KR100737947B1 - eNOS MUTATIONS USEFUL FOR GENE THERAPY AND THERAPEUTIC SCREENING - Google Patents

Abstract

The present invention is directed to new NOS variants or mutants comprising structural alterations at the site of Akt dependent phosphorylation. Altered NOS proteins or peptides, especially human eNOS proteins or peptides, Akt proteins or polypeptides and their encoding nucleic acid molecules include diseases with restenosis, hypertension, atherosclerosis, heart failure, diabetes, and angiogenesis after angioplasty It is useful as a gene therapy to treat diseases. NOS proteins and peptides are also useful in methods for screening for agents that modulate NOS activity.

NOS mutation, eNOS, gene therapy

Description

ENOS MUTATIONS USEFUL FOR GENE THERAPY AND THERAPEUTIC SCREENING} Useful for Gene Therapy and Therapeutic Screening

1A-1B are wild type Akt, but Akt, which is not kinase inactive, is intended to increase NO release from cells expressing cell membrane related eNOS. In FIG. 1A, COS cells were transfected with plasmids for eNOS in the presence or absence of Akt or kinase inactive Akt (K179M), and production of NO (analyzed as NO ₂ ⁻ ) was measured by chemiluminescence. In FIG. 1B, COS cells were transfected with various NOS plasmids as described above. In both FIGS. 1A and 1B, the values for NO ₂ ⁻ production were subtracted from the levels obtained from cells transfected only with β-galactosidase cDNA. Inset shows protein expression in whole cell filtrate. Data is mean ± SEM, n = 3-7 experiments; * Indicates p <0.05.

2A-2D are phosphorylation of eNOS by Akt in vitro and in vivo . In FIG. 2A, COS cells were transfected with HA-Akt or HA-Akt (K179M), and the filtrate was immunoprecipitated and subjected to in vitro kinase reaction with histone 2B (25 mg) or recombinant eNOS (3 mg) as substrate. Was placed. The top panel shows the incorporation of the ³² P substrate and the bottom panel shows the amount of substrate by Coomassie staining of the gel. The double serine mutant of wild type or eNOS (eNOS S635 / 1179) labeled ³² P in FIG. 2B was affinity purified from transfected COS and radiographed (top panel) or Western blotting (top blotting). Bottom panel). The graph data in FIG. 2B reflects the amount of labeled protein relative to the amount of eNOS responding to a particular antigen in the gel. In FIG. 2C, labeled eNOS was digested into peptides separated by trypsin and RP-HPLC. The top chromatogram shows the major labeled trypsin peptide moving with the unlabeled synthetic phosphopeptide standard (bottom chromatogram). Insets are shown by the same mass ions as the linear mode MS (top) and phosphopeptide standards of the labeled peptide. In FIG. 2D, recombinant wild type eNOS or eNOS S1179A was purified and placed in an in vitro kinase reaction with recombinant Akt as the same amount (2.4 mg) was disclosed in the methods. The top panel in FIG. 2D shows the integration into ³² P eNOS and the bottom panel shows the amount of substrate by Coomassie staining of the gel. The graph data (n = 3) reflects the amount of labeled eNOS relative to the amount of eNOS (Kumasi) in the in vitro kinase reaction.

3 is evidence that serine 1179 is functionally important for Akt stimulated NO release. COS cells were transfected with plasmids for wild type eNOS or eNOS mutants in the presence or absence of Akt, and expression of protein and production of NO (analyzed as NO ₂ ⁻ ) were measured. Interestingly, the structure with the S1179 mutation to A was not activated by Akt and the mutation of S1179 to D gained function. In A, data are mean ± SEM for experiments 4-7; * Indicates significant difference (p <0.05).

4A-4C show that Akt regulates basic and stimulated production of NO in endothelial cells. In FIG. 4A, BLMVEC was infected with adenovirus structures (β-gal, myr Akt and AA-Akt as controls) and the amount of NO ₂ ⁻ produced over 24 hours was measured (n = 3). Inset shows expression of eNOS and Akt. In FIG. 4B, the filtrate from BLMVEC infected with adenovirus was incubated with various concentrations of free calcium and NOS activity was measured (n = 3 experiments). In FIG. 4C, BLMVEC was stimulated with VEGF (40 ng / ml) for 30 minutes following infection with adenovirus as above and NO ₂ ⁻ release was quantified by chemiluminescence. Data was expressed as VEGF stimulated release of NO ₂ ⁻ after subtracting basal levels. Data is mean ± SEM, n = 4; * Indicates significant difference (p <0.05).

5A and 5B show the purity and dimer / monomer ratio of wild type and eNOS S1179D. In a and b, SDS-PAGE analysis was performed on a 7.5% polyacrylamide gel stained with Coomassie blue. Molecular weight standards (lane 1) and their sizes are shown on the left in kDA. Wild-type eNOS (lane 2) and eNOS S1179D (lane 3) (1 μg each) were degraded as shown by the arrowheads. In b, proteins (2 μg each) were digested on SDS-PAGE done at 4 ° C. Lane 1 was the molecular weight standard. Rain samples of wild type and eNOS S1179D were digested in lanes 2 and 3, respectively. In lane 4, wild type eNOS was boiled in SDS indicator buffer.

6A and 6B eNOS S1179D has a higher NO production rate (a) and reductase activity (b) than wild type eNOS. In a, the rate of NO generated from wild-type (●) and S1179D (○) eNOS was measured using a hemoglobin capture assay as a function of L-arginine concentration, and the data was double reciprocal plot). In b, DCIP and cytochrome c analysis was done in the presence or absence of CaM. Values are mean ± SE, n = 4-6 measurements. Similar results were obtained with at least three enzyme preparations. Significant differences (p <0.05) between wild type and S1179D eNOS are indicated by asterisks.

In FIGS. 7a and 7b the NOS activity (a) and NADPH-dependent reductase (b) are increased for eNOS S1179D as compared to wild type enzyme. Hemoglobin capture (a) and NADPH-dependent cytochrome c reduction (b) assays were performed for both wild type and S1179D eNOS. In a, NO production rates were measured in the presence of all NOS cofactors (wild type (filled symbol) and S1179D (empty symbol) eNOS). The rate of cytochrome c reduction was performed in the absence of BH4 (a) for arginine and wild type (circular) and S1179D (triangle) in the presence (filled symbol) or absence (empty symbol) of 120 nM calmodulin. The value is mean ± SE and n = 3-6 measurements from at least three enzyme preparations.

In FIGS. 8A-8D the calmodulin- and calcium-dependent and reductase activities of NOS were slightly enhanced for S1179D eNOS. Calmodulin-dependent hemoglobin capture (a) and cytochrome c reduction (b) were performed for both wild type (filled symbols) and S1179D eNOS (empty symbols). The rate of NO production detected by hemoglobin capture was in the presence of all NOS cofactors, while cytochrome c reduction was performed in the absence of arginine and BH4. In c and d the same experiment was carried out in the presence of increasing concentrations of free calcium. Insets at c and d show calcium-dependent turnover of S1179D and wild-type eNOS in both NO production and cytochrome c analysis. Maximum turnover was as follows for wild type and S1179D eNOS, respectively: a 22 and 43 min ⁻¹ ; b, 620 and 1400 min 1; c, 58 and 100 min 1; And d, 1930 and 3810 min ⁻¹ . Values are mean ± SE and n = 3-6 measurements from at least three enzyme preparations.

EGTA-initial inactivation of NOS in FIGS . 9A and 9B is reduced in S1179D eNOS. Hemoglobin capture (a) and reductase assay (b) were performed as previously described with the following modifications. The reaction was monitored for 1 minute to measure the initial rate; EGTA was then added to the reaction mixture and the rate was monitored for an additional 1 minute. The free calcium concentration in the reaction was 200 μM and the amount of EGTA added was such that the final concentrations were chelators with 0, 200, 400 and 600 μM. Specific activity is normalized to 100% for wild type and S1179D eNOS. Values are mean ± SE and n = 3-6 measurements from at least three enzyme preparations. nd indicates no detection activity for wild-type eNOS.

Cross Reference to Related Application

This application claims the benefit of US Provisional Application No. 60 / 129,550, filed April 16, 1999, which is incorporated herein by reference in its entirety.

Poet for Federal Support

The present invention results in part from studies funded by the following federal grant monies: HL 57665 and HL 61371.

Technical field

The present invention relates to novel NOS variants or mutants comprising structural alterations at Akt dependent phosphorylation sites. Altered NOS proteins or peptides and their encoded nucleic acid molecules have diseases with post angioplasty restenosis, hypertension, atherosclerosis, heart failure, diabetes mellitus, and deficient angiogenesis. It is useful as a gene therapy agent for the treatment of diseases including.

Atherosclerosis and vascular thrombosis are major causes of morbidity and mortality and lead to coronary artery disease, myocardial infarction, and seizures. Atherosclerosis begins with endothelial degeneration that aligns blood vessels. Endothelial degeneration may eventually result in endothelial damage caused by ingestion of some, oxidized low density lipoprotein (LDL) cholesterol. Rupture due to such damage can lead to thrombosis and occlusion of blood vessels. In the case of coronary arteries, rupture due to complex injuries can prompt the development of myocardial infarction, while in the case of carotid arteries, seizures may occur subsequently.

In atherosclerotic coronary heart disease, endothelial dysfunction can reduce the production of vasodilators such as nitric oxide. Myocardial ischemia is caused by coronary arterial stenosis, which restricts flow, or by endothelial dysfunction when autoregulated vasodilation is interrupted. In both cases, arterial blood flow cannot increase further in proportion to increasing oxygen demand. Among other situations, myocardial ischemia has a constant oxygen demand but is fast blocked by coronary artery spasm, rapid evolution of the underlying atherosclerotic plaque that reduces the diameter of the coronary lumen, and / or blocked by platelet aggregates. It may occur when there is a first decrease in the amount of blood flow established through intermittent microvessels.

Balloon angioplasty is usually used to reopen the narrowed blood vessel with a plaque. Although balloon angiogenesis has a high probability of opening blood vessels, this method often invades the endothelium during practice, causing blood vessel injuries. These injuries move and spread the smooth muscle cells of the blood vessels to the damaged area, resulting in a lesion, known as endometrial hyperplasia or restenosis. These new injuries recur in three to six months after angioplasty in a significant proportion of patients.

In atherosclerosis, thrombosis and restenosis, normal blood vessel function is also lost, causing blood vessels to contract rather than expand. Excessive vasoconstriction narrows the lumen of the vessel, limiting blood flow. It can cause symptoms such as angina (if a heart artery is involved), or transient cerebral ischemia (ie, a "minor seizure", if cerebrovascular is involved). This abnormal vascular function (excessive vasoconstriction or inadequate vasodilation) also occurs in other disease states. High blood pressure (high blood pressure) is caused not only by thickening of the blood vessel walls, especially smaller circulating blood vessels, but also by excessive vasoconstriction. This process not only adversely affects pulmonary blood vessels, but can also cause pulmonary (lung) hypertension. Other abnormalities known to be associated with excessive vasoconstriction or inappropriate vasodilation include transplant atherosclerosis, congestive heart failure, toxemia of pregnancy, Raynaud's phenomenon, Prinzmetal's angina (coronary vasospasm), cerebral vasospasm, hemolytic-uremia and impotence.

