INTRODUCTION

At the Institute of Chemistry of Natural Compounds (currently the Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, RAS) with the participation of Vadim Tikhonovich Ivanov in the 1960–1970s, a topochemical approach was developed which made it possible to purposefully create new structural types of biologically active molecules. This approach involved reversing the direction of acylation, replacing ester and amide bonds, and changing the configuration of asymmetric centers. The possibilities of the developed approach were clearly demonstrated in the determination of the chemical and spatial structure of such antibiotics as valinomycin and enniatin, as well as in the study of the ion transport they carry out through biological membranes. These works have received wide recognition in the world, as evidenced by their presentation at international congresses and publications in prestigious high-ranking journals (see, for example, [1]). As part of the PhD thesis of V.I. Tsetlin, the applicability of this approach to the creation of effective inhibitors and substrates of proteolytic enzymes was tested. Then, in the laboratory of V.T. Ivanov, work was carried out with natural protein neurotoxins: analysis of the spatial structure, and development of methods for selective modification and study of the interaction of toxins with their target—the nicotinic acetylcholine receptor (nAChR). The next step was the analysis of the interaction of nAChRs with α‑conotoxins, which was carried out at the Laboratory of Neuropeptide Reception (headed by the author of this review) in collaboration with V.T. Ivanov’s Laboratory of Peptide Chemistry.

It has been established that a change in the configuration of individual amino acid residues in linear peptides affects the efficiency of their interaction with the proteolytic enzymes chymotrypsin and pepsin [2]. The most interesting results were obtained for cyclopeptides: it was previously believed that peptide bonds in cyclic peptides are inaccessible for cleavage by proteolytic enzymes, and cyclopeptides can only play the role of inhibitors; however, in our work, a series of cyclic peptides was synthesized containing a Leu-Tyr fragment sensitive to the action of pepsin and chymotrypsin, but differing in the number of Gly residues in the peptide (4–8). As a result, it was found that hexa- and octapeptides are inhibitors, while cyclodecapeptide turned out to be a substrate that was superior in efficiency to the then available linear substrates of these enzymes [3]. Modern computer modeling methods make it possible to design ligands with the desired structure and functional properties based on NMR databases and X-ray diffraction analysis of the spatial structures of peptide and protein compounds, as well as their complexes with enzymes, receptors, and other targets. However, it should be noted that the synthesis of peptide analogs with retro- and retro-enantio structure and their cyclization continue to be used, for example, to obtain peptides resistant to proteolysis, as well as for peptides used for intracellular delivery of various compounds attached to them [4]. The same approach was applied to the synthesis of a proteolysis-resistant peptide that interacts with the transferrin receptor and is able to penetrate the blood–brain barrier [5]. Retro-enantio-peptides are also considered as promising candidate compounds for preventing the penetration of the HIV-1 virus (human immunodeficiency virus type 1) [6]. Since neurotoxins from snake venom will be discussed below, it is appropriate to note a recent work in which retro-enantio-peptide fragments of crotalicidin from rattlesnake venom are considered as possible antimicrobial agents [7].

The next stage of work under V.T. Ivanov’s guidance was the study of the conformation of natural snake protein neurotoxins and the study of the topography of their binding to the target, nAChR. Works in the IBCh RAS on toxin topics were initiated by Academician Yu.A. Ovchinnikov. In the laboratory of V.T. Ivanov, a series of selectively labeled derivatives of snake neurotoxins was synthesized, and in collaboration with the laboratory led by V.F. Bystrov, using the 1H-NMR method, the first information was obtained on the spatial structure of the neurotoxin, in particular, the proximity of loops II and III of the neurotoxin was established [8]. In those years, the complete structure of such a protein could not be established by 1H-NMR, but a year later, the complete spatial structure of the related neurotoxin was determined by X-ray diffraction analysis [9], in which the location of the three loops of the neurotoxin was characterized in detail (because of which later the name of these neurotoxins appeared as “three-loop”); our conclusion was also consistent with the crystal structure.

In Ivanov’s laboratory, natural α-neurotoxins and their chemically modified derivatives were studied by various spectral methods [10]. Of fundamental importance was our first reduction of the long-type natural α-neurotoxin and the developed conditions for its subsequent reoxidation with complete restoration of the initial toxicity [11].

