We performed a computer search for the DSIP homologous structures in the protein data bases and found the KND peptide (WKGGDNASGE), which was closely structurally related to DSIP, corresponding to the 324–332 fragment of the human lysine-specific histone demethylase 3B (EC 1.14.11), and possibly was a prototype of the real endogenous DSIP. Previously, we compared some biological effects of DSIP and KND. Antioxidant, anticonvulsant, and behavioral effects of KND proved to be more pronounced than those of DSIP, suggesting the higher efficacy and therapeutic potential of KND and its derivatives. In this study, we synthesized new analogs of DSIP and KND and examined their detoxifying action after the treatment with the cisplatin cytostatin in comparative experiments in vivo on rodents. KND and several its analogs were found to decrease the cisplatin effects on the induced changes in masses of target organs. The correcting effects of the studied peptides on the cisplatin-induced hematological abnormalities, such as the changes in the leukocyte content in blood and thrombocytopenia, were revealed after their intranasal administration to animals in low doses. A decrease in a pronouncement of histological deviation from a norm was shown in the tissues of liver, kidney, and spleen. A significant hepatoprotective and nephroprotective effects of the studied peptides were also observed. Thus, the revealed antitoxic properties of DSIP, KND, and their analogs, could be a basis for the creation of drugs of lower toxicity in the anticancer treatment and therapy of medicinal hepatitis.

С целью изучения потенциальных возможностей применения аналогов пептида дельта-сна (DSIP) в онкологии синтезированы 16 пептидов, отличающихся по структуре от DSIP заменами 1–2 а.о. Исследованы антиоксидантные свойства пептидов in vitro и их детоксицирующая активность in vivo в модели токсикоза, вызванного цитостатиком цисплатином, широко применяемым в терапии опухолевых заболеваний. Показано, что практически все исследованные аналоги DSIP проявляют прямую антиоксидантную активность, причем аналог ID-6 обладал активностью более высокой, чем DSIP, и сравнимой с таковой для витамина С и ?-каротина. Данный аналог продемонстрировал также наиболее выраженный среди исследуемых пептидов детоксицирующий эффект по отношению к действию цисплатина, что проявлялось как в снижении до 17% гибели животных от острой токсичности, по сравнению с 50–67% в контроле, так и в улучшении ряда биохимических показателей крови – снижении активности ферментов аспартат- и аланинаминотрансфераз и концентрации конечных продуктов азотистого обмена: креатинина и мочевины. Таким образом, пептиды семейства DSIP могут оказаться перспективными средствами для снижения токсических эффектов цитостатиков, применяемых в онкологии.

Sixteen peptides differing in structure from the delta sleep inducing peptide (DSIP) by one to two substitutions of amino acid residues have been synthesized to study the possibility of their application in oncology. The antioxidant properties of the peptides in vitro and their detoxifying activity in vivo have been examined on a model of toxicosis caused by cisplatin, a cytostatic drug widely used in the therapy of tumor diseases. It has been shown that almost all DSIP analogues examined exhibit a direct antioxidant activity, with the activity of the ID-6 analogue being higher than that of DSIP and comparable with that of vitamin C and β-carotene. This analogue shows the most pronounced detoxifying activity toward the action of cisplatin, which manifests itself as a decrease in the death of animals from acute toxicity to 17% compared to 50–67% in the control and the restoration of some biochemical parameters of blood, in particular, a decrease in the activity of the enzymes, aspartate and alanine aminotransferases, and the concentration of the end products of nitrogen exchange: creatinine and urea. Thus, the peptides of the DSIP family may appear promising agents for decreasing the toxic effects of cytostatics used in oncology.

Synthetic peptides corresponding to the 59–72, 60–72, and 61–72 sequence of human interleukin 2 (IL-2) with an acetyl group and methoxygroup on their N- and C-termini, respectively, were shown to exhibit pronounced hepatoprotective properties. These peptides neutralized the toxic effect of such toxins as tetrachloromethane and galactosamine on liver in the in vivo experiments. The action of these peptides resulted in normalization of the duration of thiopental narcosis of the experimental animals and the level of hepatospecific enzymes in their blood. The hepatoprotective activity of the most active peptide (59–72) proved to be similar to that of prednisolone (the well-known anti-inflammatory agent), whereas the bestatine cytotoxic dipeptide had no such hepatoprotective effect. A target of the peptide hepatoprotective effect was shown to be the preliminary activated macrophages. The action of the most active (59–72)-peptide probably induced transformation of the classically activated macrophages into their state close to the alternative activation.

