Agonists of hypophysis adenylate cyclase peptide-activator receptor-3(r3)(pacap) and methods for their pharmacological using
FIELD: medicine, endocrinology, biochemistry, peptides.
SUBSTANCE: invention represents new peptides that act in vivo as stimulators of insulin secretion by pancreas beta-cells in glucose-dependent regimen. Such peptides as enhancers of insulin secretion stimulate insulin secretion by insula cells in rats in vitro and in vivo. Proposed peptides represent a new way for treatment of patients with reduced secretion of endogenous insulin, in particular, for treatment of diabetes mellitus type 2. In particular, invention represents polypeptide taken among the specific group VIP/PACAP-related polypeptides or their functional equivalents. Also, invention claims method for preparing both recombinant and synthetic peptides. The advantage of invention involves new peptides that can be used as stimulators of insulin secretion.
EFFECT: improved and valuable medicinal properties of peptides.
47 cl, 4 tbl, 10 dwg, 18 ex
The technical field to which the invention relates.
The present invention relates to a new identified polypeptides and the use of such polypeptides for therapeutic purposes. More specifically, the polypeptides of the present invention is applicable to stimulate insulin secretion by beta cells of the pancreas in a glucose-dependent mode, which thereby provides a possible way to treat those subjects who have a metabolic disorder such as diabetes or impaired glucose tolerance, which is a pre-diabetic condition.
Diabetes is characterized by impaired glucose metabolism, which, among other things, is manifested in the increase of glucose in blood of patients-diabetics. The underlying specified defects classify diabetes into two major groups: type 1 diabetes or insulin-dependent diabetes mellitus (IDDM), which occurs when the patient experiences a lack of beta cells in the pancreas that produce insulin, and type 2 diabetes or insulin-dependent diabetes mellitus (NIDDM), which is present in patients with impaired function of beta-cells and altered activity of insulin.
Patients with type 1 diabetes type now being treated with insulin, while the majority of patients with type 2 diabetes type lacats means which stimulate the function of beta cells, or by using tools that increase the susceptibility of tissue of patients to insulin. Over time, almost half of patients with type 2 diabetes type lose the ability to respond to these funds, then they are moved on insulin. Drugs currently used in treatment of diabetes of the 2nd type are:
Inhibitors α-glucosidase (PRECOSE®, VOGLIBOSETMand MIGLITOL®). Inhibitors α-glucosidase reduce the movement of glucose after eating a meal due to delay glucose absorption from the intestine. These medicines are safe and provide treatment of patients with diabetes, as expressed in weak and moderate. However, in the scientific literature reported side effects on the gastrointestinal tract.
The insulin sensitizers. The insulin sensitizers - these are medicines which increase the body's response to insulin. Thiazolidinedione, such as REZULINTM(troglitazone), activate γ-receptor PPAR and modulate the activity of a group of genes that are not yet fully defined. Although these medications are effective, they are associated with hepatotoxicity. Due to the toxic effect on the liver REZULIN was withdrawn from the pharmaceutical market.
Tools that increase insulin secretion (sulfonylureas and other means, to the E. through active ATP-dependent potassium channel). SPS provide the standard treatment of diabetes of the 2nd type, which has a fasting glucose from weak to moderate. Limitations on the application of SPS associated with possible induction of hypoglycemia, weight gain and a high frequency of primary and secondary cases of unsuccessful treatment. In 10-20% of the initial treatment of patients unable to detect a significant effect of treatment (primary cases of unsuccessful treatment). Secondary cases of unsuccessful treatment reveal another 20-30% of patients after 6 months of use SFM. Insulin therapy becomes necessary for 50% receiving SFM after 5-7 years of treatment (A.J.Scheen et al., 1989, Diabetes Res. Clin. Pract., 6, 533-543).
GLUCOPHAGE™ (Metformin hydrochloride) is biguanides, which reduces the amount of glucose in the blood by inhibiting the release of glucose from the liver and strengthen the peripheral uptake and utilization of glucose. This agent is effective in reducing blood glucose in patients with mild to moderate severity and has no side effects associated with weight gain or possible induction of hypoglycemia. However, GLUCOPHAGE has a number of side effects, including gastrointestinal disorders and acidosis. GLUCOPHAGE is contraindicated for diabetics older than 70 years and patients with impaired kidney or liver. Finally, GLUCOPHAGE is characterized by the same frequency per the ranks and secondary cases of unsuccessful treatment, what and engineering.
Insulin is prescribed after diet, exercise and oral medications are not able to adequately control the level of glucose in the blood. This treatment has the disadvantages associated with the need for injections, the possibility of hypoglycemia and a set of excess weight.
Taking into account the existing problems in modern methods of treatment therapy of type 2 diabetes type needs new approaches. In particular, new methods of treatment to maintain normal (glucose-dependent) insulin secretion. These new drugs should have the following characteristics: dependence of the stimulation of insulin secretion from glucose, i.e. the induction of insulin secretion only in the presence of high glucose in the blood; low frequency of primary and secondary cases of unsuccessful treatment and preservation of functioning islet cells. The strategy for the development of new methods of treatment described here is based on the signal mechanism involving cyclic adenosine monophosphate (camp) and its influence on insulin secretion.
Cyclic AMP is a key regulator of the process of insulin secretion. Increasing the amount specified signaling molecule stimulates the closure of potassium channels with subsequent activation mechanism proteinsin the s-A. Closing potassium channels causes depolarization of the cells and subsequent opening of calcium channels, which in turn leads to the exocytosis of insulin granules. In the absence of glucose and low concentrations there is a slight secretion of insulin or she is absent (A.Weinhaus et al., 1998, Diabetes, 47, 1426-1435). Tools that increase the secretion, such as peptide-activator pituitary adenylate cyclase (RASAR) and GLP-1, use of camp for the regulation of insulin secretion in a glucose-dependent mode (.Komatsu et al., 1997, Diabetes, 46, 1928-1938). Tools that increase insulin secretion, mechanism of action which is based on the increase of camp, for example, GLP-1 and RACER, also can increase the synthesis of insulin in addition to insulin secretion (G.Skoglund et al., 2000, Diabetes, 49, 1156-1164; P.Borboni et al., 1999, Endocrinology, 140, 5530-5537).
RACER is a potent stimulator of glucose-dependent insulin secretion by beta cells of the pancreas. Were described three different types of receptors RACER (R1, R2, and R3) (A.Harmar et al., 1998, Pharmacol. Reviews, 50, 265-270). RACER does not show selectivity for the type of receptor with comparable activity and affinity to all three types of receptors. R1 can be found primarily in the CNS, whereas R2 and R3 are more widely distributed. R2 localized in the Central nervous system, as well as in the liver, lungs and intestines. R3 is in the Central nervous system, pancreas, skeletal muscles, with whom rdte, kidney, adipose tissue, testis and stomach. Recent research has confirmed that R3 is responsible for the secretion of insulin by beta cells (N.Inagaki et al., 1994, Proc. Natl. Acad. Sci. USA, 91, 2679-2683). Insulinotropic action RACER mediated GTP-binding protein Gs. The accumulation of intracellular camp, in turn, activates nonspecific cation channels in beta-cells, increasing the CA++and stimulates the exocytosis of insulin-containing secretory granules.
RACER is the latest representative of the superfamily, metabolic, neuroendocrine and neurotransmitter peptide hormone whose action is mediated by a mechanism of signal transmission with the participation of camp (Arimura, 1992, Regul. Peptides, 37, 287-303). Bioactive peptides are released from the biosynthetic precursor in two molecular forms - in the form of a 38-amino acid peptide (RASAR-38) and/or 27-amino acid peptide (RASAR-27) with aminirovanie With ends (Arimura, CIT. above).
The highest concentration of the two forms of the peptide found in the brain and testes (review Arimura, CIT. above). A shorter form of the peptide - RASAR-27 - shows 68% of the structural similarities with vasoactive intestinal polypeptide (VIP). However, the distribution of RASER and VIP in the Central nervous system indicates that these structurally related peptides have different neurotransmitter function (Kves et al., 1991, Neuroendocrinology, 54, 159-169).
Recent studies have shown a variety of biological actions RACER-38 - from participation in reproductive processes (McArdle, 1994, Endocrinology, 135, 815-817) to the ability to stimulate insulin secretion (Yada et al., 1994, J.Biol. Chem., 269, 1290-1293).
Vasoactive intestinal peptide (VIP) is a peptide of 28 amino acids, which was first isolated from the upper part of the small intestine boar (Said & Mutt, 1970, Science, 169, 1217-1218; U.S. patent No. 3879371). This peptide belongs to the family of structurally related small polypeptides, which includes helderman, secretin, somatostatin and glucagon. The biological effects mediated by VIP activation localized on cell membrane proteins, receptors involved in signal system with the participation of intracellular camp. These receptors source were known as VIP-R1 and VIP-R2, however, it was subsequently established that they are the same receptors that PACAP-R2 and PACAP-R3. VIP shows comparable levels of activity and action in respect of PACAP-R2 and PACAP-R3.
To improve the stability of the VIP in the lung fluid of humans with a series of options VIP (Bolin et al., 1995, Biopolymers, 37, 57-65) was formed in order to increase the propensity of this peptide to the formation of spiral and reduce its proteolytic destruction. The replacement was carried out according to the provisions of amino acids 8, 12, 17 and 25 to 28, for which b is La shows the importance for binding to the receptor. Moreover, the sequence "GGT" was attached as a label to the end of the mutant variants of VIP with the hope of more effective up the spiral. Finally, for further stabilization of the helix was synthesized a number of cyclical variations (U.S. patent No. 5677419). Although described efforts were not aimed at achieving receptor selectivity, this has resulted in two similar (denoted here as R3P0 and R3P4), which had a more than 100-fold selectivity against PACAP-R3 (Gourlet et al., 1997, Peptides, 18, 403-408; Xia et al., 1997, J.Pharmacol. Exp. Ther., 281, 629-633).
GLP-1 is secreted by L-cell of the intestine after eating and functions as a hormone incretin (i.e. it stimulates dependent glucose output insulin from the beta cells of the pancreas). He is a peptide consisting of 37 amino acids, which are differentially expressed with the gene of glucagon, which is determined by the type of tissue. For GLP-1 were obtained clinical data to demonstrate the beneficial effect of increasing camp levels in beta-cells. Infusion of GLP-1 to patients with trudnoizvlekaemymi diabetes of the 2nd type normalize their levels of fasting blood glucose (.Gutniak et al., 1992, New England J.Med., 326, 1316-1322), and longer infusion improved the function of beta cells to the level characteristic of normal subjects (J.Rachman et al., 1996, Diabetes, 45, 1524-1530). In a recent message, which was shown, that GLP-1 improves the ability of beta cells to respond to glucose in subjects with impaired glucose tolerance (MVpp et al., 1998, Diabetes, 47, 1259-1265). However, all these effects are short-term because of the short time half-life of the peptide. Recently, the company Novo Nordisk has suspended clinical trials of GLP-1. It was reported that this failure was attributed to the very short half-life of the peptide in plasma, comprising a few minutes.
EXENDIN-4TM. The company Amylin Pharmaceuticals conducts clinical trials phase I drug EXENDIN-4 (AC2993) - 39-amino acid peptide that was originally identified in Gila monsters. Recently tests have passed in the second phase. Amylin reported preclinical results, indicating 4-hour efficiency and effectiveness in animal models after subcutaneous, oral and intranasal introduction of AC2993. However, at doses of 0.2 and 0.3 µg/mg often suffered from headaches, postural hypotension, diarrhea and vomiting.
There is a need for an improved peptide, which would have had glucose-dependent activity RASAR, GLP-1 or EXENDIN-4 to enhance the secretion of insulin and also showed weaker side effects.
Brief description of the invention
The present invention represents a new polypeptides that function in vivo as agonists of the receptor for PACAP-R3 (on the - R3) and effective in the treatment of diseases and conditions that can be improved by means possessing the activity of agonists R3. Preferred polypeptides of the present invention are selective agonists R3, with higher performance in relation to R3 from R2 and R1. As an example, but not to limit, these polypeptides stimulate insulin synthesis and its secretion by beta cells of the pancreas in a glucose-dependent mode with a subsequent decrease in the level of glucose in plasma. It was shown that these polypeptides-amplifiers insulin secretion stimulated insulin secretion, islet cells of rat and human in vitro and in vivo. Unlike RACER-27 these polypeptides-amplifiers secretion also reduce the level of glucose in vivo to a greater extent than the control fillers after provocation glucose.
The polypeptides of the present invention provide a new way of treating patients, for example, metabolic disorders such as those caused by reduced secretion of endogenous insulin, in particular diabetes of the 2nd type, or patients with impaired glucose tolerance, which is a pre-diabetic condition, and with a weak change in insulin secretion.
