. Neurosci. 2:320-330). In another embodiment the invention, the viral vector is a vector-based attenuating of adenovirus, such as the vector described Statford-Perricaudet et al. (J.Clin.Invest.90:626-630 (1992)). In another embodiment of the invention the specified vector is a vector on the basis of a defective adeno-associated virus (Samulski et al. (1987) J. Virol. 61:3096-3101; Samulski et al. (1989) J.Virol. 63:3822-3828).

The present invention also covers the use of directed delivery into the cell not only by delivery of the transgene into the coronary artery or the femoral artery, but also through the use of tissue-specific promoters. For example, when attaching a tissue-specific transcriptional regulatory sequences of the light chain 2 myosin left ventricle (MLC[2V]) or the heavy chain of myosin (MHC) to the transgene, such as NOS genes of the present invention in adenoviral constructs, expression of the transgene is limited to the myocytes of the ventricles of the heart. The efficiency of gene expression and the degree of specificity provided by the promoters MLC[2V] and MTL with lacZ, were determined using recombinant adenoviral system of the present invention. Expression specific to the heart, was described previously by Lee et al. (J. Biol.Chem. 267:15875-15885 (1992)). The MLC promoter[2V] consists of 250 TPN and easily fits under limited what I packaging of adenovirus 5. The promoter of the heavy chain of myosin, a well-known strong promoter transcription is a suitable alternative promoter that is specific for the heart, and is less than 300 BP are also Suitable promoters cells of smooth muscles, such as alpha-SM22 promoter (Kemp et al., (1995) Biochem. J.310 (Pt 3):1037-43) and the promoter of alpha-SM actin (Shimizu et al. (1995) J. Biol. Chem. 270 (13):7631-43). Other promoters such as the promoter, troponin-C, despite the high efficiency and quite small, do not have adequate tissue specificity. When using promoters MLC[2V] or MNF and delivery of the transgene in vivo it is obvious that only one cardiac miocic (i.e. without concomitant expression in endothelial cells, smooth muscle cells and fibroblasts of the heart) can provide an adequate expression of NOS protein.

The restriction of expression of the myocytes of the heart also has advantages from the point of view of applicability of gene transfer for the treatment of clinical myocardial ischemia. When restricting the expression of the heart area, you can avoid possible undesirable effects of angiogenesis in neserbeznyh tissues such as the retina. In addition, all of the cells cardiac myocytes is likely to provide the long-lasting transgene expression, since these cells do not undergo rapid update; and so the expression is not what should be reduced by division and cell death, as it usually happens in the case of endothelial cells. Endothelialization promoters are already in rasporyajenii for these purposes. Examples endothelialization promoters are the promoter of Tie-2 (Schlaeger et al. (1997) Proc.Natl. Acad. Sci. 1:94 (7): 3058-63), endothelin promoter (Lee et al. (1990) J.Bio.Chem. 265:10446-10450) and the eNOS promoter (Zhang et al. (1995) J.Bio.Chem. 270 (25):15320-6).

In the case of treatment of heart disease the present invention relates to the delivery of the heart vector at high titer by intracoronary or intramuscular injection, preferably by transfection of cells of all types. Diseases such as erectile dysfunction and cardiovascular disease, including myocardial infarction, myocardial ischemia, heart failure, restenosis, stenostoma, restenosis after angioplasty and damage when necessary vascular bypass surgery, can be treated with the use of transgenes NOS, preferably transgenic eNOS or nNOS.

Suitable recombinant vectors can be purified by the method of plaques in accordance with the standard method. Derived viral vectors are propagated in 293 cells, which provide functions EA and EB in trans to titles, preferably in the range of approximately 1010-1012viral particles/ml Cells can infect up to 80% of confluently and collect within 48 hours. After the wire is placed 3 cycles of freezing-thawing cell debris precipitated by centrifugation and the virus purified by ultracentrifugation in a CsCl gradient (preferably by ultracentrifugation twice in the gradient CsCl). Before in vivo injection, viral source drugs absoluut by gel filtration on a column of Sephadex such as Sephadex G25. Then the product was filtered through a 30 micron filter, which reduces the side effects of intracoronary injection of unfiltered virus (life-threatening irregular heart rhythm) and to ensure effective gene transfer. The initial preparation of the virus has the final titer in the range of 1010-1012viral particles/ml Recombinant adenovirus must have a high degree of purification without contamination by wild-type virus (potentially replicative virus). Contaminated structures can cause intense immune response in an animal host. On this basis, to remove impurities and wild-type virus can be implemented propagation and purification of the obtained virus, for example, by identifying the desired recombinants by PCR using appropriate primers, by performing two cycles of purification method plaques and by ultracentrifugation twice in CsCl gradient. In addition, problems associated with the occurrence of arrhythmias induced by the introduction of adenoviral vector to the patient, can be solved by filtering recombinant adenovirus through the filter of appropriate size held up nutricional the second injection. This strategy also allows to significantly increase the efficiency of gene transfer and expression.

The original preparation of virus can be obtained in the form of injectable preparation containing pharmaceutically acceptable carrier, such as saline, if necessary. The final title in this vector at the specified drug for injection is preferably in the range of about 107-1013viral particles and contributes to efficient gene transfer. Other pharmaceutical carriers, composition and doses described below. Adenoviral transgene constructs are delivered to the myocardium by direct injection through the coronary artery (or vascular graft) by standard methods using percutaneous catheter under fluoroscopic control in a quantity sufficient for expression of the transgene to the extent that enables highly effective therapy. This injection can be done deeply (about 1 cm) into the lumen of the coronary artery (or vessel-transplant), and preferably it can be done in both the coronary artery, as the development of collateral blood vessels in a high degree varies in each individual patient. When injecting this material directly into the lumen of the coronary artery with coronary ka is etherow the delivery of the desired gene can be quite efficient and can minimize the loss of recombinant vectors in the proximal aorta during injection. It is known that when delivered in this way, the gene expression in hepatocytes does not occur, and the viral RNA is not detected in the urine at any time after intracoronary injection. In the present invention can be used, for example, any type of coronary perfusion catheter or catheter Stack. In addition, for gene transfer NOS, and preferably eNOS or nNOS, in the artery wall can be used and other methods known in the art.

For the treatment of peripheral vascular disease, namely diseases characterized by insufficient blood supply to the legs, recombinant adenovirus expressing NOS, and preferably a peptide or protein eNOS or nNOS present invention can be delivered using a catheter inserted into the proximal part of the femoral artery or arteries, resulting in efficient gene transfer into cells of skeletal muscles along with the blood flow coming from the femoral artery.

In cases where the transgene, or a nucleic acid encoding a NOS, and preferably eNOS or nNOS, or Akt protein of the present invention are first transferred into the cells of the endothelium or vascular smooth muscle in vitro, including the patient's own cells, DNA can be transfected directly into cells (see, U.S. patent 5658565). Mainly for transfection cleto the target plasmid vector, containing a DNA sequence encoding Akt and NOS of the present invention, or biologically active fragment can be used in mediated liposomes transfection of target cells. The stability of liposomes in combination with a hermetic nature of these vesicles makes them valuable vesicles for delivery of therapeutic DNA sequences (see review Mannino &Gould-Forgerite (1988) BioTechniques 6 (7):682-690). It is known that liposomes are absorbed by the cells of many types by merging. In one of the embodiments of the invention can be used cationic liposomes containing cationic derivatives of cholesterol, such as SF-chol or DC-chol. Molecule DC-chol contains a tertiary amino group, spacer elements stem of medium length and carbamoyl linker bond, as described Gao &Huang (Biochem. Biophys. Res. Comm. 179:280-285, 1991).