The substances released by the endothelium, formerly called "endothelium derived relaxing factor" (EDRF), play an important role in inhibiting these pathological processes. EDRF is currently known to be nitric oxide (NO). NO plays many roles in human physiology, including relaxation of vascular smooth muscle, inhibition of platelet aggregation, inhibition of mitogenesis, diffusion of vascular smooth muscle, and leukocyte adhesion. Since NO is the most potent endogenous vasodilator and is highly responsible for motor-induced vasodilation in the conduit arteries, increasing NO synthesis can also improve motor skills in normal individuals and individuals with vascular disease. .

Endothelial nitric oxide synthase (eNOS) is a nitric oxide synthase (NOS) isoform responsible for maintaining body blood pressure, vascular remodeling and angiogenesis (Shesely et al ., 1996; Huang et al ., 1995; Rudic et al ., 1998; Murohara et al ., 1998). Since the lack of endothelial production of NO is an ongoing feature of atherosclerosis and vascular damage, eNOS has proven to be an attractive target for gene therapy of blood vessels. While regulation of eNOS activity is quite vague, it is known that eNOS is phosphorylated in response to various forms of cellular stimulation (Michel et al ., 1993; Garcia-Cardena et al ., 1996; Corson et al ., 1996). The role of phosphorylation and responsible kinase (s) in the regulation of nitric oxide (NO) production has not been previously identified.

To some extent, the present invention, serine / threonine protein kinase, Akt (protein kinase B) can directly phosphorylate eNOS on serine 1179 (serine 1177 in human eNOS), and mutant eNOS (S1179A) is resistant to Akt phosphorylation and activity. While this is based on the discovery that it can activate enzymes that induce NO production. Moreover, using adenovirus mediated genes, delivery-activated Akt increases basal NO release from endothelial cells and activity deficient Akt decreases VEGF stimulated NO production. Thus, eNOS is a newly described Akt substrate that links signal transduction through Akt for the release of the gaseous second messenger, NO. The present invention is also based, to some extent, on the discovery that mutant eNOS, eg, S1179D, shows an increase in NO production rate and an increase in reductase activity.

The present invention includes NOS polypeptides or proteins and their encoding isolated nucleic acid molecules, wherein the NOS polypeptide or protein comprises residue 1179 of bovine eNOS, residue 1177 of human eNOS, residue 1412 of human nNOS or human substituted amino acid residues corresponding to residue 1415 of nNOS. Preferred substitutions include amino acids with negatively charged R groups, including aspartic acid and glutamic acid.

The invention also includes NOS polypeptides or proteins and their encoding isolated nucleic acid molecules, wherein the NOS polypeptides or proteins correspond to residue 1179 of bovine eNOS, residue 1177 of human eNOS, residue 1412 of mouse nNOS, or residue 1415 of human nNOS. To include substituted amino acid residues. Preferred substitutions include amino acids having an uncharged R group such as alanine.

The present invention relates to ischemic diseases, in particular peripheral vascular disease and / or myocardial ischemia, having a lack of endogenous angiogenesis in a patient comprising the step of delivering a transgene encoding the NOS polypeptide or Akt polypeptide of the invention. A method of stimulating collateral vessel development is provided.

The invention further includes transgene animals, except humans, which express the NOS polypeptides of the invention.

Finally, the present invention comprises the steps of (a) exposing purified NOS, preferably cells expressing eNOS or nNOS, or NOS, preferably eNOS or nNOS, and Akt to an agent; And (b) identifying a medicament that modulates Akt modulating activity of NOS, which generally comprises measuring Akt modulating activity of NOS, preferably eNOS or nNOS.

Demonstration that NO production is regulated by Akt dependent phosphorylation of eNOS provides a new constitutively active eNOS mutation for use in gene therapy aimed at improving endothelial function in cardiovascular diseases associated with dysfunction in the synthesis or biological activity of NO. do. Such diseases include restenosis after angioplasty, hypertension, atherosclerosis, and heart failure, including those associated with myocardial infarction, diabetes and lack of angiogenesis. This finding also provides new therapeutic targets useful for the design of a medicament suitable for treating diseases associated with dysfunction in the synthesis or biological activity of NO.

The present invention also provides new constitutively active nNOS mutants with substituted amino acids corresponding to residue 1412 of murine nNOS or residue 1415 of human nNOS for use in gene therapy aimed at disease treatment.

Specific embodiment

Production of NOS Mutant Proteins or Polypeptides

The present invention provides NOS proteins or polypeptides, allelic variants of NOS proteins, and conservative amino acid substitutions of NOS proteins, all of which include mutations of serine residues that are Akt mediated phosphorylation sites. For example, a protein or polypeptide of the invention may comprise (1) a mutation of residue 1177 from serine to another amino acid such as alanine (Janssens et al . (1992) J. Biol . Chem . 267: 14519-14522, which Human eNOS protein, which is incorporated by reference in its entirety) and which is resistant to Akt mediated phosphorylation; (2) a mutation of residue 1179 from serine to another amino acid such as alanine (SEQ ID NO: 2 of US Pat. No. 5,498,539, which is incorporated herein by reference in its entirety), Resistant bovine eNOS protein; (3) a human nNOS protein comprising a mutation of residue 1415 from serine to another amino acid such as alanine and resistant to Akt mediated phosphorylation; (4) a murine nNOS protein comprising a mutation of residue 1412 from serine to another amino acid such as alanine; (5) a human eNOS protein comprising a mutation of residue 1177 from serine to an amino acid containing a negatively charged R group such as aspartic acid or glutamic acid, which is constitutively active and shows an increase in NO production and an increase in reductase activity; ; (6) a bovine eNOS protein comprising a mutation of residue 1179 from a serine to an amino acid containing a negatively charged R group such as aspartic acid or glutamic acid, which is constitutively active and exhibits an increase in NO production and an increase in reductase activity; ; (7) a human nNOS protein comprising a mutation of residue 1415 from a serine to an amino acid comprising a negatively charged R group such as aspartic acid or glutamic acid, which is constitutively active and exhibits an increase in NO production and an increase in reductase activity; ; (8) a murine nNOS protein comprising a mutation of residue 1412 from a serine to an amino acid containing a negatively charged R group such as aspartic acid or glutamic acid, which is constitutively active and exhibits an increase in NO production and an increase in reductase activity; ; (9) is modified to include amino acids other than serine at a position corresponding to position 1177 in human eNOS or position 1179 in bovine eNOS, position 1412 in rat nNOS, and position 1415 in human nNOS, and is resistant to Akt phosphorylation, or NOS proteins from species other than human, bovine, or murine that are constitutively active or that exhibit increased NO production and increased reductase activity. NOS mutants may also be produced by mutating other amino acids in the phosphorylation motif RXRXXS / T.

The present invention provides constitutively active NOS polypeptides, preferably eNOS or nNOS, which show an increase in NO production and reductase activity and which comprise mutations in the serine residues at the Akt mediated phosphorylation site. Also conservative variants such as substitution, deletion and insertion mutants of these NOS polypeptides exhibiting increased NO production and reductase activity within the skill of the art. As used herein, conservative variants refer to the activity of constitutively active NOS, preferably eNOS or nNOS, or to the reductase activity of constitutively active NOS, preferably eNOS or nNOS, to produce NO. It refers to a change in the amino acid sequence that does not adversely affect. Substitution, insertion, or deletion can adversely affect constitutively active NOS polypeptides when the altered sequence affects the activity of constitutive NOS, resulting in no increase in NO production and increased reductase compared to wild type NOS. It is said to have no activity. For example, the overall charge, structure or hydrophobicity / hydrophilicity of the constituent NOS can be altered without adversely affecting the activity of the constituent NOS. Thus, the amino acid sequence of a NOS polypeptide can be altered without adversely affecting the activity of the NOS, for example to make the polypeptide more hydrophobic or hydrophilic.

As used herein, a mutant or variant of a "constitutively active" NOS, modified or isolated from its original source, refers to a NOS protein, preferably eNOS or nNOS, which refers to residue 1177 or bovine in human NOS. NOS produces NO at higher rates than native NOS containing serine in its unphosphorylated form at the amino acid residue corresponding to residue 1179. Preferred constitutively active variants include amino acids having a negatively charged R group, such as aspartic acid or glutamic acid, at the amino acid residue corresponding to serine at position 1177 in human eNOS or position 1179 in bovine NOS.

The present invention comprises NOS protein or polypeptide, allelic variants of NOS protein, and substituted amino acid residues corresponding to residue 1177 of bovine eNOS, residue 1179 of human eNOS, residue 1412 of murine nNOS, and residue 1415 of human nNOs. Conservative amino acid substituents of the NOS protein are provided, wherein the substituted amino acid residues comprise an uncharged R group such as alanine.

The NOS protein of the invention, preferably eNOS or nNOS protein, may be in isolated form. As used herein, a protein is meant to be separated when physical, mechanical or chemical methods are used to remove the protein from the cellular components generally associated with the protein. One skilled in the art can readily use standard purification methods to obtain isolated proteins.

Also included in the present invention are NOS peptides that span the Akt phosphorylation site corresponding to residue 1179 in bovine eNOS, residue 1177 in human eNOS, residue 1412 in murine nNOS, or residue 1415 in human nNOS. The peptide may comprise a serine at the phosphorylation site, and preferably comprises a substitution of serine at a position corresponding to residue 1179 in bovine eNOS, residue 1177 in human eNOS, residue 1412 in rat nNOS, or residue 1415 in human nNOS. You may. The substituents include, but are not limited to, amino acids having an R group similar to serine at the phosphorylation site, such as aspartic acid or glutamic acid. The substituents also include amino acids having non-negative R groups such as alanine. Peptides spanning the site may be about 3, 5, 7, 10, 12, 15, 17, 20, 25, 30, 40, 50 or more amino acids in length.

NOS proteins, polypeptides or peptides of the invention may be characterized by a nucleotide triplet encoding a serine corresponding to position 1177 in human eNOS, position 1179 in bovine eNOS, residue 1412 in rat nNOS, or serine at residue 1415 in human nNOS. It may be prepared by any means available, including recombinant expression from modified NOS cDNA to replace or alter. To mutate nucleotide triplets encoding serine residues, any available technique can be used, such as homologous recombination, site specific mutagenesis or PCR mutagenesis (Sambrook et al ., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989). Starting material cDNAs encode human and bovine eNOS cDNAs that also encode eNOS proteins of other animal species, including, but not limited to, rabbits, mice, murine, pigs, sheep, horses, and non-human primate species. cDNAs.

