S. D. Varfolomeev

Molecular modeling of butyrylcholinesterase inhibition by cresyl saligenin phosphate

Presumably, aerotoxic syndrome is caused by the impact of tricresyl phosphate (TCP), a lubricating oil additive for aircraft engines, on the human body. The most toxic tri-ortho-isomer of this compound is metabolized in the body to give cresyl saligenin phosphate (CSP), which inhibits butyrylcholinesterase (BChE) giving phosphoserine. In this case, BChE acts as a stoichiometric bioscavenger irreversibly binding CSP and as a biomarker of exposure to low doses of TCP. The mechanism of CSP interaction with BChE at the initial stage of formation of the enzyme—inhibitor complex was studied by molecular modeling techniques. The results indicate that BChE interaction with the (R)-enantiomer of CSP in the most stable envelope conformation of the saligenin ring is most preferable. Comparison of the results obtained using different computation methods demonstrated that the best agreement with experimental data can be achieved by combining standard molecular docking methods with quantum mechanics methods for more accurate structure preparation.

Enzyme active sites: bioinformatics, architecture, and mechanisms of action

A comparative analysis of the amino acid sequences of some enzymes which comprise superfamilies of enzymes belonging to different classes was carried out. Based on the amino acid sequence alignment for enzymes belonging to different classes with the use of the information entropy as a criterion, the amino acid residues involved in the catalytic portion of the active site are demonstrated to be most conservative. The rating scale for conservativeness of amino acids in enzymes is created. Glycine and aspartic acid are the most commonly occurring conservative amino acids essential for the catalysis. The role of aspartic acid and histidine in the mechanism of molecule activation in the catalytic site is considered using hydrolases as examples. The role of glycine, proline, and cysteine in the structural organization of the active sites is discussed.

Role of Protein Dimeric Interface in Allosteric Inhibition of N-Acetyl-Aspartate Hydrolysis by Human Aspartoacylase

The results of molecular modeling suggest a mechanism of allosteric inhibition upon hydrolysis of N-acetyl-aspartate (NAA), one of the most abundant amino acid derivatives in brain, by human aspartoacylase (hAsp). Details of this reaction are important to suggest the practical ways to control the enzyme activity. Search for allosteric sites using the Allosite web server and SiteMap analysis allowed us to identify substrate binding pockets located at the interface between the subunits of the hAsp dimer molecule. Molecular docking of NAA to the pointed areas at the dimer interface predicted a specific site, in which the substrate molecule interacts with the Gly237, Arg233, Glu290, and Lys292 residues. Analysis of multiple long-scaled molecular dynamics trajectories (the total simulation time exceeded 1.5 μs) showed that binding of NAA to the identified allosteric site induced significant rigidity to the protein loops with the amino acid side chains forming gates to the enzyme active site. Application of the protein dynamical network algorithms showed that substantial reorganization of the signal propagation pathways of intersubunit communication in the dimer occurred upon allosteric NAA binding to the remote site. The modeling approaches provide an explanation to the observed decrease of the reaction rate of NAA hydrolysis by hAsp at high substrate concentrations.

Proteomic analysis of exhaled breath condensate for diagnostics of respiratory system diseases

Study of proteomic composition of exhaled breath condensate (EBC) is a promising non-invasive method for diagnostics of respiratory system diseases in patients. In this study the EBC proteomic composition of 53 donors, including patients with different respiratory system diseases has been investigated. Cytoskeletal keratins type II (1, 2, 3, 4, 5, 6) and cytoskeletal keratins type I (9, 10, 14, 15, 16) were invariant for all samples. Analyzing the frequency of occurrence of proteins in different groups of examined patients, several categories of proteins have been recognized: proteins found in all pathologies (dermcidin, alpha-1- microglobulin, SHROOM3), proteins simultaneously found in two groups (CSTA, LCN1, JUP, PIP, TXN), and proteins specific for a particular group (PRDX1, annexin A1/A2). The EBC analysis by HPLC-MS/MS can be used for identification of potential protein markers specific for inflammatory pulmonary diseases of infective origin (pneumonia) as well as for non-infectious diseases such as chronic obstructive pulmonary disease (COPD).

Three Faces of N-Acetylaspartate: Activator, Substrate and Inhibitor of Human Aspartoacylase

Hydrolysis of N-acetylaspartate (NAA), one of the most concentrated metabolites in brain, catalyzed by human aspartoacylase (hAsp) shows a remarkable dependence of the reaction rate on substrate concentration. At low NAA concentrations, sigmoidal shape of kinetic curve is observed, followed by typical rate growth of the enzyme-catalyzed reaction, whereas at high NAA concentrations self-inhibition takes place. We show that this rate dependence is consistent with a molecular model, in which N-acetylaspartate appears to have three faces in the enzyme reaction, acting as activator at low concentrations, substrate at moderate concentrations, and inhibitor at high concentrations. To support this conclusion we identify binding sites of NAA at the hAsp dimer including those on the protein surface (activating sites) and at the dimer interface (inhibiting site). Using the Markov state model approach we demonstrate that population of either activating or inhibiting site shifts the equilibrium between the hAsp dimer conformations with the open and closed gates leading to the enzyme active site buried inside the protein. These conclusions are in accord with the calculated values of binding constants of NAA at the hAsp dimer, indicating that the activating site with a higher affinity to NAA should be occupied first, whereas the inhibiting site with a lower affinity to NAA should be occupied later. Application of the dynamical network analysis shows that communication pathways between the regulatory sites (activating or inhibiting) and the gates to the active site do not interfere. These considerations allow us to develop a kinetic mechanism and to derive the equation for the reaction rate covering the entire NAA concentration range. Perfect agreement between theoretical and experimental kinetic data provides strong support to the proposed catalytic model.

