Synthesis of Short Peptides with Corticotropin-Like Structure and Study of Their Stress-Protector Activity
Tech Area / Field
- MED-DRG/Drug Discovery/Medicine
8 Project completed
Senior Project Manager
Weaver L M
Institute of Bioorganic Chemistry (Branch), Russia, Moscow reg., Puschino
- Institute of Highly Pure Biopreparations, Russia, St Petersburg
- CHUM Centre de Recherche, Canada, QC, Montreal\nQueen's University / Department of Chemical Engineering, Canada, ON, Kingston
Project summaryThe objectives of the project: synthesis of a set of short corticotropin-like peptides and study of their effect on non-specific resistance of laboratory animals subjected to various stress factors.
Stress – is one of the general non-specific neuro-humoral body responses to the attack of “extreme” irritants (stressors), which is mainly determined by the function of hypothalamus, hypophysis and adrenal cortex (Selye H). The physiology and pathology of exposure to stress. Montreal, 1950; Selye H. The stress of life. N. Y., 1956). Stress serves a protective function and is aimed at the re-establishing of the disturbed homeostasis. It differs from the strain of hearing, vision and muscle tone, since it is not related to any particular system (visual, muscular etc.). Clinically stress manifests itself as a general adaptation syndrome. The resulting changes in the body are so constant that they can be regarded as specific for this kind of syndrome, despite the fact that stress by itself is not specific by its etiology. It should be noted that pathogenic effect of any agent includes specific action on a tissue, non-specific local effect and non-specific effect mediated by hypophysial-adrenal system. According to the opinion of Selye - the founder of the stress theory, there is no disease that is not influenced by adaptation syndrome and, vice versa, there is no disorder representing a pure adaptation illness.
The action of stressors results in enhanced secretion of hypothalamic corticotropin-releasing hormone that through the portal blood vessels system enters the anterior lobe of hypophysis and stimulates adrenocorticotropic hormone (corticotropin) secretion. Corticotropin enters the blood, reaches the adrenal cortex and stimulates glucocorticoid production in its cells. The main vitally important action of glucocorticoids under stress conditions is to provide the body rapidly with glucose by activating gluconeogenesis in the liver. On the other hand, these hormones play a permissive role in catecholamines’ action on nonstriated muscles of vessels. Under stress conditions adrenaline and noradrenaline are released from adrenal core and as a result, the centralization of blood circulation occurs, that is increased blood delivery to muscles and decreased – to peripheral tissues. This effect of catecholamines takes place only in the presence of glucocorticoids (cortisol). Increased secretion of adrenal hormones due to prolonged or excessive action of stressor can result in pathological processes (necrosis, suppression of inflammatory reactions that are necessary to restrict infection spreading, etc.) and adaptation syndrome progression. A growth of psycho-emotional pressure and deterioration of ecological situation – all these factors result in the enhancement of stress and decline in adaptation capacities of modern man. Therefore, one of the most important tasks of modern biology and medicine is the construction of novel effective and safe agents capable of elevating adaptation potential of the human body under the influence of various extreme factors.