In another embodiment of the invention relating to the use of liposomal technology, viral or non-viral vector containing a DNA sequence encoding a biologically active fragment of the NOS protein, preferably a protein fragment of eNOS or nNOS, delivered to the target cell by transfection with lipofectamine (Bethesda Research Laboratory). Lipofectamine is a liposomal composition comprising in a ratio of 3:1 from poly-lipid 2,3-dialerace-N-[2-(sperminated)ethyl]-N,N-dimethyl-1-Pro is onlinetreatment (DOPSA) and neutral lipid valeriebertinellibio (DOPE).

Other non-viral methods of gene delivery include, but are not limited to, (a) direct infection "bare" (deproteinizing) DNA; (b) transfection of cells, mediated by calcium phosphate [CA3(RHO4)2]; (C) transfection of cells of the host mammal by electroporation; (d) DEAE-dextroposition transfection of cells; (e) indirect polybrene shipping; (f) fusion of protoplasts; (g) microinjection; (h) transformation mediated polylysine, with subsequent transfer of genetically modified cells back to the mammalian host.

The production of transgenic animals

The present invention also relates to transgenic animals containing mutant gene NOS, and preferably a mutant gene for eNOS or nNOS, described in this application. Transgenic animals are genetically modified animals, which was experimentally introduced recombinant, exogenous or cloned genetic material. This genetic material is often called a "transgene". The nucleic acid sequence of the transgene in the case of formation of NOS can be integrated either in the locus of the genome, where this particular sequence of nucleic acid is not usually present, either in the normal locus for the transgene. This transgene can comp the better their sequences of nucleic acids, derived from the genome of the same species or from species different from the species of the animal target.

The term "transgenic animal embryonic cell line" means a transgenic animal whose germ cell line was introduced genetic modification or genetic information, resulting in this transgenic animal was given the ability to transfer genetic information to the offspring. If such offspring really has some or all of these changes, or genetic information, it is also transgenic animals.

Change or genetic information may be alien to this animal belongs to the recipient, alien only for the specific recipient, or it can represent the genetic information already present in the recipient. In the latter case, modified or introduced gene may be expressed differently than the native gene.

Transgenic animals can be produced by various methods, including transfection, electroporation, microinjection, gene delivery in embryonic stem cells and infection with recombinant virus and a retrovirus (see, for example, U.S. patent No. 4736866; U.S. patent No. 5602307; Mullins et al. (1993) Hypertension 22 (4):630-633; Brenin et al. (1977) Surg. Oncol. 6(2)99-110; Tuan (ed), Recombiant Gene Expression Protocols, Methods in Molecular Biology No. 62, Humana Press (1977)).

Was produced a number of recombinant or transgenic mice, including mice that Express activated oncogenic sequences (U.S. patent No. 4736866); which Express T-antigen of simian SV40 (U.S. patent No. 5728915); have no expression of interferon regulatory factor 1 (IRF-1) (U.S. patent No. 5731490); which is detected dopaminergic dysfunction (U.S. patent No. 5723719); which Express at least one human gene implicated in the regulation of blood pressure (U.S. patent No. 5731489), who find greater similarity to the symptoms, usually present in Alzheimer's disease, occurring in natural conditions (U.S. patent No. 5720936); which have a lower potential for mediating cell adhesion (U.S. patent No. 5602307); which have the gene for bovine growth hormone (Clutter et al. (1996) Genetics 143 (4):1753-1760); or which have the ability to generate full humoral response to the introduction of human antibodies (McCarthy (1997) The Lancet 349(9049): 405).

Although mice and rats are a preferred animals for most experiments using transgenes, however, in some cases it is preferable or even necessary to use alternative kinds of animals. In addition to mice ways using transgenes were successful is used on other animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and Guinea pigs (see, for example, Kim et al. (1997) Mol.Reprod. Dev. 46(4):515-526; Houdebine (1995) Reprod. Nutr. Dev. 35 (6):609-617; Petters (1994) Reprod. Pertil. Dev. 6(5):643-645; Schnieke et al. (1997) Science 278 (5346):2130-2133; and Amoah (1977) J.Animal Science 75 (2):578-585).

The method of introduction of nucleic acid fragments in recombinant competent mammalian cells may be any method that favors cotransformation many molecules of nucleic acid. Detailed description of the methods of producing transgenic animals is easily accessible to every expert, including descriptions in U.S. patent No. 5489743 and in U.S. patent No. 5602307. In addition, well-developed method of production of NOS-transgenic animals. So, for example, were produced transgenic mice that Express inducible or sverkhekspressiya wild-type eNOS (see Ohashi et al. (1998) J.Clin. Invest. 102 (12):2061-71; and Drurnmond. et al. (1988) J.Clin.Invest. 102 (12):2033-4). These methods can be used for producing transgenic mice that Express mutant NOS of the present invention.

Analyses of therapeutic screening

The discovery of the fact that the phosphorylation of eNOS regulates its activity, allows you to develop screening tests for the identification of agents that modulate Akt-regulated activity or the expression of NOS, and preferably eNOS and nNOS. Can be used in any suitable format, including analysis of the in vivo transgenic animals, the analysis of proteins in vitro analyses of cell cultures and highly effective format.

In many programs, screening drugs, which are tested libraries of compounds, it is desirable to carry out high-efficiency analyses to maximize the number of compounds tested in a given period of time. The analyses that are carried out in cell-free systems, which can be carried out with purified or ProcessName proteins, can be often preferred as "primary" screening, which can be developed for rapid detection and relatively easy detection of modification in molecular targets, mediated by the test compound. In addition, the effects of cellular toxicity and/or bioavailability of the test compound can mostly not be taken note of in the in vitro system, because the analysis focuses mainly on the assessment of the impact of drug on the molecular target that can be manifested in the inhibition of, for example, binding between molecules.

Analysis based on cell or tissue culture, can be carried out, for example, by seeding cells CS-7 (100 mm-Cup) and transfection of these cells with plasmids for NOS (7,-30 mg) and Akt (1 mg) using calcium phosphate. To balance all transpency can be cotransfection expressing the vector for cDNA β-galactosidase. Twenty-four to forty-eight hours after transfection the expression of corresponding proteins (40-80 mg) can be confirmed by the analysis method, the Western blot using antibodies (mAb) to eNOS (9D10, Zymed), mAb to ON (SA, Boehringer Mannheiem), polyclonal antibodies (b) to iNOS (Zymed Laboratories) or mAb to nNOS (Zymed Laboratories).

