Department of Biological Testing

All publications (show selected)

Arkady Murashev

A novel approach to bacterial expression and purification of myristoylated forms of neuronal calcium sensor proteins.

Laboratory of pharmacokinetics

The well-established procedures for preparation of native-like myristoylated forms of recombinant neuronal calcium sensor (NCS) proteins via their bacterial co-expression with N-myristoyl transferase from Saccharomyces cerevisiae often yield a mixture of the myristoylated and non-myristoylated forms. We report a novel approach to preparation of several NCSs, including recoverin, GCAP1, GCAP2, neurocalcin δ, and NCS-1, ensuring their nearly complete N-myristoylation. The optimized bacterial expression and myristoylation of the NCSs is followed by a set of procedures for separation of their myristoylated and non-myristoylated forms using a combination of hydrophobic interaction chromatography steps. We demonstrate that the refolded and further purified myristoylated NCS-1 maintains its Са2+-binding ability and stability of tertiary structure. The developed approach is generally suited for preparation of other myristoylated proteins.


  1. Vladimirov VI, Baksheeva VE, Mikhailova IV, Ismailov RG, Litus EA, Tikhomirova NK, Nazipova AA, Permyakov SE, Zernii EY, Zinchenko DV (2020). A Novel Approach to Bacterial Expression and Purification of Myristoylated Forms of Neuronal Calcium Sensor Proteins. Biomolecules 10 (7), 1–22

De Novo Design and Chemical Synthesis of an Antibacterial Peptide

Laboratory of Biological Testing,  Group of Peptide Chemistry

Aiming to design a novell class of antibacterial peptides chemical synthesis of a conjugate consisting of two functionally-active peptide fragments was proposed. The first one possesses amyloidogenic properties toward a parent cytosolic protein of bacteria and the second one belons to a family of cell penetrating peptides. We presumed that the cell-penetrating part of the conjugate would deliver the amyloidogenic fragment accross the bacterial cell membrane into cytosol where this fragment induced polymerisation of the cytosolic protein. This in turn would lead to the death of a bacterial cell. The approach described was tested using conjugates consisting of a number of amyloidogenic fragments of protein S1 from Thermus thermophilus and Tat cell penetrating peptide. One of the conjugates exhibited considerable antibacterial activity comparable to kanamycin. Preliminary studies aiming to shed light on the mechanism of conjugate action at the molecular level indicate that the mode of the interaction of the conjugate with the bacteria is more complex than the presumed one described above, however the existance of the intracellular target for the conjugate is not exluded.


  1. Kurpe SR, Grishin SY, Surin AK, Selivanova OM, Fadeev RS, Dzhus YF, Gorbunova EY, Mustaeva LG, Azev VN, Galzitskaya OV (2020). Antimicrobial and amyloidogenic activity of peptides synthesized on the basis of the ribosomal S1 protein from thermus thermophilus. Int J Mol Sci 21 (17), 1–19

Natural polyamines – novel modulators of the proteasome activity

Laboratory of pharmacokinetics,  Laboratory of hormonal regulation proteins

Anionic buffers such as phosphate or carbonate inhibit proteasome activity during alkalization. Importantly, supplementation of a carbonate–phosphate buffer with spermine counteracts carbonate-driven proteasome stalling in alkaline conditions, predicting an additional physiological role of polyamines in maintaining the metabolism and survival of cancer cells.

Three-finger neurotoxins and new analogues of bradykinin-potentiating peptides were found in the venom of vipers for the first time, using transcriptomics and quantitative proteomics

Laboratory of ligand-receptor interactions,  Laboratory of Biological Testing,  Laboratory of molecular toxinology

From Azemiops feae viper venom, we have isolated previously azemiopsin, a new neurotoxin, that inhibited the nicotinic acetylcholine receptor. To characterize other A. feae toxins, quantitative proteomics was used and 120 unique proteins were identified. In total, identified toxins represented 14 families, among which bradykinin-potentiating peptides with unique amino acid sequences were found and manifested biological activity in vivo. Proteomic analysis also revealed non-conventional three-finger toxins belonging to the group of toxins with neurotoxic activity. This is the first indication of the presence of three-finger neurotoxins in viper venoms. In parallel, transcriptome analysis of the venom gland was carried out by the next generation sequencing, and additionally identified 206 putative venom transcripts. Together, the study unveiled the venom proteome and venom gland transciptome of A. feae which in general resemble those of other snakes from the Viperidae family. However, new toxins not found earlier in viper venom and including three-finger toxins and unusual bradykinin-potentiating peptides were discovered.