At present in the Laboratory of protein chemistry and Laboratory of biological testing of Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry studies on properties and mechanism of action of corticotropin-like peptides (immunocortin and leucocorticotropin) are carried out. In the early 1980s a group of scientists from Institute for Biological Research (California, USA) (Julliard J.H. et al. Science, 1980, 208, 183-185) trying to isolate corticotropin from human placenta extract, used immobilized antibodies to this hormone as affinity adsorbents. A 50 kDa protein was thereby isolated; it was found to be a heavy (H) chain of immunoglobulin G (IgG). Elucidation of the causes of such an effect led to the discovery of corticotropin-like sequence in the H-chain. It was found that 9-22 fragment of variable part of IgG1 H-chain (AEVKKPCSSVKVSC) was homologous (36%) to corticotropin aminoacid sequence 11-24 (KPVGKKRRPVKVYP). The authors of the present project have synthesized and studied the decapeptide VKKPGSSVKV, corresponding to the sequence 11-20 of the variable part of human IgG1 H-chain (termed immunocortin). The results obtained indicate that immunocortin displays neurotropic and immunosuppressive activity in vitro. Similar to corticotropin, immunocortin binds with high affinity to the hormone receptors on synaptic brain membranes, lymphocytes and macrophages of rats and mice and activates adenylate cyclase of target cells (Mitin Y.V., Navolotskaya E.V. et al. Int. J. Peptide Protein Res., 1993, 41, 517-521; Lepikhova T.N., Navolotskaya E.V., Zargarova T.A. et al. Rus. J. Immunol., 1998, 3, (3-4), 266-269;. Peptides, 2000, 21, 353-357; Navolotskaya E.V., Zargarova T.A. et al. Biochemistry (Moscow), 1999, 64. (7), 758-764; Rus. J. Bioorgan. Chem., 2000, 26 (1), 27-33; Immunol. Lett., 2000, 72, 93-99). Corticotropin-like sequence was also found in human, bovine, rabbit and rat pro-interleukin-1a (pIL-1a): fragment 81-88 of pIL-1a (GK-VLKKRR) has 60% homology with corticotropin fragment 10-18 (GKPVGKKRR). The authors of the project have synthesized the octapeptide GKVLKKRR (termed leucocorticotropin). An investigation of its activity revealed that, similar to immunocortin, it binds with high affinity to corticotropin receptors on mouse splenocytes and macrophages and activates adenylate cyclase of these cells (Zav’yalov V.P., Navolotskaya E.V. et al. Immunol. Lett., 1995, 46, 125-128). Thus, immunocortin and leucocorticotropin have mechanism of action identical with corticotropin: peptides bind to the hormone receptors and activate adenylate cyclase of target cells.
Collaborative research conducted by scientists from Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences and laboratory of radio-biophysics (Lomonosov Moscow State University, Biology Department) demonstrated that immunocortin affects the production of 11-oxycorticosteroids (CS) in rat adrenals in vivo. Single intramuscular injection of immunocortin (10 mg/kg) resulted in CS content reduction in adrenals and blood twofold within 1 h and threefold within 24 h. The significant CS content reduction in adrenals within 24 h after the injection of immunocortin allows conclusion about complex systemic effect of the peptide in the whole body. The results obtained make it reasonable to study the effect of immunocortin as well as its fragments and analogs on the content of corticosteroids and catecholamines in the brain, blood and adrenals in normal conditions and under various kinds of stress.
The researchers from the laboratory of radio-biophysics over a period of years investigate the role of biogenic amines and corticosteroids in pathophysiology and biochemistry of stress. They have established animal models to study changes in the content of these compounds in tissues after hemorrhagic shock, acute hypobaric hypoxia, hypo- and hyperthermia. The determining role of corticosteroids- catecholamines system in the conditions of hemorrhagic shock and hypoxy has been demonstrated [Deev L.I., Goncharenko E.N. et al. Biull. Eksp. Biol. Med. (Russian) 1997, 124 (7), 50-52; Antonova S.V., Akhalaia M.Ya., Goncharenko E.N. et al. Biull. Eksp. Biol. Med. (Russian) 1997, 124 (10), 400-402; Platonov A.G., Goncharenko E.N., Kudriashova N.Yu. Biull. Eksp. Biol. Med. (Russian) 1999, 128 (11), 518-520]. The authors of the present project have studied the possibility to use semax (a synthetic analog of corticotropin fragment 4-10 that possesses prominent anti-hypoxy properties) for the correction of changes in biogenic amines system (serotonin in the brain stem and spleen, adrenaline and noradrenaline in the adrenals) during resuscitation period after hemorrhagic shock. It has been shown that a single injection of semax early after resuscitation allows correction of the changes taking place during the early stage (1-7 day), but do not influence the changes occurring during the late stage (28 day) [Bastrikova N.A., Goncharenko E.N., Kudriashova N. Yu. Patol. Fiziol. Eksp. Ter. (Russian) 1999, 3, 19-22.]