Synthesis and investigation of the peptides stimulating bactericidal activity of macrophages
Tech Area / Field
- MED-DRG/Drug Discovery/Medicine
3 Approved without Funding
Institute of Bioorganic Chemistry (Branch), Russia, Moscow reg., Puschino
- Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany
- University of Maryland / School of Medicine, USA, MD, Baltimore\nCHUM Centre de Recherche, Canada, QC, Montreal
Project summaryThe proposed research project is directed towards the solution of one of the most important problems of modern biology and medicine – the creation of novel effective and safe agents for the protection against bacterial infections.
Objective of the project: Synthesis of short peptides, agonists of non-opioid β-endorphin receptor, and development on their basis agents enhancing resistance to microbial infection.
The most effective currently available approach to the treatment of infectious diseases is the use of antibiotics. The last generation of antibiotics are highly potent and relatively low toxic and low allergic. Nevertheless, the prolonged application may cause a number of serious complications. The microorganisms may acquire the resistance even to "strong" antibiotics. and the antibiotic therapy inevitably leads to a decrease or complete loss of the sensitivity of the organism to these drugs. This continuous "arms race" results in permanent needs of novel and more potent drugs. The encounter of the immune system with antigens is a determining factor for its maturation and proper functioning. The presence of some bacteria in the organism is tonic for the immune system. On the other hand, the intestinal dysbacteriosis inevitable after an oral administration of antibiotics leads to the weakness of immune system that opens the way for viral infections and allergies.
We have synthesized three peptides: SLTCLVKGFY (the fragment 364-373 of the IgG heavy chain), VKGFY, and cyclo(VKGFY), referred to as immunorphin (IMN), pentarphin (PNT) and cyclopentarphin (cPNT), respectively. We found that 1 nM of these peptides significantly increased the adhesion and spreading of murine peritoneal macrophages and their bactericidal activity in vitro, as it has been shown by phagocytosis of Salmonella typhimurium virulent strain 415 (peptides were about two orders more potent than patented stimulator of phagocytosis tuftsin). We found that the effects of IMN, PNT and cPNT on macrophages are mediated by naloxone-insensitive (non-opioid) receptor for β-endorphin. Our studies have shown that these peptides, when given intraperitoneally at the physiological concentrations, are non-immunogenic and non-toxic for mice (LD50 > 2500 mg/kg). (International Patent WO03061683A1 Publication Date: 31.07.2003). The degradation of the peptides yields amino acids. Thus, the listed above peptides in combination with antibiotics is likely able to reduce the therapeutic doses and, hence, the negative side effects of the antibiotics.
In the frames of the project we plan to
- synthesize a set of short peptides (no less than 50 linear and cyclic analogs of IMN and β-END with the length of 3-5 amino acid residues);
- select by receptor binding assay the peptides with a high affinity to the non-opioid β-endorphin receptor on mouse peritoneal macrophages;
- study the possibility to use the peptides with a high affinity for the enhancement of a resistance of laboratory animals to a variety of macrophage-based infections, such as Mycobacterium tuberculosis, Salmonella typhimurium, Staphylococcus aureus. Based on the analysis of the correlation between structure and macrophage-stimulating activity of peptides, a conclusion will be made about the main requirements on their structure.
Major research tasks:
- Synthesis of a set of short, linear, and cyclic β-endorphin-like peptides and analysis of their affinity for murine macrophage non-opiod β-endorphin receptor in vitro.
- Study of the ability of high-affinity peptides to stimulate bactericidal activity of macrophages during phagocytosis of Salmonella typhimurium virulent strain
415 in vitro.
- Investigation of the effect of peptides possessing by the most phagocyte-stimulating activity in vitro on survival of laboratory animals infected by widely-distributed microbial infections (Mycobacterium tuberculosis, Salmonella typhimurium, Staphylococcus aureus).
Expected Results and their applications
Results of basic research and theoretical importance:
- Data for the influence of synthetic peptides, agonists of non-opioid β-endorphin receptor, on functional activity of murine peritoneal macrophages in vitro and in vivo.
- Data for the correlation between structure and macrophage-stimulating activity of synthetic peptides, agonists of non-opioid β-endorphin receptor.
Results of applied and commercial significance:
- Recommendations on the applications of the synthetic β-endorphin-like peptides to enhance the resistance of human and animals against the pathogenic microorganisms.
- Conclusion about the main requirements for the structure of β-endorphin-like peptides with the bactericidal-stimulating activity of macrophages.
- Short linear and cyclic peptides enhancing resistance to microbial infection.
Technical Approach and Methodology
To solve the tasks of the project a complex of modern methods of investigation will be used:
Peptides will be synthesized by both solid-phase and classical methods of peptide synthesis. Rough product will be purified first by low-pressure chromatography and then by preparative HPLC. The purity of desirable compounds will be assessed by analytical reverse phase HPLC, and the structure will be confirmed by amino acid analysis and mass-spectroscopy. The purity of the synthesized products must be no less than 99%. The spatial structure of active analogs will be analyzed by two-dimensional NMR-spectroscopy, CD and UV-spectroscopy methods. Radioligand analysis will be used for the estimation of affinity of the peptides to non-opioid receptor of mouse peritoneal macrophages. At first [3H]IMN will be prepared. The peptides with highest affinity will be chosen for further investigations.
The phagocytosis of Salmonella typhimurium virulent strain 415 by mouse peritoneal macrophages in vitro will be used as a model for the selective influence of peptides on phagocytic activity of macrophages. We will use in this study the virulent strain of Salmonella typhimurium 415 with typical morphological and functional properties. The LD50 was approximately 100 bacterial cells per mouse injected intraperitoneally. Peritoneal macrophages will be obtained from male BALB/c mice, 10-12 weeks old. The peptides with the most phagocyte-stimulating activity will be selected for investigations in vivo.
The experiments in vivo will be carried out in the animal house, licensed for the work with Mycobacterium tuberculosis, Salmonella typhimurium and Staphylococcus aureus in accordance with the specially developed schemes and protocols.
Meeting ISTC Goals and Objectives
The actualization of this project will make a valuable contribution to the development of fundamental and applied biology and medicine, integration of the scientists from our institutions in the world scientific community and also will give the possibility to our scientific groups to achieve economical independence due to the elaboration and development of the technologies in the given project. At the same time, the project will contribute into the civil conversion of the scientists from the Institute of Immunological Engineering, who fulfilled earlier the State orders of the Ministry of Defense, to give them possibility to redirect their scientific interests and to apply their scientific experience for carrying out of fundamental and applied investigations.
Role of Foreign Collaborators/Partners
Consistent with the scope of activities of the project proposal the collaboration with the foreign scientists will include:
- Information exchange in the course of project implementation;
- Discussions of the experimental data obtained in the course of the project execution;
- Collaborative research works in the scope of project activities.