This review systematized modern conceptions of the most frequently occurring types of macrophage activation. Mechanisms of induction and regulation of these three types of activation were discussed. Any macrophage population was shown to easily change its properties depending on its microenvironment and concrete biological situation (functional plasticity of macrophages). Many intermediate functional states of macrophages were described, along with the most pronounced and well-known activation states (classical activation, alternative activation, and type II activation). These intermediate states were characterized by various compositions of their biological properties, including elements of all the three aforementioned types of activation. Macrophage activity was shown to be regulated by a complex network of interrelated cascade mechanisms.

A comparative study of secondary specificities of enteropeptidase and trypsin was performed using peptide substrates with general formula A-(Asp/Glu) n -Lys(Arg)-↓-B, where n = 1-4. This was the first study to demonstrate that, similar to other serine proteases, enteropeptidase has an extended secondary binding site interacting with 6-7 amino acid residues surrounding the peptide bond to be hydrolyzed. However, in the case of typical enteropeptidase substrates containing four negatively charged Asp/Glu residues at positions P2-P5, electrostatic interaction between these residues and the secondary site Lys99 of the enteropeptidase light chain is the main factor that determines hydrolysis efficiency. The secondary specificity of enteropeptidase differs from the secondary specificity of trypsin. The chromophoric synthetic enteropeptidase substrate G5DK-F(NO2)G (kcat/Km = 2380 mM–1·min–1) is more efficient than the fusion protein PrAD4K-P26 (kcat/Km = 1260 mM–1·min–1).

The three-dimensional structure of the Fab fragment of a monoclonal antibody (LNKB-2) to human interleukin-2 (IL-2) complexed with a synthetic antigenic nonapeptide, Ac-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-OMe, has been determined at 3.0 A resolution. In the structure, four out of the six hypervariable loops of the Fab (complementarity determining regions [CDRs] L1, H1, H2, and H3) are involved in peptide association through hydrogen bonding, salt bridge formation, and hydrophobic interactions. The Tyr residues in the Fab antigen binding site play a major role in antigen-antibody recognition. The structures of the complexed and uncomplexed Fab were compared. In the antigen binding site the CDR-L1 loop of the antibody shows the largest structural changes upon peptide binding. The peptide adopts a mostly alpha-helical conformation similar to that in the epitope fragment 64-72 of the IL-2 antigen. The side chains of residues Leu 66, Val 69, and Leu 70, which are shielded internally in the IL-2 structure, are involved in interactions with the Fab in the complex studied. This indicates that antibody-antigen complexation involves a significant rearrangement of the epitope-containing region of the IL-2 with retention of the alpha-helical character of the epitope fragment.

Antigen-binding fragments (Fab) of mouse monoclonal antibodies to human interleukin-2 were obtained in preparative quantities by a modified procedure. These Fab-fragments were shown to be homogeneous according to the isoelectric focusing method. Various monocrystals of these free Fab-fragments and their complexes with the antigenic peptide corresponding to the 59-72 sequence of interleukin-2 were obtained. These were shown to be suitable for X-ray and were preliminarily studied by X-ray.

The peptide C-I-6 is a modified fragment (sequence 59-72) of human interleukin-2 and stimulates regeneration of hepatic cells and tissues. Study of the possible embryotoxic and teratogenic properties of C-I-6 showed that in some cases it induces the following statistically significant changes: reduces the number of viable fetus, increases the occurrence of hydronephrosis and the number of external and internal hematomas, increases the weight and size of the embryo. It is concluded that intraperitoneal administration of C-I-6 causes a mild embryotoxic and teratogenic effect in rats.

For the study of the antigenic structure and function of human interleukin 2, the peptides corresponding to its 60-72 sequence were synthesized by conventional methods of peptide chemistry in solution. To enhance the stability of the synthetic peptides towards the proteolysis and to remove their terminal charges, we acetylated their NH2 groups and esterified with methanol their carboxyls. Some of these peptides were converted from being cytotoxic to possessing strong growth-stimulating activity for the preliminary activated macrophages both in vitro and in vivo. The biologically active peptides were also shown to enhance regeneration-reparation processes in liver and skin.