Twenty-four to forty-eight hours after transfection the medium process for measuring the level of nitrite (NO2-), a stable decomposition product of NO in aqueous solution by NO-specific chemiluminescence as described Sessa et al., 1995. The environment is subjected to deproteinization and samples containing NO2-, is refluxed in glacial acetic acid containing sodium iodide. Under these conditions, NO2-quantitatively recovered to NO, the amount of which is estimated at chemiluminescent detector after reaction with ozone in NO analyzer (Sievers, Boulders, CO). In all experiments, the controls can be obtained by inhibiting the release of NO2-using an inhibitor of NOS. In addition, the release of NO2-from cells transfected with cDNA β-galactosidase can be deducted to account for background levels NO 2 -present in the serum or in the environment. Accumulation of cGMP in COS cells can also be used in bioanalysis on the production of NO, as has already been described. In alternative format conversion3H-L-arginine3H-L-citrulline can be used to determine NOS activity in the lysates of COS cells or endothelial cells, as described previously (Garcia-Cardena et al., 1998).

For studies of phosphorylation in vivo COS cells can be transfected with a cDNA of wild-type or S635 (control), bovine eNOS 1179A, D or E, human eNOS 1177D or E, rat nNOS 1412D or E, human nNOS 1415D or E, and HA-Akt during the night. 36 hours after transfection, the cells are placed in enriched cialisbuynow serum and containing no phosphate minimal supportive environment Dulbecco, to which was added 80 µci/ml32P-phosphoric acid, for 3 hours. The cell sample can be pretreated with wortmannin (500 nm) does not contain phosphate medium for 1 hour before and during tagging. Then the lysates collected, NOS solubilizers and partially purified using affinity chromato-graphy on ADP-sepharose, as described earlier, and after electrophoresis in LTO-page (7.5 percent) enable32P in NOS visualize through autoradiography, and the number of NOS protein is confirmed by analysis by the method of Western blot on OS.

For studies of phosphorylation in vitro recombinant NOS, purified from E.coli, eNOS, purified from another source, or peptides NOS, including the site of Akt-phosphorylation, incubated with Akt wild-type or inactive kinase Akt, immunoprecipitation from transfected COS cells. Briefly, proteins or peptides NOS incubated with32P-g-ATP (2 ml, specific activity 3000 CI/mmol), ATP (50 mm), DTT (1 mm) in buffer containing HEPES (20 mm, pH 7.4), MnCl2(10 mm), MgCl2(10 mm) and immunoprecipitated Akt, for 20 minutes at room temperature.

In experiments evaluating the phosphorylation in vitro NOS wild-type and mutant recombinant NOS Akt (1 mg), purified from infected with baculovirus SF9 cells, incubated with wild-type NOS, bovine eNOS S1179A, human eNOS S1177A, bovine eNOS S1179D or E, human eNOS S1177D or E, rat nNOS S1412D or E, or human nNOS S1415D or E, using basically the same conditions that were described above. Proteins can be separated by electrophoresis in LTO-SDS page, and the inclusion of32P and the amount of protein determined by staining Kumasi, as described above.

The above screening tests that allow you to analyze Akt-dependent phosphorylation or activation of NOS, and preferably eNOS or nNOS, can be used for screening agents a wide range. So, e.g. the, agents that inhibit the dephosphorylation of NOS inhibitors phosphatase) at the amino acid corresponding to serine 1179 in bovine eNOS, residue 1177 in human eNOS, residue 1412 in rat nNOS, or residue 1415 in human nNOS, can be used as therapeutic molecules. Similarly, agents that activate Akt or imitate the website Akt-phosphorylation on eNOS, can be used as therapeutic molecules.

Agents that have been analyzed above methods may be arbitrarily selected, or directionally selected or designed. Used herein, an agent is randomly selected when the agent was randomly selected without considering the specific sequences involved in the binding of the protein of the present invention, taken separately or with its associated substrates, with its binding partners etc. Example of a random selection of agents is the use of a chemical library or a combinatorial peptide library, or media for culturing of the organism.

Used herein, the agent is directionally selected or designed, if this agent was not chosen arbitrarily, and taking into account the sequence of the target site and/or its conformation in connection with the action of this agent. So, for example, directed the selected peptide agent can be a peptide, amino acid sequence which is similar to the website Akt-phosphorylation in NOS, and in particular peptides or small molecules that mimic the state of phosphorylation of NOS.

Agents of the present invention can be, for example, peptides, small molecules, derivatives of vitamins as well as carbohydrates. For any specialist it is obvious that the structural nature of the agents of the present invention does not have any restrictions.

Peptide agents of the present invention can be obtained by standard methods of solid-phase (or synthesis in solution synthesis of peptides known in the art. In addition, DNA encoding these peptides may be synthesized using commercially available equipment for synthesis of oligonucleotides and produced recombinant methods using standard systems for production of recombinant DNA. The production methods of solid-phase peptide synthesis is necessary in the case when you want to turn on not encoded by the genome data of amino acids.

Another class of agents of the present invention are antibody having immunoreactivity with the main provisions in the proteins of the present invention. Agents in the form of antibodies obtained by immunization of suitable mammalian peptides containing antigenic region, i.e. h is STI proteins, which are aimed at these antibodies.

The use of agents identified as agents modulating the activity of eNOS

The agents of the present invention, such as agents that inhibit the dephosphorylation of NOS inhibitors phosphatase) at the amino acid corresponding to serine 1179 in bovine eNOS, residue 1177 in human eNOS, residue 1412 in rat nNOS, or residue 1415 in human nNOS, as well as agents that activate Akt or imitating the website Akt-phosphorylation on NOS, can be introduced parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, intradermally or transbukkalno. Alternative or simultaneous introduction can be carried out orally. Enter the dose will depend on the age, health and weight of the recipient, the type of concurrent treatment, if performed, frequency of administration and the desired effect. As described below, there are many methods that can be easily adapted for the introduction of these agents.

The present invention also relates to compositions containing one or more agents of the present invention. Although the input number can vary for each individual, however, each specialist can determine the optimal ranges of effective amounts of each component. The usual dose is 0.1-100 mg/kg of weight t is La. The preferred dose is 0.1-10 mg/kg of body weight. The most preferred dose is 0.1-1 mg/kg of body weight.

In addition farmacologicas active agent compositions of the present invention may contain suitable pharmaceutically acceptable carriers, including excipients and additives, which facilitate processing of the active data connection with obtaining drugs that may be pharmaceutically acceptable for delivery to the desired site. Suitable compositions for parenteral administration are aqueous solutions of the active compounds in water-soluble form, for example in the form of water-soluble salts. In addition, can be entered suspensions of the active compounds as appropriate oily suspensions for injection. Suitable lipophilic solvents or carriers are fatty oils, for example sesame oil, or synthetic esters of fatty acids, for example etiloleat or triglycerides. Aqueous suspension for injection may contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. This suspension may also, but not necessarily, contain stabilizers. To encapsulate the agent for delivery into the cell can also be used liposomes.

The pharmaceutical is Skye composition for systemic injections of the present invention can be prepared for intracolonic, parenteral or topical administration. In fact, all these three types of compositions can be used simultaneously to achieve systemic injections of the active ingredient.

Suitable compositions for oral administration are hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and their forms of controlled release.

When implementing the methods of the present invention compounds of the present invention can be used separately or in combination, or in combination with other therapeutic or diagnostic agents. In some preferred versions of the invention, the compounds of the present invention can be introduced along with other compounds typically prescribed for these conditions according to generally accepted medical practice, such as anticoagulant agents, thrombolytic agents, or other antithrombotic agents, including platelet aggregation inhibitors, tissue plasminogen activators, urokinase, PUK, streptokinase, heparin, aspirin and warfarin. Compounds of the present invention can be used in vivo typically mammalian, such as human, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.

