Subject of the research:“Construction of new-age medicines on basis of peptide antibiotics — molecular factors of innate immunity”
Leader of the research: the director of ESC T.V. Ovchinnikova.
At the moment medicine faces with a problem of pathogens’ resistance to antibiotics. Intensive rising of resistant bacterial infections makes search of new medicines be necessary. Research at the SEC IBCh are targeted to a complex structural and functional study of new natural peptide antibiotics, mechanisms of their functioning as molecular factors of inherent immunity, to development of production technologies of their recombinant and synthetic analogs, to preclinical tests and design on their base of new-age antibiotics.
Discovery of versatile biological functions of endogenous antimicrobial peptides (AMP) shows that these natural antibiotics are perspective molecules for creation of new antibiotics and have ability to inactivate a wide spectrum of microorganisms, including bacteria, fungi, protozoa, tunicary viruses. AMP as molecular factors of inherent immunity work as mediators of phagocytic, inflammatory and stress processes. In view of AMPs’ antibacterial and immunomodulatory activity presence, a lot of foreign pharmaceutical companies (in USA, Canada, France, the Netherlands, etc.) began creation of new antibiotics on their base. Successful clinical trials were passed by peptide antibiotics for treatments of sepsis, meningitis, pneumonia, gastric ulcer, candidiasis, mucositis, gingivitis, acne, impetigo and for healing of burns and wounds complicated with repeated infection.
Research at the ESC are aimed to discovery, purification, study of structure, biological characteristics and molecular mechanisms of natural peptide antibiotics’ action, development of methods of their production and to preclinical tests. Objects of research are bacterial, fungi, vegetable and animal AMPs, including those of bacteria Bаcillus licheniformis, fungi Emericellopsis salmocynnemata and Emericellopsis minima, lentil Lens culinaris, nereides Arenicola marina, jellyfish Aurelia aurita, sturgeon Acipencer guldenstadti, tortoise Emys orbicularis, toads Bufo bufo gargarisan, etc. Particularly, from coelomic cell of nereides Arenicola marina new peptides were extracted, which have high antibacterial activity in relation to gram-positive and gram-negative bacteria and yeast fungi; they were named “arenicins”. Determined by us full primary structure of processed arenicins and genes of their predecessors lets come to the conclusion that we revealed new family peptides, which don’t belong to any known family of AMP. A new effective technology of a heterologous expression was created, strain-producers were engineered, new methods of extraction and purification of genetically engineered arenicins were developed. Few patents of Russian Federation were acquired for original structure of arenicins and their production methods. At the same time fundamental research on arenicins activities are being continued, so as detailed study of their biological activity mechanisms and preclinical tests with animal models. Discovery, structural and functional research of other groups of peptide antibiotics are being continued too. The matters being under the study are original and patentable. The final aim of project is creation of new effective medicines’ prototypes series on base natural peptide antibiotics and biotechnological platform for their production.
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Novel antimicrobial peptides from ancient marine invertebrates (2018-12-03)
As a part of the study of antimicrobial peptides (AMPs) from animal species, conducting at the Science-Educational Centre of the IBCh RAS, novel host-defense cationic peptides from ancient marine invertebrates were found – nicomicin-1 and -2 from the small Arctic polychaeta Nicomache minor and polyphemusin III from the horseshoe crab Limulus polyphemus, and their structural and biological properties were studied. The peptides were expressed in the bacterial system, and their spatial structure was analyzed. Nicomicins are unique among polychaeta AMPs scaffolds, combining an amphipathic N-terminal α-helix and C-terminal extended part with a six-residue loop stabilized by a disulfide bridge. This structural arrangement resembles C-terminal Rana-box motif observed in the α-helical host-defense peptides isolated from frog skin. Nicomicin-1 exhibited strong in vitro antimicrobial activity against Gram-positive bacteria at submicromolar concentrations. The main mechanism of nicomicin-1 action is based on membrane damage but not on the inhibition of bacterial translation. The structural analysis of prepronicomicins reveals that the BRICHOS domain does not exclusively participate in biosynthesis of β-hairpin polychaeta AMPs, but could also be a part of precursor of α-helical AMPs, namely nicomicins. Polyphemusin III is β-hairpin AMP that caused fast permeabilization of the cytoplasmic membrane of human leukemia cells HL-60. Flow cytometry experiments for annexin V-FITC / propidium iodide double staining revealed that the caspase inhibitor, Z-VAD-FMK, did not abrogate disruption of the plasma membrane by polyphemusin III. Our data suggest that polyphemusin III disrupts the plasma membrane integrity and induces cell death that is apparently not related to apoptosis. In comparison to known polyphemusins and tachyplesins, polyphemusin III demonstrates a similar or lower antibacterial effect, but significantly higher cytotoxicity against human cancer and transformed cells in vitro.