. These results combined with a large body of literature data concerning application of peptide preparations for the correction of hemorrhagic shock and hypoxia consequences, suggest that peptides are able to restore the balance of all systems of the body. Nowadays bioactive peptides, the derivatives of natural peptide hormones, are regarded as potential new type therapeutic agents. Such peptide properties as rapid effect in the body, non-toxicity and ultimate degradation to single aminoacid level have now aroused considerable interest among biologists and medical circles all over the world. Unfortunately, polyfunctionality of peptide hormones limits their use as therapeutic agents. Therefore, for a construction of new drugs based on natural peptide hormones it is necessary to synthesize their selectively acting analogs resistant to enzyme degradation. In the frames of the present project we plan to: 1) synthesize a set of peptides with corticotropin-like structure (no less than 50 linear and cyclic analogs of corticotropin fragment 11-24, immunocortin and leucocorticotropin with the length of 2-14 aminoacid residues);
2) select by preliminary testing the peptides exhibiting the most affinity to corticotropin receptors in rat adrenals; 3) study the possibility to use high-affinity peptides for the correction of corticosteroids- catecholamines system disturbance after acute hemorrhagic shock, hypobaric hypoxia, hypo- and hyperthermia, induced in rats. Relying on the analysis of the correlation between structure and stress-protective activity of corticotropin-like peptides, a conclusion will be made about the main requirements for their structure.
One of the most important adaptive reactions is autoregulation of diaminoxydase (histaminase) activity. This enzyme catalyzes desamination of histamine and prevents its accumulation in toxic amounts. The authors of the project have shown that under stress conditions as a result of body adaptation ability reduction the balance of histamine-diaminoxydase (DAO) system disturbs [Akhalaia M.Yu., Goncharenko E.N., Kudriashova N.Yu. et al. Radiats Biol. Radioecol. (Russian) 2001, 41 (1), 56-58]. The capacity to maintain a steady state of histamine-DAO system under stress is regarded as one of the indicators of adaptation abilities of the body. A particular importance for adaptation to functional overload has the condition of cardiovascular system; for its normal functioning a tolerance of histamine-DAO system is necessary. In this connection in the frames of the project we plan to investigate the ability of corticotropin-like peptides to make corrections in histamine-DAO system in myocardium of rats subjected to hypo- and hyperthermia.
According to current concepts, radiation stress is a special kind of stress reaction of the body with no receptors for external irritant – ionizing radiation. Obviously free radicals resulting from radioactive irradiation initiate the reactions of lipids peroxidation and trigger, by negative feedback mechanism, an activation of stress-realization systems, hypersecretion of biogenic amines and corticosteroids. The scientists from the laboratory of radio-biophysics have found that 137Cs g-radiation in low doses (0.06 cGy/day) influences the level of catecholamines and corticosteroids in mouse adrenals [Goncharenko E.N. et al. Radiats Biol. Radioecol. (Russian) 2000, 40 (2), 160-161]. The content of these compounds changed similarly: within 1-2 days after irradiation they increased, and within 9 days -statistically proven decrease (about 30%) was found. In the frames of the project we plan to study the effect of high-affinity corticotropin-like peptides on changes in catecholamines and corticosteroids content in adrenals of mice subjected to low doses of ionizing radiation. The most active peptides will be recommended for further study as correctors of radiological hazard, caused by low doses of radioactive irradiation, when non-specific component (stress) plays an important role in the body reaction to radiation.
Thus, investigations in the frames of the present project will make it possible to prepare, study and propose for pre-clinical trials short corticotropin-like peptides, improving the body tolerance to various stress factors (hypo- and hyperthermia, hypoxia, hemorrhagic shock, ionizing radiation).