An important task was to obtain information about which areas the neurotoxin contacts with its target, nAChR. For this, in Ivanov’s laboratory, using selective chemical modification, a series of derivatives was obtained containing one spin or fluorescent label on the identified residues of α-neurotoxin II from the venom of the Central Asian Naja oxiana cobra. Involvement of the Swedish scientist Evert Karlsson, who first purified the nAChR receptor from the Torpedo marmorata electric ray by affinity chromatography on a snake toxin column provided us with this receptor and allowed us, by using fluorescence and electron paramagnetic resonance (EPR) methods in collaboration with the laboratory of V.F. Bystrov, for the first time to identify a number of amino acid residues of a neurotoxin in contact with the receptor [12]. It should be noted that at that time there was no information about the spatial structure of the receptor itself.

At present, X-ray diffraction and cryoelectron microscopy data are available for muscle-type nAChRs from the electric organ of the Torpedo stingray and for some nAChRs of the neuronal type. Crystal structures of complexes of α-neurotoxins with models such as acetylcholine-binding protein (AChBP), which mimics the ligand-binding domain of nAChR, as well as with heterologously expressed ligand-binding domains of the α1 and α9 subunits of nAChRs, have previously been made with α-bungarotoxin, a long-type neurotoxin; complexes with full-length Torpedo and α7 nAChRs were later resolved with the same toxin. The above study of the binding topography was performed by us on a short-type neurotoxin; the presented toxins somewhat differ from long-type neurotoxins in selectivity of binding to different types of nicotinic receptors. However, the cryoelectronic structure of the Torpedo receptor in combination with a short-type neurotoxin was established quite recently [13]. As in the case of α-bungarotoxin, the central loop II of the neurotoxin plays a major role in binding. However, in the previously established structures, the side loop III of α-bungarotoxin did not come into contact with the receptor, but in the case of a short neurotoxin, it is essential for interaction. We drew the conclusion about the role of this loop in earlier works, which was also noted by the authors of the cited paper [13].

In addition to fluorescent and spin-labeled derivatives, various photoactivated derivatives of short and long types of neurotoxins were later obtained in the Laboratory of Neuropeptide Reception (headed by V.I. Tsetlin). At the same time, not only the α-subunits of the Torpedo receptor, but also its other subunits, were found to be involved in the binding of α-neurotoxins, and in collaboration with Professor F. Hucho (Free University, Berlin), one of the contact points in the receptor itself was identified [14].

Our interest in animal venoms was not limited to their protein components. Thus, the peptide apamin is present in bee venom, the target of which is Ca2+-activated K+-channels. In the laboratory of V.T. Ivanov, various options for the synthesis of biologically active peptides were developed, and radioactive derivatives of this peptide were obtained for the first time [15].

In modern studies of nicotinic receptors, α-conotoxins, neurotoxic peptides from the Conus venomous marine mollusks, play an important role. They not only make it possible to distinguish between muscle and neuronal nAChR subtypes, but also serve as excellent identifiers for individual neuronal nAChR subtypes. In cooperation with Ivanov’s laboratory, a series of different α-conotoxins was synthesized [16], their photoactivated derivatives were obtained, and for the latter photoinduced contacts were established with all subunits of the Torpedo receptor [17]. An important achievement was the first establishment of the crystal structure of α-conotoxin in a complex with an acetylcholine-binding protein [18], carried out in a joint study with Dutch scientists who discovered this protein, which acts as an excellent structural model of the ligand-binding domains not only of nAChR, but also of other receptors of the family ligand-gated channels.

One more example of cooperation with Ivanov’s laboratory should be mentioned: we are talking about bacteriorhodopsin, a light receptor and a proton channel. We were the first to apply the method of tritium planigraphy to a membrane protein, which made it possible to distinguish the regions of the polypeptide chain located on the surface from the regions located inside the membrane by the level of incorporated radioactivity [19].

Works on toxin topics started in the laboratory of peptide chemistry, headed by V.T. Ivanov, continue quite successfully at the present time in the department of molecular neuroimmune signaling (headed by V.I. Tsetlin), in cooperation mainly with other departments of our institute, as well as with foreign laboratories. Proteomic and transcriptomic studies are carried out in the laboratory of molecular toxinology (headed by Prof. Yu.N. Utkin): Bungarus multicinctus, new analogs of α-bungarotoxin were discovered, which, unlike it, are able to distinguish between two orthosteric centers in nAChR of Torpedo californica [20]. Detailed studies of the venoms of a number of vipers (including those living in Russia) were carried out and, in particular, the various phospholipases A2 contained in them were characterized [21]. Recently, in collaboration with the Gamaleya Research Institute of Epidemiology and Microbiology, it was found that some dimeric phospholipases A2 from viper venom exhibit virucidal activity, preventing the interaction of the receptor-binding domain of the S-protein of the SARS-CoV-2 virus with the ACE2 cell receptor and destroying the lipid bilayer of the virus [22].

