Computer-aided Evaluation of Polyvalent Medications' Pharmacological Potential. Multiphytoadaptogen as a Case Study.

Many human diseases including cancer, degenerative and autoimmune disorders, diabetes and others are multifactorial. Pharmaceutical agents acting on a single target do not provide their efficient curation. Multitargeted drugs exhibiting pleiotropic pharmacological effects have certain advantages due to the normalization of the complex pathological processes of different etiology. Extracts of medicinal plants (EMP) containing multiple phytocomponents are widely used in traditional medicines for multifactorial disorders' treatment. Experimental studies of pharmacological potential for multicomponent compositions are quite expensive and time-consuming. In silico evaluation of EMP the pharmacological potential may provide the basis for selecting the most promising directions of testing and for identifying potential additive/synergistic effects. Multiphytoadaptogen (MPhA) containing 70 major phytocomponents of different chemical classes from 40 medicinal plant extracts has been studied in vitro, in vivo and in clinical researches. Antiproliferative and anti-tumor activities have been shown against some tumors as well as evidence-based therapeutic effects against age-related pathologies. In addition, the neuroprotective, antioxidant, antimutagenic, radioprotective, and immunomodulatory effects of MPhA were confirmed. Analysis of the PASS profiles of the biological activity of MPhA phytocomponents showed that most of the predicted anti-tumor and anti-metastatic effects were consistent with the results of laboratory and clinical studies. Antimutagenic, immunomodulatory, radioprotective, neuroprotective and anti-Parkinsonian effects were also predicted for most of the phytocomponents. Effects associated with positive effects on the male and female reproductive systems have been identified too. Thus, PASS and PharmaExpert can be used to evaluate the pharmacological potential of complex pharmaceutical compositions containing natural products.

[Possibilities of in silico estimations for the development of pharmaceutical composition phytoladaptogene cytotoxic for bladder cancer cells]. / Vozmozhnosti otsenok in silico dlia razrabotki farmkompozitsii fitoladaptogen, tsitotoksichnoi dlia kletok raka mochevogo puzyria.

Based on the prediction of biological activity spectra for several secondary metabolites of medicinal plants using the PASS computer program and validation in vitro of the predictions results the priority direction of the pharmaceutical composition Phytoladaptogene (PLA) development was determined. PLA is a complex of structurally diverse small organic compounds including biologically active substances of phytoadaptogenes (ginsenosides from Panax ginseng, rhodionin from Rhodiola rosea and others) compiled considering previously developed pharmaceutical compositions. Two variants of the pharmaceutical composition were studied: - the major and minor variants included 22 and 13 compounds, respectively. The probability of activity exceeds the probability of inactivity for 1400 out of 1945 pharmacological effects and mechanisms predicted by PASS for the major variant of PLA. The wide range of predicted activities is mainly due to the low structural similarity of constituent compounds. An in silico prediction indicates the possibilities of antitumor properties against bladder, stomach, colon, ovarian and cervical cancers both for minor and major PLA compositions. It was found that the highest probability values of activity were predicted for three mechanisms: apoptosis agonist, caspase-3 stimulant, and transcription factor NF-κB inhibitor. According to the PharmaExpert program they are associated with the antitumor effect against bladder cancer. Experimental validation was using the human bladder cancer cell line RT-112. The results of the MTT test have shown that the cytotoxicity of the major PLA variant is higher than that of the minor PLA variant. In vitro experiments performed using two methods (double staining with annexin V and propidium iodide and detection of active caspase-3 in cells) confirmed that the death of bladder cancer cells occurred via the apoptotic mechanism. The data obtained correspond to the results of the prediction and indicate advantages of the major PLA composition. Thus, PLA can become the basis for the development of a drug with the antitumor activity against bladder cancer. The antitumor activity predicted by PASS for other cancers may be the subject of further studies.

NMR relaxation parameters of methyl groups as a tool to map the interfaces of helix-helix interactions in membrane proteins.