Among acid-sensitive ion channels (ASIC) the ASIC1a and ASIC3 subunits are the most attractive pharmacological targets. The inhibition of these channels by specific ligands can treat many socially significant diseases, such as chronic and pathological pain, ischemic stroke, and Parkinson's disease. Sevanol, a natural lignan isolated from Thymus armeniacus, inhibits the activity of ASIC1a and ASIC3 isoforms and has a significant analgesic and anti-inflammatory effect. However its production by chemical synthesis is a labor and material consumption process from many stages. The structure-functional investigation  allowed to minimize the structure of Sevanol and to keep both analgesic activity in animal models, and ASICs inhibitory effect for analogs. The location of Sevanols binding site in the Central vestibule of ASIC1a was predicted in mathematical modeling experiment and its competition with the FRRF-amide peptide for this binding site was proved by electrophysiology. Sevanols analogues had a significant analgesic and anti-inflammatory effect in animal models by various administration routes- intravenous or intramuscular (parenteral methods), as well as intranasal or oral (non-invasive methods). Cozy synthesis method developed for each analogs allow us to recommend these molecules as potential prodrugs, since the absence of side effects identified at this moment together with oral activity are potent competitive advantages of new molecules to existent analgesic drugs. The results were published in Pharmaceuticals (Basel) journal.

Created by: Osmanov D.I., Vladimirov A.A., Koshelev S.G., Andreev, Y.A., Kozlov S.A. from laboratory of neuroreceptors and neuroregulators, Belozerova, O.A., Kublitski V.S. from laboratory of metabolic pathways chemistry, Chugunov A.O., Efremov R.G. from laboratory of biomolecular modeling, Palikov V.A., Palikova, Y.A., Shaikhutdinova E.R., Dyachenko I.A. from laboratory of biological examination, and  Gvozd A.N. from scientific center of biomedical technologies of the Federal medical-biological Agency.

The physiological effect of two bisbenzylisoquinoline alkaloids having activity on ASIC1a

Laboratory of bioengineering of neuromodulators and neuroreceptors,  Laboratory of Biological Testing,  Laboratory of neuroreceptors and neuroregulators

The ASIC1a is the most sensitive subtype of acid-sensing ion channel in the cell membrane, and it plays an important role in the excitation of neurons of CNS. Long time the ligands to this ASIC subtype are under intense attention for the development of drugs for pain relief, as well as protectors from strokes and neurodegenerative diseases. In in vitro experiments on heterologically expressed ASIC1a channels, the action of two bisbenzylisoquinoline alkaloids from plants was studied by electrophysiological method of two-electrode potential fixing on oocyte cells.

The alkaloid lindoldhamine extracted from the leaves of Laurus nobilis L. significantly inhibited the ASIC1a channel’s response to physiologically-relevant stimuli of pH 6.5–6.85 with IC50 range 150–9 µM, but produced only partial inhibition of that response to more acidic stimuli. In mice, the intravenous administration of lindoldhamine at a dose of 1 mg/kg significantly reversed complete Freund’s adjuvant-induced thermal hyperalgesia and inflammation; however, this administration did not affect the pain response to an intraperitoneal injection of acetic acid. Thus, it was shown not only a prospective of plant alkaloids using for a pain relief, but was indirectly confirmed the involvement of the ASIC1a channels of the peripheral nervous system in the generation of a pain response to mild acidification.

The structural analogue named daurisoline, unlike lindoldamine, did not inhibit the activation of the ASIC1a channel by protons, but produced the second peak component of the ASIC1a current. This second peak manifested with a 2.5 seconds delay after the first fast respond followed by completely desensitization with the same kinetics as the main peak. The presence of second current components was specific characteristic of ASIC2 and ASIC3 subtypes early, but this component is sustained, that last all time while the acid stimulus presented. The discovery of the second component of ASIC1a current allows us to declare the common mechanism of opening and desensitization for all ASICs, which will be interesting to determine in further experiments.

Mechanism of the nonopioid β-endorphin action on the activity of the hypothalamic–pituitary–adrenal axis

Laboratory of hormonal regulation proteins,  Laboratory of toxicology in vitro

The binding of β-endorphin to a nonopioid receptor on the anterior pituitary cells has been found to increase the expression of inducible NO-synthase, which leads to a decrease in the secretion of ACTH and corticosterone. Thus, it has been shown that the nonopioid β-endorphin receptor and its agonists are involved in the regulation of the activity of the hypothalamic–pituitary–adrenal axis at the level of the pituitary and adrenal glands.