In particular, in one aspect of the present invention presents a polypeptide selected the C group, consisting of SEQ ID nos 11-14, SEQ ID NO 18, SEQ ID NO 21-26, SEQ ID NO 32-36; SEQ ID NO 40-53; SEQ ID NO 57-61; SEQ ID NO 63-99; SEQ ID NO 102-119; SEQ ID NO 121-137; SEQ ID NO 139-177; SEQ ID NO 179, 180, SEQ ID NO 183-202, 322-341, as well as fragments, derivatives and variants that exhibit at least one biological function, which essentially coincides with that of the polypeptides listed in SEQ ID NO ( generally referred to as "polypeptides of the present invention"), including their functional equivalents. The preferred option of the present invention is a polypeptide selected from the group consisting of SEQ ID NO 12, 18, 21-26, 32-35, 41, 43-53, 63, 66, 70-92, 94-99, 102-104, 107, 109, 112-119, 121-137, 139, 140, 142-156, 156-174, 187, 322-341, as well as fragments, derivatives and variants that exhibit at least one biological function, which is essentially identical with that of the polypeptides listed in SEQ ID NO. The preferred variant of the present invention is a polypeptide selected from the group consisting of SEQ ID NO 18, 24, 25, 32, 33, 43-50, 52, 53, 70-87, 92, 98, 99, 104, 107, 112-114, 129-131, 137, 140, 144, 147-151, 156-159 and 161-173, 323, 324, 326, 327, 335, 338, 341 and fragments, derivatives and variants that exhibit at least one biological function, which is essentially identical with that of the polypeptide, listed in SEQ ID NO. The most preferable variant of the present invention is a polypeptide selected from the group consisting of SEQ ID NO 18 32, 43, 45, 47, 50, 52, 71, 72, 83, 86 and 87, as well as fragments, derivatives and variants that exhibit at least one biological function, which is essentially identical with that of the polypeptides listed in SEQ ID NO.
Another alternative embodiment of the present invention are polynucleotide, which encodes polypeptides of the present invention, and the corresponding vectors and cells of the hosts required for recombinant expression of the polypeptides of the present invention. These polynucleotide sequences include those identified as SEQ ID NO 204, 207-211, 214-230 and 232-321. Preferred polynucleotide include those identified as SEQ ID NO 204, 207-209, 215, 217-230, 232-234, 237, 239, 242-268, 270-281 and 284-321. More preferred polynucleotide include those identified as SEQ ID NO 207, 218-224, 226-230, 234, 237, 242-244, 258-260, 266, 268, 272, 275-279, 284-287, 289-301 and 303, 304, 306, 307, 318 and 321. The most preferred polynucleotide include those identified as SEQ ID NO 217, 221 and 226.
Antibodies and antibody fragments that selectively bind to polypeptides, also provided by the present invention. Such antibodies can be used to identify polypeptides of the present invention and can be identified and generated using procedures well known in the art, the key those ways, which is similar to that described below in example 17.
The invention is a method of treating diabetes and/or other diseases or conditions, carried out with the use of the polypeptides of the present invention, preferably based on a function of the polypeptides of the present invention as agonists R3, mammal, comprising introducing said mammal a therapeutically effective amount of any of the polypeptides of the present invention or any polypeptide that is active against R3, for example, SEQ IN NO 5 and 9.
Also claimed methods for producing polypeptides of the present invention, as recombinant or synthetic.
Brief description of drawings
The figure 1 shows the amino acid sequence of the polypeptides SEQ ID NO 11 to 14, SEQ ID NO 18, SEQ ID NO 21-26, SEQ ID NO 32-36, SEQ ID NO 40-53, SEQ ID NO 57-61, SEQ ID NO 63-99, SEQ ID NO 102-119, SEQ ID NO 121-137, SEQ ID NO 139-177, SEQ ID NO 179, 180, SEQ ID NO 183-202 and SEQ ID NO 322-341, which are claimed polypeptides.
The figure 2 shows the comparison of the sequences of mutant variants of VIP and native polypeptides VIP and PACAP38, GLP-1, EXENDIN-4, and examples of R3-Emiratele polypeptides. Conservative residues are in bold and shaded in dark grey color, while conservative substitutions are shaded in light gray color.
The figure 3 shows the restriction maps is a typical plasmid encoding GST-chimeric peptide.
In figures 4A-4B shows graphs illustrating the effect of GLP-1 or R3P3 on insulin secretion, islet cells of the rat in vitro.
Figure 5 shows a graph showing the effect of peptide R3P3 on the excretion of glucose.
The figure 6 shows a graph showing the effect of RASER and related polypeptides on the water content in the intestines of mice Balb/C.
Figure 7 shows a chart that indicates that a dose of 1 nmol/kg R3P3, R3P12, R3P13 or GLP-1 enhances excretion of glucose in rats after subcutaneous injection.
The figure 8 shows the polynucleotide sequence of SEQ ID NO 54-56 and 203-301 that encode the polypeptides of the present invention.
The first 6 nucleotides represent site recognition by the restriction enzyme BamHI, after which there are 12 nucleotides coding for website recognition "IEGR" factor Ha. The last 6 nucleotides represent site recognition by restrictase Xhol or EcoRI, a 6 nucleotides in front of them encode two stop codon. The nucleotides between the site of factor XA and stop codons encode the amino acid sequence of the corresponding polypeptide. The nucleotides between the two restriction sites to clone on the relevant restriction sites in the vector pGEX-6P-1 (Amersham Pharmacia Biotech). Non SEQ ID nos given in parentheses.
The figure 9 shows the influence RASAR, VIP and selective agonists of the receptor on heart rate in dogs, in consciousness (see example 15).
The figure 10 shows the detection R3P66 method of TYPHOID using polyclonal antibodies produced in rabbits, immunogenic C-terminal sequence R3P66 (Ac-CRKQVAAKKYLQSIKNKRY-COOH).
A detailed description of the preferred options
The present invention represents a new polypeptides and fragments, derivatives and variants exhibiting at least one biological function, which is essentially identical with that of the polypeptide of figure 1 (collectively, the "polypeptides of the present invention"). The polypeptides of the present invention function in vivo as agonists R3 or, in other words, used for the prevention and/or treatment of such diseases or conditions as diabetes, asthma, hypertension, problems in the reproductive sphere in men, including sperm motility in men, cardiovascular disease, ulcers, and other conditions identified here, or operate any other way as described here below. Preferred polypeptides of the present invention should stimulate insulin secretion by beta cells of the pancreas in a glucose-dependent mode.
The polypeptides of the present invention are agonists R3. Prepost is positive they are election - agonists R3 is at least 10-fold selectivity against R3 from R2 and/or R1. More preferably they are selective agonists R3 with at least 100-fold selectivity against R3 from R2 and/or R1. Most preferably they stimulate the secretion of insulin in the plasma glucose-dependent mode without induction of stagnation or increase the level of glucose in plasma, which is a contraindication for treatment, such as type 2 diabetes type. In addition, preferably, the polypeptides of the present invention was selective agonists of the receptor R3 that thereby defines, for example, increased output of insulin in the plasma, but was not selective with respect to other receptors, which are responsible for such unpleasant or dangerous side effects like water retention in the gastrointestinal tract, and/or adverse cardiovascular effects, such as increased heart rate. It is now established that R3-mediated secretion of insulin does not cause hypoglycemia, and R2 activation causes the release of glucose into the plasma, which is contraindicated in the treatment of type 2 diabetes type, and water retention in the gastrointestinal tract, and activation of R1 leads to cardiovascular manifestations such as increased frequencies of the heart.
The polypeptides of the present invention represent a new way of treating patients with reduced secretion of endogenous insulin or impaired glucose tolerance, in particular diabetes of the 2nd type.
RASAR, VIP, GLP-1 and Exendin-4 are polypeptides capable of stimulating insulin secretion in a glucose-dependent mode. However, by itself this fact does not guarantee reduction of glucose levels in vivo. Since it is known that RASAR binds to receptors PACAP-R1, -R2 and-R3, and VIP, as it is known, is associated with PACAP receptor-R2 and-R3, it was thought that they may have similar conservative structural properties. Following multiple comparison shows the ratio of primary structures:
(where a one-letter symbols of amino acids can be found in Zubay, 1988, "Biochemistry", 2d ed., MacMillan Publ., New York, p.33, and decoded below). The polypeptides of the present invention (Fig 1) is closest to the VIP in terms of their primary structure except for SEQ ID NO 57-61, 66-69 and 176, 177, 179, 180, 183-202, which is closest to RASAR.
The authors of the present invention created a new polypeptide, which is an agonist of the receptor R3, preferably selective agonist R3, and/or exhibiting selective glucose-dependent action on enhancing insulin secretion and selective activation of re is of aptara PACAP-R3 actually leads to a glucose-dependent mechanism of insulin secretion by beta cells of the pancreas, with a corresponding reduction of glucose levels in vivo. In light of the above, the inventors first investigated the structure RACER-27 and VIP to determine the residues that are most likely to be responsible for receptor selectivity. It is known that RASER and VIP do not reduce the level of glucose in vivo, but, on the contrary, stimulate the release of glucose from the liver. It has been shown that activation of R2 increases glucose levels in plasma in vivo. Previously received a variety of mutant variants and RACER, and VIP that had different goals. For example, serial deletions of RESAR-27 and RACER-38 at both ends confirmed the importance of both end sections for binding to the receptor (Gourlet et al., 1995, Eur. J.Pharm., 287, 7-11; Gourlet et al., 1996, Regul. Peptides, 62, 125-130). Binding to membranes of rats brain and adenylyl cyclase activity of chimeric mutant variants of the PACAP-27/VIP showed the importance of N-terminal residues of RASAR for recognition PACAP-R1 (Ando et al., 1996, Biomed. Pept. Proteins Nucleic Acids, 2, 41-46). Increasing the basicity Leul7-PACAP27 and Leu17-VIP due to mutations K15R, K20R and K21R and completion With the end of the sequence GKR has led to increased duration relaxants activity in the trachea of the Guinea pig, which presumably is determined by the protection from binding to heparin (Kashimoto et al., 1996, Ann. NY Acad. Sci., 805, 505-510). It has been shown (Gourlet et al., 1996, Biochim. Biophys. Acta, 1314, 267-273)that Q16R-mutant variant VIP and RASAR shows a higher affinity for sravnenie is with the corresponding native polypeptide in proportion PACAP-R2 and R1. High-affinity selective R2 agonist was formed (Gourlet et al., 1997, Peptides, 18, 1539-1545) by obtaining substituted chimeric peptide [K15,R16,L27]VIP(1-7)/GRF(8-27). Acylation at the N end and replacing D-Phe2such a selective agonist led to the formation of powerful electoral R2 antagonist (Gourlet et al., 1997, Peptide, 18, 1555-1560). Mutant variants Y22L and Y22A VIP, but not Y22F, show less affinity in respect of PACAP-R3, suggesting the importance of having the aromatic group in position 22 to bind to the receptor R3, but not for binding to the receptor R2 (Gourlet, 1998, Eur. J.Biochem., 348, 95-99). Helderman and helopeltis - VIP-like peptides isolated from the venom of the salivary glands of lizards, is a show about a 100-fold selectivity in respect of the PACAP receptor-R3 (Gourlet, 1998, Ann. NY Acad. Sci., 865, 247-252). Photoaffinity tagging of RESAR-27 by replacing F6 and Y22 on p-benzoyl-L-phenylalanine (pBz) or K15, K and K on pBz2suggested that K15 and F22 closer to PACAP-R1 than F6, C and C (Cao et al., 1997, Eur. J.Biochem., 244, 400-406; Cao et al., 1998, Ann. NY Acad. Sci., 865, 82-91).
The authors of the present invention discovered a number of polypeptides, which cause stimulation of insulin secretion in a glucose-dependent mode and lead to lower glucose levels in vivo. These polypeptides have some similarity with VIP and RAZAR. In particular, multiple comparison showed the following:
However, in the scientific or patent literature there is no information which would indicate that the selected modification sequences VIP and RASAR result in a polypeptide with the ability to stimulate insulin secretion in a glucose-dependent mode and to reduce the concentration of glucose in plasma.
Some of the terms used in this application will be determined now, and other terms as their introduction. Single-letter designation of specific amino acids, the amino acids and three-letter abbreviations are: A - alanine (Ala); - cysteine (Cys); D - aspartic acid (Asp); E is glutamic acid (Glu); F is phenylalanine (Phe); G is glycine (Gly); H is histidine (His); I is isoleucine (Ile); K is lysine (Lys); L - leucine (Leu), M - methionine (Met); N is asparagine (Asn); P - Proline (Pro); Q is glutamine (Gln); R - arginine (Arg); S - serine (Ser); T is threonine (Thr); V - valine (Val); W is tryptophan (Trp); Y is tyrosine (Tyr).