In the case of soluble proteins, chemical shift mapping is used to identify the intermolecular interfaces when the NOE-based calculations of spatial structure of the molecular assembly are impossible or impracticable. However, the reliability of the membrane protein interface mapping based on chemical shifts or other relevant parameters was never assessed. In the present work, we investigate the predictive power of various NMR parameters that can be used for mapping of helix-helix interfaces in dimeric TM domains. These parameters are studied on a dataset containing three structures of helical dimers obtained for two different proteins in various membrane mimetics. We conclude that the amide chemical shifts have very little predictive value, while the methyl chemical shifts could be used to predict interfaces, though with great care. We suggest an approach based on conversion of the carbon NMR relaxation parameters of methyl groups into parameters of motion, and one of such values, the characteristic time of methyl rotation, appears to be a reliable sensor of interhelix contacts in transmembrane domains. The carbon NMR relaxation parameters of methyl groups can be measured accurately and with high sensitivity and resolution, making the proposed parameter a useful tool for investigation of protein-protein interfaces even in large membrane proteins. An approach to build the models of transmembrane dimers based on perturbations of methyl parameters and TMDOCK software is suggested.

Effect of Phytoadaptogen Administration during Early Ontogeny on Lifespan and Somatic Status of CBA Mice with High Incidence of Tumors.

We studied the influence of nontoxic phytoadaptogen complex on the lifespan and somatic status (body weight, coat state, and motor activity) of CBA mice predisposed to spontaneous hepatomas. Administration of the complex phytoadaptogen during the first month of postnatal ontogeny increased mean animal lifespan by 17.1% (p<0.001) and median of survival by 25.6% (p<0.001) and promoted maintenance of satisfactory physical status of CBA mice during spontaneous hepatocarcinogenesis.

Morphological Studies of Hepatocellular Carcinomas in Male CBA Mice with High Liability to Cancer under the Effect of Phytoadaptogen.

Treatment of CBA mice predisposed to cancer with a complex phytoadaptogen in the therapeutic and preventive modes led to the appearance of moderate and low-differentiation hepato-cellular carcinomas infiltrated by leukocytes. Destructive signs were detected in tumor tissue.

Effects of Phytoadaptogen on Hepatoma Development in Cancer-Prone CBA Mice.

The incidence, size, and number of hepatocarcinomas decreased in cancer-prone male CBA mice under the effect of preventive and therapeutic treatment with a complex phytoadaptogen.

[The Qualitative Analysis of the Amide Derivative of HLDF-6 Peptide and Its Metabolites with the Use of Tritium- and Deuterium-Labeled Derivatives].

The goal of the study was to elaborate the pharmacokinetics methods of the amide derivative of peptide HLDF-6 (TGENHR-NH2) and its range of nootropic and neuroprotective activity is wide. The hexapeptide 41TGENHR46 is a fragment of the HDLF differentiation factor. It forms the basis for the development of preventive and therapeutic preparations for treating cerebrovascular and neurodegenerative conditions. Pharmacokinetic and molecular mechanisms of the action of the HLDF-6 peptide were studied using tritium- and deuterium-labeled derivatives of this peptide, produced with the use of the high-temperature solid-state catalytic isotope exchange reaction (HSCIE). This reaction was employed to produce the tritium-labeled peptide [3H]TGENHR-NH2 with a molar radioactivity of 230 Ci/mmol and the deuterium-labeled peptide [2H]TGENHR-NH2 with an average deuterium incorporation equal to 10.5 atoms. It was shown by the NMR spectroscopy that the isotope label distribution over the labeled peptide's molecule was uniform, which allowed qualitative analysis ofboth the peptide itself and its fragments in the organism's tissues to be conducted. The newly developed pharmacokinetics method makes it possible to avoid almost completely losses of the peptides under study due to biodegradation during the analysis of tissues. These labeled peptides were used in mice, rats and rabbits to study the pharmacokinetics of the peptide and to calculate the values of its principal pharmacokinetic parameters. Characteristics of its pharmacokinetic profile in the blood were obtained, the hypothesis of pharmacokinetics linearity tested, its metabolism analyzed and its bioavailability value, 34%, calculated. It has been shown that the studied TGENHR-NH2 peptide shows high resistance to hydrolysis in the blood plasma, with dipeptidyl aminopeptidases making the largest contribution to its hydrolysis.