1H-NMR spectra of the interleukin-2 synthetic fragment Ac-Leu66-Glu-Glu-Val-Leu-Asn-Leu72-OCH3 in the presence or absence of the monoclonal antibody were analysed. The data obtained are consistent with an extended unordered conformation of the free peptide. Measurements of NOESY cross-peak intensities allowed us to determine the spatial structure of the peptide bound to the antibody. The peptide has an amphiphilic surface with hydrophobic and hydrophilic amino acid side chains clustered on the opposite sides of its alpha-helical-like structure. The hydrophobic and hydrophilic clusters are located on the opposite sides of the bound peptide's surface. The hydrophobic side chains contact the antibody surface, while the hydrophilic ones are oriented into the solvent (T. A. Balashova et al. (1991) Bioorgan. Khim. (USSR), v. 17, p. 1470-1486). Hydrolysis of the methyl ester slowly ocurs in the presence of the antibody. This process does not alter the conformation of the peptide bounded with the antibody, though decreases the peptide's affinity to the antibody.

Proton signals for nine synthetic peptide fragments of human interleukin-2 (region 59-78) were assigned for aqueous solutions both of pure peptides and their mixtures with LNKB-2 monoclonal antibody. The nonspecific magnetization transfer (NOE) between the antibody or its Fab-fragment and the peptides was studied upon large excess of free peptide over bound peptide. NOE spectra using modified pulse sequence, enabling to eliminate broad signals and achieve higher (peptide signal)/noise ratio were obtained. The saturation transfer experiments indicated that methyl groups of amino acid residues corresponding to Leu66,70,72, Val69 and Ala73 in interleukin-2 contact with the antibody binding site. Thus, the hydrophobic interactions are of major importance for the LNKB-2-IL-2 peptide complexes. The minimal IL-2 fragment which can still bind to LNKB-2 monoclonal antibody is -Leu70-Asn71-Leu72-.

The effect of a modified peptide of the middle part of the human interleukin-2 molecule (C-1-6) on the growth of transplantable sarcoma 180 of mice was studied. C-1-6 injected intraperitoneally in the dose of 0.5, 5 and 50 mug 4 times every other day was shown to stimulate tumor growth and enhance angiogenesis induced by the tumor tranpslanted intracutaneously. Stimulation of tumor growth was most apparent when the agent was given late (on day 4) after transplantation. Supernatants of murine peritoneal macrophages activated by 1 mug/ml solution of C-1-6 proved capable of stimulating tumor growth, too. It is suggested that the stimulating effect of C-1-6 on tissue regeneration be investigated.

The effect of a modified peptide of the middle part of the human interleukin-2 molecule (C-1-6) on the growth of transplantable sarcoma 180 of mice was studied. C-1-6 injected intraperitoneally in the dose of 0.5, 5 and 50 micrograms 4 times every other day was shown to stimulate tumor growth and enhance angiogenesis induced by the tumor transplanted intracutaneously. Stimulation of tumor growth was most apparent when the agent was given late (on day 4) after transplantation. Supernatants of murine peritoneal macrophages activated by 1 microgram/ml solution of C-1-6 proved capable of stimulating tumor growth, too. It is suggested that the stimulating effect of C-1-6 on tissue regeneration be investigated.

Proton signals for nine synthetic peptide fragments of human interleukin-2 (region 59-78) were assigned for aqueous solutions both of pure peptides and their mixtures with LNKB-2 monoclonal antibody. The nonspecific magnetization transfer (NOE) between the antibody or its Fab-fragment and the peptides was studied upon large excess of free peptide over bound peptide. NOE spectra using modified pulse sequence, enabling to eliminate broad signals and achieve higher (peptide signal)/noise ratio were obtained. The saturation transfer experiments indicated that methyl groups of amino acid residues corresponding to Leu66,70,72, Val69 and Ala73 in interleukin-2 contact with the antibody binding site. Thus, the hydrophobic interactions are of major importance for the LNKB-2-IL-2 peptide complexes. The minimal IL-2 fragment which can still bind to LNKB-2 monoclonal antibody is -Leu70-Asn71-Leu72-.

Synthesis of peptides corresponding to the 59-72 and 25-36 sequences of human IL-2 is reported. The former peptide, which had been shown to be immunogenic in the protein molecule, was prepared to obtain antipeptide antibodies for isolation and purification of the recombinant IL-2. We located the epitope at the C-terminus of this peptide. In accordance with the IL-2 secondary structure and hydrophilicity profile analysis, the 25-36 fragment was chosen as the potential epitope. The peptides were synthesized by conventional methods in solution, conjugated with a protein carrier, and polyclonal rabbit antisera were obtained. Antibodies against both peptides were shown to be specific to human IL-2 in ELISA. Besides, monoclonal antibodies to IL-2 recognized in ELISA the 59-72 peptide, suggesting the epitope located in this region to be main one in the protein molecule.