The term "polynucleotide encoding a polypeptide" encompasses polynucleotide, which includes only coding for a polypeptide sequence, and polynucleotide, which includes additional coding and/or noncoding sequence. Also, the present invention concerns polynucleotides that hybridized described here above sequences, if they are characterized by at least about 70%, pre is respectfully at least about 90%and more preferably at least about 95%sequence identity. The present invention applies in particular, polynucleotides encoding polypeptides that hybridized in hard conditions described here above polynucleotide. In this text, the term "stringent conditions" refers to "stringent conditions for hybridization". Preferably, hybridization should occur only when there is at least about 90%and preferably about 95-97%sequence identity. Polynucleotide that hybridic described here above polynucleotide in a preferred embodiment encode polypeptides which retain essentially the same biological function or activity as the Mature polypeptide encoded by the cDNA molecules.
The terms "functional equivalent" and "essentially the same biological function or activity" means that the level of biological activity 30-100% or more of the biological activity, which demonstrates the polypeptide with which a comparison is made when the biological activity of each of the polypeptides is determined in the same procedure. For example, a polypeptide that is functionally equivalent to the polypeptide of figure 1, the polypeptide, which in the case of radioimmunoassay test scintillation approximation (RIA SPA) camp, on Sanogo in the particular example 16, shows the accumulation of camp in the cell line Cho expressing the receptor for PACAP/VIP-R2 (PACAP-R3) of a person.
The polypeptide of the present invention, which is an agonist R3 is a polypeptide that exhibits approximately 30-100% or more of the maximum agonist activity against PACAP27-R3 when tested using the Protocol of example 16. Preferred polypeptides of the present invention, which are selective agonists R3 compared to PACAP receptor-R2 and R1 are those polypeptides which exhibit the ratio of agonist activity R3 to activity in relation to R2 about 10:1 or higher, and more preferably about 100:1 or higher, and/or have attitude agonist activity R3 compared with activity against receptor R1 about 10:1 or higher, and more preferably about 100:1 or higher when the polypeptide is tested in accordance with the Protocol of example 16 using cells expressing the appropriate receptors.
"Stringent hybridization conditions" refer to the incubation during the night two fragments polynucleotides that they hybridized at 42°C in a solution containing 50% formamide, 5×SSC (750 mm NaCl, 75 mm sodium citrate), 50 mm sodium phosphate (pH 7,6), 5×denhardt's solution, 10% doctranslate and 20 microgram/ml denatured, subjected to shaking DNA salmon sperm with the next washing the filters in 0.1× SSC at about 65°C.
The terms "fragment", "derivative" and "variant" when used with respect to polypeptides with figure 1 indicate the fragments, derivatives and variants of polypeptides which retain essentially the same biological function or activity of such polypeptides according to, further described below.
Similar includes propolypeptide, consisting of an amino acid sequence of the polypeptide of the present invention. Active polypeptide according to the present invention can be derived from additional amino acids that make up a complete polypeptide molecule, with the participation of natural processes in vivo or using procedures well known in the art, for example, catalytic or chemical cleavage. For example, a 28-amino acid native peptide VIP in vivo is expressed in the form of significantly more long polypeptide, which is then processed in vivo to release a 28-amino acid active Mature peptide.
The fragment is part of a polypeptide that essentially retains the same functional activity, which is identified in the models in vivo, described later in this text.
Derived encompasses all modifications of the polypeptide, which essentially retain the functions described herein and includes additional structure and related to her function, for example, Paglierani polypeptides, which are characterized by a higher half-life, chimeric polypeptides that have the properties of specificity in respect of any target or complementary activities, such as toxicity specific target in accordance with the described hereafter.
The polypeptides of the present invention can be recombinant polypeptides, natural purified polypeptides or synthetic polypeptides.
The fragment, derivative or variant of the polypeptide of the present invention may be (i) those in which one or more amino acid residues is replaced by conservative or non-conservative amino acid residues (preferably conservative amino acid residue)and such substituted amino acid residue may be encoded or not encoded by the genetic code; or (ii) those in which one or more amino acid residues include the replacement group; or (iii) those in which the Mature polypeptide chemerisov with another compound so that the compound increases the half-life of the polypeptide (for example, polyethylene glycol); or (iv) those in which additional amino acids chemerisov with Mature polypeptide so that they form a leader or secretory sequence or the selected, used for purification of the Mature polypeptide, or propolypeptide sequence; or (v) those in which the polypeptide sequence chemerisov with larger polypeptide, for example, human albumin, an antibody or a fragment of the Fc, with the purpose of renewal. Such fragments, derivatives or variants and analogs are considered as falling in the scope clear to the experts in the art based on the foregoing in this text.
Preferably derivatives of the present invention should include conservative amino acid substitutions (defined hereafter), produced by one or more predicted, preferably non-key amino acid positions. "Non-core" amino acid residue refers to a residue that can be changed in the sequence of wild-type protein without altering the biological activity, while the "core" amino acid residue for the biological activity necessary. "Conservative amino acid substitution" is a substitution in which the amino acid residue is substituted with amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (in the example, lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Nonconservative substitutions should not be made by a conservative amino acid residues or amino acid residues within the conservative protein domain, such as the remains of 19 and 27, as these residues are essential for activity of the protein, such as activity against R3 and/or selectivity with respect to R3. Fragments or biologically active portion encompass polypeptide fragments suitable for use as a drug, to generate antibodies, as a research reagent and the like. Fragments include peptides having amino acid sequences essentially similar to or derived from the amino acid sequence of the polypeptide of the present invention and exhibiting at least one activity of such a polypeptide but which include fewer amino acids than described in this text polnorazmernyi polypeptides. Typically, biologically active portion includes a domain or motif with at least one activity of the polypeptide. A biologically active portion of the polypeptide may be a peptide, for example, consists of 5 or more amino acids. Such biologically active portions can be obtained synthetically or using recombinant methods and can be tested for one or more functional activities of a polypeptide of the present invention using the methods described in this application and/or known in the art.
Moreover, the preferred derivatives of the present invention comprise the Mature polypeptide, which were chemerisov with another compound so that the compound increases the half-life of the polypeptide and/or reduce potential immunogenicity of the polypeptide (for example, polyethylene glycol "PEG"). In the case of "Paglierani" chimerization polypeptide on the PEG can be carried out using any method known to specialists in this field of technology. For example, Pegylation can be done first by making mutant cysteine in the polypeptide and then by site-specific derivatization with PEG-maleimide. Cysteine can be added to the end of the peptides (see, for example, Tsutsumi et al., 2000, Proc. Natl. Acad. Sci. USA, 97(15), 8548-8553).
the variants of the polypeptide of the present invention encompass polypeptides, characterized by an amino acid sequence essentially the same as the amino acid sequence of rooms SEQ ID NO with figure 1 or its domain. The term "essentially similar" refers to a first amino acid sequence that includes a sufficient or minimum number of identical or equivalent amino acid residues as compared to the second amino acid sequence such that the first and second amino acid sequences have a common structural domain and/or common functional activity. For example, amino acid sequence, including common structural domain that is identical to at least 45%, preferably about 75-98%, defined here as essentially similar. Preferred options should be essentially the same as the amino acid sequence of the preferred polypeptides of the present invention. Variants include variants of the polypeptide encoded by polynucleotides that hybridize with polynucleotides of the present invention or its complement under stringent conditions. Such variants generally retain the functional activity of the polypeptides of the present invention. Libraries of fragments of polynucleotides can be used to create a heterogeneous sample of fragments for screening posleduyushego selection. For example, a library of fragments can be generated by treating the double-stranded PCR fragment of polynucleotide a nuclease under conditions where the gap occurs in a single molecule only once, followed by denaturation of double-stranded DNA, denaturaciei DNA with the formation of double-stranded DNA which can include sense/antisense pairs various broken products, removing single-stranded fragments from the newly formed duplexes by treatment with S1 nuclease and legirovaniem the resulting libraries of fragments in the composition expressing vector. Using this method it is possible to form an expression library that encodes N-terminal and internal fragments of various sizes of the composition of the polypeptide of the present invention.
Variants include polypeptides that differ in amino acid sequence due to mutagenesis. Variants that function as agonists R3, can be identified by screening for the activity of the agonist R3 comprehensive libraries Malinov, for example, the truncated mutant variants of the polypeptides of the present invention.
In one embodiment, the heterogeneous library of analogues is created by combinatorial mutagenesis at the level of nucleic acids and it is encoded heterogeneous Genn is th library. Heterogeneous library of variants can be obtained, for example, catalytically by ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of possible variants of amino acid sequences is expressed as individual polypeptides, or alternatively, in a group larger chimeric proteins (e.g., for presentation at ragovoy surface), which includes a set of sequences. There are a number of methods that can be used to generate libraries of potential options on the material of a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, followed by legirovaniem synthetic gene into a composition suitable expressing vector. Use of a degenerate set of genes provides in one mixture, of all of the sequences encoding the desired set of potential variant sequences. Methods of synthesis of degenerate oligonucleotides is well known in the art (see, for example, Narang, 1983, Tetrahedron, 39, 3; Itakura et al., 1984, Annu. Rev. Biochem., 53, 323; Itakura et al., 1984, Science, 198, 1056; Ike et al., 1983, Nucl. Acid Res., 11, 477).
In the art, the number of known methods for screening gene products of composition combine the priori libraries, generated by point mutations or truncation, and screening cDNA libraries for gene products having a selected property. Such methods can be adapted for rapid screening of the gene libraries generated by the combinatorial mutagenesis polypeptide agonist R. the Most widely used methods, which are suitable for high-throughput screening large gene libraries typically include cloning the gene library into competent replication expressing vectors, transforming appropriate cells obtained library vectors and expression pereaminirovanii genes under conditions in which detection of a desired activity provides the selection of the vector encoding the gene product was detected. Recursive comprehensive mutagenesis (REM), a mathematical method that enhances the frequency of functional mutations in the libraries, can be used in combination with the test screening to identify desirable options.
The present invention also provides chimeric or hybrid polypeptides. Examples are those polypeptides of the present invention, which is described in SEQ ID NO 18 and 172, which is the chimeras target sequence of the pancreas "SWCEPGWCR" (D.Rajotte et al., 1998, J.Clin. Invest., 102, 430-437) with SEQ ID NO 8 and 32 corresponding is O. The sequence of a target formed so as to localize the delivery of the polypeptide in the pancreas and to minimize possible side effects. The polypeptides of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, for example, be isostere peptides, and may include other amino acids than the 20 amino acids, which is genetically determined. The polypeptides can be modified either by natural processes, such as posttranslational processing, or by using methods of chemical modification, are well known in the art. Such modifications are described in detail in textbooks and specialized monographs, as well as in the extensive scientific literature. Modifications can affect any part of the polypeptide, including the peptide skeleton, side chains of amino acids and N - or C-terminal parts. It should be clear that the same type of modification may be present in the same or varying degrees at different sites in a given polypeptide. Also, this polypeptide can include many types of modifications. The polypeptides may be branched, for example, as a result of obyedinenie, and they may be cyclic, with or without branching. Cyclic, branched and RA is extensive cyclic polypeptides may result from the natural post-translational processes or can be obtained by the methods of synthesis. Modifications include acetylation, acylation, ADP-ribosylating, amidation, covalent joining flavina, covalent joining of heme, covalent joining of nucleotide or nucleotide derivative, covalent joining of a lipid or lipid derivative, covalent joining phosphotidylinositol, cross-linking, cyclization, the formation of disulfide bonds, demethylation, formation of covalent cross-links, formation of cysteine, formation of Pyroglutamate, formirovanie, γ-carboxylation, glycosylation, formation of GPI-anchors, hydroxylation, iodination, methylation, monitorowanie, oxidation, Pagalilauan, proteolytic cleavage, phosphorylation, prenisolone, racemization, selenopyran, sulfation mediated transport RNA, the addition of amino acids to proteins, for example, marginalrevenue, and obyedenyatsya (see, for example, "Proteins, Structure and Molecular Properties", 2d ed., ..Creighton, W.H.Freeman and Co., New York, 1993; "Posttranslational Covalent Modification of Proteins", B.C.Johnson ed., Academic Press, New York, 1983, pp. 1-12; Seifter et al., 1990, Meth. Enzymol., 182, 626-646; Rattan et al., 1992, Ann. NY Acad. Sci., 663, 48-62 per).
The polypeptides of the present invention encompass polypeptides of figure 1 corresponding to SEQ ID NO 11 to 14, SEQ ID NO 18, SEQ ID NO 21-26, SEQ ID NO 32-36, SEQ ID NO 40-53, SEQ ID NO 57-61, SEQ ID NO 63-99, SEQ ID NO 102-119, SEQ ID NO 121-137, SEQ ID NO 139-77, SEQ ID NO 179, 180, SEQ ID NO 183-202, 322-341, and sequence characterized by "significant," the variability of their sequences. "Minor variation" should cover any variant sequence, a substitution or a deletion, which essentially maintain at least one biological function of polypeptides of the present invention, preferably the activity of the agonist R3, more preferably the activity selective agonist R3, and most preferably displayed here activity on insulin secretion. Such functional equivalents are preferably can include polypeptides, which are characterized by at least about 90%identity to the polypeptide of figure 1, and more preferably at least 95%identity to the polypeptides of figures 1 and even more preferably at least 97%identity to the polypeptide of figure 1, and also include portions of such polypeptides having essentially the same biological activity. However, any polypeptide, with minor variability of the amino acid sequence of the polypeptide of figure 1, which shows the functional equivalence as described later in this text, is included in the description of the present invention.