As used herein, a nucleic acid molecule encoding a NOS protein or polypeptide of the invention, preferably an eNOS or nNOS protein or polypeptide, is "isolated" when said nucleic acid molecule is substantially separated from contaminating nucleic acid encoding another polypeptide in the source of the nucleic acid. It is said. "

The invention also provides fragments of encoding nucleic acid molecules. As used herein, a fragment of an encoding nucleic acid molecule refers to a small portion of the entire protein that encodes a sequence. The size of the fragments will be determined by the intended use. For example, if a fragment is selected to encode an active portion of the protein, the fragment will have to be large enough to encode a functional region of the protein, including the Akt phosphorylation site. If the fragment is to be used as a nucleic acid probe or PCR primer, the fragment length at that time is such that relatively few false positives are obtained during probing / priming to regions that span or are adjacent to the NOS Akt phosphorylation site. Is selected.

Fragments (i.e., synthetic oligonucleotides) of the encoding nucleic acid molecules of the invention, which are used as probes or specific primers for polymerase chain reaction (PCR), or used to synthesize the gene sequence encoding proteins of the invention, are described in chemical techniques. , For example, Matteucci, et al . 1981 J. Am. Chem . Soc . It can be easily synthesized by the phosphotriester method of 103: 3185-3191 or using an automated synthesis method. In addition, larger DNA segments are readily available by known methods, such as the synthesis of a group of oligonucleotides that define various modular segments of a gene, followed by ligation of the oligonucleotides to create a completely altered gene. Can be prepared.

The encoding nucleic acid molecules of the present invention may be further modified to include detectable labels for diagnostic and probe purposes. These various labels are known in the art and can be readily used with the encoding molecules disclosed herein. Suitable labels include, but are not limited to, biotin, nucleotides labeled with radioisotopes, and the like. One skilled in the art can use any known label to obtain a labeled encoding nucleic acid molecule.

The present invention also provides recombinant DNA molecules (rDNAs) comprising such a NOS coding sequence. As used herein, rDNA molecules are DNA molecules that have undergone molecular manipulation. Methods for generating rDNA molecules are known in the art, see, for example, Sambrook et al., Molecular Cloning (1989). In a preferred rDNA molecule, the coding DNA sequence is functionally linked with expression control sequences and / or vector sequences.

The selection of vectors and / or expression control sequences to which one of the encoding sequences of the group of proteins of the invention is functionally linked is known, as is known in the art, such that certain functional properties, eg, protein expression and host to be transformed, are known. Depends directly on the cell. The vectors contemplated by the present invention may also be involved in the replication or insertion into the host chromosome, preferably in expression, of the structural genes contained at least in the rDNA molecule.

Expression control elements used to regulate expression of functionally linked protein encoding sequences are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, other regulatory elements. Preferably, the inducible promoter is readily regulated, such as in response to nutrients in the medium of the host cell.

In one embodiment, a vector containing a coding nucleic acid molecule is used for autonomous replication and maintenance of prokaryotic replicons, ie, non-chromosomal recombinant DNA molecules in prokaryotic host cells, such as transformed bacterial host cells. It will include DNA sequences with the ability to engage. Such replication units are known in the art. In addition, a vector comprising a prokaryotic replicating unit may also include a gene whose expression provides a detectable marker such as drug resistance. Typical bacterial drug resistance genes are those that provide resistance to ampicillin or tetracycline.

Vectors containing prokaryotic replication units may also include prokaryotic or bacteriophage promoters that may be involved in the expression (transcription and translation) of coding gene sequences in bacterial host cells such as E. coli . Promoters are expression control elements formed by DNA sequences that enable transcription for binding and development of RNA polymerases. Promoter sequences that can coexist with bacterial hosts are typically provided in plasmid vectors containing restriction sites suitable for insertion of the DNA segments of the invention. Typical vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories (Richmond, Calif.), And pPL and pKK223 available from Pharmacia, Piscataway, NJ.

Expression vectors that can coexist with eukaryotic cells, preferably expression vectors that can coexist with vertebrate cells, can also be used to form rDNA molecules comprising coding sequences. Eukaryotic cell expression vectors are known in the art and are available from several commercial sources. Typically, vectors are provided that contain suitable restriction sites for insertion of a given DNA segment. Typical vectors are pSVL and pKSV-10 (Pharmacia), pBPV-1 / pML2d (International Biotechnologies, Inc.), pTDT1 (ATCC, # 31255) and similar eukaryotic expression vectors.

Eukaryotic cell expression vectors used to construct the rDNA molecules of the invention may further comprise selectable markers, preferably drug resistance selection markers, which are effective in eukaryotic cells. Preferred drug resistance markers are genes whose expression indicates neomycin resistance, ie neomycin phosphatase ( neo ) gene (Southern et al . (1982) J. Mol. Anal. Genet. 1; 327-341). Alternatively, selectable markers may be present on separate plasmids and the two vectors are selected by co-transfection of the host cell and selected by incubation in the appropriate drug for the selectable marker.

The present invention further provides host cells transformed or transfected with nucleic acid molecules encoding the NOS protein of the invention, preferably the eNOS or nNOS protein. The host cell can be a prokaryotic or eukaryotic cell. Eukaryotic cells useful for the expression of the proteins of the invention are not limited as long as the cell line is compatible with cell culture and with the propagation of expression vectors and the expression of gene products. Preferred eukaryotic host cells include, but are not limited to, yeast, insect and mammalian cells, preferably vertebrate cells such as from mouse, rat, monkey or human cell lines. Preferred eukaryotic host cells are Chinese hamster ovary (CHO) cells available as CCL61 from ATCC, NIH Swiss mouse embryo cells NIH / 3T3, baby hamster kidney cells available as CRL 1658 from ATCC hamster kidney cells (BHK), and similar eukaryotic tissue culture cell lines. Any prokaryotic host can be used to express rDNA molecules encoding the proteins of the invention. Preferred prokaryotic hosts are E. coli, particularly for constitutively active NOS mutants.

Suitable cell hosts are transformed or transfected with the rDNA molecules of the present invention by conventional methods, which depend on the type of vector used and the host system used. And an electrophoretic method and the salt treatment is conventionally used, with regard to transformation of prokaryotic host cells, for example, Cohen et al., (1972 ) Proc. Natl . Acad . Sci. USA 69 : 2110; And Maniatis et al ., Molecular Cloning, A Laboratory Mammal , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). For transformation of vertebrate cells with vectors comprising rDNA, electrophoretic, cationic lipid or salt treatment methods are commonly used, see Graham et al . (1983) Virol . 52 : 456; Wigler et al ., (1979) Proc . Natl . Acad . Sci . See USA 76 : 1373-76. Successfully transformed or transfected cells, ie, cells comprising the rDNA molecules of the invention, can be identified by known techniques, including selection for selectable markers. For example, cells resulting from the introduction of rDNA of the present invention can be cloned to produce single colonies. Cells from these colonies can be harvested and lysed, and their DNA content is shown in Southern (1975) J. Mol . Biol . 98 : 503 or Berent et al . (1985) Biotech . The presence of rDNA can be tested using proteins produced from cells analyzed via methods such as those described by 3 : 208, or immunological methods.

The present invention further provides a method for producing NOS protein, preferably eNOS protein or nNOS protein of the invention using the nucleic acid molecules disclosed herein. Under general conditions, the production of recombinant forms of proteins typically involves the following steps. Nucleic acid molecules encoding the proteins of the invention are first obtained. If the coding sequence is followed by introns, it is entirely suitable for expression in any host. The nucleic acid molecule is then preferably operably linked with a suitable control sequence, as described above, to form an expression unit comprising a protein open reading frame. The expression unit is used to transform or transfect the appropriate host and the transformed or transfected host is cultured under conditions that allow the production of recombinant protein. Optionally, the recombinant protein is isolated from the medium or cell; Recovery and purification of the protein may not be necessary if it can tolerate impurities.

Each of the following steps can be done in a variety of ways. For example, a given coding sequence may be obtained from genomic fragments and may be used directly in a suitable host. Construction of expression vectors usable for various hosts is accomplished using appropriate replication units and control sequences as described above. Control sequences, expression vectors, and transformation or transfection methods depend on the type of host cell used to express the gene and have been described in detail above. Appropriate restriction sites can be added at the end of the coding sequence to provide an excisable gene for insertion into the vector, if not normally available. Those skilled in the art can readily adapt any host / expression system known in the art to produce recombinant proteins for use with the nucleic acid molecules of the present invention.

Gene therapy

Any suitable gene delivery system with the appropriate gene expression system using the most appropriate delivery route is achieved by the present invention. For example, the NOS mutations or variant genes of the present invention, preferably the eNOS or nNOS mutations or variant genes, or Akt genes may be involved in the production of proteins encoded in vitro or in vivo, such as cardiomyocytes (and skeletal muscle cells). Including, it may be delivered to the heart (or skeletal muscle). Particularly useful are human eNOS, which comprise amino acids with negatively charged R groups, such as aspartic acid or glutamic acid, at positions corresponding to serine 1177 in human Akt genes and NOS mutants, preferably human eNOS. Routes for administering NOS mutations or variant genes include, but are not limited to, intravascular injection, intramuscular injection, intraperitoneal injection, intradermal injection and intraarterial injection.

The adenovirus gene delivery system offers several advantages: (a) Adenovirus can accommodate relatively large DNA inserts; (Ii) can be grown to high titers; (Iii) can infect a wide variety of mammalian cell types; (Iii) can be used with a number of available vectors, including other promoters. In addition, adenovirus can be administered by intravenous injection because it is suitable for blood flow. Preferred transport vectors are helper-independent replication deficient human adenovirus 5, but other means of transport are available and may be used, including the delivery of nucleic acids directly to the cells of interest (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; Harv Heart Lett (1999) 9 (8) : 5-6; and Nabel et al . (1999) Nat. Med . 5 (2): 141-2).

Using the adenovirus 5 system, over 60% transfection frequency was shown by one coronary injection in vivo (Giordano and Hammond (1994) Clin . Res . 42: 123A). Non-replicating recombinant adenovirus vectors are particularly useful for transfecting coronary endothelial and cardiomyocytes, so that they are transfected very effectively after coronary artery injection. Non-replicating recombinant adenovirus vectors are also useful for transfecting certain cells of the peripheral vascular system (see US Pat. No. 5,792,453, which is fully incorporated herein by reference).