[Preparation of Transmembrane Fragments Growth Hormone Receptor GHR in a Cell-Free Expression System for Structural Studies].

Growth hormone somatotropin and its membrane receptor GHR, belonging to a superfamily of the type I receptors possessing tyrosine kinase activity, are involved in the intercellular signal transduction cascade and regulate a number of important physiological and pathological processes in humans. Binding with somatotropin triggers a transition of GHR between two alternative dimer states, resulting in an allosteric activation of JAK2 tyrosine kinase in the cell cytoplasm. Transmembrane domain of GHR directly involved in this complex conformational transition. It has presumably two dimerization interfaces corresponding to the "unliganded" and the active state of GHR. In order to study the molecular basis of biochemical signal transduction mechanism across the cell membrane, we have developed an efficient cell-free production system of a TM fragment of GHR, which contains its TM domain flanked by functionally important juxtamembrane regions (GHRtm residues 254-298). The developed system allows to obtain -1 mg per 1 ml of reaction mixture of 13C- and 15N-isotope-labeled protein for structural and dynamic studies of the GHR TM domain dimerization in the membrane-mimicking medium by high-resolution heteronuclear NMR spectroscopy.

Integrins LFA-1 and Mac-1 and cytokines IL-6 and IL-10 in high-cancer mice under the influence of phytoadaptogen.

The expression of leukocyte integrins LFA-1 and Mac-1 and cytokines IL-6 and IL-10 was studied in mice predisposed to spontaneous hepatocarcinomas. The efficacy of a phytoadaptogen in correcting these parameters was evaluated. The role of adhesive interactions between immune cells and target cells in the recovery of antitumor regulatory mechanisms was estimated.

[Experimental approach to the gene therapy of motor neuron disease with the use of genes hypoxia-inducible factors].

Motor neuron disease (MND), or amyotrophic lateral sclerosis, is a fatal neurodegenerative disorder characterized by a progressive loss of motor neurons in the spinal cord and the brain. Several angiogenic and neurogenic growth factors, such as the vascular endothelial growth factor (VEGF), angiogenin (ANG), insulin-like growth factor (IGF) and others, have been shown to promote survival of the spinal motor neurons during ischemia. We constructed recombinant vectors using human adenovirus 5 (Ad5) carrying the VEGF, ANG or IGF genes under the control of the cytomegalovirus promoter. As a model for MND, we employed a transgenic mice strain, B6SJL-Tg (SOD1*G93A)d11 Gur/J that develops a progressive degeneration of the spinal motor neurons caused by the expression of a mutated Cu/Zn superoxide dismutase gene SOD1. Delivery of the therapeutic genes to the spinal motor neurons was done using the effect of the retrograde axonal transport after multiple injections of the Ad5-VEGF, Ad5-ANG and Ad5-IGF vectors and their combinations into the limbs and back muscles of the SOD1(G93A) mice. Viral transgene expression in the spinal cord motor neurons was confirmed by immunocytochemistry and RT-RCR. We assessed the neurological status, motor activity and lifespan of experimental and control animal groups. We discovered that SOD1(G93A) mice injected with the Ad5-VEGF + Ad5-ANG combination showed a 2-3 week delay in manifestation of the disease, higher motor activity at the advanced stages of the disease, and at least a 10% increase in the lifespan compared to the control and other experimental groups. These results support the safety and therapeutic efficacy of the tested recombinant treatment. We propose that the developed experimental MND treatment based on viral delivery of VEGF + ANG can be used as a basis for gene therapy drug development and testing in the preclinical and clinical trials of the MND.

[Investigation of multyphytoadaptogene anti-radiation activity in mice].