As is known in the art, "the gathering of the creation" between two polypeptides is determined by comparing the amino acid sequence and its conservative amino acid substitutes of one polypeptide to the sequence of the second polypeptide. Such conservative substitutions include that described above and Dayhoff, 1978, "The Atlas of Protein Sequence and Structure", 5, and Argos, 1989, EMBO J. 8, 779-785. For example, conservative substitutions are amino acids belonging to one of the following groups:
- Ala, Pro, Gly, Gln, Asn, Ser, Thr;
Is Cys, Ser, Tyr, Thr;
- Val, Ile, Leu, Met, Ala, Phe;
Is Lys, Arg, His;
Is Phe, Tyr, Trp, His, and
- Asp, Glu.
The present invention also relates to vectors which include polynucleotides of the present invention, host cells that have been genetically transformed with vectors of the present invention, and obtaining polypeptides of the present invention recombinant methods. Cell owners can be genetically transformed (transpulmonary or transformed, or transliterowany) with vectors of the present invention, which may be, for example, a cloning vector or expressing vector. The vector can take the form of, for example, a plasmid, a viral particle, a phage, etc. Transformed cell hosts can be precultural in standard nutrient media modified in accordance with the activating promoters or method of selection of transformants. Culturing conditions, such as temperature, pH and the like, correspond to those previously used to host cells selected for expression, and will be clear to skilled in about the Asti equipment. Polynucleotide according to the present invention can be used to obtain a polypeptide using recombinant methods. Thus, for example, the polynucleotide sequence can be incorporated into any of various expression systems, in particular, vectors or plasmids for expression of the polypeptide. Such vectors include chromosomal, achromosome and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; yeast plasmids; vectors derived from combinations of plasmids and ragovoy DNA, viral DNA, for example, virus, cowpox virus, adenovirus, poxvirus birds and virus false rabies. However, any other vector or plasmid may be used, if only they were capable of replication and viable in the cell host.
The appropriate DNA sequence may be embedded in the structure of the vector using different procedures. In General, the DNA sequence is built by a suitable restriction site using methods known in the art. These methods and more are considered as well-known specialists in this field of technology. The DNA sequence in expressing the vector is functionally attached to a suitable sequences to control gene expression (promoter)for synthesis of an Mr is K. As typical examples of such promoters may be mentioned: LTR promoter or SV40, promoter, lac or trp E. coli, the promoter PLλ-phage and other promoters, for which the known regulation of gene expression in prokaryotic or eukaryotic cells or their viruses. Also expressing vector includes initiating broadcast of the binding site on the ribosome and the terminator of transcription. The vector may include sequences that are suitable for amplificating expression. In addition, expressing the vectors preferably include a gene determining phenotypic trait for selection of transformed host cells, such as dehydropeptidase, or resistance to neomycin for cultures of eukaryotic cells, or such as resistance to tetracycline or ampicillin in E.coli. The vector containing the appropriate DNA sequence as described here above, as well as an appropriate promoter or regulatory sequence, can be used to transform a suitable host, so that the owner could Express the protein. As typical examples of suitable hosts may be mentioned: bacterial cells, such as E. coli, Salmonella typhimurium, Streptomyces; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as Cho, COS or elanoma Boyes; adenoviruses; plant cells, etc. the Choice of a suitable host, as you can assume, will be clear to a person skilled in the field of technology with the objectives set out in this text.
The present invention also includes recombinant constructs comprising one or more sequences, in the broad sense described in the text. Designs include a vector such as a plasmid or viral vector, into which a sequence of the present invention was incorporated in a direct or reverse orientation. In a preferred aspect of this variant embodiment design additionally includes regulatory sequences, including, for example, the promoter is functionally attached to the sequence. Large numbers of suitable vectors and promoters are known to experts in the art and commercially available. The following vectors are given by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, phagescript, psiX174, pBluescript SK, pBsKS, pNHSa, pNH16a, pNH18a, pNH46a (Stratagene); pTRC99A, pKK223-3, pKK233-3, pDR540, PRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXTl, pSG (Stratagene), pSVK3, pBPV, pMSG, PSVL (Pharmacia). However, it can be used any other plasmid or vector, if only they were able to replicate and survive in the host. Promoter regions can be selected from any desired gene using vecto the s CAT (chloramphenicolchloramphenicol) or other vectors, with selective markers. The two relevant vectors are RCC-8 and RSM. Specifically mentioned bacterial promoters are the lacI, lacZ, T3, T7, gpt, PRthat λ-phage and trp. Eukaryotic promoters include pretani the CMV promoter, the promoter timedancing of herpes simplex virus, early and late promoters of SV40, LTRS of retroviruses and the promoter metallothionein-1 mouse. The selection of the appropriate vector and promoter is well understood specialists in this field of technology.
The present invention also relates to host cells carrying the above-described construction. A host cell may be a cell of higher eukaryotes, such as a cell of the mammal or cell of the lower eukaryotes, such as yeast cell or a host cell can be a prokaryotic cell such as a bacterial cell. The introduction of design into the host cell can be accomplished by transfection with calcium phosphate, transfection using DEAE-dextran, or electroporation (L.Davis, M.Dibner, I.Battey, 1986, Basic Methods in Molecular Biology"). Structures in the cells of the host can be used in the standard manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the present invention can be produced synthetically using standard peptide synthesizers.
Reliabilty can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be used to produce such proteins using RNAS derived from DNA constructs of the present invention. Appropriate cloning and expressing vectors for use with prokaryotic and eukaryotic organisms-hosts are described in Sambrook et al., 1989, "Molecular Cloning: A Laboratory Manual", 2d edition. Cold Spring Harbor, NY: the contents of this manual are included here for information in the form of bibliographic references.
Transcription of DNA encoding the polypeptides of the present invention, in higher eukaryotes increased due to the embedding in vector enhancer sequence. Enhancers are CIS - acting elements of DNA usually about 10-300 base pairs (BP)that act on the promoter, enhancing transcription. Examples are the enhancer of SV40 virus in the field of late gene from customers start replication (nucleotides 100-270), enhancer of early cytomegalovirus promoter, enhancer of polyomavirus from late gene from customers start replication and adenovirus enhancers. In General, recombinant repressiruyuschy vectors should include sites start replication and selective markers for transformation of a host cell, for example, a gene of resistance to ampici the Lin E. coli and the gene TRP1 S.cerevisiae, and a promoter derived from intensively expressed gene, with the aim of ensuring transcription of downstream structural sequence. Such promoters can be derived from the operons encoding, inter alia, enzymes of glycolysis, such as 3-phosphoglycerate (PGK), α-factor, acid phosphatase, or heat shock proteins. Heterologous structural sequence is collected in appropriate phase with sequences of translation initiation and termination, including, preferably, a leader sequence capable of secretion of translated protein into periplasmatic space or the extracellular environment. Optional heterologous sequence can encode a chimeric protein comprising N-terminal identification peptide imparting desired properties, e.g., stabilization or simplified purification of expressed recombinant product.
Applicable for use in bacteria expressing vectors design by embedding the structural DNA sequence that encodes a desired protein together with suitable translation, initiation and termination signals in the functional phase of reading with an active promoter. The vector must include one or more phenotypic selective markers, and the site is achala replication to ensure maintenance of the vector and, if desired, to provide amplification within the host organism. Suitable prokaryotic hosts for transformation are E. coli, Bacillus subtilis. Salmonella typhimurium and various species included in the genera Pseudomonas, Streptomyces, and Staphylococcus, although can also be selected and other hosts. Applicable expressing vectors for use in bacteria may include a selective marker and bacterial website start replication originating from the composition of the commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercially available vectors include, for example, RCC-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These fragments of the skeleton pBR322 combined with an appropriate promoter and the structural sequence to be Express.
After transformation of a suitable strain of host cells and culturing of the strain host to a suitable density of cells selected promoter derepression appropriate method (for example, change of temperature or chemical induction)and cells are cultured for an additional period of time. Typically, cells are harvested by centrifugation, destroy physical or chemical means, and the crude extract gave leave for the further purification. Microbial cells used for expression of proteins can be destroyed using any standard method, including cyclic freezing-thawing, exposure to ultrasound, mechanical disruption, or use of agents that lyse cells.
Culture system various mammalian cells can also be used for expression of recombinant protein. Examples of expression systems, mammalian cell lines are COS-7 fibroblasts kidney green monkeys, described by Gluzman, 1981, Cell, 23, 175, and other cell lines capable of Express compatible vector, for example, cell lines S, T, Cho, HeLa, and KSS. Expressing vectors for mammalian cells should include the site of the beginning of replication, a suitable promoter and enhancer, and necessary binding sites on the ribosome, the site of polyadenylation, donor and acceptor splicing sites, sequence termination of transcription and 5'-flanking retranscribing sequence. DNA sequences derived from the genome of SV40 virus, for example, the site of the beginning of replication, early promoter, enhancer, splicing sites and sites poliakrilovaya SV40, can be used to provide the necessary retranscribing genetic elements.
The polypeptides of the present invention which may be isolated and purified from cultures of recombinant cells using methods used here, including precipitation with ammonium sulfate or ethanol, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, chromatography on hydroxyapatite and lectinology chromatography. If necessary, can be applied to the phase protein refolding to complete the spatial configuration of the Mature protein. Finally, as the final stages of purification can be applied to high-performance liquid chromatography (HPLC).
The polypeptides of the present invention can be a product of chemical synthesis procedures or can be produced using recombinant technologies in prokaryotic or eukaryotic host (for example, cultured cells, bacteria, yeast, higher plants, insects and mammals). Depending on which of the host body in the procedure of recombinant production of the polypeptides of the present invention can be glycosylated carbohydrates mammalian or other eukaryotic or may not be glycosylated. The polypeptides of the present invention may also include the initial methionine residue. Selected or purified polypeptide of the present invention or biologically active portion essentially free from other the CSOs cellular material or culture medium in the case of production it using recombinant technology, or substantially free from chemical precursors or other chemicals in the case of chemical synthesis. Preferably the selected polypeptide according to the present invention is essentially free of cellular material and contains less than about 30% (by dry weight) polipeptides or polluting material. When the polypeptide of the present invention or biologically active portion receive recombinant means, preferably the culture medium is less than about 30% of the volume of the polypeptide preparation. When the present invention is carried out by means of chemical synthesis, the preparations preferably contain less than about 30% by dry weight of chemical precursors or not related to the invention of chemical substances.
The polypeptides of the present invention can be effectively allocated as described in the specific examples hereinafter. The preparation of purified polypeptide is pure, at least about 70%; preferably the drug is clean at 85-99%. The purity of drugs can be assessed using any method known in the art, such as electrophoresis LTO-polyacryl-amide gel or mass spectroscopy, liquid chromatography.
Polynucleotide sequence encoding the polypeptide according to N. the present invention, can be synthesized in whole or in part, using chemical methods well known in the art (see, e.g., Caruthers et al., 1980, Nucl. Acids Res. Symp. Ser., 215-223; Horn et al., 1980, Nucl. Acids Res. Symp. Ser., 225-232). Polynucleotide that encodes the polypeptide can then be cloned into a composition expressing vector for expression of the polypeptide.
As should be clear to a person skilled in the technical field advantage may be obtaining the nucleotide sequences encoding the polypeptide, including naturally occurring codons. For example, codons preferred for a particular prokaryotic or eukaryotic host can be selected to increase the intensity of expression of the polypeptide or obtain RNA transcript having desirable properties, such as half-life, which is more in comparison with that in the transcript, obtained from the sequence found in natural conditions.
The nucleotide sequence described in this text, can be designed using methods known in the art, to change the coding for the polypeptide sequences for various purposes, including, but not limited to, changes that modify the formation, processing and/or e is cpressey of the polypeptide or mRNA product. To convert the nucleotide sequences can be used "mixing" DNA using random fragmentation and PCR re-Assembly of gene fragments and synthetic oligonucleotides. For example, site-specific (directed) mutagenesis can be used to introduce new restriction sites, change the settings of glycosylation changes of preference codons, making splicing variants, making mutations, etc.
Alternatively, the polypeptides of the present invention can be produced using chemical methods to synthesize the corresponding amino acid sequence, such as by direct peptide synthesis using solid-phase methodologies (see, for example, Merrifield, 1963, J.Amer. Chem. Soc., 85, 2149-2154; Roberge et al., 1995, Science, 269, 202-204). Synthesis of polypeptides can be performed using manual techniques or automatically. Automated synthesis can be performed, for example, using a peptide synthesizer model A from Applied Biosystems (Perkin-Elmer). Optional fragments of the polypeptide can be synthesized separately and combined using chemical methods with obtaining polnorazmernoi molecule.