The adenovirus vectors used in the present invention can be constructed by the rescue recombination technique disclosed in Graham et al . (1988) Virology 163: 614-617. In brief, eNOS transgenes are cloned in a shuttle vector that includes promoters, polylinkers and some partial flanking adenoviral sequences from which the E1A / E1B gene has been removed. As a shuttle vector, the plasmid pAC1 ( Virology 163: 614-617, coding for the left terminal portion of the human adenovirus 5 genome (Virology 163: 614-617, 1988), excluding the initial protein encoding E1A and E1B sequences essential for viral replication) 1988) (or analogues) and plasmid ACCMVPLPA ( J. Biol . J. Biol . Chem . 267: 25129-25134, 1992). The use of plasmid pAC1 or ACCMVPLA facilitates the cloning process. The shuttle vector is then co-transfected into plasmids containing the entire human adenovirus 5 genome, which is too long to be encapsidated with protein membranes in 293 cells. Co-transfection may be performed by calcium phosphate sedimentation or lipofection ( Biotechniques 15: 868-872, 1993). Plasmid JM17 encodes the entire human adenovirus 5 genome by adding a portion of the vector pBR322 containing genes for ampicillin resistance (4.3 kb). Although JM17 encodes all of the anenovirus proteins to make mature virus particles, it is too long to wrap with a protein membrane (40 kb for 36 kb for the wild type). In a small subset of co-transfected cells, structural recombination between a transgene containing a shuttle vector such as plasmid pAC1 and a plasmid with the entire adenovirus 5 genome such as plasmid pJM17 lacks the E1A / E1B sequence, and The secondary genome, which includes the transgene but loses additional sequences such as the pBR322 sequence during recombination, provides a recombinant genome that is small enough to be encased in a protein membrane. CMV driven beta-galactosidase encoding adenovirus HCMVSP1 lac Z ( Clin . Res . 42: 123A, 1994) is used to assess the efficiency of gene delivery using X-gal treatment. Can be used.

In other embodiments, the gene encoding NOS, preferably eNOS or nNOS, may be introduced in vivo through a weakened or lacking DNA virus, which virus is for example herpes simplex virus (HSV), papillomavirus. ), Epstein Barr virus (EBV), adenovirus and adeno-associated virus (AAV), but are not limited thereto. Preferred are deficient viruses that are wholly or almost entirely lacking viral genes. The deficient virus is not contagious after being introduced into the cell. The use of defective, vital vectors allows for administration to cells of a particular localized region, regardless of the fact that the vector can infect other cells. Thus, certain loci, for example, in the brain or spinal cord, are specifically targeted to the vector. In certain embodiments, deficient herpes virus 1 (HSV1) vectors may also be used (Kaplitt et al . (1991) Molec . Cell . Neurosci . 2: 320-330). In another embodiment, the viral vector is a weakened adenovirus vector, such as the vector disclosed by Stratford-Perricaudet et al. ( J. Clin . Invest 90: 626-630 (1992)). In another embodiment, the vector is a deficient adeno-associated virus vector (Samulski et al . (1987) J. Virl . 61: 3096-3101; Samulski et al . (1989) J. Virol . 63: 3822-3828).

The present invention also contemplates the use of cell targeting not only by transporting the transgene to the cell coronary artery, or the femoral artery, but also, for example, by the use of tissue specific promoters. For example, by transfecting the tissue specific transcriptional regulatory sequences of the left ventricular myosin light chain-2 (MLC [2V]) or myosin heavy chain (MHC) to a transgene, such as the NOS gene of the invention in the structure of an adenovirus, a transgene Expression is limited to ventricular myocytes. The degree of specificity and efficiency of gene expression by the MLC [2V] and MHC promoters according to lac Z were measured using the recombinant adenovirus system of the present invention. Cardiac specific expression has already been reported by Lee et al . ( J. Biol . Chem . 267: 15875-15885 (1992)). The MLC [2V] promoter consists of 250bp and is easily adapted into adenovirus-5 packaging constraints. Myosin heavy chain promoters are known as strong promoters of transcription and provide suitable alternative cardiac specific promoters and are comprised of less than 300 bp. SM22 alpha promoter (Kemp et al ., (1995) Biochem J 310 (Pt 3): 1037-43) and SM alpha actin promoter (Shimizu et al . (1995) J Biol Smooth muscle cell promoters such as Chem 270 (13): 7631-43) can also be used. Other promoters, such as the troponin-C promoter, are very effective and small enough, but lack adequate tissue specificity. By using a MLC [2V] or MHC promoter and transporting the transgene in vivo, only cardiac myocytes (no secondary expression in endothelial cells, smooth muscle cells, and fibroblasts in the heart) provide adequate expression of the NOS protein. something to do.

Expression limited to cardiomyocytes also has an advantage with regard to the utility of gene delivery for the treatment of clinical myocardial ischemia. By limiting expression to the heart, it avoids potentially harmful effects on angiogenesis in non-cardiac tissues such as the retina. In addition, with respect to cells of the heart, myocytes may provide the longest transgene expression because the cells do not undergo rapid rotation; Expression therefore occurs in endothelial cells but is not reduced by cell division and death. Endothelial specific promoters are already used for this purpose. Examples of endothelial specific promoters include the Tie-2 promoter (Schlaeger et al . (1997) Proc. Natl Acad Sci 1; 94 (7): 3058-63), the endothelin promoter (Lee et al . (1990) J. Biol . Chem . 265: 10446-10450), and the eNOS promoter (Zhang et al . 1995) J Biol . Chem 270 (25): 15320-6).

The present invention involves the targeting of the heart by intracoronary or intramuscular injection with a high titer vector with respect to the treatment of heart disease, and it is now desirable to transfect all cell types. Diseases such as erectile dysfunction and cardiovascular diseases including myocardial infarction, myocardial ischemia, heart failure, restenosis, stent stenosis, post angiogenesis stenosis, and bypass graft failure are NOS transgenes, preferably eNOS or nNOS. It may also be treated as described using a transgene.

Successful recombinant vectors can be plaque purified according to standard methods. The resulting viral vector is preferably propagated over 293 cells that provide E1A and E1B functions in trans to have a titer in the range of about 10 ¹⁰ -about 10 ¹² virus particles / ml. Cells are harvested 48 hours after infection with 80% confluence. After three freeze-thaw cycles, cell debris is pelleted by centrifugation and the virus is purified by CsCl gradient ultracentrifugation (double CsCl gradient ultracentrifugation is preferred). Prior to in vivo injection, viral stock is desalted by gel filtration through Sepharose columns such as G25 Sephadex. The product is then filtered through a 30 micron filter, thereby reducing the deleterious effects of intra-coronary injection of unfiltered virus (life threatening cardiac arrhythmias) and promoting effective gene transfer. The resulting viral colony has a final viral titer in the range 10 ¹⁰ -10 ¹² virus particles / ml. Recombinant adenovirus should be highly purified as not a wild type (potentially repeatable) virus. Impure structures can cause a vigorous immune response in the host animal. In this regard, breeding and purification may be performed to confirm successful recombination, eg, by PCR with appropriate primers, and to block contaminants and wild-type viruses by performing two plaque purifications and double CsCl gradient ultracentrifugation. . In addition, problems associated with cardiac arrhythmias caused by injecting adenovirus vectors into a patient can be avoided by filtration of the recombinant adenovirus through a filter of the appropriate size prior to intracoronary injection. This strategy also appears to substantially improve gene delivery and expression.

The viral colony may be in the form of an injectable preparation comprising, for example, a pharmaceutically acceptable carrier such as saline when necessary. The final titer of the vector in the injectable article is preferably in the range of about 10 ⁷ -about 10 ¹³ virus particles to enable effective gene delivery. Other pharmaceutical carriers, formulations and dosages are described below. The adenovirus transgene construct is sufficient for transgene expression to allow for highly effective treatment, and can be used for direct intracoronal (or graft vascular) injection using standard percutaneous catheter based methods under fluoroscopic guidance. By the myocardium. It is injected deep into the lumen of the coronary artery (or graft) (approximately 1 cm in the lumen of the artery) and is preferably injected into both coronary arteries because the growth of the bypass vessels can vary significantly within each patient. By injecting the material directly into the lumen of the coronary artery by a coronary catheter, it is possible to target the gene fairly effectively and minimize the loss of the recombinant vector to the proximal aorta during the injection. When transported in this way, gene expression does not occur in hepatocytes and viral RNA cannot continue to be found in urine after intracoronary injection. For example, various coronary catheter, or Stack perfusion catheter, can be used in the present invention. In addition, other techniques known to those skilled in the art can also be used to deliver NOS genes, preferably eNOS or nNOS, to the arterial wall.

For the treatment of peripheral vascular disease, a disease characterized by insufficient blood supply to the legs, the recombinant adenovirus expressing the NOS of the present invention, preferably eNOS or nNOS, peptide or protein, can be used in the proximity of the femoral artery or arteries. It may be carried by a catheter inserted into the site, thus transferring the gene into cells of skeletal muscle that receives blood from the femoral artery.

The nucleic acid or transgene encoding the NOS, preferably eNOS or nNOS, or Akt protein of the invention is first delivered ex vivo to endothelial or vascular smooth muscle cells, including the cells of the patient, the DNA directly into the cell. May be transfected (see US Pat. No. 5,658,565). In general, to transfect target cells, plasmid vectors comprising DNA sequences encoding Akt or NOS of the present invention or biologically active fragments thereof may be utilized for liposome mediated transfection of target cells. The stability of liposomes, linked to the impermeability of these vesicles, makes the liposomes a useful vehicle for the transport of therapeutic DNA sequences (for review, Mannino and Gould-Forgerite (1988) BioTechniques 6 (7): 682- See 690). Liposomes are known to be taken up in many cell types by fusion. In one embodiment, cationic liposomes including cationic cholesterol derivatives, such as SF-chol or DC-chol, may be utilized. DC-chol molecules are composed of tertiary amino groups, medium length spacer arms and carbamoyl linker bonds, as described by Gao and Huang ( Biochem , Biophys . Res. Comm . 179: 280-285, 1991). carbamoyl linker bond).

In another embodiment of the use of liposome technology, a vector of a viral or nonviral lineage comprising a DNA sequence encoding a biologically active NOS protein fragment, preferably an eNOS or nNOS protein fragment, is a target according to lipofectamine. The cells are transferred to the target cells by transfection (Bethesda Research Laboratory). Lipofectamine is a polylipogenic lipid 2,3 dioleyloxy-N- [2 (sperminecarboximido) ethyl] -N, N-dimethyl-1-propanaminitrim fluoroacetate (polycationic lipid 2,3 dioleyloxy 3: 1 liposome formation of -N- [2 (sperminecarboxymido) ethyl] -N, N-dimethyl-1-propanaminiumtric fluroacetate (DOPSA) and neutral lipid dioleoly-phosphatidylethanolamine (DOPE) to be.

Other nonviral modes for gene delivery include (a) direct injection of exposed DNA; (b) calcium phosphate [Ca ₃ (PO ₄ ) ₂ ] mediated cell transfection; (c) mammalian host cell transfection by electrophoresis; (d) DEAE-dextran mediated cell transfection; (e) polybrene mediated delivery; (f) protoplast fusion; (g) microinjection; And (h) polylysine mediated transformation with genetically treated cells delivered back to the mammalian host.

