Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.9, #3)

Pharmacological agents and transport nanosystems based on plant phospholipids by N. V. Medvedeva; V. N. Prozorovskiy; D. V. Ignatov; O. S. Druzilovskaya; V. A. Kudinov; E. O. Kasatkina; E. G. Tikhonova; O. M. Ipatova (205-216).
A new generation of plant phosphatidylcholine (PCh)-based pharmacological agents has been developed under academician A.I. Archakov leadership at the Institute of Biomedical Chemistry (IBMC). For their production a unique technology allowing to obtain dry lyophilized phospholipid nanoparticles up to 30 nm was elaborated. The successful practical application of PCh nanoparticles as a drug agent may be illustrated by Phosphogliv (oral and injection formulations). Being developed at IBMC for the treatment of liver diseases, including viral hepatitis, Phosphogliv (currently marketed by the “Pharmstandard” company) is approved for clinical application in 2000, and is widely used in medical practice. Based on the developed and scaled in IBMC technology of preparation of ultra small size phospholipid nanoparticles without the use of detergents/surfactants and stabilizers another drug preparation, Phospholipovit, exhibiting pronounced hypolipidemic properties has been developed. Recently completed preclinical studies have shown that PCh nanoparticles of 20–30 nm activate reverse cholesterol transport (RCT) and in this context it is more active than well known foreign preparation Essentiale. Phospholipovit is now at the stage of clinical trials (phase 1 completed). PCh was also used as a basis for the development of a transport nanosystem with a particle size of 20–25 nm in diameter and also for incorporation of drug substances from various therapeutic groups. Using several drugs substances as an example, increased bioavailability and specific activity were demonstrated for the formulations equipped with such transport nanosystem. Formulations equipped with the transport nanosystems have been developed for such pharmacological agents as doxorubicin, rifampicin, budesonide, chlorin E6, prednisolone, and others.
Keywords: phosphatidylcholine; phospholipid nanoparticles; drug delivery nanosystem; targeted delivery

The way to the peptide vaccine against hepatitis C by E. F. Kolesanova; B. N. Sobolev; A. A. Moysa; E. A. Egorova; A. I. Archakov (217-227).
In order to overcome the problem of genetic variability of hepatitis C virus (HCV) envelope proteins during vaccine development, we have used the so-called “reverse vaccinology” approach defined as “from genome to vaccine.” In the context of this approach we have created a database of amino acid sequences of HCV proteins, performed an analysis of the viral genome, and identified several highly conserved regions in HCV envelope proteins. These regions demonstrated low antigenic activity within full-length proteins and viral particles: antibodies against these regions were found not in all hepatitis C patients. However, among highly conserved regions, two regions, containing a wide set of potential T-helper epitope motifs were identified. Using solid-phase peptide synthesis we have generated several synthetic peptide constructs, which consist of two highly conservative sites of the HCV envelope protein E2 joint by a linker; one of these sites contained a set of T-helper epitope motifs. In experiments on laboratory animals the resultant peptide constructs exhibited immunogenicity comparable to the immunogenicity of the protein molecules and were able to induce generation of antibodies, which specifically bound HCV envelope proteins. Immunization with certain constructs resulted in generation of antibodies that were able to bind HCV from the blood plasma of hepatitis C patients. Based on these peptide constructs an original preparation of the synthetic peptide vaccine against hepatitis C is now under development.
Keywords: hepatitis C; vaccine; virus; envelope proteins; antigens; peptides