Newly synthesized polypeptide can be substantially purified using high-resolution zhidkosti the th chromatography (see, for example, Creighton, 1983, "Proteins: Structures and Molecular Principles", W.H.Freeman&Co., New York, NY). The composition of the synthetic polypeptide of the present invention can be confirmed by using amino acid analysis or sequencing, for example, using the procedure of degradation on Adminu (see Creighton, CIT. above). In addition, any portion of the amino acid sequence of the polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins with obtaining a variant polypeptide or chimeric polypeptide.
The polypeptides of the present invention because of the ability to stimulate insulin secretion, islet cells of the pancreas in vitro and to reduce the level of blood glucose in vivo can be used for the treatment of type 2 diabetes type (insulin-independent diabetes). The polypeptides can be used for prevention in subjects with impaired glucose tolerance to prevent the development of type 2 diabetes type. In addition, the polypeptides of the present invention can be used for the treatment of asthma (Bolin et al., 1995, Biopolymer, 37, 57-66; U.S. patent No. 5677419) (it is shown that the polypeptide R3PO active relaxation of smooth muscles of Guinea-pig trachea); induction of low blood pressure (VIP induces hypotension, tachycardia and tide kravik the patient's face-asthmatics (Animage & P.S.Sever, 1986, Peptides, 7, 279-280; A.Morice et al., 1983, The Lancet II, 1225-1227), problems of male reproductive function (Y.Slow et al., 1999, "Effects of vasoactive intestinal peptide on human sperm motility". Arch. Androl., 43 (1), 67-71); as protivopolozhnogo and neuroprotective agents (D.E. Brenneman et al., 1998, "VIP neurotrophism in the central nervous system" multiple effectors and identification of a femtomolar-acting neuroprotective peptide", Ann. NY Acad. Sci., 865, 207-212); cardiotoxic to the attacks of ischemia (R.Kalfin et al., 1994, "Protective role of intracoronary vasoactive intestinal peptide in ischemic and reperfused myocardium", J.Pharmacol. Exp. Ther., 268 (2), 952-958; D.K.Das et al., 1998, "Coordinated role of vasoactive intestinal peptide and nitric oxide in cardioprotection", Ann. NY Acad. Sci., 865, 297-308); and finally as antiulcer funds (Tuncel et al., 1998, "The protective effect of vasoactive intestinal peptide (VIP) on stress-induced gastric ulceration in rats", Ann. NY Acad. Sci., 865, 309-322).
The polypeptides of the present invention can be used in combination with a suitable pharmaceutical carriers for preparation of pharmaceutical compositions for parenteral administration. Such compositions contain a therapeutically effective amount of the polypeptide and a pharmaceutically acceptable carrier or excipient. This carrier is thus not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. Form of the drug must meet the route of administration. The invention is a pharmaceutical package or kit containing the E. one or more containers, filled with one or more components of the pharmaceutical compositions of the present invention. In connection with these tanks may be marking in the form specified by the Federal Agency regulating the manufacture, use or sale of pharmaceuticals or biological products, so that such labeling reflects approval by the Agency of manufacture, use or sale for the introduction of man. In addition, the polypeptides of the present invention can be used in combination with other therapeutic agents.
The pharmaceutical compositions can be introduced in a standard way, such as oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The pharmaceutical composition is administered in a quantity which is effective for the treatment and/or prophylaxis of the specific indication. In General, they are administered in the amount of at least about 350 ng (0.1 nmol) per 1 kg of body weight, and in most cases they need to be entered in the quantity not exceeding about 35 μg (10 nmol) per 1 kg of body weight per day. In most cases, the dosage is from about 0.1 μg/kg to about 100 mg/kg of body weight daily, taking into account the routes of administration, symptoms and other Specified parameters do not account for the biologist the economic accessibility of the peptide in vivo: in this case, can be used more or less to achieve the desired effective dose. Specialist in the art will be able to determine through experimental dosages or other standard approaches the total number that you want to use to achieve an effective dose.
The polypeptide of the present invention can also be used in accordance with the present invention by expression of such polypeptide in vivo, which is often referred to as "gene therapy". Thus, for example, cells can be constructed using polynucleotides (DNA or RNA)encoding the polypeptide ex vivo, and then constructed cells administered to the patient, which treatment with the polypeptide. Such methods are well known in the art. For example, cells can be designed using procedures known in the art, by use of a retroviral particle containing RNA which encodes a polypeptide according to the present invention.
Local delivery enhancers of insulin secretion using gene therapy can deliver a therapeutic agent to the site of the target, namely in the pancreas. For example, specific to the pancreas, the promoter used to create a model of murine tumor of the beta cells of the pancreas (D.Hanahan, 1985, "Heritable formation of pancreatic beta-cell tumors in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes", Nature, 315 (6015) 115-122).
Provides gene therapy methodology both in vitro and in vivo. The number of known methods of transfer potential therapeutic genes in specific cell populations: see, for example, Mulligan, 1993, "The basic science of gene therapy", Science, 260, 926-931. These methods include:
1) direct gene transfer - see, for example, Wolff et al., 1990, "Direct gene transfer into mouse muscle in vivo. Science, 247, 1465-1468;
2) DNA transfer via liposomes (see, for example, Caplen et al., 1995, "Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis", Nature Med., 3, 39-46; Crystal, 1995, "The gene as a drug", Nature Med., 1, 15-17; Gao& Huang, 1991, "A novel cationic liposome reagent for efficient transfection of mammalian cells", Biochem. Biophys. Res. Commun., 179, 280-285;
3) DNA transfer using retroviruses: see, for example, KAU et al., 1993, "In vivo gene therapy of hemophilia B: sustained partial correction in factor IX-deficient dogs", Science, 262, 117-119; Anderson, 1992, "Human Gene Therapy", Science, 256, 808-813;
4) the transfer of DNA using DNA-viruses. Such DNA viruses are adenoviruses (preferably the vectors on the basis of Ad-2 or Ad 5), herpes viruses (preferably the vectors based on herpes simplex virus and parvovirus (preferably the vectors on the basis of defective or non-Autonomous parvoviruses, more preferably vectors based on adeno-associated viruses, most preferably a vector based on AAV-2): see, for example. Ali et al., 1994, "The use of DNA viruses as vectors for gene therapy". Gene Therapy, 1, 367-384; U.S. patent No. 4797368 included here for information in view of the bibliographic references, and U.S. patent No. 5139941 included here for information in the form of bibliographic references.
The choice of a particular vector system for transferring the gene of interest is dictated by a number of factors. One of the important factors is the nature of the cell population target. Although retroviral vectors have been studied in detail and found their place in a large number of gene therapy applications, such vectors are in General not suitable to infect non-dividing cells. In addition, retroviruses have oncogenic potential. However, recent developments in the field of lentiviral transfer vector-based can overcome some of these limitations: see Naldini et al., 1996, "In vivo gene delivery and stable transduction of non-dividing cells by a lentiviral vector. Science, 272, 263-267.
Retroviruses from which can be derived retroviral plasmid vectors mentioned above are thus not limited to, leukemia virus mouse Malone, virus, spleen necrosis, retroviruses such as rous sarcoma virus, the virus sarcoma of Harvey, leukosis virus of birds, leukosis virus gibboney monkeys, human immunodeficiency virus, adenovirus, virus, myeloproliferative sarcoma virus tumor of the breast. In one variant embodiment, the retroviral plasmid vector is derived from leukemia virus mouse molony.
The advantage of adenoviruses is because they are characterized by Shiro the named range of hosts, can infect resting or permanently differentsirovaniya cells, such as neurons or hepatocytes, and, apparently, are not carcinogenic: see, for example. Ali et al., CIT. above, str. It is believed that adenoviruses do not integrate into the genome of the host cell. Due to their extrachromosomal existence, the risk of insertional mutagenesis significantly reduced: Ali et al., CIT. above, str.
Adeno-associated viruses (AAV) have similar benefits as vectors based on adenoviruses. However characterized AAV site-specific integration into chromosome 19 of the human (Ali et al., CIT. above, str).
In a preferred variant embodiment, DNA encoding a polypeptide amplifier insulin secretion according to the present invention, is used in gene therapy of diseases such as diabetes.
In accordance with this variant gene therapy using DNA that encodes a polypeptide amplifier insulin secretion or mutant variants of the present invention, applied to a patient in need thereof simultaneously with the diagnosis or immediately thereafter.
To a person skilled in the art should be understood that any suitable gene therapy vector encoding a polypeptide amplifier insulin secretion, DNA or DNA fragment, derivative or variant of the polypeptide amplifier s is ecii insulin, can be used in accordance with this alternative embodiment. Known methods of constructing such vectors: see, for example, W.F.Anderson, 1998, "Human gene therapy". Nature, 392, 25-30; I.M.Verma & N.Somia, 1998, "Gene therapy - promises, problems, and prospects". Nature, 389, 239-242. Introduction of a vector comprising DNA polypeptide amplifier insulin secretion in the website-the target can be carried out using known methods.
The vector includes one or more promoters. Suitable for use promoters are thus not limited to, the retroviral LTR; the SV40 promoter and the promoter of cytomegalovirus (CMV) of a person described in Miller et al., 1989, Biotechniques, 7 (9), 980-990, or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the promoters of histone genes, polymerase-III β-actin). Other viral promoters that can be used are thus not limited to, adenoviral promoters, promoters timedancing (TC) and the promoters of parvovirus B19. The choice of a suitable promoter will be clear to experts in the art based on the described in the text.
The sequence of the nucleic acid encoding the polypeptide of the present invention, is under the control of a suitable promoter. Suitable promoters that can be used are, who eat most are not limited to, adenoviral promoters, such as the main promoter of the late gene of adenovirus, or heterologous promoters, such as cytomegalovirus (CMV) promoter, the promoter respiratory syncytial virus (RSV), inducible promoters, such as the MMT promoter, the promoter metallothionein, the promoters of heat shock, the albumin promoter, the ApoAl promoter, promoters globin genes of man, the promoters of viral timedancing, such as the promoter timedancing of herpes simplex virus, retroviral LTRS (including the modified retroviral LTR, described herein above), the promoter of the gene β-actin and the promoters of genes of human growth hormone. The promoter may be native promoter under the control of which is the gene encoding the polypeptide.
Retroviral plasmid vector used for transduction of packaging cell lines with the aim of obtaining cell lines-producers. Examples of packaging cells which may be transliterate are thus not limited to, cell lines RE, RE, ψ-2, ψ-AM, RA, kzt19-14, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAml2 and DAN described in Miller, 1990, Human Gene Therapy, 1, 5-14, which in full is included here for information in the form of bibliographic references. To transducible packaging cells with the vector, you can use any method known in the art. Such methods t Auda, thus not limited to, electroporation, the use of liposomes and the precipitation of calcium phosphate. In an alternative embodiment, the retroviral plasmid vector may be packaged in a liposome or connected with lipid and then introduced to the recipient. Cell line-producers generates infectious retroviral vector particles which bear sequence(ti) nucleic acids encoding the polypeptides. Such retroviral vector particles then can be used for transduction of eukaryotic cells both in vitro and in vivo. Traduzione eukaryotic cells will Express the sequence(ti) a nucleic acid encoding the polypeptide. Eukaryotic cells that can be transducible are thus not limited to, embryonic stem cells, embryonal carcinoma cells, and hematopoietic stem cells, hepatocytes, fibroblasts, cultured myoblasts, keratinocytes, endothelial cells and epithelial cells of the bronchi. Separate gene therapy approach is the so-called "transcreations therapy," in which cells of a patient treated ex vivo with the aim of induction of its own "buried" chromosomal genes to produce protein of interest after the return made to the patient. Transcriptionstar therapy implies that the patient has h rmally chromosome set, required for activation. Transcriptionstar therapy involves the introduction of a promoter or other exogenous regulatory sequences capable of activating target genes, the composition of the chromosomal DNA of the patient's cells ex vivo cultivation and selection of cells that are actively producing the protein, and then reverse the making of activated cells in the body of the patient, assuming that after that they fully "settle down". Then "activated gene" cells produce the protein of interest for a sufficient time, perhaps throughout the life of the patient. U.S. patent No. 5641670 and 5733761 describe this principle: they fully included here for information in the form of bibliographic references.
With the aim of better understanding the present invention provides the following examples. These examples are given solely for illustrative purposes and are not intended to be limit the scope of the present invention. Everything mentioned in this publication are included in full for the information in the form of bibliographic references.
Example 1. Method of isolation of the islets of Langerhans in the rat
Rats Sprague-Dawley (275-320 g) was used as donor islets of Langerhans. Briefly, the pancreas was filled with 10 ml of cold rekonstruirovany Liberase RI (Boehringer Mannheim), with whom Bireli and incubated with an additional 5 ml of enzyme solution in a water bath for 30 minutes. The tissue suspension was twice washed with cold Hanks buffer with 10% of PTS (Gibco), resuspendable in 8 ml of 25% ficoll (Sigma) and then layered ficoll 23%, 20% and 11%, 5 ml each. After centrifugation the islets of Langerhans in the layer of 20% was collected, washed twice with cold Hanks buffer with 10% PTS and resuspendable in culture medium RPMI-1640 with 10% PTS (Sigma).