The following working examples, in particular, illustrate preferred embodiments of the present invention and should not be construed as limiting the description of the invention. Other common configurations will be obvious to every specialist.

Examples

In examples 1-12 were carried out following the procedure.

Transfection of cells: cDNA bovine eNOS, human iNOS, rat nNOS in pcDNA3 and-labeled wild-type Akt, Akt (K179M) or myr-Akt in pCMV6 generated standard cloning methods. myr-nNOS in pcDNA3 was generated by PCR with the inclusion of new aminobenzo that contains a consensus site N-monitorowania eNOS (MGNLKSVG, SEQ ID N0:l), connected with the preservation of the reading frame to a second amino acid nNOS-coding sequence. In preliminary experiments in COS cells, the structure was subjected to N-monitorowania by including3H-myristic acid, whereas native nNOS was not subjected to such monitorirovania, which received approximately 60% of the total protein targeting to the membrane fraction of cells, whereas only 5-10% of nNOS were membrane-associated in COS cells. Mutation of the alleged sites of Akt-phosphorylation in eNOS was generated using the set for siteprovides Quick Change mutagenesis (Stratagene) according to the accordance with the manufacturers ' instructions. All mutants were confirmed by DNA sequencing. Cells COS-7 were sown (100 mm-Cup) and transferrable plasmids NOS (7.5 to 30 mg) and Akt (1 mg) plasmids using calcium phosphate. To balance all transpency was used expressing the vector for cDNA β-galactosidase. Twenty-four to forty-eight hours after transpency the expression of corresponding proteins (40-80 mg) was confirmed by the analysis method Western blotting using antibodies (mAb) to eNOS (9D10, Zymed), mAb to ON (SA, Boehringer Mannheiem), polyclonal antibodies (pAb) to iNOS (Zymed Laboratories) or mAb against nNOS (Zymed Laboratories).

The release of NO from transfection cells COS: 24-48 hours after transpency environment were processed for measurement of nitrite (N02-), a stable decomposition product of NO in aqueous solution, using an NO-specific chemiluminescence as described Sessa et al., 1995. Environment subjected deproteinization and samples containing NO2-that was boiled under reflux in glacial acetic acid containing sodium iodide. Under these conditions, NO2-quantitatively recovered to NO, the amount of which was estimated at chemiluminescent detector after reaction with ozone in NO analyzer (Sievers, Boulders, WITH). In all experiments, the release of NO2-inhibited by the NOS inhibitor. Cu is IU the release of NO 2-from cells transfected with cDNA β-galactosidase, subtracted to account for background levels of NO2-present in the serum or in the environment. In some experiments, the accumulation of cGMP in COS cells was used in bioanalysis on the production of NO, as has already been described.

Tests for activity NS: Transformation3H-L-arginine3H-L-citrulline was used to determine NOS activity in the lysates of COS cells or endothelial cells, as described previously (Garcia-Cardena et al., 1998).

Studies of phosphorylation in vivo and in vitro: studies of phosphorylation in vivo COS cells can be transfected with a cDNA of wild-type or S635, eNOS 1179A and HA-Akt during the night. 36 hours after transfection, cells were placed in enriched cialisbuynow serum and containing no phosphate minimal supportive environment Dulbecco, to which was added 80 µci/ml32P-phosphoric acid, for 3 hours. Some cells were pre-treated with wortmannin (500 nm) does not contain phosphate medium for 1 hour before and during tagging. Then the lysates were collected, NOS was solubilizers and partially purified using affinity chromatography on ADP-sepharose, as described earlier, and after electrophoresis in LTO-page (7.5 percent) enable32R NOS visualized by autoradiography, and the number of white is and eNOS was confirmed by analysis using Western blotting on eNOS. For studies of phosphorylation in vitro of recombinant eNOS, isolated from E. coli were incubated with Akt wild-type or inactive kinase Akt, immunoprecipitation from transfected COS cells. samples were incubated with32P-g-ATP (2 ml, specific activity 3000 CI/mmol), ATP (50 mm), DTT (1 mm) in buffer containing HEPES (20 mm, pH 7.4), MnCl2(10 mm), MgCl2(10 mm), immunoprobe targeted Akt, for 20 minutes at room temperature.

In experiments to assess the phosphorylation of eNOS in vitro wild-type and mutant eNOS recombinant Akt (1 mg), purified from infected with baculovirus SF9 cells, were incubated with wild-type NOS or eNOS S1179A (2.4 mg, purified from E. coli) using basically the same conditions that were described above. Proteins were separated by electrophoresis on LTO-SDS page, and the inclusion of32P and the amount of protein was determined by staining Kumasi, as described above.

In studies on the identification of labeled peptide eNOS immunoprecipitated Akt was incubated with recombinant eNOS, as described above. The sample was subjected to electrophoresis in LTO-SDS page, and the band with eNOS hydrolyzed in the gel and the obtained trypticase fragments were purified using RP-HPLC. Then monitored the mass of the peptide and the inclusion of32P and noticeable labeled peak then analyzed using mass spectrometry. In other experiments, p is tidy, the appropriate possible customers Akt-phosphorylation, synthesized, purified by HPLC and confirmed by mass spectrometry (W.M. Keck Biotechnology Resource Center, Yale University School of Medicine). The wild-type peptide represented 1174RIRTQSFSLQERHLRGAVPWA1194 (SEQ ID NO:2), and the mutant peptide was identical except that S1179 was replaced by alanine. In kinase reactions in vitro, carried out mainly as described above, were incubated with peptides (25 mg) with recombinant Akt (1 mg). Then the products of the reactions caused spots on phosphocellulose filters and the number of incorporated phosphate was measured on the Cherenkov counter.

Infection with adenovirus and the release of NO in endothelial cells: Microvascular endothelial cells bovine lung (BLMVEC) were cultured in 100 mm dishes (for analyses on the basal release of NO and activity of NOS)or in 96-well plates (for stimulated release of NO), as described previously (Garcia-Cardena et al., 1996a). BLMVEC were infected with adenovirus at a multiplicity of infection of 200 containing β-galactosidase 29, labeled inactive by phosphorylation mutant of Akt (AA-Akt; Alessi et al., 1996) or carboxy-terminal-tagged constitutively active Akt (myr-Akt) for 4 hours. The virus was removed and cells were left to recover for 18 hours in complete medium. In preliminary experiments using virus containing the th β -galactosidase, these conditions were optimal for the infection 100% crops. To measure the production of basal levels of NO in the medium was collected 24 hours after the initial virus infection. To measure the stimulated release of NO cells were washed in serum-free medium and then stimulated with VEGF (40 ng/ml) for 30 minutes. In some experiments, 24 hours after infection with adenovirus was determined by the degree of dependence NOS from calcium. Infected cells were literally in the buffer for the analysis of NOS containing 1% NP40, and soluble in the detergent material is used for the activity measurement. Lysates were incubated with EGTA-buffered calcium with obtaining the appropriate quantity of free calcium in the incubation.

Statistics: Data were expressed as mean + srcpath. The comparison was performed using a bilateral student test or analysis of variance (ANOVA) using the criterion of post-hoc (lat. - "after that"), if necessary. Differences were considered significant at p<0,05.