Electrochemical methods in biomedical studies by V. V. Shumyantseva; T. V. Bulko; E. V. Suprun; A. V. Kuzikov; L. E. Agafonova; A. I. Archakov (228-243).
Own experimental studies on the development of highly sensitive methods of electrochemical analysis applicable for biochemical research in the postgenomic era as well as electrochemical sensor systems for analysis of various biological objects have been summarized. Electroanalysis of catalytic activity of cytochrome P450 resulted in the development of a system for screening potential substrates and/or inhibitors of this class hemoproteins, as well as biologically active compounds modulating the catalytic function of this protein. The study of kinetics of bioaffinity troponin I/anti-troponin I (antibody to TnI) interactions in human plasma resulted in the development of a highly sensitive piezoelectric immunosensor, performing direct registration of biochemical interactions based on the difference of the kinetic parameters of specific and nonspecific bioaffinity interactions without additional administration of labels and without chemical modifications. The developed methods of direct registration of the electrochemical activity of bacterial cells Escherichia coli JM109 are applicable for real time evaluation of antibacterial activity of drug substances; this requires minimal volumes of cells (106 CFU/electrode). Special attention is paid to experimental data on preparation of polymers with molecular imprints (molecularly imprinted polymers, MIP) as analogues of antibodies and biorecognizing elements, carrying selective complementary analytes binding based on the “lock and key” principle.
Keywords: electrochemical biosensors; molecularly imprinted polymers (MIP); cytochrome P450; myoglobin; cardiac markers; bacterial cells

AFM-based technologies as the way towards the reverse Avogadro number by T. O. Pleshakova; I. D. Shumov; Yu. D. Ivanov; K. A. Malsagova; A. L. Kaysheva; A. I. Archakov (244-257).
Achievement of the concentration detection limit for proteins at the level of the reverse Avogadro number determines the modern development of proteomics. The review considers a possibility of approximating the reverse Avogadro number by using nanotechnological methods: AFM-based fishing with mechanical and electrical stimulation, nanowire detectors, and other methods. The ability of AFM to detect, quantify, visualize and characterize physicochemical properties of proteins at concentrations up to 10–17–10–18 M is demonstrated. The combination of AFM-fishing with mass-spectrometry allows to identify proteins not only in pure solutions, but also in multi-component biological fluids (serum). Special attention is paid to analysis of possibilities to improve the biospecific fishing efficiency by the use of SOMAmers in both AFM and nanowire systems. The paper also provides criteria for evaluation of the sensitivity of fishing-based detection systems. The fishing efficiency depending on the detection system parameters is estimated. The practical implementation of protein fishing depending on the ratio of the sample solution volume and the surface of the detection system is discussed with emphasis on advantages and disadvantages of the modern promising nanotechnological methods of protein detection implemented on the basis of these schemes.
Keywords: atomic force microscopy; nanowires; low-abundant proteins; biosensor

Physico-chemical methods for studying amyloid-β aggregation by S. P. Radko; S. A. Khmeleva; E. V. Suprun; S. A. Kozin; N. V. Bodoev; A. A. Makarov; A. I. Archakov; V. V. Shumyantseva (258-274).
Alzheimer’s disease is the most prevalent neurodegenerative pathology. According to the amyloid cascade hypothesis, transition of the amyloid-β peptide (Aβ) from the monomeric form to the aggregated state is a key event in pathogenesis of the Alzheimer’s disease. The mechanism of Aβ aggregation is intensively studied in vitro, by means of synthetic peptides and various physico-chemical methods allowing evaluation of size, molecular structure, and morphology of the formed aggregates. The review considers both the wellknown and recently introduced physico-chemical methods for analysis of Aβ aggregation, including microscopy, optical and fluorescent methods, electron paramagnetic resonance, electrochemical and electrophoretic methods, gel-filtration, and mass spectrometric methods. Advantages and disadvantages of these methods are considered. Special attention is paid to the unique possibility of simultaneous analysis of both Aβ monomers and its oligomers as well as large aggregates by means of atomic force microscopy or fluorescence correlation spectroscopy. The high detection sensitivity of the latter method provides opportunity for investigating the aggregation process in Aβ solutions of low peptide concentrations. Among mass spectrometric methods, the ion mobility mass spectrometry is considered as a method enabling to obtain information about both the spectrum of Aβ oligomers and their structure. Simultaneous employment of several methods providing complementary data about Aβ aggregates is the best experimental approach for studying the process of Aβ aggregation in vitro.
Keywords: Alzheimer’s disease; amyloid-β; aggregation; analytical methods