Caveolin-1 as a regulator of neuronal calcium sensor proteins in phototransduction system.

Laboratory of pharmacokinetics

Caveolin-1 is the major regulatory protein of detergent resistant membranes (DRM), which associating with regulation of signaling activity in different organism systems. Rod cell membranes contain cholesterol-rich DRM, which accumulate visual cascade proteins. In this study, photoreceptor Ca2+-binding proteins recoverin, NCS1, GCAP1, and GCAP2, belonging to neuronal calcium sensor (NCS) family, were recognized as novel caveolin-1 interacting partners. We demonstrated that all studied proteins co-precipitate with -from rod outer segment membranes, and can directly interact with caveolin-1. Pull-down assay, surface plasmon resonance spectroscopy and isothermal titration calorimetry data indicate that there is interaction with full-length caveolin-1 N-terminal domain (1-101) and caveolin-1 scaffolding domain (81-101). Interestingly that this interaction occurs only in absence of calcium ions, what is supported by surface localization of caveolin-1 interaction site in Ca2+-free NCS proteins state. Caveolin-1 increase Ca2+-sensitivity of recoverin, and as a consequence, makes its inhibitory activity to rhodopsinkinase more pronounced, but not interfere with recoverin-­rhodopsinkinase interaction. Amount of free Ca2+, required for this process, consider caveolin­1 influence, become in good agreement with physiological conditions of photoreceptor cell. GCAP-2 is upregulated by caveolin-1 in Ca2+-free state, which increase guanylate cyclase activity. It seems that there is a common mechanism of interaction between caveolin-1 and NCS proteins in Ca2+-free state. For recoverin increasing of Ca2+-sensitivity is due to the stabilization of the open conformation of its second Ca2+-binding domain EF2. Obtained data suggest that at low calcium NCS proteins are compartmentalized in photoreceptor rafts via binding to caveolin-1, what enhances their activity or ensures their faster responses on Ca2+-signals.


  1. Vladimirov VI, Zernii EY, Baksheeva VE, Wimberg H, Kazakov AS, Tikhomirova NK, Nemashkalova EL, Mitkevich VA, Zamyatnin AA, Lipkin VM, Philippov PP, Permyakov SE, Senin II, Koch KW, Zinchenko DV (2018). Photoreceptor calcium sensor proteins in detergent-resistant membrane rafts are regulated via binding to caveolin-1. Cell Calcium 73, 55–69

Development of efficient solid-phase synthesis methods for the preparation of peptides that possess immunosuppresive activity

Laboratory of Biological Testing,  Laboratory of pharmacokinetics,  Group of Peptide Chemistry

The relative efficacies of several synthesis methods have been investigated aiming to prepare target peptides that are quite potent in a test for suppression of the experimental autoimmune encephalomyelitis. Toward this goal chemical yields of the target peptides as well as side product distributions in the samples of the peptides obtained using various methods have been evaluated and quantitatively characterized. A variation of Fmoc/tBu methodology have been found to be the most efficient providing targets peptides in the highest chemical yield since it allowed to use a broader range of  activated amino acid derivatives. Side products of the amino acid doubling were detected among the side products of chemical synthesis. Some of the amino acid doubling side products made the HPLC purification step to be complicated. Nevertheless the synthetic methods developed allowed the preparation of the target peptides in sufficient quantities.

Multiple sclerosis is a chronic autoimmune disease with neurological pathology. The dominant role of immunological processes in the development of the disease dictates the need for medications that specifically minimize the activity of immune processes. The peptides A8AMS and mA8AMS, homologous to the fragment of the human IgG VH domain, have been shown to act in vitro and in vivo, effectively reducing the symptoms of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. The results provide new opportunities for the development of peptide drugs for the treatment of multiple sclerosis.

Molecular mechanism of the non-opioid beta-endorphin action

Laboratory of hormonal regulation proteins,  Laboratory of pharmacokinetics,  Laboratory of toxicology in vitro

Using a synthetic peptide octarphin (TPLVTLFK), a selective agonist of non-opioid beta-endorphin receptor, data on the molecular mechanism of the non-opioid action of the hormone have been obtained. It was found that the non-opioid effect of beta- endorphin on the target cell is realized by the following way: increase in the inducible NO synthase expression → increase in NO production → increase in the activity of soluble guanylate cyclase → increase in the intracellular level of cGMP.