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The aim of this work is to verify the possibility of transport of 26 biologically active ultrashort peptides (USPs) into cells via LAT and PEPT family transporters. Molecular modeling and computer-assisted docking of peptide ligands revealed that the size and structure of ligand-binding sites of the amino acid transporters LAT1, LAT2, and of the peptide transporter PEPT1 are sufficient for the transport of the 26 biologically active di-, tri-, and tetra-peptides. Comparative analysis of the binding of all possible di- and tri-peptides (8400 compounds) at the binding sites of the LAT and PEPT family transporters has been carried out. The 26 biologically active USPs systematically showed higher binding scores to LAT1, LAT2, and PEPT1, as compared with di- and tri-peptides, for which no biological activity has been established. This indicates an important possible role which LAT and PEPT family transporters may play in a variety of biological activities of the 26 biologically active peptides under investigation in this study. Most of the 26 studied USPs were found to bind to the LAT1, LAT2, and PEPT1 transporters more efficiently than the known substrates or inhibitors of these transporters. Peptides ED, DS, DR, EDR, EDG, AEDR, AEDL, KEDP, and KEDG, and peptoids DS7 and KE17 with negatively charged Asp - or Glu - amino acid residues at the N-terminus and neutral or positively charged residues at the C-terminus of the peptide are found to be the most effective ligands of the transporters under investigation. It can be assumed that the antitumor effect of the KE, EW, EDG, and AEDG peptides could be associated with their ability to inhibit the LAT1, LAT2, and PEPT1 amino acid transporters. The data obtained lead to new prospects for further study of the mechanisms of transport of USP-based drugs into the cell and design of new antitumor drugs.

Peptides are characterized by their wide range of biological activity: they regulate functions of the endocrine, nervous, and immune systems. The mechanism of such action of peptides involves their ability to regulate gene expression and protein synthesis in plants, microorganisms, insects, birds, rodents, primates, and humans. Short peptides, consisting of 2-7 amino acid residues, can penetrate into the nuclei and nucleoli of cells and interact with the nucleosome, the histone proteins, and both single- and double-stranded DNA. DNA-peptide interactions, including sequence recognition in gene promoters, are important for template-directed synthetic reactions, replication, transcription, and reparation. Peptides can regulate the status of DNA methylation, which is an epigenetic mechanism for the activation or repression of genes in both the normal condition, as well as in cases of pathology and senescence. In this context, one can assume that short peptides were evolutionarily among the first signaling molecules that regulated the reactions of template-directed syntheses. This situation enhances the prospects of developing effective and safe immunoregulatory, neuroprotective, antimicrobial, antiviral, and other drugs based on short peptides.

Peptide KE exhibits immunoprotective, geroprotective, and oncostatic activities and stimulates functional activity of fibroblasts. The KE motif is present in amino acid sequences of some cytokines and peptide hormones functionally similar to KE peptide. However, the relationship between the presence of KE motif and protein functions on the scale of known human proteome has not yet received sufficient attention. The incidence of bioregulatory peptide KE in proteins of various functional groups constituting human proteome is studied. The study is carried out with the use of the available data on the human proteome (UniProt portal) comprising 20,417 proteins. The levels of KE motifs were maximum in cytoplasmic and nuclear proteins, while the presence of KE in the membrane and all other proteins was the minimum. KE peptide molecules released from nuclear proteins during limited proteolysis can bind to DNA and regulate gene expression.

The applying of many drugs in elderly and old people often is the reason of liver dysfunction. Thereat, the risk of liver fibroid induration, acute and chronic hepatitis increases during aging. It is the reason to find new, effective and harmless hepatoprotectors. In the review is shown the data of hepatoprotective, immunoprotective and antiageing properties of liver polypeptide complex (Ventvil) and KEDA tetrapeptide (Lys-Glu-Asp-Ala, Livagen). In liver pathology experimental models (liver fibroid induration, acute and chronic hepatitis) in amimals and in vitro was shown high efficiency of Ventvil and KEDA peptide. Ventvil and KEDA peptide had concordant effects - normalized immune and antioxidant status, restored liver function during hepatitis. It was demonstrated, that maximal hepato- and immunoprotective effect of peptides verified in aging.

Neurodegenerative diseases are a heterogeneous group of nervous system pathologies. They are found mainly in people of an older age group. The incidence of neurodegenerative diseases is continuously growing due to an increase in the average life expectancy of the population. At the moment, there are no effective and safe treatments for neurodegenerative diseases, which are most often diagnosed at the stage of decompensation, when therapy is ineffective and does not bring positive outcomes. Most of the currently used drugs act only symptomatically. The review provides analyzed data and information about the prospects of using peptide bioregulators as neuroprotectors with high physiological activity and low immunogenicity.