- (2018). Cytotoxic Potential of the Novel Horseshoe Crab Peptide Polyphemusin III. Mar Drugs 16 (12),
- (2018). Novel Antimicrobial Peptides from the Arctic Polychaeta Provide New Molecular Insight into Biological Role of the BRICHOS Domain. Mar Drugs 16 (11),
Dimerization of β-hairpin antimicrobial peptides plays a key role in the cytotoxicity but not in the antibacterial activity (2017-12-03)
In this investigation we performed the structure-activity relationship study of arenicin-1 to design less cytotoxic analogs of the peptide. More than 50 modified arenicin-1 analogs were obtained using methods of directed mutagenesis and heterologous expression in bacteria. The comparative evaluation of the impact of different biophysical parameters such as hydrophobicity, net charge, and β-hairpin length was carried out. Taken together, the obtained data suggest that dimerization rather than high hydrophobicity is the key reason of the cytotoxicity of natural β-hairpin antimicrobial peptide. The pronounced bactericidal activity against pathogens including extensively drug resistant ones as well as a wider therapeutic window makes arenicin analogs promising broad-spectrum antibacterial agents.
- (2017). Dimerization of the antimicrobial peptide arenicin plays a key role in the cytotoxicity but not in the antibacterial activity. Biochem Biophys Res Commun 482 (4), 1320–1326
- (2017). A therapeutic potential of animal β-hairpin Antimicrobial Peptides. Curr Med Chem 24 (17), 1724–1746
- (2016). Bioengineering and functional characterization of arenicin shortened analogs with enhanced antibacterial activity and cell selectivity. J Pept Sci 22 (2), 82–91
- (2015). Design of antimicrobial peptide arenicin analogs with improved therapeutic indices. J Pept Sci 21 (2), 105–113
The mechanism of lipid binding and transfer by plant lipid-transfer proteins (2017-12-03)
The lentil lipid transfer protein, designated as Lc-LTP2, was isolated from the Lens culinaris seeds. The protein belongs to LTP1 subfamily and consists of 93 amino acid residues. Its spatial structure contains four α-helices (H1-H4) and a long C-terminal tail. Here, we report the ligand-binding properties of Lc-LTP2. The fluorescent TNS binding assay revealed that the Lc-LTP2 affinity for saturated and unsaturated fatty acids was enhanced with a decrease in acyl chain length. Measurements of boundary potential in planar lipid bilayers and calcein dye-leakage in vesicular systems revealed preferential interaction of Lc-LTP2 with the negatively charged membranes. Lc-LTP2 more efficiently transferred anionic dimyristoyl-phosphatidylglycerol (DMPG) than zwitterionic dimyristoyl-phosphatidylcholine (DMPC). NMR experiments confirmed the higher affinity of Lc-LTP2 for anionic lipids and the ones with smaller volumes of hydrophobic chains. The acyl chains of the bound lyso-palmitoyl-phosphatidylglycerol (LPPG), DMPG, or dihexanoyl-phosphatidylcholine molecules occupied the internal hydrophobic cavity, while their head groups protruded into aqueous environment between H1 and H3 helices. The spatial structure and backbone dynamics of the Lc-LTP2/LPPG complex were determined. The internal cavity was expanded from ~600 to ~1000 А3 upon the protein ligation. Another entrance into the internal cavity, restricted by the H2-H3 interhelical loop and C-terminal tail, appeared to be responsible for the Lc-LTP2 attachment to the membrane or micelle surface and probably played an important role in the lipid uptake determining the ligand specificity. Our results confirmed previous assumption regarding the membrane-mediated antimicrobial action of Lc-LTP2 and afforded molecular insight into its biological role in the plant.
- (2017). Ligand Binding Properties of the Lentil Lipid Transfer Protein: Molecular Insight into the Possible Mechanism of Lipid Uptake. Biochemistry 56 (12), 1785–1796
- (2017). Plant pathogenesis-related proteins PR-10 and PR-14 as components of innate immunity system and ubiquitous allergens. Curr Med Chem 24 (17), 1772–1787
- (2016). A novel lipid transfer protein from the pea Pisum sativum: Isolation, recombinant expression, solution structure, antifungal activity, lipid binding, and allergenic roperties. BMC Plant Biol 16 (1), 107
- (2016). Lipid Transfer Proteins As Components of the Plant Innate Immune System: Structure, Functions, and Applications. Acta Naturae 8 (2), 47–61
- (2016). A novel lipid transfer protein from the dill Anethum graveolens L.: Isolation, structure, heterologous expression, and functional characteristics. J Pept Sci 22 (1), 59–66