Example 1: Akt modulates the production of NO from eNOS

To investigate the possibility that the inhibitory effector kinase PI-3, Akt, can directly affect the production of NO, cells COS-7 (which does not Express NOS) has been cotransfection with eNOS and Akt wild-type (HA-Akt), or inactive kinase Akt (Ha-Akt K79M), and the accumulation of nitrite (NO2-) was measured by NO-specific chemiluminescence. Transfection of eNOS increased accumulation of NO2-that is , the action is noticeably increased when cotransfection with wild-type Akt, but not with the inactive option kinase (figa). Identical results were obtained using cGMP as a biological analysis of biologically active NO. In these experimental conditions Akt was catalytically active, as determined using the assays, Western blotting using phospho-Akt-specific antibody (AB)(which recognizes serine 473; data not shown) and analysis on the activity of Akt (see, figa). Transfection of constitutively active form of Akt (myr-Akt) has led to an increase in cGMP accumulation (reviewed in COS cells) from 5.5±of 0.8 to 11.6±0.9 pmol cGMP/mg protein in cells transfected with one of eNOS or eNOS with myr-Akt, respectively), whereas the inactive kinase Akt did not have any impact on the accumulation of cGMP (5,8±0.8 pmol cGMP, n=4 experiments). As shown in the insert in the lysates of COS cells expressibility equal levels of eNOS and Akt, which allows to make the assumption that Akt modulates eNOS and thereby increases the production of NO in basal conditions.

eNOS is a doubly acylated, periperi the mini-membrane protein, which is sent to the Golgi apparatus and plasma membrane of endothelial cells (Liu et al., 1997; Garcia-Cardena et al., 1996a; Shaal et al., 1996), and compartmentalization necessary for the efficient production of NO in response to agonist stimulation (Sessa et al., 1995; Liu et al., 1996; Kantor et al., 1996). In order to assess whether membrana compartmentalisation to activate eNOS via Akt, cells COS-7 cotranslational cDNA for Akt and quantitatively assessed monitorowanie, palmitoylation defective mutant of eNOS (G2A eNOS and NO release. As can be seen from FIGU, Akt activates reallymoving form of eNOS, suggesting that the functional interaction of both proteins is required for their compartmentalization into the membrane (Downward et al., 1998). Then it was determined whether Akt to activate structurally similar, but differ soluble isoforms of NOS, neuronal and inducible NOS (nNOS and iNOS, respectively). Cotransfected Akt forms nNOS and iNOS did not lead to further increase in the level of NO release, demonstrating the specificity of Akt compared to eNOS. However, the addition of N-monitorowania to nNOS in order to strengthen its interaction with biological membranes, resulted in stimulation of nNOS with Akt in the same type as was observed in eNOS, which suggests that both isoforms can be receptive to the mi to activation of the kinase Akt when zakalivanie in the membrane.

Example 2: Production of mutations eNOS

The above experiments suggest that Akt, probably through phosphorylation of eNOS can modulate NO release from intact cells. Indeed, the two alleged motive Akt-phosphorylation (RXRXXS/T) present in eNOS (serine 635 and 1179 in bovine eNOS or serine and 633 1177 in human eNOS), and one motive is present in nNOS (serine 1412 in rat nNOS and 1415 in the human nNOS), and obvious motives were not detected in iNOS. In order to determine whether eNOS potential substrate for Akt-phosphorylation in vitro, COS cells were transferrable HA-Akt or HA-Akt (K179M), and kinase activity was assessed using recombinant eNOS as a substrate. As seen on figa, active kinase phosphorylates histone 2A and eNOS (69±2.9 and 115,4±3.8 pmol ATP/nmol of substrate, respectively, n=3), whereas inactive Akt does not contribute to a significant increase in phosphorylation of histone or eNOS. To determine whether the alleged sites of Akt-phosphorylation in eNOS is responsible for the inclusion of32P, two serine were motivovany to be replaced with alanine residues, and the ability of Akt to stimulate eNOS phosphorylation of wild type and mutant was assessed in intact cells of COS. Transfected cells were labeled32P-orthophosphate, eNOS was partially purified using affin the Oh chromatography on ADP-sepharose and quantitatively evaluated the state of phosphorylation and protein level. As can be seen from FIGU, coexpressed with Akt leads to a 2-fold increase in eNOS phosphorylation compared with restimulating cells. Pre-treatment of eNOS/Akt-transfected cells wotmania leads to the abolition of Akt-induced increase in phosphorylation. In addition, mutation of Surinov 635 and 1179 their replacement by alanine residues abolishes Akt-dependent eNOS phosphorylation, which suggests that these residues may serve as a potential Sagami phosphorylation in intact cells.

To identify residues directly phosphorylated Akt, eNOS wild-type cells were then incubated with immunodeciency Akt, and phosphorylation sites were determined by HPLC with subsequent MALDi-mass spectrometry (MALDi-MC). As can be seen from figs previously32P-labeled trypticase phosphopeptide was elyuirovaniya together with synthetic phosphopeptides (amino acids 1177-1185 with phosphoserine in position 1179) and had identiry mass ions, as determined by linear MILLISECONDS. Using monitoring by MALDi-MC reflective type as labeled trypticase peptide and standard phosphopeptide has detected a loss of HP3O4that pointed to the fact that trypticase peptide was phosphorylated. In addition, mutation S1179 replace it with And led to the reduction of the level of Akt-dependent phospho what helirovanie eNOS compared with the wild-type protein (fig.2D). Identical results were obtained using peptides (amino acids 1174-1194)occurring protein from wild-type or eNOS S1179A, as substrates for recombinant Akt (wild-type peptide was included 24,6±3.7 nmol phosphate/mg compared to alanine mutant peptide, which included 0,22+0.02 nmol phosphate/mg; n=5). Overall, these data suggest that eNOS is a substrate for Akt and that the main phosphorylation site is serine 1179 (serine 1177 in human eNOS).

Then, the authors present invention was evaluated the functional significance of the proposed site Akt-phosphorylation in serine 635 and identified site serine 1179. Transfection of COS cells double mutant eNOS S635/1179A reverses Akt-dependent release of NO. Mutation of serine 635 and replaced with alanine does not reduce the release of NO, whereas eNOS S1179A reverses Akt-dependent activation of eNOS (figure 3). These results suggest that the series 1179 plays a functionally important role for the release of NO. Mutation of serine 1179 in aspartic acid (eNOS S1177D) to replace the negative charge provided by adding phosphate, partially mimics the activation state induced Akt (S1177D in eNOS person). All the mutants obtained by site-directed mutagenesis, expressibility in a high degree (see box Western blots) and the protected catalytic NOS activity in cell lysates (in COS cells, transfected only eNOS, NOS activity was 85,3±27,0, 71,9±2,9, 80,8±23,2 and 131,8±to 36.7 pmol generated L-citrulline/mg protein from lysates of COS cells expressing wild type eNOS S1179A, S635, A and S1179D, respectively, n=3 experiments).