Short peptides are applied for supporting skin function during ageing, because they can permeate the intact stratum corneum of the epidermis and affect the cells of the dermis. Short peptides are part of natural metabolism of cells and many of them have geroprotective properties. In the review we are considering the base sorts of peptides that are used for normalized skin fibroblasts function: matrikines, carnosine, collagen peptides, cytokine and growth factor analogs, defensins, immunoprotective peptides and polyfunctional peptides. Polyfunctional peptides (AcSDKP, KED, AEDG, AED) have geroprotective properties, slow apoptosis and stimulate skin cell proliferation, also increase functional activity of skin fibroblasts, normalize intracellular matrix hemostasis. Polyfunctional peptides are the antioxidants and immunoprotectors and can activate microcirculation in dermis. Peptide regulation of skin function during ageing are the fast-developing and prospective area in molecular gerontology.

Cortexin is a clinically approved cerebral cortex polypeptide complex in calves. The mechanism of cortexin action is not understood well. Two cortexin derivatives, short peptides EDR and DS with neuroprotective activity, were synthesized. According to the data of molecular modeling, these peptides are able to bind to the histone H1.3 protein. This can affect the conformation of histone H1.3, which leads to a change in the chromatin structure in the loci of some genes, in particular Fkbp1b encoding the FK506-binding protein. Electrophysiological processes associated with the Ca^(2+) exchange are disturbed in the pyramidal neurons of the hippocampus during aging of the brain. The Fkbp1b gene encodes peptidyl-prolyl cis-trans isomerase, regulating the release of calcium ions from the sarcoplasmic and endoplasmic reticulum of neurons. The activation of the Fkbp1b gene transcription under treatment with short peptides can promote the synthesis of its protein product and the activation of the Ca^(2+) release from organelles of the sarcoplasmic and endoplasmic reticulum of neurons, which, in turn, can lead to an increase in the functional activity of neurons.

In review it is shown data about polypeptide vessel complex, extracted from calf vessel and named Slavinorm® (Vasolin). Slavinorm® had therapeutic effects in cardio-vascular age related pathology in animal models. Slavinorm® prevents atherosclerosis, normalizes lipid metabolism, lipid peroxidation parameters, station of congenital and adaptive immunity, callicrein-kinins system, vessel and trombocitary hemostasis, blood coagulability, fibrinolysis, activates vessel wall reparation. Slavinorm® has positive effects in other age-related diseases (arthritis, pancreatitis, respiratory distress).

More than a quarter of the elderly and senile age population suffers from kidney pathology. For this reason, a prophylaxis of kidney diseases with the safe and effective nephroprotectors is a priority of gerontology. An influence of polypeptide kidney complex (PKC), peptides AED, EDL, AEDG on the functional state of old rats kidneys was studied in research. Administration of PKC, peptides AED and EDL increased diuresis by 1,2-1,4 times. PKC and peptide AED reduced urine protein level and protein excretion by 1,5-2,8 times. PKC, peptides AED and EDL increased distal sodium transport by 1,2-1,3 times. Peptides AED and EDL increased sodium excretion by 1,3 and 1,6 times, respectively. Renal effects of peptide AEDG resulted in a reduction of glomerular filtration rate by 21%, decrease in urine protein level by 3,1 times and protein excretion - by 2,5 times. Peptide AEDG reduced absolute sodium reabsorption by 1,3 times and increased distal sodium transport by 1,4 times. Realization of glomerular-tubular and tubular-tubular balances is verified by correlation between glomerular filtration rate (GFR) and absolute sodium reabsorption, proximal and distal sodium reabsorption. In kidney tissue a stimulation of the antioxidant enzymes activity on the background of inhibition of the peroxidation processes intensity was observed, which in complex with morphological data evidences the absence of nephrotoxic effects. PKC, peptides AED, EDL and AEDG may be considered as nephroprotective agents in kidney aging.

We have discovered motives of short-chain epigenetically active peptides in some proteins of long-lived African mole rat Heterocephalus glaber. These epigenetic regulators are located in the protein structure between lysine and arginine residues, thus facilitating their release in limited proteolysis. Some of these epigenetic regulators are not found in the proteins of short-lived species - Norway rat Rattus norvegicus and house mouse Mus musculus.