Transgenic  Production of animals

As described herein, transgenic animals comprising a mutant NOS gene, preferably a mutant eNOS or nNOS gene, are also included in the present invention. Transgenic animals are genetically altered animals in which recombinant, exogenous or cloned genetic material has been transferred into the animal by experiment. Such genetic material is often called "transgene". In the form of NOS, the nucleic acid sequences of the transgene may be mixed at the locus of the genome where a particular nucleic acid sequence is not otherwise normally found, or at the normal locus for the transgene. The transgene may consist of nucleic acid sequences derived from the genome of the same species or from genomes of species different from the species of the target animal.

The term "germ cell line transgenic animal" refers to the ability of a transgenic animal to introduce genetic changes or genetic information into a germ line cell, thereby delivering the genetic information to the offspring. Say transgenic animals. If the progeny have little or all of said change or genetic information, it is also a transgenic animal.

The change or genetic information may be independent of the species of the animal to which the recipient belongs, may be only independent of a particular individual recipient, or may be genetic information that the recipient already has. In the last case, the altered or introduced gene may be expressed differently from the original gene.

Transgenic animals can be produced by a variety of other methods including transfection, electrophoresis, microinjection, gene labeling in embryonic hepatocytes and recombinant viral and retroviral infections (eg, US Pat. No. 4,736,866; U.S. Pat.No. 5,602,307; Mullins et al . (1993) Hypertension 22 (4): 630-633; Brenin et al. (1997) Surg. Oncol. 6 (2) 99-110; Tuan (ed.), Recombinant Gene Expression Protocols , Methods in Molecular Biology No. 62, Humama Press (1997).

Numerous recombinant or transgene mice have been produced and expressing activated oncogin sequences (US Pat. No. 4,736,866); Expressing an apes SV 40 T-antigen (US Pat. No. 5,728,915); Lack of expression of interferon regulatory factor 1 (IRF-1) (US Pat. No. 5,731,490); Exhibiting dopaminergic dysfunction (US Pat. No. 5,723,719); Expressing at least one human gene that participates in blood pressure control (US Pat. No. 5,731,489); Showing similarities to those present in naturally occurring Alzheimer's disease (US Pat. No. 5,720,936); Reduced ability to modulate cellular adhesion (US Pat. No. 5,602,307); Possessing bovine growth hormone genes (Clutter et al . (1996) Genetics 143 (4): 1753-1760); Or those capable of generating a complete human antibody response (McCarthy (1997) The Lancet 349 (9049): 405).

Mice and rats are the animals of choice for the best transgenic experiments, but in some cases it is desirable or even necessary to use other animal species. Transgenic processes have been successfully used in a variety of non-murine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (eg, Kim et al . (1997). Mol.Reprod. Dev. 46 (4): 515-526; Houdebine (1995) Reprod.Nutr. Dev. 35 (6): 609-617; Petters (1994) Reprod.Fertil. Dev. 6 (5): 643-645; Schnieke et al . (1997) Science 278 (5346): 2130-2133; and Amoah (1997) J. Aimal Science 75 (2): 578-585).

The introduction of nucleic acid fragments into mammalian cells suitable for recombination is possible by methods that favor the simultaneous transformation of multiple nucleic acid molecules. Detailed procedures for producing transgenic animals will be readily available to those skilled in the art, including those disclosed in US Pat. No. 5,489,743 and US Pat. No. 5,602,307. In addition, the production of NOS transgenic animals is considerably developed. For example, transgenic mice have been produced that induce or overexpress wild-type eNOs (Ohashi et al. (1998) J. Clin. Invest. 102 (12): 2061-71; and Drummond et al. (1998). J. Clin. Invest. 102 (12): 2033-4). These methods can also be used to produce transgenic mice expressing the NOS mutants of the invention.

Therapeutic Screening Analysis

The discovery that phosphorylation of eNOS modulates its activity allows development of screening assays to identify Akt regulated NOS, preferably eNOS or nNOS, agents that modulate activity or expression. Any available format may be used, including in vivo transgenic animal assays, ex vivo protein based assays, cell culture assays, and high throughput formats.

In many drug screening programs that test libraries of compounds, high throughput assays are preferred to maximize the number of compounds investigated at a given time. Assays performed in acellular systems, such as those that can be derived from purified or semi-purified proteins, can be brought about to allow for rapid development and relatively easy change detection in molecular targets mediated by test compounds. In that sense they can often be encouraged as "primary" screens. Moreover, the effects of the cytotoxicity and / or bioavailability of the test compound may generally be ignored in an in vitro system, and instead the assay may be evident in the inhibition of binding, for example between injections. The main focus is on the effect of the drug on molecular targets.

Analysis of the cell or tissue culture basis can also be done by, for example, plating COS-7 cells (100 mm dish) and transfecting with NOS (7.5-30 mg) and Akt (1 mg) plasmid using calcium phosphate. To balance all transfections, expression vectors for β-galactosidase cDNA may be cotransfected. 24 to 48 hours after transfection, the expression of the appropriate protein (40-80 mg) was eNOS mAb (9D10, Zymed), HA mAb (12CA5, Boehringer Mannheim), iNOS pAb (Zymed Laboratories) or nNOS mAb (Zymed). Laboratories) may be confirmed by Western blot analysis.

24 to 48 hours after transfection, as described (Sessa et al ., 1995), by NO specific chemiluminescence, stable nitrite (NO ₂ ⁻ ), stable breakdown product of NO in aqueous solution The medium may also be treated for the determination of). The protein is deproteinized from the medium and the sample containing NO ₂ ⁻ is refluxed in glacial acetic acid containing sodium iodine. Under these conditions, NO ₂ ⁻ is quantitatively reduced to NO quantified by a chemiluminescence detector after reacting with ozone in a NO analyzer (Sievers, Boulders, CO). In all experiments, the control can be prepared by inhibiting NO ₂ ⁻ release by use of a NOS inhibitor. In addition, NO ₂ ⁻ release from cells transfected with β-galactosidase cDNA may be subtracted from the control for background levels of NO ₂ ⁻ found in serum or medium. CGMP accumulation in COS can also be used as a bioassay for NO production as described. In other formats, the conversion of ³ HL-arginine to ³ HL-citrulline has already been described (Garcia-Cardena et al ., 1998), to measure NOS activity in COS cells or endothelial cell lysates. May be used.

For in vivo phosphorylation studies, COS cells were either wild type or S635 (control), bovine 1179A, D or E eNOS, human 1177D or E eNOS, rat 1412D or E nNOS, human 1415D or E nNOS and HA-Akt May be transfected overnight. 36 hours after transfection, cells were dialyzed serum rich phosphate-free Dulbecco's minimum essential supplemented with 80 μCi / ml of ³² P orthophosphate for 3 hours. medium). Cell aliquots may be pretreated with wortmannin (500 nM) in phosphate-free medium for 1 hour and during labeling. The filtrate was then harvested, the NOS dissolved and partially purified by ADP Sepharose Affinity Chromatography as described previously and ³² P integration into NOS was determined by radioautomography after SDS-PAGE (7.5%). It was visualized and the amount of NOS protein was confirmed by western blotting on NOS.

For in vitro phosphorylation studies, recombinant NOS purified from E. coli , eNOS purified from other material sources, or NOS peptides spanning Akt phosphorylation sites are incubated with wild-type or kinase inactive Akt immunoprecipitated from transfected COS cells. . In brief, the NOS protein or peptide is ³² P g-ATP in a buffer containing HEPES (20 mM, pH = 7.4), MnCl ₂ (10 mM), MgCl ₂ (10 mM) and immunoprecipitated Akt for 20 minutes at room temperature. (2 ml, specific activity 3000 Ci / mmol), ATP (50 mM), DTT (1 mM).

In experiments investigating the in vitro phosphorylation of wild type and mutant NOS, recombinant Akt (1 mg) purified from baculovirus infected SF9 cells was wild type, S1179A bovine eNOS, S1177A human eNOS, S1179D or E bovine eNOS, S1177D or E human eNOS, S1412D or E murine nNOS, or S1415D or E human nNOS are incubated essentially using the conditions described above. The protein may be degraded by SDS-PAGE and ³² P integration and the amount of protein may be measured by Coomassie staining as described above.

The screening assay, which analyzes Akt dependent phosphorylation or activation of NOS, preferably eNOS or nNOS, may also be used to screen for a wide variety of agents. For example, agents that inhibit the dephosphorylation of NOS at the amino acids corresponding to serine 1179 in bovine eNOS, residue 1177 in human eNOS, residue 1412 in rat nNOS, or residue 1415 in human nNOS (phosphatase inhibitors) are useful treatments. It may be a molecule. Similarly, agents that activate Akt or resemble Akt phosphorylation sites on eNOS may be useful therapeutic molecules.

Agents analyzed in this manner may be arbitrarily selected or rationally selected or designed. As used herein, a medicament refers to one that is randomly selected when the medicament is arbitrarily selected without considering the particular sequence involved in association with only the protein of the invention or its associated substrate, binding partner, and the like. Examples of any selected agents are the use of chemical libraries or peptide combination libraries, or growth broth of organisms.

As used herein, a medicament refers to something that has been reasonably selected or designed when selected based on a sequence of the site of interest and / or not on any consideration of its shape related to the action of the medicament. For example, a reasonably selected peptide agent may be a peptide whose amino acid sequence is similar to the Akt phosphorylation site in NOS, in particular a peptide or small molecule similar to the NOS phosphorylation state.

The medicament of the invention can be, for example, peptides, small molecules, vitamin derivatives, and also carbohydrates. Those skilled in the art will readily appreciate that there are no restrictions as to the structural properties of the medicaments of the present invention.

Peptide medicaments of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis, as is known in the art. In addition, the DNA encoding these peptides can be synthesized using commercially available oligonucleotide synthesis means and recombinantly produced using standard recombinant production systems. If a nongenic encoding amino acid is to be included, production using solid phase peptide synthesis is necessary.

Another class of agents of the invention are antibodies that immunoreact with the critical positions of the proteins of the invention. Antibody medicaments are obtained by immunization of suitable mammalian subjects with peptides, which comprise portions of the protein intended to be targeted by the antibody, as antigenic regions.

eNOS  By adjusting the activity Identified  Use of drug

Agents of the invention such as phosphatase inhibitors which inhibit dephosphorylation of NOS at amino acids corresponding to serine 1179 in bovine eNOS, residue 1177 in human eNOS, residue 1412 in rat nNOS, or residue 1415 in human nNOS And also agents that activate Akt or resemble Akt phosphorylation sites on NOS can be administered via the parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or oral route. It can be administered orally or otherwise. The amount administered will depend on the age, health and weight of the recipient, the type of co-treatment, frequency of treatment, if any, and the nature of the desired effect. As described below, there are many ways that can be readily adapted to administering such agents.