In order to determine mediates whether Akt release of NO from endothelial cells, the endothelial cells of the capillary vessels bovine lung (BLMVEC) were infected with adenoviruses expressing activated Akt (myr-Akt), defective in the activation of Akt (AA-Akt) or β-galactosidase as a control, we measured the accumulation of NO. As can be seen from tiga, myr-Akt stimulates basal production of NO from BLMVEC, while cells infected β-galactosidase or defective in activation of Akt, was released low levels of NO, which was approaching the limits of detection. These data, together with similar results obtained for COS cells, suggest that the phosphorylation of eNOS via Akt is sufficient to regulate the production of NO in the residual levels of calcium. Indeed, the NOS activity measured in lysates from myr-Akt-positive BLMVEC demonstrated that the sensitivity of this enzyme to activation by calcium, analyzed at a fixed concentration of calmoduline, is higher relative to the activity of NOS, nab is demoi in BLMVEC, infected with the indicated virus containing β-galactosidase (pigv). It is interesting to note that the sensitivity to calcium NOS activity in cells infected with deficient activation of Akt, was largely suppressed compared with cells infected with myr-Akt and β-galactosidase.

It is known that VEGF-treatment of endothelial cells promotes activation of Akt 23 and the release of NO through a mechanism partially blocked by inhibitors of PI kinase-3 (Papapetropoulos et al., 1997). To determine the functional relationship between VEGF as agonist release of NO and Akt activation BLMVEC were infected with adenoviruses for myr-Akt, AA-Akt or β-galactosidase, and conducted a quantitative assessment of VEGF-stimulated release of NO. As can be seen from figs, infection of endothelial cells by enzyme myr-Akt leads to increased VEGF-regulated NO production, whereas AA-Akt inhibits the release of NO. These results suggest that Akt is involved in the communication signal, necessary for both basal and stimulated production of NO in endothelial cells.

Overall, these data demonstrated that Akt can fosforilirovanii eNOS on serine 1179 (serine 1177 human eNOS) and that this phosphorylation enhances the ability of this enzyme to generate NO.

Example 3: Materials and m is methods

eNOS Constructs and protein purification - Bovine eNOS wild-type plasmid pCW expressed with groELS in the cells of BL21 E. coli as described previously (Martasek et al., 1996). Mutant eNOS S1179D for expression in E. coli was generated as follows. eNOS S1179D in pcDNAS (Fulton et al., 1999) was digested with Xhol/XbaI, was subcloned into the same sites of eNOS in pCW and coexpression with groELS. Selection of recombinant eNOS was performed as described previously (Roman et al., 1995; Martasek et al., 1999) with the following modifications. eNOS was suirable with 2',5'-D-sepharose or 10 mm NADPH, 10 mm 2'AMR. The number of samples was estimated using the maximum optical density at 409-412 nm, with an extinction coefficient for the heme content of 0.1 μm-1cm-1. The purity of the samples was determined by electrophoresis in 7.5% of the LTO-SDS page followed by staining of Kumasi. Electrophoresis in SDS page with LTOs at low temperature was carried out similarly except that the samples are not boiled, and the electrophoresis was carried out at 4°suspension of ice water (Klatt et al., 1995). In experiments in which the cofactors NOS (L-arginine, calmodulin and NADPH) was subjected to titration, cleaning and storing is not performed, and the incubation was carried out as described below.

Analysis on the activity of NOS - Production of NO was determined by analysis on the capture of hemoglobin (Kelm et al., 1988). Briefly, the reaction mixture contained eNOS (0.5-2.5 m in the g), oxyhemoglobin (8 μm), L-arginine (100 μm), VN (5 μm), CaCl2(120 mm), calmodulin (120-200 nm) and NADPH (100 μm) in HEPES buffer (50 mm), pH 7.4. When determining the value of EC50for calcium in the case of eNOS to the above reaction mixture is modified as follows: MOPS buffer (10 mm, pH 7,6), KCl (100 mm) and Myself (250 nm) were replaced. Under these conditions the free calcium was calculated using the program WinMAXC version 1.8 (Stanford University) with Kd=2,2×10-8M. Exact concentration of free calcium was achieved by mixing appropriate proportions of initial solutions in 10 mm K2EGTA and 10 mm CaEGTA (Molecular Probes). The NOS activity was traced to a linear dependence for 2 minutes at 401 nm, and the production of NO was calculated on the basis of optical density using an extinction coefficient of 60 mm-1cm-1. All reactions were carried out at 23°and each value was represented by data from 3-8 experiments. To determine the concentration califoolya was used extinction coefficient 0,0033 μm-1cm-1at 276 nm. In this method, the production of NO is completely blocked by the addition of nitro-L-arginine (1 mm). When inactivation of eNOS was determined by adding to the reaction mixture EGTA (200-800 μm)for 1 minute after initiation of the reaction with NADPH was added to the chelator. Identical conditions were used for p and evaluating the reductase activity of NADPH-cytochrome C. These reaction mixture contained Himself (120 nm) and CaCl2(200 μm) in 0.5-ml volume with eNOS (0/5 µg).

The conversion of L-arginine to L-citrulline was analyzed as described previously Bredt and others (1990). Briefly, samples (0.25 to 2 μg) were incubated for 3-10 minutes at 23°With the following reaction mixture: 3 pmol L-[3H]arginine (55 CI/mmol), 10-300 μm arginine, 1 mm NADPH, 120-200 nm of calmoduline, 2 mm CaCl2and 30 μm VN in a final reaction volume of 50-100 μl. The reaction was completed by addition of 0.5 ml of 20 mm HEPES, pH 5.5, containing 2 mm EGTA and EDTA. The reaction mixture was applied onto the resin Dowex AG50WX8, and passing the stream were recorded on a liquid scintillation analyzer, Packard 1500.

Analysis on the reductase activity is the Reductase activity of NADPH-cytochrome C and recovering 2,6-dichlorophenolindophenol (DCIP) was defined as the change in optical density at 550 nm, as described previously Martasek et al. (1999) and Masters et al. (1967), using the extinction coefficient 0,021 μm-1cm-1as for cytochrome C and DCIP. Briefly, the reaction mixture (1 ml) contained either cytochrome C (90 μm); DCIP (36 μm), HEPES buffer (50 mm), pH of 7.6, NaCl (250 mm), NADPH (100 μm), calmodulin (120 nm) and CaCl2(200 μm), or other substances, as indicated. After adding eNOS monitored response within 60 seconds (at 23°). If inactivation of the reductase activity was determined by addition of EGTA (200-800 μm), then th is ez 1 minute after initiation of the reaction was added to the chelator and monitored the reaction for another 1 minutes The reaction mixture contained HEPES buffer (50 mm), pH 7,6; Himself (120 nm) and CaCl2(200 μm) and the reaction was initiated with NADPH (100 μm). In the experiments, which were evaluated by EGTA-inactivation of eNOS, to simulate the conditions used in the experiments on the trapping of hemoglobin, NaCl was added. Adding NOS inhibitors did not affect the rate of cytochrome C reduction (data not given). Definition EC50for calcium in the case of eNOS was determined as described above in the analysis to capture the hemoglobin.

Data analysis and statistics All data are expressed as mean ± srcvar. Determination was carried out at least three replicates with at least three different batches of enzymes for each data series. Enzyme wild-type and mutant enzymes were purified at the same time to control variations in the activity of various drugs. Statistical significance was determined using t-test, t-test and differences were considered significant at p<0,05.