In primary culture of mouse hippocampal neurons, peptide EDR (200 ng/ml) under conditions of amyloid synaptotoxicity (a model of Alzheimer's disease) increased the number of mushroom spines by 71% and returned this parameter to the normal level. Under the same conditions, tripeptide KED (200 ng/ml) increased the number of mushroom spines in hippocampal neurons by 20%. Tripeptide EDR can be recommended for further experimental study as a candidate neuroprotective agent for prevention and treatment of Alzheimer's disease.

EDL peptide produced a nephroprotective effect on experimental models gentamycin-induced nephropathy and ischemia/reperfusion kidney injury in rats. The nephroprotective effect of EDL peptide manifested in prevention of oliguria and retention azotemia, a decrease in proteinuria and sodium excretion, prevention of critical decrease in activities of antioxidant enzymes, suppression of LPO, and normalization of energy supply to kidneys cells. Our findings confirm the prospects of further studies of the nephroprotective properties of peptide EDL in various pathologies of the kidneys.

Immunohistochemical and morphometric analysis showed that epithalamin and epithalon produced similar effects on the functional morphology of the spleen in pinealectomized rats. Both peptides prevented hyperplasia of lymphoid cells in follicular germinative centers induced by pinealectomy and potentiated the decrease in extramedullary hemopoiesis. These findings confirm the data on functional relationships between the pineal gland and immune system. The effects of epithalamin and epithalon on cell and tissue homeostasis in the spleen of old pinealectomized rats can be regarded as a manifestation of the general regulatory effect of these peptides.

Synthetic peptides (cytogens) Cortagen, Epithalon, Livagen, and Vilon stimulated the growth of explants from rat brain cortex, subcortical structures, liver, and thymus, respectively, in organotypic cultures. These peptides produced tissue-specific effects: they stimulated the growth of explants from tissues, whose cytomedins (peptide complexes) were used for chemical synthesis.

Objectives: The review on our own data on the effect of the pineal peptide preparation Epithalamin on free radical processes in rodents and humans is presented in this paper., Results: The activity of Cu, Zn-superoxide dismutase (SOD) was found decreased in the brain of aged rats (30 months old) by 46.8% as compared to young animals. Concentration of Schiff's bases in the brain also went down with age (by 13.6%), while the level of dien conjugates (DC) and protein peroxidation (PPO) remained unchanged. General antioxidation activity (AOA) in the brain also remained stable with age. The liver of aged rats showed significant increase of Schiff's bases (by 27.1%) and PPO products (by 109.2%) and considerable decrease of SOD activity. The level of DC and general AOA in the liver remained unchanged with age. Considerable elevation of protein and lipid peroxidation products contents was registered in the blood serum of aged rats. At the same time, general AOA and SOD activity remarkably decreased. The results obtained evidence from both significant age-related alterations in the activity of free radical processes in animal organism and organic peculiarities of their dynamics. Application of peptide drug epithalamin suppressed significantly the intensity of peroxide chemoluminescence in the blood serum (2.8-fold) and lipid peroxide oxidation (LPO) expressed in the considerably decreased DC contents (4,1-fold). The contents of Schiff's bases showed only a tendency towards decrease (by 14.4%, p > 0.05) and PPO level remained unchanged. Epithalamin administration was followed by considerable (by 36.6%, p < 0.01) increase of general AOA and increased SOD activity (by 19.7%) in males. Epithalamin decreased significantly the contents of conjugated hydroperoxides and ketodienes in tissues of D.melanogaster females, increased catalase activity in drosophila males and females, and increased SOD activity in males of D.melanogaster by 41%. Humans reveal significant age-related decrease of antioxidation defence indices., Conclusion: Epithalamin administration to patients with age-related pathology eliminates imbalance in prooxidation and antioxidation systems.

Lys-Glu in vitro stimulated interleukin-2 gene expression in mouse spleen lymphocytes. This effect depended on peptide concentration and duration of treatment. It is hypothesized that this peptide is the shortest regulatory fragment promoting the transport of trans-acting factors into the nucleus. It can not be excluded that Lys-Glu is a structural component of trans-acting factor active centers, which are necessary for the activation of interleukin-2 gene transcription in lymphocytes.

Effects of synthetic pineal tetrapeptide L-Ala-L-Glu-L-Asp-L-Glu (Epithalon) on specific catalase activity and the content of conjugated hydroperoxides in highly inbred Drosophila melanogaster lines differing in reproductive functions were studied. It was shown that Epithalon is a potent modulator of the antioxidant defense, whose biological activity 1000-fold surpasses that of the complex pineal peptide preparation Epithalamin.