The invention also provides compositions containing one or more agents of the invention. Although individual needs vary, determination of the optimum range for an effective amount of each component is within the skill of the art. Typical dosages include 0.1 to 100 mg / kg body weight, and preferred dosages include 0.1 to 10 mg / kg body weight. Most preferred dosages include 0.1 to 1 mg / kg body weight.

In addition to pharmacologically active agents, the compositions of the present invention may be prepared in suitable pharmaceutical forms, including auxiliaries and excipients which facilitate the process of processing the active compound into a pharmaceutically usable preparation for delivery to the site of action. It may also contain an acceptable carrier. Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example water-soluble salts. In addition, suspensions of the active compounds may be administered as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides. Aqueous injection suspensions may include substances that increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. Optionally, the suspension may also include a stabilizer. Liposomes can also be used to wrap a protein membrane with a medicament for delivery into cells.

Pharmaceutical formulations for systemic administration in accordance with the present invention may be formulated for enteral, parenteral or topical administration. In fact, all three types of formulations can be used to simultaneously achieve systemic administration of the active component.

Formulations suitable for oral administration include tablets, elixirs, suspensions, syrups or inhalants and controlled controlled release forms thereof, including hard or soft gelatin capsules, pills, coated tablets. .

In practicing the methods of the invention, the compounds of the invention may be used alone or in combination, or in combination with other therapeutic or diagnostic agents. In certain preferred embodiments, the compounds of the invention are anticoagulant agents, thrombolytic agents, or platelet aggregation inhibitors, tissue plasminogen activators, urokinases, prourokinases. May be co-administered with other compounds typically prescribed for these conditions in accordance with generally accepted medical practice, such as streptokinase, heparin, aspirin, or other antithrombotics, including warfarin. . The compounds of the present invention can usually be utilized in vivo or ex vivo in mammals such as humans, sheep, horses, cattle, pigs, dogs, cats, mice and mice.

The following examples specifically point to preferred embodiments of the invention and are not to be construed as limiting the remainder of the disclosure in any way. Other generic forms will be apparent to those skilled in the art.

Example

The following procedure was employed in Examples 1-2.

Cell Transfection : Bovine eNOS, human iNOS, murine nNOS cDNAs in pcDNA3 and HA-tagged wild type Akt, Akt (K179M) or myr-Akt in pCMV6 were generated by standard cloning. myr-nNOS in pcDNA3 is generated by PCR and the eNOS N-myristoylation consensus site (MGNLKSVG, SEQ ID NO: 1) fused in the frame for the second amino acid of the nNOS coding sequence A new amino terminus containing was mixed. In preliminary experiments in COS cells, these constructs were N-myristolised based on the integration of ³ H-myristic acid, whereas the original nNOS was not so treated and targeted at the membrane portion of the cell. About 60% of the protein, while only 5-10% of nNOS was connected to the membrane in COS cells. Mutations in putative Akt phosphorylation sites in eNOS were generated using the Quick Change site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. All mutants were verified by DNA sequencing. COS-7 cells were plated (100 mm dish) and transfected with NOS (7.5-30 mg) and Akt (1 mg) plasmid using calcium phosphate. To balance all transfections, expression vectors for β-galactosidase cDNA were used. 24 to 48 hours after transfection, the expression of the appropriate protein (40-80 mg) was eNOS mAb (9D10, Zymed), HA mAb (12CA5, Boehringer Mannheim), iNOS pAb (Zymed Laboratories) or nNOS mAb (Zymed). Laboratories) was confirmed by Western blot analysis.

NO release from transfected COS cells: 24 to 48 hours after transfection, the medium was nitrite (NO ₂ ⁻ ), aqueous solution by NO specific chemiluminescence as described (Sessa et al ., 1995). In order to determine the stable breakdown product of NO. The medium was deproteinized and the sample containing NO ₂ ⁻ was refluxed in glacial acetic acid containing sodium iodine. Under these conditions, NO ₂ ⁻ was quantitatively reduced to NO quantified by a chemiluminescence detector after reacting with ozone in a NO analyzer (Sievers, Boulders, CO). In all experiments, NO ₂ ⁻ release could be inhibited by NOS inhibitors. In addition, NO ₂ ⁻ release from cells transfected with β-galactosidase cDNA was subtracted from the control for background levels of NO ₂ ⁻ found in serum or medium. In some experiments, cGMP accumulation in COS was used as a biological assay for NO production as described.

NOS Activity Assay : The conversion from ³ HL-arginine to ³ HL-citrulline was used to measure NOS activity in COS cells or endothelial filtrates as previously described by Garcia-Cardena et al . (1998).

In Vivo and Ex vivo Phosphorylation Studies : For in vivo phosphorylation studies, COS cells were transfected overnight with cDNA for wild type or S635, 1179A eNOS and HA-Akt. 36 hours after transfection, cells were placed in minimal essential medium of Dulbeco-phosphate-free phosphate Dulbecco supplemented with 80 μCi / ml of ³² P orthophosphoric acid for 3 hours. Some cells were pretreated with wortmannin (500 nM) in phosphate-free medium for 1 hour and during labeling. The filtrate was harvested, eNOS was dissolved and partially purified by ADP Sepharose Affinity Chromatography as described previously and ³² P integration into eNOS was visualized by radioautomography after SDS-PAGE (7.5%). The amount of eNOS protein was confirmed by western blotting on eNOS. For in vitro phosphorylation studies, recombinant eNOS purified from E. coli was incubated with wild type or kinase inactive Akt immunized from transfected COS cells. eNOS was dissolved in ³² P g-ATP (2 ml, specific activity 3000 Ci) in a buffer containing HEPES (20 mM, pH = 7.4), MnCl ₂ (10 mM), MgCl ₂ (10 mM) and immunoprecipitated Akt at room temperature for 20 minutes. / mmol), ATP (50 mM), DTT (1 mM).

In an experiment investigating the in vitro phosphorylation of wild-type and mutant eNOS, recombinant Akt (1 mg) purified from baculovirus infected SF9 cells was used wild-type or S1179A eNOS (2.4 mg) using essentially the conditions described above. , Purified from E coli). Protein was degraded by SDS-PAGE and ³² P integration and the amount of protein was measured by Coomassie staining as described above.

In the study identifying labeled eNOS peptides, immunoprecipitated Akt was recombinant eNOS cultured as described above. The sample was spread on SDS-PAGE, the eNOS band was sorted on the gel, and the resulting trypsin fragment was purified by RP-HPLC. Peptide mass and ³² P integration were investigated and protruding labeled peaks were also analyzed by mass spectrometry. In another experiment, peptides corresponding to potential Akt phosphorylation sites were synthesized, purified by HPLC and verified by mass spectrometry (WMKeck Biotechnology Resource Center, Yale University School of Medicine). The wild type peptide was 1174RIRTQSFSLQERHLRGAVPWA1194 (SEQ ID NO: 2) and the mutant peptide was identical except that S1179 was changed to alanine. In vitro kinase reactions were essentially as described above and incubated peptide (25 mg) with recombinant Akt (1 mg). The reaction was then measured by Serenkov counting the amount of phosphate filtered and mixed on the phosphocellulose filter.

Adenovirus Infection and NO Release in Endothelial Cells : As described previously in bovine pulmonary microvascular endothelial cells (BLMVEC) in 100 mm dishes (for basal NO release and NOS activity analysis) or in C6 well plates (for stimulated NO). (Garcia-Cardena et al., 1996a). BLMVEC is a 200 MOI adenovirus, HA-tagged inactive phosphorylation mutant Akt (AA-Akt; Alessi et al ., 1996) or carboxyl terminal HA-tagged, containing β-galactosidase 29 for 4 hours. It was infected with constitutively active Akt (myr-Akt). Virus was removed and cells left for recovery in complete medium for 18 hours. In preliminary experiments with β-galactosidase virus, these conditions were optimal for infecting 100% of the culture. Media was collected for NO release 24 hours after initial infection with virus for measurement of basal NO production. To measure stimulated NO release, cells were then washed with serum free medium and then stimulated with VEGF (40 ng / ml) for 30 minutes. In some experiments, the calcium phosphate dependence of NOS was measured 24 hours after adenovirus infection. Infected cells were lysed in NOS assay buffer containing 1% NP40, and wash solution material used for activity. The filtrate was incubated with EGTA buffered calcium to obtain an appropriate amount of free calcium in the culture.

Statistics : Data are expressed as mean ± SEM. Comparisons were made using a two-tailed Student's t-test or ANOVA with a post-hoc test where appropriate. The difference was considered significant as p <0.05.

Example  One: Akt end eNOS NO production from production

To investigate the possibility that the downstream effector of PI-3 kinase, Akt, can directly affect NO production, COS-7 cells (which do not express NOS) were eNOS and wild type Akt (HA). -Akt), or kinase inactive Akt (HA-Akt K179M) and was cotransfected and accumulation of nitrite (NO ₂ ⁻ ) was measured by NO specific chemiluminescence. Transfection of eNOS results in markedly enhanced effects by increased NO ₂ ⁻ accumulation and cotransfection of wild type Akt, but not the kinase inactive variant (FIG. 1A). The same results are obtained using cGMP as a biological assay for biologically active NO. Under these experimental conditions, Akt was catalytically active as measured by Western blotting and Akt activity assays using phospho-Akt specific Ab (recognized as serine 473; not shown) (see FIG. 2A). ). Transfection of the constitutively active form of Akt (myr-Akt) ranges from cGMP accumulation (analyzed in COS cells) from 5.5 ± 0.8 to 11.6 ± 0.9 pmol cGMP / mg protein (eNOS alone or eNOS and myr-, respectively). While kinase inactive Akt does not affect cGMP accumulation (5.8 ± 0.8 pmol cGMP, n = 4 experiments). As shown in the inset, the same levels of eNOS and Akt expressed in COS cell filtrates indicate that Akt regulates eNOS to increase NO production under basic conditions.

eNOS is a dual acylated peripheral membrane protein targeted in the Golgi region and in the plasma membrane of endothelial cells (Liu et al ., 1997; Garcia-Cardena et al ., 1996a; Shaal et al ., 1996) classification is required for the effective production of NO in response to agonist challenge (Sessa et al., 1995; Liu et al., 1996; Kantor et al., 1996). To examine whether eNOS activity by Akt requires membrane sorting, COS-7 cells were cotransfected with cDNA for Akt and myristoylation, palmitoylation defective mutants of eNOS ( G2A eNOS) and NO release were quantified. As shown in FIG. 1B, the fact that Akt does not activate the unacylated form of eNOS indicates that the classification of both proteins on the membrane is required for its functional interaction (Downward et al ., 1998). Next, it was examined whether Akt could activate neuronal and inducible NOS (nNOS and iNOS, respectively), which are structurally similar but distinct soluble NOS isoforms. The simultaneous transfection of Akt with nNOS and iNOS did not further increase NO release, indicating the specificity of Akt for eNOS. However, in order to enhance interaction with biological membranes, the addition of N-myristolation sites to nNOS resulted in Akt stimulation of nNOS in a manner similar to that shown by eNOS, with both isoforms immobilized. When sensitive to activation by Akt kinase.