Results

Expression and purification of eNOS - eNOS wild type and S1179D eNOS expressed and isolated from E. coli. From 1.6 liters of culture were usually allocated approximately 2.5-4.0 mg samples using chromatography on 2'5'-ADP-sepharose 4B. As can be seen from tiga, both enzymes had a purity >90%, defined on the basis of staining Kumasi. These results are typical, as can be seen from seven independent the preparations for wild-type eNOS, and S1179D eNOS received sequentially. Both enzymes were present mainly in their dimeric form, as was shown by electrophoresis in SDS page with LTOs (pigv).

eN05 S1179D is a big NO-sitesnow and the reductase activity

than wild-type eNOS - Then eNOS activity of wild type and S1179D eNOS was compared by measuring the rate of production of NO. eNOS S1179D found a higher number of turns (under optimal conditions) compared with the wild-type enzyme (84±6 compared to 27±1 min-1n=6 individual and pairwise enzyme products). The Km values for drugs with L-arginine were similar to values obtained for drugs eNOS wild type and S1179D eNOS (figa, 1,8 compared with 2.5 μm, respectively; see table 1).

Table 1

Kinetic parameters for wild-type eNOS and eNOS S1179D
Substrate/CofactorAnalysisDetermination of the catalytic activityWild-typeS1179D
ArginineCapture hemoglobinKcat27±1 min-184±6 min-1
  Km 2.5 μm
 Capture hemoglobinKcat17±1 min-153±3 min-1
  Km8 mcm36 microns
 Cytochrome CKcat+CaM460±18 min-1840+59 min-1
  Kcat-CaM70±5 min-1290±9 min-1
  Km+Itself0,75 µm1,9 µm
NADPH -CaM0.40 micronof 2.0 μm
 L-citrullineKcat22±2 min-143±2 min-1
  EC508 nm7 nm
 Cytochrome CTocat620±78 min-11140±75 min-1
Himself  EU5013 nm21 nm
 Capture hemoglobinKcat58±1 min-1100±3 min-1
 (100 mm KCl)EC50310 nm250 nm
 Cytochrome C (100 mm KCl)Kcat1909±33 min-13798±54 min-1
Ca2+EC50290 nm220 nm

Since the rate of flow of electrons from the reductase domain in oxygenase domain is essential for NOS catalysis, we estimated the increased activity of eNOS S1179D in order to determine whether this increase is attributed to the increase in reductase activity. When assessing recovery as DCIP, and cytochrome C was observed a significant increase in activity S1179D compared with the activity of wild-type eNOS (pigv). In addition, this increase was paid in the presence of Himself, which contributed to the increase in the total activity of both enzymes. If S1179D basal recovery of cytochrome C in the absence Itself was 4 times more than in the case of wild-type eNOS. The value Itself is stimulated recovery C is tohama with was higher for S1179D eNOS (749± 35 and 1272±55 min-1for the wild type and S1179D eNOS, respectively, n=3-5); however, 8x-stimulation with Himself observed for wild-type eNOS compared with 3 stimulation for S1179D eNOS.

Then determined, produces whether eNOS S1179D more superoxide, which should restore cytochrome C, compared with wild-type eNOS. As expected, any inhibiting superoxide-dismutase cytochrome C reduction was not observed (as an indicator of the generation of superoxide anion in the absence of Himself as that previously reported for wild-type eNOS (86±6, compared with 95±8 min-1and for S1179D eNOS (278+9 compared to 288±7 min-1; n=3-5). However, in the presence of the superoxide dismutase reduces the level of cytochrome C reduction as in the case of wild-type eNOS (610±51 compared to 866±8 min-1), and in the case of eNOS S1179D (1179±43 compared to 1518±19 min-1). The relative reduction in the activity of cytochrome C in the presence of superoxide dismutase was similar for both enzymes (30% for wild-type eNOS and 22% for S1179D eNOS), suggesting that increased reductase activity of eNOS S1179D compared with the activity of wild-type eNOS (as shown by the analysis on the recovery of cytochrome C) is not caused by the lack of binding of the enzyme.

NADPH-dependent formation of NO and the reductase act is vnesti not differ between wild-type eNOS and eNOS S1179D - Since the binding site with NADPH is located in the immediate vicinity of Ser-1179 in eNOS, was evaluated NADPH-dependent production of NO and recovery of cytochrome C. eNOS S1179D has a higher maximum speed (Kcat)than wild-type eNOS, which is indicated by the production of NO, is analyzed in the presence of Himself (figa). The increase in Kcatassociated with 4-fold increase in the Km for NADPH in the case of eNOS S1179D compared with wild-type eNOS (36 and 8 μm, respectively). Tocatfor NADPH-dependent cytochrome C reduction in the absence of Himself above for eNOS S1179D than for wild-type eNOS (290 and 70 min-1respectively; figv). In the case of cytochrome C reduction in the presence of Himself To acatsignificantly higher for S1179D eNOS compared with wild-type eNOS (840 460 min-1respectively). The Km values for NADPH was not changed depending on the absence or presence Itself (and 0.40 and 0.75 μm for wild-type eNOS and 2.0 and 1.9 for S1179D eNOS in otsutstvie and in the presence, respectively).

Value EC50for he Himself remained unchanged for eNOS wild type and S1179D eNOS in order to determine cause increased activity of eNOS S1179D changes in the affinity of the enzyme towards Himself, the kinetics of NOS activity and cytochrome C reduction was analyzed in the presence of all NOS cofactors in excessive quantities depending the barb on the concentration Itself. Kcatfor Myself-activation activity of NOS was 22 min-1for wild-type eNOS and 43 min-1for S1179D eNOS. The conversion of these data, normalized for differences in Kcatrevealed a small shift in the curve to the left for S1179D eNOS and small differences in the values EC50for Himself, which is consistent with the previously available data on phospho-eNOS (Mitchell et al., 1999). Value EC50was 8 nm for wild-type and 7 nm for S1179D eNOS (figa). Was measured NADPH-mediated recovery of cytochrome C. Tocatto activate using the cytochrome C reduction was about 2 times higher for S1179D eNOS compared with the wild-type enzyme (1140 and 620 min-1for S1179D eNOS and eNOS wild type, respectively). Conversion of the data, normalized for differences in Kcatrevealed small differences in the values EC50for Himself in the case of eNOS wild type and S1179D eNOS (21 I comparison with 13 nm; figv).

Comparison of activation with calcium and inactivation of eNOS In the previous experiments, it was demonstrated that "the apparent sensitivity to calcium eNOS was increased in cells expressing either a large number of phospho-eNOS or eNOS S1179D that gave reason to assume that phosphorylation leads to a change in the affinity of the activation of calcium/Cam (Dimmeler et al., 1999; Fulton et al., 1999). As can be seen from figs, after normaliza the AI for differences in maksimalno activity dependence on calcium were slightly increased for S1179D eNOS (p< of 0.05, two-way analysis of variance). Size EU50for calcium in the case of eNOS wild type and S1179D eNOS also slightly differed (310 and 250 nm of calcium, respectively), as determined by production of NO (in the presence of 250 nm Itself). As can be seen from the insert in figs, increasing the concentration of free calcium actually leads to increased turnover of eNOS S1179D more largely, than this can be observed in the case of the wild-type enzyme. In addition, the values of EC50for calcium, based on the recovery of cytochrome C, were similar to values obtained when measuring the production of NO (fig.8D; 290 and 220 nm for eNOS wild type and S1179D eNOS, respectively). And in this case, the value of Vmax for calliphoridae activation of turnover eNOS S1179D exceeded this value for the wild-type enzyme (Fig.4D, insert).