Immunomodulatory molecule L-Glu-L-Trp was isolated from natural calf thymic peptide complex Thymalin by reverse-phase high performance liquid chromatography. On the basis of the synthesized dipeptide a pharmaceutical was designed containing this compound, which later receives the brand name Thymogen. The agent activated T-cell differentiation, T-cell recognition of peptide-MHC complexes, induced changes in intracellular composition of cyclic nucleotides, and activated neutrophilic chemotaxis and phagocytosis. The effect of dipeptide on survival, life span and spontaneous tumor development was studied in female rats. Seventy-six, five-month-old outbred female rats were randomly subdivided into two groups and were subcutaneously injected with 0.2 ml of normal saline (controls, 32 rats) or with 5 micrograms/rat of the dipeptide L-Glu-L-Trp, dissolved in 0.2 ml of saline (44 rats), 5 times per week for 12 months. Animals were monitored up to their natural death and all the tumors discovered were studied microscopically. Mean life span of rats in both groups was similar but that of 10% maximum survived control rats constituted 949 +/- 16.1 days, whereas in the dipeptide-treated rats this value was 1048 +/- 21.1 days (P < 0.001). Six out of 44 rats treated with the drug survived over the maximum life span of control rats (965 days). The aging rate indicated as alpha in the Gompertz equation, was 0.0071 days-1 in controls and 0.0041 days-1 in rats exposed to L-Glu-L-Trp. Total tumor incidence was 1.5 times lower (P < 0.01), malignant tumor incidence 1.7 times lower (P < 0.01), and hematopoietic malignancies (leukemias and lymphomas) 3.4 times lower (P < 0.02) in rats exposed to the dipeptide in comparison with controls. Thus, treatment with L-Glu-L-Trp delayed aging rate and decreased spontaneous tumor incidence in rats.

Treatment with pineal peptide preparation epithalamin was followed by the increase of the mean lifespan of female D. melanogaster, SHR mice, C3H/Sn mice and LIO rats by 11-31% (P < 0.05). Ninety percent mortality as well as maximum lifespan were increased in fruit flies, C3H/Sn mice and rats. Mortality rate was decreased by 52% in D. melanogaster, by 52% in rats, by 27% in C3H/Sn mice. It did not change in SHR mice exposed to epithalamin. Treatment with the pineal peptide increased MRDT in flies, C3H/Sn mice and rats. It has been shown that epithalamin increased synthesis and secretion of melatonin in rats and inhibits free radical processes in rats and in D. melanogaster. It is suggested that antioxidative properties of epithalamin lead to increased lifespan of three different animal species.

Natural thymic peptides have been isolated from calf thymus by mild acid extraction. Pharmaceutical containing natural peptides (Thymalin) was put into practice as immunocorrector. One of the immunomodulatory molecules (L-Glu-L-Trp) has been isolated from Thymalin by reversed-phase high performance liquid chromatography. Pharmaceutical containing this agent (Thymogen) was designed on the base of synthesized dipeptide. A novel immunomodulatory dipeptide was synthesized and termed Vilon. Both natural and synthetic pharmaceuticals activated T-cell differentiation, T-cell recognition of peptide-MHC complexes, induced the changes in intracellular composition of cyclic nucleotides and cytokine [interleukin (IL-2), interferon (IFN)] excretion of blood lymphocytes. Synthetic dipeptides activated neutrophil chemotaxis and phagocytosis. They had no influence on antioxidant response in thymocytes in comparison with natural peptides. Thymalin and Thymogen were used in persons with chronic pathology and immune dysfunction. The results indicate that thymic peptides participate in the regulating mechanisms of inflammatory processes as cytokine antagonists and show the difference between natural and synthetic products. It is important for the drugs designed to prevent immune dysfunction development.

