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Synthesis of New Immunomodulators

#A-500


Chemical Synthesis of New Immunomodulators of Brain. Biochemical Mechanisms of Their Action and Implementation in Medicine

Tech Area / Field

  • CHE-SYN/Basic and Synthetic Chemistry/Chemistry
  • BIO-CHM/Biochemistry/Biotechnology
  • MED-DRG/Drug Discovery/Medicine

Status
3 Approved without Funding

Registration date
07.04.2000

Leading Institute
Institute of Biochemistry, Armenia, Yerevan

Collaborators

  • Bowling Green State University / Department of Chemistry, USA, OH, Bowling Green\nUniversität Tübingen / Physiologisch-Chemisches Institut, Germany, Tübingen

Project summary

During past decades a significant progress is observed in studying biological active peptides of brain: neuropeptides. That area of studies refers to the priority ones in modern bioorganic chemistry, biochemistry, pharmacology, and medicine. Creation of new drug preparations on the basis of native peptides is a step to the future evolutionary medicine. Those preparations are of new type released by the organism in the process of evolution in response to various negative exterior effects such as stress, pain, fear, etc. Evolutionary approach in the medicine attracts attention by the fact that the endogenous peptides in difference from traditional drug remedies, do not possess accompanying impact. They are rapidly split in the organism and manifested the effect mainly at pathologies.

It is for many years in the Department of Neurohormones Biochemistry of H.Buniatian Institute of Biochemistry, NAS RA, headed by Prof. Armen Galoyan there are carried out basic studies on isolation of new peptide neurohormones from the hypothalamus and neurohypophysis, deciphering of their structures, as well as revelation of neuroendocrine and neurochemical mechanisms of regulation by these peptides of visceral and endocrine organs.

There were isolated and chemically identified a number of new viscerotropic neurohormones, as well as biologically active compounds. They can be used for the regulation of cardiovisceral system (namely, coronary circulation ) and metabolism of visceral organs. Among newly discovered polypeptides of hypothalamus there was revealed a peptide system of regulation of the actvity for Ca2+ -CaM-dependent enzymes, particularly, cAMP and cGMP PDE, MLCK, calcineurine, 5’-nucleotidase, etc. These peptides are constrictors for coronary vessels. Coronary dilatory glycopeptides are as antagonists for the mentioned polypeptides in reference to coronary vessels tonus. Thus, there are revealed basic mechanisms for the regulation of coronary circulation by peptides of hypothalamus.

Intogether with cardioactive neurohormones discovered by A.Galoyan, of a certain role are studies on polypeptides with immunomodulatory activity. Thus, from the neurosecretory granules of the hypothalamus and neurohypophysis Galoyan A.A. succeeded to isolate and identify immunomodulators. Neurophysine II appeared to be their precursor. From the neurosecretory granules of the neurohypophysis and hypothalamus there was isolated for the first time and identified interleukin 1, interleukin 1, interleukin 2, 6, as well as tumor necrosis factor (TNF-). The primary structures of the latter are fully deciphered. It was shown that these peptides possess the properties of activators of the immune system on one hand, and on the other hand the ability to defence the neurones at various disturbances of nerve system (hemisection of the spinal cord, neurotoxicosis, effect of snake venom, etc.). The experimental data testify that these new polypeptides, e.g. immunomodulators of brain, are of importance for chemical medicine at treatment of neurodegenerative, immune, and other diseases (Alzheimer’s disease, Parkinson’s disease, psychical diseases, etc.). Chemical synthesis of the mentioned polypeptides will create wide possibilities as for multiprophyle scientific studies, as well as for the treatment of the above indicated human diseases.

There is an intention to carry out researches on studying the structure-activity relationship of the known peptide - Delta Sleep Inducing Peptide (DSIP; Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu). DSIP is a neuropeptide with a wide spectrum of neuromodulatory activity . The literary data (as well as our own ones) testify DSIP to increase stability against stress, to normalize biochemical and clinical changes in the organism induced by stress, to be effective at treatment of opioid intoxication and symptoms of withdrawal . There are data on DSIP immune stimulating activity. There is high correlation between DSIP-like immunoreactivity in plasma in suicidal patients with major depressive disorders and shizophrenic volunteers.In difference from all the other known peptides DSIP possesses a unique capability to penetrate through blood brain barrier which is very important at creation of a new preparation based on it, and is an important limitation for the other neuropeptides.

Taking into account the above mentioned and the fact that recently a number of new polypeptides-immunomodulators were discovered in the Department of Neurohormones Biochemistry, Institute of Biochemistry NAS RA, which are not identified yet by anyone, hence the goal of the present project is:

· To work out for the first time methods of chemical synthesis for a number of new polypeptides with immunomodulating activity which will be capable to supply high homogeneity.

· To realize the synthesis of the above mentioned peptides in preparative quantities purified analytically (homogeneity according to data of analytical HPLC, H1-NMR, mass-spectroscopy, and amino acid analysis).

· To study biological activity and biochemical mechanisms of action of the synthetized polypeptides. It is supposed to study pharmacological, biochemical and physiological mechanisms of action.

· To synthetize new structural analogues of DSIP having an aim to increase enzymatic stability of native peptide, namely the analogues with substitution in DSIP structure for D- amino acids and unnatural amino acids.