Molecular polymorphism of human enzymes as the basis of individual sensitivity to drugs. Supercomputer-assisted modeling as a tool for analysis of structural changes and enzymatic activity of proteins

The nature of individual sensitivity to drugs associated with molecular polymorphism of human enzymes is discussed. The influence of molecular polymorphism on the activity of key human esterases, in particular, cholinesterases and carboxylesterase, responsible for hydrolytic metabolism of ester-containing drugs, is analyzed. A method was developed, which involves supercomputer-assisted modeling as a tool for assessment of molecular mechanism of the impact of point mutations on the catalytic activity of enzymes. This work is a part of a study aimed at elaboration of the concept and methods of personalized medicine.

Computer simulation in molecular medicine and drug design

Achievements in genomics and proteomics, as well as the exponential growth of computing power, ensure qualitatively new opportunities in understanding the molecular nature of “living matter” and, as a consequence, the extension of the capabilities of medicine. This paper considers two scientific fields in which high-throughput computing is especially effective. The first is computer-aided design of drugs, and the second is analysis of human molecular polymorphism and individual sensitivity to drugs. General provisions are illustrated with specific examples, including the development of a new class of antimyasthenic drugs—cholinesterase inhibitors, research in the molecular polymorphism of brain aspartoacylase, the clarification of the nature of Canavan disease, and the study of individual sensitivity to drugs—cholinesterase inhibitors.

DIAGNOSIS OF RESPIRATORY DISEASES USING THE PROTEOMIC ANALYSIS OF EXHALED BREATH CONDENSATE

Investigation of exhaled breath condensate (EBC) is a noninvasive diagnostic method in respiratory diseases. The objective of this study was to compare EBC protein spectrum in healthy volunteers and in patients with chronic obstructive pulmonary disease (COPD), pneumonia and lung cancer (NSCLC), as well as to assess a role of proteomic analysis of EBC for diagnosis and differential diagnosis of these diseases. Methods . We examined 18 patients with COPD, 13 patients with community-acquired pneumonia, 26 patients with lung cancer and 24 healthy non-smoking volunteers. EBC was collected using ECoScreen system (VIASYS Healthcare, Germany) and a standardized method. EBC-samples were lyophilized, hydrolyzed and analyzed by HPLC and tandem mass spectrometry. To identify proteins, we used Mascot (Matrix Science, UK) and IPI-human (version 3.82) databases provided by the European Bioinformatics Institute. Results . Proteomic analysis of EBC identified more than 300 different proteins; most of them were types I and II cytoskeletal keratins. Cytokeratin 5, 6, and 14 concentrations in EBC of NSCLC patients were significantly higher than that in healthy volunteers. Dermcidin, immunoglobulin alpha, kininogen, cytoplasmic actin, serum albumin, and Zn-alpha2-glycoprotein were identified in EBC of healthy volunteers and patients with COPD and pneumonia. High concentration of peroxiredoxin in EBC of COPD patients could be due to severe oxidative stress. High levels of acute-phase and hypoxia proteins (annexins A1 and A2, HSP90B, cystatins M and B, collagen and histones fragments) were detected in EBC of pneumonia patients. Also, β- и α-subunit of hemoglobin, nuclear ubiquitin casein (NUCKS), POTEE, high mobility group protein (HMG-I/HMG-Y) and lactoferrin were identified in EBC of NSCLC patients. Conclusion . We found that EBC in healthy nonsmokers and in patients with COPD, pneumonia and NSCLC had characteristic protein spectrum. Most of the identified proteins could be used for diagnosis of these diseases.

Supercomputer technologies for structural-kinetic study of mechanisms of enzyme catalysis: A quantum-chemical description of aspartoacylase catalysis

The results of modeling of the complete catalytic cycle of aspartoacylase-catalyzed N-acetylaspartate hydrolysis by the combined quantum mechanics/molecular mechanics method and with the use of umbrella sampling replica-exchange molecular dynamics simulations are reported. It has been shown that the decrease in the high-energy barriers of rate-limiting stages is achieved through the preceding equilibrium stages, such as proton transfer and conformational changes. General features of the catalytic behavior of enzymes have been formulated.

High-potential electrolytes for supercapacitors: Lithium polyaspartate

It was shown that widening the electrochemical window and considerable increase in the electrical energy stored by pseudocapacitor (supercapacitor) type devices can be attained by inclusion of polymeric electrolytes, such as lithium polyaspartate.