Example  2: eNOS  Production of mutations

The experiments imply that Akt by phosphorylation of eNOS can modulate NO release from progenitor cells. In fact, two putative Akt phosphorylation motifs (RXRXXS / T) are present in eNOS (serine 635 and 1179 in bovine eNOS or serine 633 and 1177 in human eNOS) and one motif is present in nNOS (serine 1412 and human in rats). 1415) nNOS Unclear motifs were found in iNOS. To examine whether eNOS is a potential substrate for in vitro Akt phosphorylation, COS cells were transfected with HA-Akt or HA-Akt (K179M) and kinase activity was assessed using recombinant eNOS as substrate. As shown in FIG. 2A, active kinase phosphorylates histones 2B and eNOS (69.3 ± 2.9 and 115.4 ± 3.8 pmol of ATP / nmol substrate, n = 3, respectively), while inactive Akt significantly inhibits histone or eNOS phosphorylation. Do not increase. To determine if the putative Akt phosphorylation site in eNOS is responsible for the integration of ³² P, the two serines were mutated to alanine residues and the ability of Akt to stimulate wild-type and mutant eNOS phosphorylation was examined in native COS cells. It became. Transfected cells were labeled with ³² P-orthophosphate, eNOS was partially purified by ADP-Sepharose affinity chromatography, and phosphorylation status and protein levels were quantified. As shown in FIG. 2B, co-expression of Akt doubles phosphorylation of eNOS compared to stimulated cells. Pretreatment of eNOS / Akt transfected cells with wortmannin eliminated an increase in Akt induction in phosphorylation. Moreover, mutations to alanine residues at serine 635 and 1179 eliminated Akt dependent phosphorylation of eNOS, indicating that these residues could serve as potential phosphorylation sites in original cells.

To directly identify residues phosphorylated by Akt, wild-type eNOS was incubated with immunopurified Akt and phosphorylation sites were measured by HPLC and then by MALDi-mass spectrometry (MALDi-MS). As shown in FIG. 2C, the primary ³² P labeled trypsin phosphopeptide co-elutes with the synthetic phosphopeptide (amino acids 1177-1185 with phosphoserine at position 1179) and the same mass ions as measured by linear mode MS Has Using reflectron mode MALDi-MS monitoring, the labeled trypsin peptide and standard phosphopeptide showed loss of HP ₃ O ₄ , indicating that the trypsin peptide was phosphorylated. In addition, mutation of S1179 to A significantly reduced Akt-dependent phosphorylation of eNOS compared to wild type protein (FIG. 2D). The same result was derived from wild type or eNOS S1179A as substrate for recombinant Akt (wild type peptide mixed with 24.6 ± 3.7 nmol phosphate / mg compared to alanine mutant peptide mixed with 0.22 ± 0.02 nmol phosphate / mg; n = 5) Obtained using the peptide (amino acids 1174-1194). Collectively, these data show that eNOS is a substrate for Akt and the primary site of phosphorylation is serine 1179 (serine 1177 in human eNOS).

Next, the functional significance of the putative Akt phosphorylation site in serine 635 and the site identified in serine 1179 was examined. Transfection of COS cells with double mutant eNOS S635 / 1179A eliminates Akt dependent NO release. Mutation of serine 635 to alanine does not reduce NO release but eNOS S1179A eliminates Akt dependent activation of eNOS (FIG. 3). These results indicate that serine 1179 is functionally important for NO release. The mutation of serine 1179 to aspartic acid (eNOS S1179D) to replace the negative charge supplied by the addition of phosphate resembles the activation state induced by Akt (S1177D in human eNOS). All site-related mutants were fully expressed (see inset Western blot) and maintained NOS catalytic activity in the cell filtrate (in COS cells transfected with eNOS only, NOS activity was determined by wild type, S1179A, S635, 1179A and eNOS S1179D, respectively). 85.3 ± 27.0, 71.9 ± 2.9, 80.8 ± 23.2 and 131.8 ± 36.7 pmol produced L-citrulline / mg protein from the filtrate of expressing COS cells, n = 3 experiments).

To test whether Akt mediates NO development from endothelial cells, bovine lung microvascular endothelial cells (BLMVECs) are activated Akt (myr-Akt), activation deficient Akt (AA-Akt), or β-galactosidate as a control. It was infected with adenovirus expressing an aze and the accumulation of NO was measured. As shown in FIG. 4A, myr-Akt stimulates basal NO production from BLMVEC, while cells infected with β-galactosidase or activation deficient Akt released low levels of NO close to the limit of detection. Together with similar results in COS cells, these data indicate that Akt phosphorylation of eNOS is sufficient to regulate NO production at the remaining levels of calcium. Indeed, NOS activity measured in filtrates from myr-Akt infected BLMVEC was analyzed in BLMVECs infected with β-galactosidase virus, with enzyme susceptibility to activation by calcium, analyzed in fixed calmodulin concentrates. It is shown to be increased compared to the NOS activity shown (FIG. 4B). Interestingly, calcium susceptibility of NOS activity in cells infected with activation deficient Akt was significantly inhibited compared to both myr-Akt and β-galactosidase infected cells.

Treatment of endothelial cells with VEGF is known to activate Akt 23 and the release of NO through the instrument is partially hampered by inhibitors of PI-3 kinase (Papapetropoulos et al ., 1997). To examine the functional link between VEGF as an antagonist for NO release and activation, BLMVEC was infected with adenovirus for myr-Akt, AA-Akt or β-galactosidase and VEGF stimulated NO release was quantified. . As shown in FIG. 4C, infection of endothelial cells according to myr-Akt increases VEGF proliferative NO production while AA-Akt decreases NO release. These results suggest that Akt participates in the signal transduction events required for both basic and stimulated NO production in endothelial cells.

Collectively these data show that Akt can phosphorylate eNOS on serine 1179 (serine 1177 in human eNOS) and that phosphorylation increases the enzyme's ability to produce NO.

Example  3

Materials and methods

eNOS Constructs and Protein Purification—In plasmid pCW, wild-type bovine eNOS was expressed as previously described as groELS in E. coli BL21 cells (Martasek et al ., 1996). S1179D mutant eNOS for expression in E. coli was generated as follows. eNOS S1179D (Fulton et al ., 1999) in pcDNA 3 was digested with XhoI / XbaI, subcloned into the same site of eNOS in pCW and co-expressed with groELS. Isolation of recombinant eNOS was performed as previously reported (Roman et al ., 1995; Martasek et al ., 1999) with the following modifications. eNOS was eluted from 2'5'-ADP Sepharose with 10ml NADPH or 10mM 2'-AMP. The amount of eNOS was quantified using a peak absorbance of 409-412 nm with an extinction coefficient for a heme content of 0.1 μM ⁻¹ cm ⁻¹ . Purity of eNOS was measured by 7.5% SDS-PAGE followed by Coomassie staining. Low temperature SDS-PAGE was performed in the same manner, except that the sample was not boiled and electrophoresis was performed on a slurry of ice / water at 4 ° C. (Klatt et al ., 1995). In experiments in which NOS cofactors (L-arginine, calmodulin and NADPH) were titrated, they were omitted from the purification and storage of the enzyme and incubated as described below.

Assay for NOS Activity —NO production was measured by hemoglobin capture assay as described (Kelm et al ., 1988). In brief, the reaction mixture was eNOS (0.5-2.5 μg), oxyhemoglobin (8 μM), L-arginine (100 μM), BH ₄ (5 μM), CaCl ₂ (120 μM) at HEPES buffer (50 mM), pH 7.4. ), Calmodulin (120-200 μM) and NADPH (100 μM). In determining the calcium EC50 value for eNOS, the reaction mixture was changed as follows: MOPS buffer (10 mM, pH 7.6), KCl (100 ml) and CaM (250 nM). Under these conditions, free calcium was calculated using a WinMAXC program, version 1.8 (Stanford University), with a Kd of 2.2 × 10 ⁻⁸ M. Accurate free calcium concentration was achieved by mixing the appropriate proportions of 10 mM K ₂ EGTA and 10 mM CaEGTA colonies solutions (Molecular Probes). NOS activity was monitored for linearity at 401 nm over 2 minutes, and NO production was calculated based on the change in absorbance using an extinction coefficient of 60 mM ⁻¹ cm ⁻¹ . All reactions were performed at 23 ° C. and each data point represents 3-8 observations. An extinction coefficient of 0.0033 μM ⁻¹ cm ⁻¹ at 276 nm was used to measure calmodulin concentration. Production of NO using this method was completely blocked by the addition of nitro-L-arginine (1 mM). When the inactivation of eNOS was measured by the addition of EGTA (200-800 μM) to the reaction mixture, the chelator was added 1 minute after the start of the reaction with NADPH. The same conditions were used when NADPH-cytochrome c reductase activity was examined. These reactions included CaM (120 nM) and CaCl ₂ (200 μM) at 0.5-mL volumes with eNOS (0.5 μg).

The conversion of L-arginine to L-citrulline was analyzed as already described by Bredt et al . (1990). In brief, eNOS (0.25-2 μg) was incubated in the following reaction mixture at 23 ° C. for 3-10 minutes: 3 pmol L- [3H] arginine (55 Ci / mmol) with 50-100 μl final reaction volume. , 10-300 μM arginine, 1 mM NADPH, 120-200 nM calmodulin, 2 mM CaCl2, and 30 μM BH4. The reaction was terminated by addition of 0.5 ml 20 mM HEPES, pH 5.5, including 2 mM EGTA and EDTA. The reaction mixture was placed on a Dowex AG50WX8 and the flow-through was calculated with a Packard 1500 liquid scintillation analyzer.

Analysis of Reductase Activity —Cytochrome as NADPH-cytochrome c reductase activity and 2,6-dichlorophenolrindophenol (DCIP) reduction have already been described by Martasek et al . (1999) and Master et al . (1967). Absorbance change at 550 nm was measured using an extinction coefficient of 0.021 μM ⁻¹ cm ⁻¹ for both c and DCIP. In short, the reaction mixture (1 ml) comprises cytochrome c (90 μΜ); DCIP (36 μM), HEPES buffer (50 mM), NaCl (250 mM), NADPH (100 μM), calmodulin (120 nM) and CaCl 2 (200 μM); Or other materials as indicated. The reaction was monitored for 60 seconds (at 23 ° C.) after addition of eNOS. When inactivation of reductase activity was measured by the addition of EGTA (200-800 μM), a chelator was added 1 minute after the start of the reaction and monitored for an additional 1 minute. The reaction included HEPES buffer (50 mM), CaM (120 nM), and CaCl ₂ (200 μM) at pH 7.6 and was initiated with NADPH (100 μM). No NaCl was added in the experiments that examined the EGTA inactivation of eNOS similar to the conditions used in the hemoglobin capture experiment. The addition of NOS inhibitor did not affect the rate of cytochrome c reduction (not shown). Measurement of calcium EC50 for eNOS was performed as described above for hemoglobin capture assay.