Example 2. Testing methodology-induced peptide increased levels of insulin in rats
Wistar rats fasted overnight (17 hours), and then they were forced not to move phenobarbital (0.1 ml per 100 g body weight). Glucose (0.4 g/kg, dissolved in saline with 1% bovine serum albumin (human) peptide (dissolved in saline with 1% bovine serum albumin (human) or without were injected with intravenously into the tail vein.
After 1 minute after injection in rats took the eye the blood sample, and 50-100 μl of plasma was tested for insulin levels using a set of Linco RIA kit (Linco Research Inc., St. Charles, MO).
Example 3. The method of determining the effect of peptides on intraperitoneal glucose tolerance in rats
Wistar rats fasted overnight, and then they were forced not to move phenobarbital. In rats took ocular blood samples (time 0), and the peptide (in 1% human albumin) were injected with the tail vein. After five minutes of 1 g/kg glucose (physiological solution) were injected with inside rusinko, and rats took ocular blood samples after 15, 30 and 60 minutes. The levels of plasma glucose was determined using an auto-analyzer Technicon Axon (Bayer Diagnostics Division of Bayer Corp., Tarrytown, NY), operating in accordance with the program SM4-2143F90 "Glucose".
Example 4. The method of determining the effect of the peptides on the water retention in the intestine in rats
Male rats fasted for 24 hours, and drinkers of water they were taken 2-3 hours before the start of the experiment. The peptide or saline were injected with rats subcutaneously without anesthesia. 10 minutes after the dose rats were killed with carbon dioxide and small intestine were cut and weighed (1). The intestine was opened by incision, water from the lumen pomahival filter paper, and the intestines were again weighed (2). The amount of water in the intestine (in grams) was determined as the difference of masses: (1) minus (2).
Example 5. The methodology of peptide synthesis
The basic procedure was used for the synthesis of some of the polypeptides of the present invention. Peptide synthesis was carried out using FMOC/tert-Putilkovo method ("Peptide Synthesis Protocol", 1994, Vol. 35, Michael W.Pennington & Ben M.Dunn) in continuous flow using PEG-polistyrenowych resins Rapp-Polymere (Rapp-Polymere, Tubingen, Germany). Upon completion of the synthesis the peptides were tsalala from the resin and removed protection using a mixture of TFA/DTT/water/triisopropylsilane� (88:5:5:2). Peptides besieged from chip off the mixture with cold diethyl ether. The precipitate washed three times with cold ether and then dissolved in 5%acetic acid followed by lyophilization. The peptides was checked by chromatography with reversed phase column (YMC-Pack ODS-AQ (YMC Inc., Wilmington, NC) system Waters ALLIANCE® (Waters Corp., Milford, MA) using water/acetonitrile with 3% TFA in a gradient from 0% to 100% acetonitrile, and using mass spectrometry MALDI mass spectrometer VOYAGER DETMMALDI (model 5-2386-00: PerSeptive BioSystems, Framingham, MA). The peptide sample was added to the matrix buffer (50:50 with distilled water and acetonitrile with 3% TFA) at a ratio of 1:1. Those peptides that do not satisfy criteria of purity >95%, was purified using chromatography with reversed-phase HPLC system, Waters Delta Prep 4000 (Waters Corp., Milford, MA).
Example 6. Cloning of peptides
Attempt recombinant protein expression VIP had previously been made to obtain ambiguous results. Motives Leu17,Gly29-VIP or Leu17,Gly29Lys30Arg31-VIP were freexpression (Simoncsits et al., 1988, Eur. J.Biochem., 178, 343-350) in the form of a C-terminal chimeras with the N-terminal part of the gene β-galactosidase in E.coli E.coli cells. Removal of methionine from 17th position removes the site of cleavage by bromine cyan. Appendix C C-end Gly or Gly-Lys-Arg was carried out in order to the fulfilling potential C-terminal amidation in vivo by the action of Pamati mammals. After cleavage of the chimeric protein with bromine cyan for methionine, made with N-all mutant variants VIP released mutant VI R cleansed, and it was shown that they exhibit activity similar to those of the native VIP, although these activities were measurable only when the peptides had a concentration of saturation. VIP was preexpansion (Raingeaud et al., 1996, Biochimie, 78, 14-25) in the form of polymer C-terminal chimeras with glutathione-3-transferase (GST) in E. coli cells. Free Monomeric or polymeric peptides VIP released by sequential cleavage by factor XA and hydroxylamine. The need for two-stage splitting led to inefficiency and to obtain a mixture of products. Thus obtained polymeric or Monomeric peptides VIP were less active than the native VIP. An improved version of the design using site cleavage only for factor XA resulted in a mutant with VIP 7-amino acid C-terminal extension and was also less active than the native VIP (Ottavi et al., 1998, Biochimie, 80, 289-293). For expression of RASAR published data to date no. To develop an efficient method for the expression of RASAR, VIP and their mutant variants of the genetic codes of these peptides were cloned as C-terminal to the GST gene with a single site cleavage by factor XA dividing m is nomercy peptide and GST. The gene encoding the site-recognition factor XA, chimerically on the DNA sequence that encodes a peptide that must be obtained, was synthesized by hybridization of two overlapping single-stranded DNA fragments (70-90-nucleotide), including the restriction sites for BamHI or XhoI directly with the 5'-side of the DNA sequence of the gene to be cloned, with subsequent DNA synthesis in the opposite chains using the large fragment of DNA polymerase I (Life Technologies Inc., Gaithersburg, MD). DNA sequence selected for each of the genes was based on reverse-transcribed designed amino acid sequence of each peptide. In some cases, the gene encoding the peptide, obtained by PCR mutagenesis (V.Picard et al., 1994, Nucleic Acids Res., 22, 2587-2591; J.Sambrook, E.F.Fritsch & T.Maniatis, 1989, "Molecular Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, New York) existing gene using the method described above. Double-stranded product is then cut by restrictase BamHI and XhoI and are ligated in the composition of the vector pGEX-6P-1 (Amersham Pharmacia Biotech), which was previously also split restrictase BamHI and XhoI. The DNA sequence of the cloned peptide genes is shown in figure 8.
For example, in the case of cloning the DNA sequences SEQ ID NO 54, 55 and 56 in the composition of the pGEX-6P-1 the following polypeptide sequence is eljnosti were expressed as chimeras with glutathione-3-transferase (GST):
RACER: IEGRHSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYQRVKNK (SEQ ID NO:2)
VIP: IEGRHSDAVFTDNYTRLRKQMAVKKYLNSILN (SEQ ID NO:1)
R3P3: IEGRHSDAVFTENYTKLRKQLAAKKYLNDLKKGGT (SEQ 10 NO:8)
Restriction map of a typical plasmid encoding GST-peptide Chimera, shown in figure 3.
Example 7. Expression and purification of recombinant peptide
The BL21 cells (Stratagene)transformed with plasmids encoding GST-peptide Chimera, were cultured at 37°to achieve OD600or =0.6-1.0 and induced with 1 mm IPTG (Life Technologies) for 2 hours at 37°C. 2 l of cells was centrifuged at 7700g within 15 minutes, weighed and stored at -20°C for at least 3 hours. A bunch of frozen cells resuspendable in 100 ml of chilled on ice FSB with 250 μl of the mixture protease inhibitors (Sigma Chemical: catalogue number P-8465) per 1 g of cells were treated with ultrasound three times for 1 minute with a 15-second intervals. Cellular debris was besieged by centrifugation at 10000g for 20 minutes. The supernatant fraction was mixed with 2 ml of 50%glutathione-sepharose resin 4B (Pharmacia) in a shaker overnight at 4°C. the Resin was centrifuged at 1500g for 15 minutes, Packed in an empty chromatography columns Poly-Prep (Bio-Rad), washed with 30 ml of the FSB and then 10 ml of buffer for factor XA (1 mm CaCl2, 100 mm NaCl and 50 mm Tris-HCl, pH 8.0). The peptides were tsalala from the column by the action of 60 units of factor XA (Pharmacia) in 1 ml buffet is for factor XA over night at 4° With and passed through the HPLC-C18 column (Beckman System Gold) using a 2-ml loop and a flow rate of 2 ml/min with the following program: 10 minutes to buffer A (0.1% of TFA in water), 30 minutes for the gradient to buffer B (0.1% of TFA in acetonitrile), 10 minutes to buffer And 10 minutes for the gradient and 10 minutes to buffer A. Peak fractions (each 1 ml) were collected and analyzed by electrophoresis in LTO-gel with 10-20% of trizina. The fractions containing the peptides of table 1, were combined and dried. Typical outputs amounted to several hundred micrograms of free peptides in 1 liter culture of E. coli. Recombinant peptides have been shown to have the same activities as their synthetic version.
Table 1 includes some of the selected polypeptides obtained in accordance with the discussion above methods peptide synthesis (example 5) or recombinant means, as described in example 7. Peptides obtained by recombinant marked with a lowercase letter "r" in front of the symbol of the peptide. Peptides derived and recombinant and synthetic pathways, indicated by an asterisk after the designation of the peptide. Peptides and R3PO R3P4 were described by Bolin et al., 1995, Biopolymers, 37, 57-66 and in U.S. patent No. 5677419.
Example 8. The secretion of insulin by the islets L is Gergana rats
Peptide R3P3 (0.1 to 100 nm) stimulates insulin secretion allocated by the islets of Langerhans of the rat glucose-dependent mode. The studies compare the effect of peptide R3P3 and GLP-1 on pancreatic cells.
The islets of Langerhans of the rat were isolated and treated with GLP-1 or R3P3 when 3 or 8 mm glucose in the culture medium in accordance with the method of isolation of islets of Langerhans of the rat, as described in example 1. As shown in figures 4A-4B, the peptide R3P3 greatly enhances insulin secretion by the islets of Langerhans, depending on the concentration, and this effect is similar to that identified for GLP-1 is known amplifier insulin secretion.
Example 9. Insulin and glucose response in vivo
As shown in tables 2 and 3, polypeptides that activate the receptor R3, also increases induced by glucose increased levels of plasma insulin compared with the action of one glucose. This elevation of insulin leads to a consistent decrease in the level of plasma glucose.
In accordance with the method of example 2 described above, starving during the night of Wistar rats were forced not to move phenobarbital, were injected with intravenous glucose with or without peptide him and took the eye the blood sample after 1 minute. Each group contained 12 rats. The graph in figure 5 shows that the polypeptide R3P3 makes us the of excretion of glucose, accompanied by an intensification of insulin secretion. The peptide or the filler was injected intravenously with subsequent glucose loading intraperitoneally as described in example 3. For some time after it was determined the level of plasma glucose. As can be seen from the graph, R3P3 significantly accelerates the elimination of glucose from the blood.
Example 10. Diarrhea side effects
As described above, in the method of example 4, starving rats were injected subcutaneously analyzed peptide (5 nmol/kg or 22 to 24 nmol/kg). Five minutes after injection of 0.3 ml of water was given orally. 5 minutes after the water dose animals were killed and determined the water content in the small intestine. As shown in figure 6, the injection of VIP with two doses led to a significant increase in the water content in the lumen of the small intestine compared to control (filler: saline). At the highest dose peptides R3 led to only a very small increase (i.e. roughly about 10%) compared to VIP. At the dose of 5 nmol/kg peptides did not cause any changes in the water content in the small intestine. The degree of water retention was used as an indicator R2 activation in vivo.
Example 11. The effect of peptides on intraperitoneal glucose tolerance in rats
Wistar rats fasted overnight, and then obtd is supported by phenobarbital. In rats took ocular blood samples (time 0) and subcutaneously injected with a peptide (in 1% human albumin). Five minutes later were injected intraperitoneally 1 g/kg glucose (physiological solution) and in rats took ocular blood samples after 30 minutes. The levels of plasma glucose was determined by auto-analyzer Axon, and the results are shown in figure 7.
From figure 7 it is seen that the glucose level in plasma increases up to 160 mg/DL compared to normal (100 mg/DL) in rats, which were injected only filler, 30 minutes after IPGTT test (test intraperitoneal glucose tolerance). In rats, which were injected peptides R3P3, R3P12 and R3P13, the increase of plasma glucose was significantly reduced, indicating that the effect of the hormone insulin. After subcutaneous administration of 1 nmol/kg effect on reduction of glucose each peptide was similar to the effect observed when an equivalent dose of GLP-1.
Example 12. Polypeptides, secreting insulin in a glucose-dependent mode
Table 2 shows a list of peptides that stimulate insulin secretion in vivo in IPGTT test or in vitro test with islets of Langerhans of the rat. As can be seen from these data, peptides enhance mediated glucose insulin secretion and in vivo and in vitro.
Plasma insulin. Data are expressed as percentages of the level of plasma insulin after 1 minute after IVGTT (0.4 g/kg glucose) pax is either with saline solution, or 0.1 nmol/kg R51, P55, R, R, or 1 nmol/kg other peptides in rats Wistar. Blood was taken from the eye, and insulin levels were measured using a kit for analysis of rat insulin Rat Insulin RIA kit (Linco Research Inc., St. Charles, MO).