To determine whether inactivation of eNOS S1179D from inactivation of the wild-type enzyme, was measured reduction in eNOS activity after the formation of the chelate complex of calcium with EGTA. In these experiments, all NOS cofactors were added in the presence of calcium (200 μm) and monitoring the production of NO was performed for 1 minute, after which were added various concentrations of EGTA and again monitored for 1 min As can be seen from tiga, adding EGTA to the enzyme wild type and S1179D eNOS resulted in the reduction of the structure of production of NO-dependent concentration. However, the production of NO from eNOS S1179D appeared to be less sensitive to the addition of EGTA; that is, the activity of wild-type eNOS was decreased much faster at low concentrations of EGTA than the activity of eNOS S1179D. The greatest differences in the activities of the enzymes was observed at a concentration of EGTA 400 μm. In addition, at 600 μm EGTA any activity for wild-type eNOS was not observed, but all the same was observed residual activity for S1179D eNOS. Similar results were obtained in analyses of the recovery of cytochrome C (Pigv), with significant differences in activity was observed when added to the reaction 400 and 600 μm chelator. However, the greatest concentration of EGTA residual reductase activity was detected for wild-type eNOS and eNOS S1179D.

In conclusion we can say that bovine endothelial synthase nitric oxide (eNOS) fosfauriliruetsa directly the protein kinase Akt by serine 1179 (Fulton et al., 1999), and human endothelial synthase nitric oxide (eNOS) fosfauriliruetsa directly by the protein kinase Akt by serine 1177 (Dimmeler et al., 1999). Mutation of residue 1179 in bovine eNOS and replace with negatively charged aspartate constitutive increases the production of nitric oxide (NO) in the absence of agonist stimulation.

It should be noted that vicepresidencia discussion and examples represent only a detailed description is of some preferred embodiments of the invention. For each specialist obvious that can be used in various modifications and equivalents that does not extend, however, beyond the scope and essence of the invention. All references, articles and patents mentioned above or below, in its entirety introduced into the present description by reference.

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1. An isolated nucleic acid molecule encoding a mutant form of the polypeptide synthetase of nitric oxide (NOS) wild-type, containing a substitution of amino acid residue corresponding to S/T motif consensus sequences RXRXXS/T polypeptide NOS wild-type, where this replacement leads to the Constitution is utive active forms of NOS, which demonstrates the increased production of NO and increased reductase activity compared with the NOS polypeptide of the wild type.

2. An isolated nucleic acid molecule according to claim 1, where the specified mutant form contains the replaced amino acid residue corresponding to residue 1179 bovine eNOS, residue 1177 of human eNOS, residue 1412 rat nNOS, or residue 1415 human nNOS.

3. An isolated nucleic acid molecule according to claim 2, where the specified substituted amino acid residue corresponding to residue 1179 bovine eNOS, residue 1177 of human eNOS, residue 1412 rat nNOS, or residue 1415 human nNOS, simulates phosphoserine.

4. An isolated nucleic acid molecule according to claim 2, where the serine corresponding to residue 1179 bovine eNOS, residue 1177 of human eNOS, residue 1412 rat nNOS, or residue 1415 human nNOS, replaced by a residue of aspartic acid or glutamic acid residue.

5. An isolated nucleic acid molecule according to claim 2, where the specified substituted amino acid contains a group R, which mimics the phosphate group.

6. The polypeptide that is encoded by an isolated nucleic acid molecule according to any one of claims 1 to 5.

7. The mutant form of the polypeptide NOS wild type containing a substitution of amino acid residue corresponding to S/T motif consensus-serial is a major RXRXXS/T polypeptide NOS wild-type, where this substitution leads to the emergence of constitutively active forms of NOS, which demonstrates the increased production of NO and increased reductase activity compared with the NOS polypeptide of the wild type.

8. The mutant form of the polypeptide NOS wild-type according to claim 7, which contains a substituted amino acid residue corresponding to residue 1179 bovine eNOS, residue 1177 of human eNOS, residue 1412 rat nNOS, or residue 1415 human nNOS.

9. The mutant form of eNOS of claim 8, where these substituted amino acids have been replaced by the residue of aspartic acid or glutamic acid residue.

10. An isolated nucleic acid molecule encoding a mutant form of the polypeptide NOS wild type selected from the group consisting of bovine eNOS, which contains the substituted amino acid residue corresponding to residue 1177; human eNOS, which contains the substituted amino acid residue corresponding to residue 1179; rat nNOS, which contains the substituted amino acid residue corresponding to residue 1412; and human nNOS, which contains the substituted amino acid residue corresponding to residue 1415, where specified substituted amino acid residue containing a non-negative charged group R.

11. An isolated nucleic acid molecule of claim 10, where the specified replaced amino the PCI-e slot residue is alanine.

12. The mutant form of the polypeptide NOS wild type selected from the group consisting of bovine eNOS, which contains the substituted amino acid residue corresponding to residue 1177; human eNOS, which contains the substituted amino acid residue corresponding to residue 1179; rat nNOS, which contains the substituted amino acid residue corresponding to residue 1412; and human nNOS, which contains the substituted amino acid residue corresponding to residue 1415, where specified substituted amino acid residue containing a non-negative charged group R.

13. The mutant form of the polypeptide NOS wild type indicated in paragraph 12, where the specified substituted amino acid residue is alanine.

14. A method of identifying an agent that modulates Akt-regulated the activity of NOS, including stage

(a) exposing NOS-expressing cells by the agent and

(b) measuring Akt-dependent NOS activity by determining whether the residue corresponding to S/T motif consensus sequences RXRXXS/T polypeptide NOS, phosphorylated or mimic phosphorylation state.

15. The method according to 14, where stage (b) includes determining the state of phosphorylation of NOS amino acid residue corresponding to residue 1179 bovine eNOS, residue 1177 of human eNOS, residue 1412 rat nNOS or OST the ku 1415 human nNOS.

16. The method according to clause 15, where NOS is activated by the specified agent.

17. The method according to clause 16, where the specified agent simulates Akt-mediated phosphorylation of eNOS.

18. The method according to 17, where the specified agent inhibits the dephosphorylation of amino acid residue corresponding to residue 1179 bovine eNOS, residue 1177 of human eNOS, residue 1412 rat nNOS, or residue 1415 human nNOS.

19. The method according to 14, where stage (b) comprises measuring the production of NO.

20. The method according to claim 19, where NO detect by measuring the concentration of nitrite or transformation3H-L-arginine3H-L-citrulline.

21. The method of identification in vitro of an agent that modulates Akt-regulated the activity of NOS, including stage

(a) exposing the protein or peptide Akt and NOS agent and

(b) measuring Akt-dependent NOS activity by determining whether the residue corresponding to S/T motif consensus sequences RXRXXS/T polypeptide NOS, phosphorylated or mimic phosphorylation state.

22. The method according to item 21, where the NOS activity was measured by determining the Akt-dependent phosphorylation status NOS.

23. The method according to item 22, where the specified peptide NOS includes the sequence of SEQ ID NO:2.

24. The method according to item 22, where in stage (b) the activity of NOS is the reductase activity.

25. The method according to item 22, where in stage (b) the activity of NOS is the I production of NO by the enzyme NOS.



 

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