It was shown previously that epithalamin delays age-related changes in reproductive and immune systems and increases the life span of mice and rats. These effects could be mediated by stimulating influences of epithalamin on synthesis and secretion of melatonin and on free radical processes. A comparative study on the effect of epithalamin and melatonin on both the life span of Drosophila melanogaster (strain HEM) and on the intensity of lipid peroxidation and activity of antioxidative enzymes in their tissues was the main aim of this work. Melatonin and epithalamin was added to the nutrition medium (100 micrograms/ml) during 2-3rd age of larvas. For survival analysis the flies were passed (five coupes per vessel) each 3-7 days. Lipid peroxidation was evaluated as the level of ketodienes (KD) and conjugated hydroperoxides (CHP) in fly tissues at the age of 11 days. Activity of Cu, Zn-superoxide dismuatse (SOD) and catalase was evaluated as well. The mean, median and maximum life span (MLS) were estimated. Mortality rate (MR) was calculated as alpha in the Gompertz equation (R = Ro (exp alpha t) and mortality rate doubling time (MRDT) as in 2/alpha. These parameters in groups of male and female flies exposed to melatonin and in male flies exposed to epithalamin were no different from the parameters for controls. However, exposure to epithalamin was followed in females by a significant increase in mean life span (by 17%, P < 0.02), of median (by 26%), of MLS by 14% and by a 2.12 times decrease of MR (P < 0.01) and MRDT (by 32%) compared with female controls. The level of CHP and KD in the tissues of male control flies was 40 and 49% less than that in females and indirectly correlates with male life span. Exposure to melatonin was followed by a decrease in the level of CHP and KD in females and the deletion of sex differences in them. Exposure to epithalamin significantly decreased the level of CHP and KD in female flies compared to controls (2.3 and 3.4 times, respectively, P < 0.001). Exposure to melatonin failed to influence the activity of catalase in males but increased it in females by 24% (P < 0.02) and failed to influence SOD activity both in males and females. Exposure to epithalamin was followed by a significant increase in activity of catalse, 20% in males and 7% in females and by an increase in SOD activity in males (41%). Thus, it was shown that exposure to epithalamin significantly increases the mean life span and MLS of female D.melanogaster and slowed down their aging rate by 2.12 times. This effect is in good agreement with the inhibiting effect of epithalamin in lipid peroxidation processes in fly tissues.

Female SHR mice, aged 3.5 or 12 months, were exposed monthly to 5-day long courses of subcutaneous injections of 0.1 mg thymus-derived or pineal gland-derived polypeptide factors (TF and PF, respectively) or 0.9% sodium chloride solution (control). PF treatment increased life span of both young and middle-aged mice by 20% and 17%, respectively, and TF increased the life span only in young mice. Both factors when administered to young mice caused a decrease in both overall tumour incidence and incidence of mammary adenocarcinomas (TF, 1.8-fold decrease; PF, 2.6-fold decrease). TF administration to mature mice did not produce any antitumour effect, whereas PF possessed certain anti-tumour activity, but the response was far less pronounced than in young animals. The results obtained give additional evidence of the geroprotective and anti-tumour effect of thymus and pineal gland-derived peptide factors. The mechanisms of action of TF and PF and perspectives of clinical use of these agents as geroprotectors are discussed.

The low-molecular-weight polypeptide factors were obtained from bovine thymus (TF), pineal gland (PF) and anterior hypothalamus (AHF). Both TF and PF administration enhanced the rejection of skin allograft and stimulated the immunological response to sheep erythrocytes in adult CBA mice. Treatment of CBA mice with AHF increased the graft survival and inhibited antibody formation to sheep erythrocytes. Chronic TF or PF administration decreased spontaneous tumor development and prolonged the life span of female C3H/Sn mice. Administration of AHF failed to influence the life span and the tumor incidence of female C3H/Sn mice. The role of immunity and hormonometabolic shifts in mechanisms of both aging and the age-associated increase in cancer incidence are discussed.

The 20 month-long treatment of female rats with daily doses of 0.1 or 0.5 mg of polypeptide pineal extract (PPE) per animal increased their lifespan by 10 and 25%, respectively, as compared with controls. At the age of 16--18 months, 38% of control rats exhibited persistent disturbances in estral function (constant estrus or repeated pseudogestations), whereas these disorders were observed in 7% of experimental animals only. After administration of PPE to 16--18 month-old female rats checked for sterility by a two-week mating, a second mating period resulted in gestation development in four out of 16 animals and deliveries, accordingly. While chronic treatment with PPE did not affect the rate of neoplasm incidence, the mean age of tumour detection in the control group was 697 days and in experimental groups it was 811 and 868 days, respectively. Certain aspects of the interrelationship of rate of ageing, lifespan and specific age pathology are discussed.

Bovine pineal polypeptide extract (PPE) exerted an anti-tumor effect on mouse-transplantable tumors: mammary cancer (RSM), squamous cell cervical carcinoma (SCC), hepatoma-22a and lympholeukemia LIO-1, and had no effect on Harding-Passey melanoma and leukemia L-1210. It was shown that PPE possessed the ability to decrease the incidence of DMBA-induced mammary adenocarcinomas in rats. The daily administration of 0.5 mg PPE prolonged the life span of rats by 25% and failed to influence spontaneous tumor development. The arguments in favor of a possible mechanism of anti-tumor action of the pineal gland are submitted. It is suggested that the anti-tumor effect of PPE may occur when the syndrome of cancrophilia is induced by tumor transplantation or chemical carcinogens.