The laboratory of ligand-receptor interactions (headed by I.E. Kasheverov, Doctor of Chemistry) analyzes the interaction of α-conotoxins with various subtypes of neuronal nAChRs, acting as targets for the search for drugs against neurodegenerative diseases. Using a new computer simulation method developed by Prof. R.G. Efremov et al., proposed and then synthesized new analogs of α-conotoxins, significantly exceeding natural compounds in affinity for α7 neuronal nAChR [23], which plays an important role in the regulation of inflammatory processes.

Together with Chinese scientists, I.E. Kasheverov et al. analyzed the crystal structures of AChBP with those α-conotoxins that have different affinities for certain subtypes of neuronal nAChR. For example, for α-conotoxin LvIA X-ray diffraction analysis of the complex with AChBP was performed, then alanine scanning of α-conotoxin and crystal structures of the selected analogs in the complex with AChBP were established. Then, computer simulation of the complex already with α3β nAChR, mutagenesis of the β2 subunit was carried out, and the effectiveness of inhibition was assessed using the electrophysiological method. As a result, contacts of the β2 subunit with the toxin responsible for its specificity to this particular receptor subtype were identified for the first time [24].

In those works conducted under the guidance of Academician V.T. Ivanov, peptides were used not only as convenient tools in a wide variety of fundamental research, but also for the development of possible drugs, which is perfectly illustrated by the creation of a widely used immunostimulating drug lycopid based on glucosaminylmuramyl dipeptide (GMDP) [25]. In joint work with Academician V.T. Ivanov, the possibility of using peptide fragments of the nAChR α-subunit as possible drug compounds for the treatment of neurodegenerative diseases was tested [26].

Peptide and protein neurotoxins from animal venoms not only serve as excellent tools for elucidating the role of the corresponding receptors in physiological and pathophysiological processes, but also open the way to the creation of new drugs. Mention should be made here of capoten (captopril), which is a modified proline residue. This drug was created over 30 years ago based on a peptide from the venom of the Brazilian snake Bothrops jaraca, which inhibits angiotensin-converting enzyme (ACE) and lowers blood pressure. Currently, there is evidence that modified derivatives of natural α-conotoxin RgIA, which targets α9/α10 nAChRs, is being tested against neuropathic pain [27]. Taking into account the significant number and important role of arginine residues in α-conotoxins, we analyzed oligoarginines of various lengths (known means for intracellular delivery of various compounds attached to them) and found that oligoarginines represent a new class of nAChR inhibitors [28]. In our recent work [29] it was shown that, similar to α-conotoxin RgIA, analgesic activity is also exhibited by octaoligoarginine R8, an effective inhibitor of α9/α10 nAChR, the synthesis of which is much simpler than that of α-conotoxins. Another example is azemiopsin, a linear peptide from the venom of the Azemiops feae viper, which does not have disulfide bonds, but is nevertheless capable of inhibiting muscle nAChRs. Conducted preclinical trials [30] have shown that azemiopsin surpasses such currently used compounds as rocuronium in its muscle relaxant activity.

CONCLUSIONS

This review shows that the studies of peptides and proteins, initiated under the guidance of Academician V.T. Ivanov (when he was a senior research fellow), were carried out quite successfully, received international recognition, and are now being actively carried out against the backdrop of rapidly developing neurochemistry and neurobiology, supported by modern genetic engineering and spectral methods. Works are widely ongoing at the Institute of Bioorganic Chemistry, RAS, including the Department of Molecular Neuroimmune Signaling (supervisor V.I. Tsetlin, student of Academician V.T. Ivanov). It should be noted that three laboratories in the department of molecular neuroimmune signaling are headed by Prof. Yu.N. Utkin, Doctor of Chemical Sciences, I.E. Kasheverov and Dr. Sci. I.V. Shelukhin, whose scientific biography began at the Institute of Bioorganic Chemistry of the Russian Academy of Sciences and is successfully continuing here.