Data Analysis and Statistics -All data were expressed as mean ± SE. At least three measurements were made with at least three different sets of enzymes for each data set. Wild-type and mutant enzymes were simultaneously purified to regulate activity changes between preparations. Statistical significance was measured using the Student's t test and p <0.05 was considered statistically significant.

result

Expression and Purification of eNOS - both wild type and S1179D eNOS were expressed and purified from E. coli. In 1.6 liters of culture, approximately 2.5-4.0 mg of eNOS was typically recovered using 2'5'-ADP Sepharose 4B chromatography. As shown in FIG. 5A, both enzymes were> 90% pure based on Coomassie staining. These results are typical, as shown from seven independent preparations for both wild type and S1179D eNOS prepared side by side. Both enzymes were mainly in dimeric form, as shown by cold SDS-PAGE (FIG. 5B).

eNOS S1179D has greater NO synthase and reductase activity than wild type eNOS -next, the activity of wild type and S1179D eNOS was compared by measuring the rate of NO production. S1179D eNOS showed greater rotation compared to wild-type enzyme (84 ± 6 vs 27 ± 1 min ⁻¹ , separate paired preparation of n = 6 enzyme). Km values by L-arginine were similar for wild type and S1179D eNOS (FIG. 6A, 1.8 vs 2.5 μM, respectively; see Table 1).

Since the rate of electrons flowing from the reductase domain to the oxygenase domain is important for NOS catalysis, the increase in S1179D eNOS activity was examined to determine if it contributed to enhancing reductase activity. When both DCIP and cytochrome c reduction were examined, a significant increase in activity was observed for S1179D compared to wild-type eNOS (FIG. 6B). Moreover, this increase was highlighted by the presence of CaM which increased the overall activity for both enzymes. Basal cytochrome c reduction was four times higher for S1179D compared to wild type eNOS in the absence of CaM. The magnitude of CaM stimulated cytochrome c reduction was greater than for S1179D eNOS (749 + 35 vs. 1272 ± 55 min ⁻¹ for wild-type and S1179D eNOS); The level of stimulation by CaM was 8-fold for wild-type eNOS compared to only 3-fold for S1179D eNOS.

Next, it was determined whether S1179D eNOS produced more superoxide than wild type eNOS, which could reduce cytochrome c. As expected, no superoxide dismutase inhibitable cytochrome c reduction was found in the absence of CaM (in superoxide anion production index) and wild type eNOS (86 ± 6 vs 95 ± 88 min). ^-1 ) and for S1179D eNOS (278 ± 9 vs 288 ± 7 min ⁻¹ , n = 3-5) as previously reported. However, in the presence of CaM, superoxide dismutase was cytochrome c reduced for both wild type (610 ± 51 vs 866 ± 8 min ⁻¹ ) and S1179D (1179 ± 43 vs 1518 ± 19 min ⁻¹ ) eNOS. Reduced the speed. The relative decrease in cytochrome c activity in the presence of superoxide dismutase was similar for both enzymes (30% for wild type and 22% for S1179D eNOS), and the increased reductase activity of S1179D compared to wild type eNOS showed It is not due to uncoupling.

NADPH -dependent NO formation and reductase activity were wild-type and S1179D it is invalid in the case of eNOS - NADPH binding site is a NADPH dependence of NO production and cytochrome c reduction doors when it lies in the immediate vicinity of eNOS in Ser-1179 was examined. S1179D eNOS had a higher maximum turnover (k _cat ) than wild type enzyme based on NO production, analyzed in the presence of CaM (FIG. 7A). Increased k _cat was associated with a four fold increase in Km for S1179D eNOS compared to wild type eNOS. In the absence of CaM, k _cat for NADPH dependent cytochrome c reduction was greater for eNOS S1179D than for wild type eNOS (290 vs. 70 min ⁻¹ , respectively; FIG. 7B). In the presence of CaM, k _cat for cytochrome c reduction was significantly higher for S1179D compared to wild type eNOSdp (840 vs. 460 min ⁻¹ , respectively). Km values for NADPH did not change in the absence or presence of CaM (0.40 and 0.75 μM for wild-type eNOS and 2.0 and 1.9 μM for S1179D eNOS, respectively, in the absence and presence of CaM).

EC50 values for CaM are wild type and S1179D were not changed between eNOS - S1179D increase the activity of the parent to determine that the contribution to the chemical modification, the NOS activity and cytochrome c reduction analysis in the presence of excess all NOS cofactors as a function of CaM concentration of the enzyme for CaM of eNOS Kinetics were examined. In the case of k _cat is wild for CaM activation of NOS activity 22 min ^-1 was the case of S1179D eNOS was 43min ^-1. The transformation of the data, normalized to the difference in k _cat , showed a slight change to the left in the curve for S1179D eNOS but a slight difference in EC50 for CaM, and reported data for phospho-eNOS (Mitchell et al. , 1999). EC50 values were 8 nM for wild type and 7 nM for S1179D eNOS (FIG. 8A). NADPH mediated cytochrome c reduction was measured. K _cat for CaM activation of cytochrome c reduction was about two times higher for S1179D eNOS compared to wild-type enzyme (1140 and 620 min ⁻¹ for S1179D and wild-type eNOS, respectively). Conversion of normalized data for the difference in k _cat revealed a small difference in EC50 values for CaM between wild type and S1179D eNOS (21 vs 13 nM; FIG. 8B).

Comparison of Calcium Activation and Inactivation of eNOS —Previous experiments showed that the “sure calcium susceptibility” of eNOS was mostly enhanced in cells expressing phospho-eNOS or S1179D eNOS, indicating that phosphorylation was increased in calcium / CaM It has been shown to alter the affinity of activation (Dimmeler et al ., 1999; Fulton et al ., 1999). As normalized for the difference in maximal activity, as shown in FIG. 8C, calcium dependence increased slightly for S1179D eNOS (p <0.05, bidirectional analysis of variability). EC50 values for the wild-type and S1179D eNOS calcium were also slightly different (310 and 250 nM calcium, respectively) and as measured by NO production (in the presence of 250 nM CaM). As shown in the inset of FIG. 8C, the increase in free calcium concentration actually increased the S1179D eNOS rotation to a greater extent than that shown with the wild type enzyme. Moreover, the EC 50 for calcium analyzing cytochrome c reduction was similar to that obtained by measuring NO production (FIG. 8D; 290 and 220 nM for wild type and S1179D eNOSdp, respectively). Again, Vmax for calcium activation of S1179D eNOS rotation was greater than wild type (FIG. 4D, inset).

To examine whether the inactivation of S1179D eNOS differs from that of wild-type enzyme, the decline in eNOS activity after chelation of calcium and EGTA was measured. In these experiments, all NOS cofactors were added in the presence of calcium (200 μM), NO production was investigated for 1 minute, then different concentrations of EGTA were added and irradiated for an additional 1 minute. As shown in FIG. 9A, the addition of EGTA to wild type and S1179D eNOS reduced NO production in a concentration dependent manner. However, NO production from S1179D eNOS was less sensitive to the addition of EGTA. In other words, wild-type eNOS activity decreased faster at lower EGTA concentrations than S1179D eNOS activity. Large differences in activity between the enzymes were seen at 400 μM EGTA. Moreover, at 600 μM EGTA, no activity was detected for wild-type eNOS, but the remaining activity was still detected for S1179D eNOS. Similar results were obtained using cytochrome c reduction (FIG. 9B), and a significant difference in activity was seen with 400 and 600 μM chelators added to the reaction. However, at the highest concentration of EGTA, the remaining reductase activity was found for both wild type and S1179D eNOS.

In summary, bovine endothelial nitric oxide synthase (eNOS) is phosphorylated at serine 1179 by direct protein kinase Akt (Fulton et al., 1999) and human endothelial nitric oxide synthase is phosphorylated at serine 1177 by direct protein kinase Akt. (Dimmeler et al ., 1999). Mutation of residue 1179 to negatively charged aspartate in bovine eNOS essentially increases nitric oxide (NO) production in the absence of antagonist muscle attack.

It is to be understood that the foregoing review and examples are merely present to detail preferred embodiments. Therefore, it will be apparent to those skilled in the art that various modifications and equivalents can be made without departing from the spirit and scope of the invention. All references, papers and patents, both above and below are hereby incorporated by reference in their entirety.

Reference

Additional references in which the entire citation is not provided in this text are fully incorporated herein.

Claims (15)

A pharmaceutical composition for promoting coronary collateral vessel development comprising a transgene encoding an Akt polypeptide as an active ingredient. The method of claim 1, Wherein said transgene is expressed from a ventricular myocyte-specific promoter, a smooth muscle cell specific promoter, or an endothelial cell specific promoter. The method of claim 2, The ventricular myocyte specific promoter is a pharmaceutical composition having a sequence of ventricular myosin light chain-2 or myosin heavy chain promoter. A pharmaceutical composition for treating peripheral-vascular deficient disease, comprising a transgene encoding an Akt polypeptide as an active ingredient. A pharmaceutical composition for treating myocardial ischemia comprising a transgene encoding an Akt polypeptide as an active ingredient. 6. The pharmaceutical composition of claim 5, wherein the expression of the transgene is limited to cardiac myocytes. The pharmaceutical composition of claim 6, wherein the expression of the transgene is limited to ventricular myocytes. The pharmaceutical composition according to any one of claims 1 to 7, wherein said transgene is encoded by a replication-defective vector. The pharmaceutical composition of claim 8, wherein the vector having replication defects is adenovirus. The pharmaceutical composition according to any one of claims 1 to 7, wherein said transgene is transfected into cells in vitro. The pharmaceutical composition of claim 10, wherein the transfected cells are delivered to a patient. A pharmaceutical composition for treating cardiovascular disease comprising a transgene encoding an Akt polypeptide as an active ingredient. The pharmaceutical composition of claim 12, wherein the cardiovascular disease is selected from the group consisting of heart failure, myocardial infarction, restenosis, post angiogenesis stenosis, stent stenosis, and bypass transplant failure. A pharmaceutical composition for treating an erectile dysfunction comprising a transgene encoding an Akt polypeptide as an active ingredient. 15. The transgene of any one of claims 1, 4, 5, 12 and 14, wherein the transgene is transported and used intravascularly, intramuscularly, intradermally, intraperitoneally, or in arteries. Pharmaceutical compositions.

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