Plasma glucose: Data expressed in percentage from the field of plasma glucose under the curve after IPGTT (1 g/kg glucose) option with the filler after a dose of 1 nmol/kg of the peptide. As reported (.Filipsson et al., 1997, J.Clin. Endocrinol. & Metabolism, 82, 3093-3098), PACAP-27 induces insulin secretion but does not affect the levels of plasma glucose. The authors of the present invention for the first time showed that this is due to the mutual influence of receptors R2 and R3 on the tendency to excretion of glucose. Selective R2 agonist [K15,R16,L27]VIP(1-7)/GRF(8-27) (P.Gourlet et al., 1997, Peptides, 18, 1539-1545), designated as R2P1, increases the level of plasma glucose by 14%, while selective R3 agonists reduce the level of plasma glucose by about 20%. Therefore, the selectivity in respect of R3 is a desirable property of drugs that should be used to achieve a reduction of glucose levels in the blood for the treatment of type 2 diabetes type.
The secretion of insulin by the islets of Langerhans. Islets of Langerhans were isolated from rats Sprague-Dawley as described in example 1 and processed either order the holder specific peptide (10 nm) for 2 hours. The concentration of insulin in the culture medium was determined using an assay kit Linco Rat Insulin RIA kit. The glucose concentration in the medium was 8 mm. Data are expressed as percentages (insulin) from the variant with the presence of only 8 mm glucose. At 3 mm glucose was not detected induced by the polypeptide of increasing concentrations of insulin: therefore, activity on insulin secretion from these polypeptides is glucose-dependent.
|Peptide||Plasma insulin (% of normal)||Plasma glucose (% of normal)/p/||The secretion of insulin by the islets of Langerhans (% of normal)|
Example 13. The pharmaceutical composition of intravenous drug
A sterile preparation for injection is prepared from 4 mg of the polypeptide SEQ ID NO 72 and 1 liter of sterile saline using the production process, well known in this technical field.
Example 14. The pharmaceutical composition of intravenous drug
A sterile preparation for injection is prepared from 400 mg of the polypeptide SEQ ID NO 174 and 1 liter of sterile saline using a production process that is well known in the art.
Example 15. Influence receptore-selective peptide agonists RACER-27, VIP and RASAR on heart rate in dogs in mind
Greyhounds placed in a bandage so as to accustom them to stay on my feet for 3 hours. Cuff cardiograph disposed around the tail of the dog.
Saline were injected with head veins and heart rate were monitored every 2 minutes to establish normal values. After 10 minutes were injected with peptide and controlled the frequency of cardiac contractions every 2 minutes for 20 minutes. If during this time the frequency of contractions was normal, gave a higher dose and controlled the frequency of contractions for 20 minutes. The area under the curve (DIC) in the first 10 minutes-induced polypeptide changes in heart rate as a percentage exceeding the PCR filler match the graph with the concentration of the polypeptide. RACER-27 and which is selective p is R1 agonist maximilan (Omega, 1997, J.Biol. Chem., 272, 966-970) show similar activity to increase the heart rate. VIP, which activates and R2, and R3, but not R1, selective R2 agonist R2P1 and selective R3 agonists R3P0, R3P3, R3P19, R3P36, R3P51 and R3P53 at least 10 times less active than RASAR-27 or maximilano. Thus, the cardiovascular effect of RESAR-27 mainly be determined by the activation of PACAP-R1. All peptides are absolute agonists in respect of the relevant receptors at a similar level of affinity. R3P0 is Roche-analogue RO 25-1553, which was shown to be a manifestation of the selectivity of PACAP-R3 at least 100 times higher than with respect to R1 and R2 (Gourlet et al., 1997, Peptides, 18, 403-408).
Example 16. SPA-test cyclic AMP
Cells SNO expressing PACAP-R3, were sown in 96-well plates (Costar) at a density of 8×104cells per well and cultured at 37°within 24 hours in the environment α-MEM with nucleosides, glutamine (Gibco BRL), 10% of PTS, 100 μg/ml penicillin/streptomycin, 0.3 mg/ml glutamine, 1 mm HEPES, 0.5 mg/ml geneticin (Gibco BRL). The medium was removed and the tablets were washed FSB. Cells were incubated with peptide in HEPES-FSB-BSA with 0.4 mg/ml soybean trypsinogen inhibitor, 0.5 mg/ml bacitracin, 100 μm IBMX for 15 minutes at 37°C. Cyclic AMP in cell extracts was evaluated quantitatively using a TEC the system for direct SPA-screening camp (Amersham Pharmacia Biotech Inc., Piscataway, NJ). The peptides shown in table 3 were tested for the activity of camp.
Table 3 shows the results of the SPA-analysis of camp in vitro in cells of Cho, transfected PACAP-R2 or PACAP-R3. IS defined as the concentration of the polypeptide, which guarantees 50% of the maximum activity RASAR-27. "NA" indicates no detectable activity. "Selectivity R3" is determined from the relationship AS when R2 IS in R3. Most of the following polypeptides design based on the VIP that has shown no activity against R1 (D.Vaudry et al., 2000, Pharmacological Reviews, 52, 269-324). Therefore, it is believed that these polypeptides do not show any activity in relation to R1. Peptides generated by the parameters of VIP include SEQ ID NO 6 to 53, 62-65 and 70-175.
|Peptide||Seq ID NO||R2 EC50, nm||R3EC50, nm||The selectivity towards R3|
|R3P66||72||100||0.4td align="center"> 250|
Example 17 Obtaining polyclonal antibodies
The synthesis of the peptide Ac-CRKQVAAKKYLQSIKNKRY-COOH was performed using a peptide synthesizer, Applied Biosystems 430A using Fmoc-chemistry in the activation of amino acids by the action of HBTU. The peptide was tsalala using a mixture 84,6% TFA, 4.4%, phenol, 4.4% water, 4.4%, thioanisole and 2.2% identicial. The crude peptide was purified using a C18 column with reversed phase with a gradient of 0.1% TFA in CH3CN. Assessment of purity was performed using a mass spectrometer (PerSeptive V Biosystems Voyager DE Pro MALDI. A cysteine residue was added to KLH using assay kit Pierce Imject Maleimide Activated mcKLH kit and the appropriate recipe (Pierce, Rockford, IL). Rabbits were immunizable using the following schemes to generate polyclonal antisera:
Day 0 - 10 ml pre-blood samples for assessment of normal the different parameters serum. 250 μg of each peptide in 1 ml of an emulsion of complete Freund adjuvant: 0.1 ml to the site 10 sites subcutaneously.
Day 14 - boosting 250 μg of each peptide in 1 ml of emulsion in incomplete Freund adjuvant: 0.1 ml to the site 10 sites subcutaneously.
Day 21 - 35-ml blood sample.
Day 35 - boosting 250 μg of each peptide in 1 ml of emulsion in incomplete Freund adjuvant: 0.1 ml to the site 10 sites subcutaneously.
Day 42 - 35-ml blood sample.
Day 56 - boosting 250 μg of each peptide in 1 ml of emulsion in incomplete Freund adjuvant: 0.1 ml to the site 10 sites subcutaneously.
Day 63 - 35-ml blood sample.
Day 77 - boosting 250 μg of each peptide in 1 ml of emulsion in incomplete Freund adjuvant: 0.1 ml to the site 10 sites subcutaneously.
Day 84 - the final blood sample.
Antibodies were analyzed using the following methods.
Tablet Immulon-III (Dynatech Laboratories Inc., Chantilly, VI) were coated with peptide-N (range 0.3-100 ng) in 100 μl EIA-covering buffer (1.6 l of 0.1 M NaHCO3+0.4 l 0.1 M Na2CO3pH of 9.5) for 3 hours at room temperature. The tablet was bottiroli 100 μl of 5% milk in TBS tween-20 (10 mm Tris, pH 8.0; 150 mm NaCl, 0.05% tween-20 (Sigma P-1379)) for 1 hour at room temperature and washed three times in TBS tween. Antibody to R3P66 added to the wells in 100 μl 5% "bloto" (TBS tween-20 + 5% milk) for 2 hours at room temperature followed by rinsing in TBS tween (wash was repeated p is tickrate). Secondary antibody (conjugate goat anti-rabbit antibody with alkaline phosphatase: BioRad) was added to the wells at a dilution of 1:1000 in 100 μl 5% "bloto" for 1 hour. The tablet is washed 5 times in TBS tween. 0.5 mg/ml p-nitrophenylphosphate (Sigma 104-105) in 100 μl of substrate buffer (1 M diethanolamine, 0.5 µm MgCl2•6H2O, pH 9,8; w/HCl) were incubated at room temperature for 1 hour to O/N. the Tablet read at OD405 using a SPECTRAmax-250 (Molecular Devices Corp., Sunnyvale, CA).
To determine recognized by the antibody produced in rabbits to the peptide Ac-CRKQVAAKKYLQSIKNKRY-COOH in accordance with example 17, the peptide R3P66 (SEQ ID NO 72)was subjected to enzyme-linked immunosorbent assay (ELISA). If the antibody recognizes the peptide, the detected signal OD405. In figure 10 it is shown that these antibodies recognize R3P66, but do not interact with homologous peptides of RESAR-27 or VIP up to a concentration of 30 µg peptide.
Example 18. Hypersensitivity of the respiratory tract in a model of acute asthma in primates
Male macaques (Masasa fascicularis)used in this analysis, contained in the conditions of constant temperature and humidity under a 12-hour light cycle. They were fed twice a day except on the day of the experiment, when they were left without food the night before the procedure. The time of the experiment water was given without restrictions.
Hypersensitivity of the respiratory tract (GFC) (the principal) was measured 1 week before testing (without impact), when the first injection of antigen and again 24 hours after injection of antigen. Induced antigen hypersensitivity of the respiratory tract was measured by the decrease in RS100(concentration methacholine required to cause a 100%increase in lung resistance) after 24 hours compared to the base value. After a 2-week vacation spent another dimension main GFC. After another week, the peptides of the present invention was administered by aerosol for 10 minutes before injection of the antigen. After 24 hours were measured again GFC, and reducing RS100as a result of processing compared with the value without processing.
The experimental procedure. On each day of experiment, animals were forced not to move with a mixture of ketamine/xylazine (70:12 mg/kg 0.1 ml/kg) directly into the cage. Under General anesthesia them transferred to the laboratory where they were placed in the position lying on their back on a heated water blanket on the trolley. In each eye inflicted eye ointment and 0.2 ml of lidocaine (2%) was sprayed into the throat and on the back of the pharynx. The jaws are held apart by using a jaw expander and provided with a cuff endotracheal tube caliber 5,0 (end of which is liberally coated xylocaine gel, 2%) was administered for holding laryngoscopies analysis. Then the animals were placed in a specially designed chair-Ogre is nichael thus, the animal was in a position small deviations ago, but in an upright sitting position, and held only a collar around the neck. The animal was surrounded vodonagrevatel veil.
Endotracheal tube was connected to an artificial respirator Harvard configured 30-35 respiratory acts per minute. Air flow was measured using pneumotachograph Fleisch, and chest pressure was determined using the pressure sensor "validyne" (as the difference between the pressure at the far end of the tube and room pressure).
Pneumotachograph and Validyne was connected to the preamplifier and then to respiratory analyzer MI2. Using primary signal flow and pressure analyzer calculated the resistance and elasticity of the respiratory tract (as well as other respiration parameters). The original 5-6-minute measurement was performed in order to verify the stability of the signal and in that the values of resistance and elasticity are measured.
The introduction of the antigen. Conducted inhalation introduction swine roundworm Ascarium suum. Available extract of pork roundworm were diluted 10 times the FSB with obtaining a solution of 1000 µg/ml Aerosol was injected under pressure through the spray of Rainbow (Puritan-Bennett)attached to the ventilator Bird mark 7A, established the 15 respiratory acts per minute. Spent 30 respiratory acts with the introduction of the antigen, and then within 15 minutes controlled sharp contraction of the bronchi.
After the injection the animal was disconnected from the ventilator, and after it was able to breathe on his own, he was freed from the chair-limiter and laid on his back on the trolley. Upon restoration of normal reflexes (eye blinking, swallowing reflex) along with the muscle tone of the limbs of the animals were returned to the vivarium.