The aim of the work was to elucidate the radioprotective activity of multyphytoadaptogene (MPA) in mice in various conditions of gamma radiation and MPA application. Males of CBA x C57BL/6 F1 mice were given 15% MPA solution with drinking water 2 weeks before the radiating (preventive application), 2 weeks before and 2 weeks after the radiating (preventive and therapeutic application) and also 2 weeks after the radiating only (therapeutic application). Animals of control groups received radiation or were given 5% ethanol solution in drinking water in the same application schemes. MPA increased the mice survival in preventive, preventive and therapeutic as well as therapeutic applications after 7.5 Gy radiation (66.7: 66.4 and 40.2% correspondingly). After 11.0 Gy radiation MPA increased the mice survival in preventive as well as preventive and therapeutic applications (75.0 and 76.9% correspondingly). MPA administration improved the somatic state, weight of animals, quality of life. MPA has no side effects. The data suggest the radioprotective activity of MPA.

Structural investigations of recombinant urokinase growth factor-like domain.

Urokinase-type plasminogen activator (uPA) is a serine protease that converts the plasminogen zymogen into the enzymatically active plasmin. uPA is synthesized and secreted as the single-chain molecule (scuPA) composed of an N-terminal domain (GFD) and kringle (KD) and C-terminal proteolytic (PD) domains. Earlier, the structure of ATF (which consists of GFD and KD) was solved by NMR (A. P. Hansen et al. (1994) Biochemistry, 33, 4847-4864) and by X-ray crystallography alone and in a complex with the soluble form of the urokinase receptor (uPAR, CD87) lacking GPI (C. Barinka et al. (2006) J. Mol. Biol., 363, 482-495). According to these data, GFD contains two ß-sheet regions oriented perpendicularly to each other. The area in the GFD responsible for binding to uPAR is localized in the flexible Ω-loop, which consists of seven amino acid residues connecting two strings of antiparallel ß-sheet. It was shown by site-directed mutagenesis that shortening of the Ω-loop length by one amino acid residue leads to the inability of GFD to bind to uPAR (V. Magdolen et al. (1996) Eur. J. Biochem., 237, 743-751). Here we show that, in contrast to the above-mentioned studies, we found no sign of the ß-sheet regions in GFD in our uPA preparations either free or in a complex with uPAR. The GFD seems to be a rather flexible and unstructured domain, demonstrating in spite of its apparent flexibility highly specific interaction with uPAR both in vitro and in cell culture experiments. Circular dichroism, tryptophan fluorescence during thermal denaturation of the protein, and heteronuclear NMR spectroscopy of ¹5N/¹³C-labeled ATF both free and in complex with urokinase receptor were used to judge the secondary structure of GFD of uPA.

[Antimutagenesis of multiphytoadaptogene in yeast saccharomyces].

Multiphytoadaptogene (MPA) consists of plant extracts components including adaptogenes. Genotoxicity analysis revealed the antimutagenic activity of MPA. MPA decreased the direct mutations frequency in ADE4-ADE8 loci induced by UV radiation and nitrous acid by 3.7 and 33 times, respectively. The lethal effect of UV radiation was inhibited when the preparation was used. MPA had no effect on replicative mutagenesis. At the same time it depressed mutagenesis caused by repair errors. The data obtained suggest the antimutagenic activity of multiphytoadaptogene is associated with postreplicative repair activation.

Bacterial and cell-free production of APP671-726 containing amyloid precursor protein transmembrane and metal-binding domains.

More than half of the mutations associated with familiar Alzheimer's disease have been found in the transmembrane domain of amyloid precursor protein (APP). These pathogenic mutations presumably influence the APP transmembrane domain structural and dynamic properties and result in its conformational change or/and lateral dimerization. Despite much data about the pathogenesis of Alzheimer's disease, the initial steps of the pathogenesis remain unclear so far. For the investigation of the molecular basis of Alzheimer's disease, we selected amyloid precursor protein fragment APP671-726 containing the transmembrane and metal-binding domains. This fragment is the substrate of the γ-secretase complex whose abnormal activity leads to the formation of amyloidogenic Aß42 peptides. This work for the first time describes a highly effective cell-free APP671-726 production method and improved method of bacterial synthesis. Both methods yield milligram quantities of isotope-labeled protein for structural study by high resolution NMR spectroscopy in membrane mimicking milieus.