· To study biological activity of new analogues of DSIP in various in vivo and in vitro test systems.

· To study physicochemical properties of new analogues of DSIP by spectral methods (NMR, CD, etc.).

In the Institute of Biochemistry of NAS RA there are real objectives for the fulfilment of the project proposed: Project manager is academician Armen A.Galoyan. He is an author of two monographs, over 400 publications, 11 patents connected with the mentioned problems .

In the Department of Neurohormones Biochemistry (headed by prof.A.Galoyan) there is founded a laboratory of chemical synthesis for neurohormones leaded by A.S.Sargsyan, who has been trained in one of the largest scientific centres of the FSU - in M.Shemyakin Institute of Bioorganic Chemistry of RAS, in the lab of Peptides Chemistry (headed by acad.V.T.Ivanov). A.S. Sargsyan has a number of publications on DSIP structure-activity relationship. Another laboratory of Chromatography and Biotechnologiy of Neurohormones exists in the Department of Neurohormones Biochemistry. There are employed methods of isolation on HPLC with reverse-phase columns. Microsequencing and mass-spectral analysis will be possible to do in Yerevan. In the Department of Neurohormones Biochemistry it is worked out a strategy for studying biological activity as well as biochemical and molecular mechanisms of peptide neurohormones (including immunomodulators discovered by Prof.A.A.Galoyan).

The Department of Neurohormones Biochemistry has a wide net of scientific co-operation with numerous centres of the world: Centre for Neurochemistry in New York (headed by Prof.Abel Lajtha); Department of Biochemistry and Molecular Biology at School of Medicine and Health Sciences in Washington (headed by Allan Goldstein); Division of Neurology, Department of Medicine Vancouver Hospital and Health Sciences Centre (Prof. Seung Kim); A.Bach Institute of Biochemistry of RAS (headed by Prof.B.F.Poglasov).

In case of financial support by ISTC it would be possible in the frames of the presented project to work out for the first time methods of chemical synthesis for recently discovered peptide immunomodulators, to obtain them in sufficient quantities to carry out investigations, to obtain highly purified (analytical grade) peptide preparations demanding international requirements. In its turn the synthesis of these peptides in preparative quantities will promote full investigation of biochemical and molecular mechanisms of their effect in norm and at various pathologies. Implementation of those new neuroendocrine preparations will give an impulse to the development of biochemistry and practical medicine. On the other hand the synthesis of new peptides – immunomodulators will create possibility to begin a new (higher) stage in their study, particularly study of relationship between the structure and function of these peptides for obtaining new analogues possessing with improved (in compare with native peptides) properties: increased stability, prolonged effect, etc.

Realization of the present project will promote further integration of scientists from Armenia to International Scientific Community. The obtained results will be published in international journals and will be reported at International Simposia and Conferences. In the process of project fulfilment there will be established a constant contact with foreign collaborators via exchange of visits and information. Realization of this project will support both basic and applied studies in the field of new preparations creation for the medicine. Successful fulfilment of the goal in the frame of the project will promote realization of national problems in the branch of basic researches and public health care. Creation of a new drug preparation will be of a commercial interest for RA, indeed as on the expences of financial income for intelectual property on one hand, and on the other hand on the expence of new industrial management and new drug production.

Present project is supported by Prof.Voelter Wolfgang from the Institute of Physiologically Active Compounds of Tubingen University (Germany) who is one of the outstanding scientists all over the world experienced in peptide synthesis. There is an agreement with Prof.W.Voelter on technical and moral promotion of the proposed project. For the scientists of the Institute of Biochemistry of NAS RA they will make available the information exchange, study of current methods for peptide synthesis, feasibility for use of modern equipment. There is an agreement to hold joint seminars and symposia, as well as aid to obtain new equipment and chemicals for peptide synthesis.

At working out methods for synthesis of peptide immunomodulators it is supposed to use two assays for peptide synthesis such as a classic method for synthesis in the solution, and a solid phase one with the use of polymer resin and automated peptide synthesator. In the process of classic method there will be used Boc/Bzl and Fmoc/OBut strategies. Certain peptide blocks will be synthetized by the method of consecutive increase of a chain from C-end using diisopropyl carbodiimide with 1-hydroxybenzotriazol (HOBT), method of activated ethers, etc. Protected peptides at intermediate stages will be purified by crystallization, their inpiduality and purity will be controlled by TLC in 4 systems of solvents and by amino acid analysis. After condensing the blocks the protected peptides will be purified from the accompanying products with HPLC in reverse-phase using preparative or semi-preparative columns. After removal of protecting groups the free peptides will be subjected to purification by complex methods including ion-exchange chromatography (DEAE-Sephadex, KM-Sephadex, etc.), gel-filtration and at last HPLC (preparative columns). By using hard phase approach synthesis there will be used chlormethylated polysterol resin containing 1% of cross-linked with pinyl benzene. In the process of synthesis there will be used Boc/Bzl, Fmoc/OBut strategy. The structure and homogeneity of the synthetized peptides will be affirmed by data of amino acid analysis, thin layer chromatography in 4 systems of solvents, H1-NMR (300 MHz) and mass-spectroscopy.


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