Computer modelling of monoamine oxidases by A. V. Veselovsky; A. S. Ivanov; A. E. Medvedev (275-282).
The article summarized results of long-term studies on active site structures of monoamine oxidase (MAO) performed in the Institute of Biomedical Chemistry (Russia) by computer modeling approaches. In mammals MAO exists in two highly homologous forms, MAO A and MAO B, distinguished by substrate specificity, inhibitor selectivity, and other characteristics. The development of approaches for active site modeling of these enzymes (with unknown three-dimensional structures) was originally based on analysis of inhibitory activity of selective inhibitors. It started from the analysis of relationships between the geometrical sizes of conformationally rigid molecules and their inhibitory activity. These studies resulted in molding of the active site structures of MAO A and MAO B. These molds reflect the sizes and shapes of active sites of these enzymes. These mold models have been used for virtual screening of molecular databases for new selective and non-selective MAO inhibitors. The generated models have been compared with three-dimensional structures of MAO A and MAO B, which appeared later.
Keywords: monoamine oxidase; MAO; computer modelling; active site; inhibitors

The possibility of using the PlasmaDeepDive™ MRM-panel in clinical diagnostics by Yu. V. Miroshnichenko; N. A. Petushkova; N. E. Moskaleva; N. B. Teryaeva; V. G. Zgoda; E. V. Ilgisonis; A. Yu. Belyaev (283-289).
Concentrations of 46 proteins have been determined in human blood plasma by the method of multiple reaction monitoring (MRM) using the PlasmaDeepDiveTM MRM-panel (Biognosys AG, Switzer land) for quantitative analysis of peptides. 18 of these proteins included into the group of proteins with higher concentrations, were also identified by method of liquid chromatography-tandem mass spectrometry (LS MS/MS) shotgun. Based on literature data it is concluded that the PlasmaDeepDiveTM MRM-panel is appli cable for studies of human plasma samples for determination of potential biomarkers of various nervous sys tem disorders.
Keywords: quantitative proteomics; marker proteins; mass spectrometry; protein panel; pathology of the central nervous system; peptide

Variability in the relative human DNA content during metagenomic analysis of gut microbiota by E. S. Kostryukova; I. Y. Karpova; A. K. Larin; A. C. Popenko; A. V. Tyaht; E. N. Ilina (290-295).
The comparative study of seven different methods for extraction of total DNA from human feces has been carried out. All these methods are recommended in protocols for metagenomic analysis of human gut microbiota. The relative abundance of human DNA in such samples registered by shotgun sequencing on a SOLiD 4 genetic analyzer has been investigated. It was shown that either initial amount of feces or a method applied for total DNA extraction insignificantly influenced the final relative human DNA proportion, which did not exceed 1% in healthy people. Invariance of this parameter allows to consider the increased proportion of human DNA in metagenomic samples as a potential marker of inflammatory bowel diseases.
Keywords: metagenomic analysis; shotgun sequencing; DNA extraction from feces

pIPredict: A computer tool for prediction of isoelectric points of peptides and proteins by V. S. Skvortsov; N. N. Alekseytchuk; D. V. Khudyakov; I. V. Romero Reyes (296-303).
The data on approximate values of isoelectric point (pI) of peptides obtained during their fractionation by isoelectric focusing can be successfully used for the creation of the pKa scale for amino acid residues. This scale can be used for pI prediction for any peptide. Such the data of peptide fractionation also provide information about various post-translational modifications (PTMs), so that the pI values may be predicted for a wide range of protein forms. In this study, pKa values were calculated using a set of 13448 peptides (including 300 peptides with PTMs significant for pI calculation). The pKa constants were estimated for N-terminal, internal and C-terminal amino acid residues separately. The comparative analysis has shown that our scale increases the accuracy of pI prediction for peptides and proteins and successfully competes with tra- ditional scales and such methods as support vector machines and artificial neural networks. The prediction performed by this scale, can be made in our program pIPredict with GUI written in JAVA as an executable jar-archive. The program is freely available for academic users at http://www.ibmc.msk.ru/LPCIT/pIPredict. The software has also the possibility of pI prediction by some other scales; it recognizes some PTMs and has the ability to use a custom scale.
Keywords: peptide; isoelectric point; posttranslational modifications; property prediction