[Investigation of multyphytoadaptogene anti-radiation efficacy in dogs experiments].

The purpose of the work was to elucidate the radioprotective efficacy of multyphytoadaptogene (MPA) in dogs in various conditions of gamma radiation and MPA application. Dogs were given 15% MPA solution with drinking water in 3,6 ml/kg dose per day 2 weeks before the radiating (preventive application), 2 weeks before and 2 weeks after the radiating (preventive and therapeutic application) as well as 2 weeks after the radiating only (therapeutic application). Animals of control groups received radiation. Dogs were exposed to 3,5 Cy acute radiation and 8,0 Gy prolonged radiation. There were no survived dogs in control groups. At the same time MPA increased dogs survival in preventive, preventive and therapeutic as well as therapeutic applications after 3,5 Gy acute radiation and after 8,0 Gy prolonged radiation. MPA improved the somatic state, interfere with leukocytes amount in blood. The data obtained suggest the radioprotective efficacy of MPA.

Modeling of presymptomatic and symptomatic stages of parkinsonism in mice.

A degradation of the nigrostriatal dopaminergic (DA-ergic) system is the key component of pathogenesis of Parkinson's disease (PD). Initial clinical symptoms appear 20-30 years after the onset of neurodegeneration, at a 70% DA depletion in the striatum and a 50% loss of nigral DA-ergic neurons. Low efficacy of the therapy might be improved if preclinical diagnostics and preventive therapy are developed. The development of appropriate experimental models should precede clinical trials. This multidisciplinary study first managed to model in mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) all together the following stages of parkinsonism: (a) the early presymptomatic stage manifested by a subthreshold degeneration of axons and DA depletion in the striatum without loss of nigral cell bodies; (b) the advanced presymptomatic stage manifested by a subthreshold degeneration of striatal axons and DA depletion and by a subthreshold loss of nigral cell bodies; (c) the advanced presymptomatic stage characterized by threshold depletion of striatal DA and a loss of DA-ergic axons and nigral cell bodies resulting in motor dysfunction. The degeneration of axons proceeds and prevails that of cell bodies suggesting higher sensitivity to MPTP of the former. Compensatory processes were developed in parallel to neurodegeneration that was manifested by the increase of the DA content in individual nigral cell bodies and DA turnover in the striatum. The developed models might be exploited for: (a) an examination of pathogenetic mechanisms not only in the nigrostriatal system but also in other brain regions and in the periphery; (b) a study of the compensatory mechanisms under DA deficiency; (c) a search of precursors of motor disorders and peripheral biomarkers in presymptomatic parkinsonism; (d) the development of preventive therapy aiming to slow down the neurodegeneration and strengthen compensatory processes. Thus, the models of the early and advanced presymptomaic stages and of the early symptomatic stage of parkinsonism were developed in mice with MPTP.

[Bacterial synthesis, purification, and solubilization of transmembrane segments of ErbB family members].

A family of epidermal growth factor receptors, ErbB, represents an important class of receptor tyrosine kinases, playing a leading role in cellular growth, development and differentiation. Transmembrane domains of these receptors transduce biochemical signals across plasma membrane via lateral homo- and heterodimerization. Relatively small size of complexes of ErbB transmembrane domains with detergents or lipids allows one to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe the effective expression system and purification procedure for preparative-scale production of transmembrane peptides from four representatives of ErbB family, ErbB1, ErbB2, ErbB3, ErbB4, for structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS as C-terminal extensions of thioredoxin A. The fusion protein cleavage was accomplished with the light subunit of human enterokinase. Several (10-30) milligrams of purified isotope-labeled transmembrane peptides were isolated with the use of a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering, CD and NMR spectroscopy. The data obtained indicate that the purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy.