Introduction peptide. The analyzed peptides were delivered via inhalation as described above. The basic solution of the peptides were diluted in sterile water to achieve a concentration of 0.5 μg in 4 μl. Based on previous measurements of the spray, as has been shown, was delivered in 4 μl of one act of breathing, and the number of respiratory acts for each animal was adjusted to deliver the exact final concentration of the spray. For each peptide the final delivered concentrations were as follows:
R3P0 (SEQ ID NO 5) - 0.6 ág/kg;
R3P76 (SEQ ID NO 82) - 3 µg/kg;
R3P82 (SEQ ID NO 88) of 1.8 mg/kg
Introduction metacholine. Curves reactions dose methacholine was built to assess the hypersensitivity of the respiratory tract. In a model of acute response measurement was performed after 24 hours and compared with a sensitivity of 7 days before the experiment is. Aerosol phosphate-saline buffer (FSB) was delivered using sprinklers in accordance with the above. The aerosol was administered for 15 respiratory acts, and then the resistance to light controlled within 10 minutes. Metafolin (Sigma) was brought to a concentration of 100 mg/ml in the FSB and such mother liquor was diluted FSB to the final concentration range from 0.1 mg/ml to 100 mg/ml in 10 minutes after administration of methacholine (0.1 mg/ml, 15 respiratory acts) conducted additional analysis. Sequential dose methacholine were injected with increasing doses with semi-log increments or until double lung resistance, or up to the maximum dose of methacholine (100 mg/kg). The bulk resistance (0%) was taken as the resistance achieved in response to the introduction of the FSB. Strengthening the resilience of the lungs (%) and dose methacholine made in a table of values, and the value of RS100(dose methacholine, causing increased resistance to 100%) was calculated according to the schedule dependence of resistance on the dose. The values obtained were converted into values of log10. The difference between RS100(the value at the 24th hour minus the base value) to experiment with the effects were compared with the data control.
Table 4 shows the parameters RS100for the three peptides. As can be seen from the above data, the peptides effectiverate caused by antigen hypersensitivity of the respiratory tract and therefore are a potential means to treat asthma.
|The parameters RS100for all three of the investigated peptides|
|R3P0 (0.6 mg/ng)||R3P76 (3,0 µg/kg)||R3P62 (1.8 µg/kg)|
|The difference log10PC100||Control||Impact||Control||Impact||Control||Impact|
|P (effect to monitor)||0.240||0.031||0.087|
|% suppression induced ODS||54||100||88||Ȋ|
|*The more negative the value, the more hyperreactive becomes an animal.|
All publications and patents mentioned in this application are included here for information in the form of bibliographic references. Various modifications and changes in the described compositions and methods of the present invention will be clear to experts in the art without deviating from the essence and scope of the present invention. Although the invention has been described in connection with specific preferred option, it should be clear that the claimed invention shall not unreasonably be limited to such specific cases. Indeed, various modifications of the above described ways embodiment of the invention that will be clear to experts in the field of molecular biology or related fields are intended to get into the volume of the following claims. Specialists in the art will understand, or plays only standard experiments they are identified numerous equivalents to the specific variants of the embodiment of the present invention described in this text. Such equivalents are considered as satisfying the following claims.
1. Polypeptide selected from the group consisting of SEQ ID NO:1114, SEQ ID NO:18, SEQ ID NO:21-26, SEQ ID NO:32-36, SEQ ID NO:40-53, SEQ ID NO:57-61, SEQ ID NO:63-99, SEQ ID NO:102-119, SEQ ID NO:121-137, SEQ ID NO:139-177, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:183-202, SEQ ID NO:322-341 and functionally equivalent fragments, derivatives and variants, the polypeptide is a selective agonist PACAP-R3 and has a selectivity regarding R3, and thus the selectivity of the polypeptide in relation R3 is higher than the selectivity of SEQ ID NO:5 or SEQ ID NO:9 with respect to R3.
2. The polypeptide according to claim 1, characterized in that the polypeptide presented in SEQ ID NO:18.
3. The polypeptide according to claim 1, characterized in that the polypeptide presented in SEQ ID NO:32.
4. The polypeptide according to claim 1, characterized in that the polypeptide presented in SEQ ID NO:43.
5. The polypeptide according to claim 1, characterized in that the polypeptide presented in SEQ ID NO:45.
6. The polypeptide according to claim 1, characterized in that the polypeptide presented in SEQ ID NO:47.
7. The polypeptide according to claim 1, characterized in that the polypeptide presented in SEQ ID NO:50.
8. The polypeptide according to claim 1, characterized in that the polypeptide presented in SEQ ID NO:52.
9. The polypeptide according to claim 1, characterized in that the polypeptide presented in SEQ ID NO:71.
10. The polypeptide according to claim 1, characterized in that the polypeptide presented in SEQ ID NO:72.
11. The polypeptide according to claim 1, characterized in that the polypeptide represented the Yong in SEQ ID NO:83.
12. The polypeptide according to claim 1, characterized in that the polypeptide presented in SEQ ID NO:86.
13. The polypeptide according to claim 1, characterized in that the polypeptide presented in SEQ ID NO:87.
14. The polypeptide according to claim 1, characterized in that the polypeptide is selected from the group consisting of SEQ ID NO:328, SEQ ID NO:331, SEQ ID NO:335, SEQ ID NO:339 and functionally equivalent fragments, derivatives and variants.
15. The polypeptide according to claim 1, characterized in that the polypeptide is selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:32, SEQ ID NO:46 and functionally equivalent fragments, derivatives and variants.
16. The polypeptide according to claim 1, characterized in that the polypeptide is selected from the group consisting of SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76 and functionally equivalent fragments, derivatives and variants.
17. The polypeptide according to claim 1, characterized in that the polypeptide is selected from the group consisting of SEQ ID NO:73, SEQ ID NO:104, SEQ ID NO:114, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:169-171 and functionally equivalent fragments, derivatives and variants.
18. The polypeptide according to claim 1, characterized in that the polypeptide is selected from the group consisting of SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:107, SEQ ID NO:130, SEQ ID NO:150, SEQ ID NO:162, SEQ ID NO:168 and functionally equivalent fragments, derivatives and variants.
19. The polypeptide according to claim 1, characterized in that h is of the specified polypeptide selected from the group consisting of SEQ ID NO:45, SEQ ID NO:82 and functionally equivalent fragments, derivatives and variants.
20. The polypeptide according to claim 1, characterized in that the polypeptide is selected from the group consisting of SEQ ID NO:328, SEQ ID NO:331, SEQ ID NO:335 and SEQ ID NO:339.
21. The polypeptide according to claim 1, characterized in that the polypeptide is selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:32 and SEQ ID NO:46.
22. The polypeptide according to claim 1, characterized in that the polypeptide is selected from the group consisting of SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:72, SEQ ID NO:74 and SEQ ID NO:76.
23. The polypeptide according to claim 1, characterized in that the polypeptide is selected from the group consisting of SEQ ID NO:73, SEQ ID NO:104, SEQ ID NO:114, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:166, SEQ ID NO:167 and SEQ ID NO:169-171.
24. The polypeptide according to claim 1, characterized in that the polypeptide is selected from the group consisting of SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:107, SEQ ID NO:130, SEQ ID NO:150, SEQ ID NO:162, and SEQ ID NO:168.
25. The polypeptide according to claim 1, characterized in that the polypeptide represents SEQ ID NO:82.
26. The polypeptide according to claim 1, characterized in that the polypeptide is selected from the group consisting of SEQ ID NO:18, SEQ ID NO:32, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:83, SEQ ID NO:86 and SEQ ID NO:87.
27. The polypeptide according to claim 1, characterized in that the polypeptide represents SEQ ID NO:82 or SEQ ID NO:88.
28. The polypeptide according to claim 1, characterized in that the polypeptide is the Wallpaper SEQ ID NO:88.
29. Polynucleotide encoding the polypeptide according to any one of claims 1 to 28, or a degenerate variant.
30. A vector containing polynucleotide on clause 29.
31. The vector according to item 30, used for installation in a cell of the host.
32. A method of obtaining a polypeptide, which implies
(a) culturing the host cell containing the vector according to item 30, under conditions suitable for expression of the specified polypeptide, and
(b) isolation of the polypeptide of the cultivated host cell.
33. The method of producing a host cell that expresses the polypeptide according to any one of claims 1 to 28, and this method involves the transformation or transfection of a host cell with the vector according to item 30.
34. A method of obtaining a polypeptide comprising
(a) culturing the cell culture on p under conditions suitable for expression of the specified polypeptide, and
(b) isolation of the polypeptide from the cell culture.
35. Pharmaceutical composition for stimulating the synthesis of insulin and its secretion by beta cells of the pancreas, containing a therapeutically effective amount of the polypeptide according to any one of claims 1 to 28 in combination with a pharmaceutically acceptable carrier.
36. The purified antibody is produced under the action of the polypeptide according to any one of claims 1 to 28, where the specified antibody specifically binds to a polypeptide according to any of the C claims 1 to 28.
37. A method of treating type II diabetes and pre-diabetic condition violations glucose tolerance, implying the introduction to the mammal a therapeutically effective amount of the polypeptide according to any one of claims 1 to 28.
38. The method according to clause 37, wherein the polypeptide is characterized by at least 10-fold selectivity against PACAP-R3 compared to PACAP-R2 or PACAP-R1.
39. The method according to clause 37, wherein the polypeptide is characterized by at least 100-fold selectivity against PACAP-R3 compared to PACAP-R2 or PACAP-R1.
40. The method according to any of p-39, characterized in that said metabolic disorder is diabetes of the 2nd type.
41. The method according to any of p-39, characterized in that said metabolic disorder is pre-diabetic state of impaired glucose tolerance.
42. The method according to any of PP-41, characterized in that therapeutically effective amount is in the range from about 0.1 μg/kg to about 1 mg/kg
43. Option vasoactive intestinal peptide, representing the polypeptide according to claim 1, selected from the group consisting of SEQ ID nos:11-14, SEQ ID NO:18, SEQ ID NO:21-26, SEQ ID NO:32-36, SEQ ID NO:40-53, SEQ ID NO:57-61, SEQ ID NO:63-99, SEQ ID NO:102-119, SEQ ID NO:121-137, SEQ ID NO:139-177, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:183-202 and its functional equivalents.
44. The method of stimulation of secretion of the Insa is in glycosidation mode in need thereof of a mammal, implying the introduction of a given mammal the polypeptide according to claim 1, selected from the group consisting of SEQ ID nos:11-14, SEQ ID NO:18, SEQ ID NO:21-26, SEQ ID NO:32-36, SEQ ID NO:40-53, SEQ ID NO:57-61, SEQ ID NO:63-99, SEQ ID NO:102-119, SEQ ID NO:121-137, SEQ ID NO:139-177, SEQ ID NO:179, SEQ ID NO:180 and SEQ ID NO:183-202.
45. A method of treating asthma in a mammal, which implies the introduction to the mammal a therapeutically effective amount of the polypeptide according to any one of claims 1 to 28.
46. The method according to paragraph 41, wherein the polypeptide is used to treat asthma.
47. The method according to paragraph 41, wherein the polypeptide used for the treatment of hypersensitivity of the respiratory tract.
28.09.1999 according to claim 2 to 8, 15, 21;
15.06.2000 on PP-13, 16-19, 22-25, 40, 41, 45 and 47;
27.09.2000 according to claims 1, 14, 20, 34, 44, and 46;
28.09.1999, 15.06.2000, 27.09.2000 on PP-33, 37-39, 42-43;
15.06.2000 and 27.09.2000 on PP and 36.
FIELD: gene engineering, in particular purification and isolation of polynucleotides.
SUBSTANCE: invention relates to purification and isolation of polynucleotides regulating mammalian gene transcription and is useful in regulation of heterologous polynucleotide expression, obtaining transgene animals, and identification of affined regulatory DNA sequences. DNA containing transcriptional regulatory DNA of hamster gene EF-1α was isolated by screening of genome library to Chinese hamster ovary (CHO-K1). Chimeric polynucleotide including isolated regulatory DNA of hamster gene EF-1α operably bonded to gene sequence encoding target protein product other than protein encoded by hamster gene EF-1α was constructed. Obtained chimeric polynucleotide is used as component of expression plasmid for transformation or transfection of host cell. To increase target gene transcription in host cell DNA containing regulatory DNA of hamster gene EF-1α was integrated into host cell genome DNA in site operably bonded to target gene. Method of present invention make it possible to increase mRNA expression level for operably bonded heterologous polynucleotides by 3-11 times.
EFFECT: increased mRNA expression of operably bonded heterologous polynucleotides.
31 cl, 3 tbl, 7 ex
FIELD: genetic engineering, molecular biology.
SUBSTANCE: invention proposes a method for detecting genes encoding membrane-bound transmembrane proteins. Method involves expression of the nucleic acid chimeric sequence in the cell-host consisting of DNA fragment encoding secreting protein that is able to bind antigen and DNA fragment to be tested; interaction of cells expressing the fused protein with antigen; selection of cells on surface of that indicated antigen is bound; isolation of recombinant vector containing in selected cells of DNA fragment to be tested and, if necessary, determination of its sequence. Also, invention proposes the developed vector constructions and comprising their sets designated for realization of the proposed method. Invention provides significant simplifying the screening process of libraries and cloning genes encoding transmembrane proteins. Invention can be used for detecting and preparing genes encoding any membrane-bound proteins used in different branches of science and practice.
EFFECT: improved isolating method, valuable biological properties of protein.
27 cl, 7 dwg, 1 tbl, 8 ex