New Immuno- and Neuromodulatory Peptides
Synthesis and Study of Novel Immuno- and Neuromodulatory Peptides, with Homologous "Hormone-Like" Sequences in Human Immunoglobulin G
Tech Area / Field
- MED-DRG/Drug Discovery/Medicine
8 Project completed
Senior Project Manager
Gremyakova T A
Institute of Bioorganic Chemistry (Branch), Russia, Moscow reg., Puschino
- Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany
- University of Turku, Finland, Turku\nTexas A&M University / College of Veterinary Medicine, USA, TX, College Station
Project summaryThe proposed research project is directed towards the solution of one of the most important problems of biology and medicine – the design of novel effective and nontoxic immunomodulating as well as neuromodulating substances.
Last years bioactive peptides, the derivatives of natural peptide hormones, have received increasing attention as potential new type therapeutic agents. The discovery of tuftsin – the tetrapeptide, originating from human immunoglobulin G (IgG) heavy chain (IgG 289-292, H-TKPR-OH) and possessing both immunoregulatory and neurotropic (namely analgesic) activities has stimulated the search for another immuno- and neuromodulating peptides of the same origin.
In the early 1980s a group of scientists from the Institute for Biological Research (California, USA) discovered that definite aminoacid sequences in IgG heavy chain are homologous to adrenocorticotropic hormone (ACTH) and b-endorphin. It was found that 364-377 fragment (SLTCLVKGFYPSDI) was homologous (40%) to b-endorphin antigenic determinant (KSQTPLVTLFKNALKN), and 9-22 fragment (AEVKKPCSSVKVSC) was homologous (36%) to ACTH aminoacid sequence 11-24 (KPVGKKRRPVKVYP). An artificial peptide (14 aminoacid residues) corresponding to b-endorphin-like sequence in IgG heavy chain was synthesized and found to interact with opioid receptors on rat brain cells. A research group from the Institute of Immunological Engineering has synthesized and partially studied both b-endorphin-like decapeptide (H-SLTCLVKGFY-OH), referred to as immunorphin, and ACTH-like decapeptide (H-VKKPGSSVKV-OH) corresponding to IgG heavy chain residues 364-373 and 11-20, respectively.
It has been shown that immunorphin competes with b-endorphin for naloxone-insensitive receptor sites on human and murine immunocompetent cells as well as on murine brain membranes. At a concentration of 1 nM immunorphin stimulated both macrophage activity and mitogen-induced proliferation of mouse splenocytes. The experimental data have revealed that the tetrapeptide Н-KGFY-OH is the minimal fragment of immunorphin which reproduces its receptor binding properties and biological activity [Zav’yalov V.P. et al. Immunol. Lett. (1996) 49, 21-26].
We have also studied the characteristics of the ACTH-like decapeptide. This peptide has been shown to compete with 125I-ACTH(13-24) (“address segment”) for binding to receptors on murine thymocytes (Ki = 5,8 nМ) and on human T-lymphoblastoid cell line MT-4 cells (Ki = 6,5 nМ). Experiments to determine the influence of the ACTH-like peptide on proliferation of thymocytes and MT-4 cells revealed that this peptide at a concentration of 1nM significantly (more than two times) reduced both mitogen-stimulated blast transformation of thymocytes and MT-4 cells proliferation. Intracerebral injection of the ACTH-like peptide to rats at doses ranging from 50 to 150 mg resulted in suppression of b-endorphin- or stress-induced analgesia. At the same time it potentiated the analgesic effect of tuftsin. Highly specific receptors for the peptide (Kd ~ 1-5 nМ) have been found on synaptic membranes of the mouse brain. Binding of the ACTH-like peptide to these receptors was accompanied by activation of adenylate cyclase and increase in intracellular cAMP content (2,5-3 times), activation of phosphokinases followed by standard reactions cascade, which is determined by enzymatic profile of target cells, and resulted in biological response [Mitin Yu.V. et al. Int. J. Peptide Protein Res. (1993) 41, 517-521; Navolotskaya E.V. et al. Immunol. Lett. (1998) in press.].
Thus, the preliminary studies indicate that both decapeptides display highly specific, saturable and reversible binding to their receptor sites on immunocompetent and neural cells and do possess certain immuno- and neuromodulatory properties.
Objectives of the project:
· To investigate the effect of b-endorphin-like, ACTH-like peptides and their modified analogs on the immunity and the central nervous system functions.
· To design novel effective immunomodulators and neuromodulators on the basis of the most active peptides.
Main tasks to solve the above problems:
· To synthesize b-endorphin-like, ACTH-like peptides and their bioactive analogs in amounts sufficient for thorough investigation.
· To investigate immunomodulatory properties and antitumor activity of b-endorphin-like, ACTH-like peptides and their derivatives.
· To examine neurotropic activity and neuroprotective effect of the peptides in vitro and in vivo , to elaborate suggestions concerning their use as regulators of brain functions.
The scientific novelty of the proposed investigation:
· Immunoregulatory and neuromodulating activities of b-endorphin-like, ACTH-like peptides and their analogs.
· Receptor sites for b-endorphin-like, ACTH-like peptides and their analogs on immune and nervous cells.
· The results obtained will shed more light on relationship between the immune and nervous systems.
· The development of synthetic peptides with valuable properties, resistant to proteolytic degradation.
· Suggestions concerning their use as immunomodulators with a broad spectrum of action (for treatment of infectious diseases, tumors and for correction of age-depressed immunity) and as regulators of brain functions.
Technical Approach and Methodology
· b-endorphin-like, ACTH-like peptides and their analogs will be synthesized by solid-phase method and purified by HPLC. Aminoacid composition will be confirmed.
· Synthesized peptides will be characterized by mass-spectroscopy, UV- and NMR-spectroscopy methods.
· Immunomodulating activity of the peptides will be analyzed by standard methods for testing humoral and cellular immunity.
· Radioligand analysis will be used for a study of receptor reactivity and binding. b-endorphin-like and ACTH-like peptides will be labelled with tritium and their tyrosine-containing analogs – with iodine-125.
· Antitumor activity of the peptides will be tested on tumor-bearing mice with transplantable tumors.
· In vitro studies will be carried out on human and rat transformed cell lines and rat organotypic hippocampal slice culture.
· In vivo studies will be carried out on appropriate animal models for human disorders.
International co-operation under the project
In order to establish new contacts and for further integration into the international scientific collaboration network, the following activities will be scheduled within the framework of this project:
· elaboration of common research strategy and collaborative programmes;
· exchange of information and materials required for successful research;
· training of personnel in specific areas of partner competence, mobility programme;
· organization of meetings and seminars with participation of collaborators;
· elaboration of assistance programme for Russian partner (consultations, advice, preparation of publications, provision of computer modelling programmes of molecular structures);
· preparation of new research projects and research of potential foreign partnership via corresponding collaborator.