Structural and dynamic study of the transmembrane domain of the amyloid precursor protein.

Alzheimer's disease affects people all over the world, regardless of nationality, gender or social status. An adequate study of the disease requires essential understanding of the molecular fundamentals of the pathogenesis. The amyloid ß-peptide, which forms amyloid plaques in the brain of people with Alzheimer's disease, is the product of sequential cleavage of a single-span membrane amyloid precursor protein (APP). More than half of the APP mutations found to be associated with familial forms of Alzheimer's disease are located in its transmembrane domain. The pathogenic mutations presumably affect the structural-dynamic properties of the APP transmembrane domain by changing its conformational stability and/or lateral dimerization. In the present study, the structure and dynamics of the recombinant peptide corresponding to the APP fragment, Gln686-Lys726, which comprises the APP transmembrane domain with an adjacent N-terminal juxtamembrane sequence, were determined in the membrane mimetic environment composed of detergent micelles using NMR spectroscopy. The structure obtained in dodecylphosphocholine micelles consists of two α-helices: a short surface-associated juxtamembrane helix (Lys687-Asp694) and a long transmembrane helix (Gly700-Leu723), both connected via a mobile loop region. A minor bend of the transmembrane α-helix is observed near the paired residues Gly708-Gly709. A cholesterol-binding hydrophobic cavity is apparently formed under the loop region, where the juxtamembrane α-helix comes into contact with the membrane surface near the N-terminus of the transmembrane α-helix.

Dimeric structure of the transmembrane domain of glycophorin a in lipidic and detergent environments.

Specific interactions between transmembrane α-helices, to a large extent, determine the biological function of integral membrane proteins upon normal development and in pathological states of an organism. Various membrane-like media, partially those mimicking the conditions of multicomponent biological membranes, are used to study the structural and thermodynamic features that define the character of oligomerization of transmembrane helical segments. The choice of the composition of the membrane-mimicking medium is conducted in an effort to obtain a biologically relevant conformation of the protein complex and a sample that would be stable enough to allow to perform a series of long-term experiments with its use. In the present work, heteronuclear NMR spectroscopy and molecular dynamics simulations were used to demonstrate that the two most widely used media (detergent DPC micelles and lipid DMPC/DHPC bicelles) enable to perform structural studies of the specific interactions between transmembrane α-helices by the example of dimerizing the transmembrane domain of the bitopic protein glycophorin A. However, a number of peculiarities place lipid bicelles closer to natural lipid bilayers in terms of their physical properties.

Bacterial Synthesis and Purification of Normal and Mutant Forms of Human FGFR3 Transmembrane Segment.

The fibroblast growth factor receptor 3 (FGFR3) is a protein belonging to the family of receptor tyrosine kinases. FGFR3 plays an important role in human skeletal development. Mutations in this protein, including Gly380Arg or Ala391Glu substitutions in the transmembrane (TM) region, can cause different disorders in bone development. The determination of the spatial structure of the FGFR3 TM domain in a normal protein and in a protein with single Gly380Arg and Ala391Glu mutations is essential in order to understand the mechanisms that control dimerization and signal transduction by receptor tyrosine kinases. The effective system of expression of eukaryotic genes in bacteria and the purification protocol for the production of milligram amounts of both normal TM fragments of FGFR3 and those with single pathogenic mutations Gly380Arg and Ala391Glu, as well as their(15)N- and [(15)N,(13)C]-isotope-labelled derivatives, were described. Each peptide was produced inEscherichia coliBL21(DE3)pLysS cells as a C-terminal extension of thioredoxin A. The purification protocol involved immobilized metal affinity chromatography and cation- and anion-exchange chromatography, as well as the fusion protein cleavage with the light subunit of human enterokinase. The efficiency of the incorporation of target peptides into DPC/SDS and DPC/DPG micelles was confirmed using NMR spectroscopy. The described methodology of production of the native FGFR3 TM domain in norma and with single Gly380Arg and Ala391Glu mutations enables one to study their spatial structure using high-resolution heteronuclear NMR spectroscopy.