Tatyana Dzimbova

Electrochemistry of coupled electron-ion transfer of a heme-like complex in an artificial organic membrane.

The electrochemistry of a heme-like complex 5,10,15,20-tetraphenylporphyriniron(III) chloride (Fe(III)-TPP-Cl) embedded in a lipophilic artificial membrane is studied by means of voltammetric techniques. The experimental system used comprises of a thin organic membrane hosting the redox probe, sandwiched between edge plane pyrolytic graphite electrode and an aqueous electrolyte solution. In the course of the voltammetric experiment the redox transformation of Fe(III)-TPP-Cl at the electrode|membrane interface is accompanied by concomitant ion transfer reactions across the membrane|water interface. The overall electrochemical process proceeds as a coupled electron-ion transfer reaction providing insight into energetic and kinetic aspects of the complex membrane charge-transfer reactions. The membrane electrochemistry of Fe(III)-TPP-Cl involves two quasireversible redox transformations typical for the Fe(III)/Fe(II) couple. Both redox processes are coupled with a chemical reactions proceeding inside the membrane as well as by complex ion transfer reactions across the membrane|water interface comprising chloride expulsion from the membrane and cation ingress from the aqueous phase.

In Vitro Assessment of the Cytotoxic Effects of Sulfo-Arginine Analogues and their Hydrazide Derivatives in 3T3 and HepG2 Cells

ABSTRACT The cytotoxic activities of sulfo-arginine analogues sArg, NsArg, and their hydrazide derivatives, sArg-CONHNH2, sArg-CONHNC6H5, sArg-CONHN(CH2CH2Cl)2, NsArg-CONHNH2, NsArg-CONHNC6H5, NsArg-CONHN(CH2CH2Cl)2, on 3T3 and HepG2 cells were examined. The substitution in the carboxylic group of sArg increases the cell growth inhibitory effects of the compounds, especially in the case of the bis-(2-chloroethyl)hydrazide substitute. Similar correlation was observed in the case of NsArg and its analogues, but here the most pronounced effect was observed with the analogue NsArg-CONHNH2.

Kyotorphin analogues containing unnatural amino acids: synthesis, analgesic activity and computer modeling of their interactions with μ-receptor

Kyotorphin (KTP; Tyr-Arg) an endogenous neuropeptide is potently analgesic when delivered directly to CNS. An effort to enhance the potency, enzymatic stability and improving bioavailability of KTP is the modification with unnatural amino acids. The aims of presented study were: (1) To synthesize new analogues of kyotorphin containing unnatural amino acids: norcanavaine (NCav) and norcanaline (NCan), structural analogues of arginine and ornithine, respectively; (2) To understand the influence of the arginine mimetics on the pharmacological properties of KTP analogues, through examination their effects on the paw pressure nociceptive threshold; (3) To find relationship between the structure and obtained biological effects of the all synthesized kyotorhin analogues, by molecular docking with μ-opioid receptor. As a result of our work four new kyotorphin analogues containing NCan and NCav were obtained. A correlation between the data from the in vivo test and docking results was found. This allows a better elucidation of the ligand-receptor interactions, the prediction of biological activity of the newly synthesized compounds, and to generate compounds with increased biological effects.

MIF-1 and Tyr-MIF-1 analogues containing unnatural amino acids: synthesis, biological activity and docking studies

Melanocyte-inhibiting factor (MIF-1) is the first hypothalamic tripeptide which has been demonstrated to act not only in the brain but also in the pituitary. Tyr-MIF-1 acts as an opiate agonist. It binds selectively and with a high affinity to the μ-opioid receptor when compared with the δ-and κ-opioid receptors. A large number of analogues of MIF-1 and Tyr-MIF-1, containing various modifiers in their structures, have been synthesized and their analgesic effect was determined by various in vivo tests. The aim of current study was: (1) to synthesize new MIF-1 and Tyr-MIF-1 analogues containing sulfoarginine (sArg) and norsulfoarginine (NsArg) in the second and third position, respectively; (2) with the help of docking procedures to find the relationship between structure and biological activity of MIF-1 and Tyr-MIF-1 analogues previously synthesized and biologically tested; (3) using found correlation to predict the biological effect of newly synthesized analogues. New analogues of MIF-1 and Tyr-MIF-1 were synthesized using methods of peptide synthesis in solution. Docking was performed with GOLD 5.0 and a correlation between the obtained docking data and the values from in vivo test was found. Some structure–activity relationships were determined. According to the correlation, we made assumptions about the biological effect of sArg and NsArg containing MIF-1 and Tyr-MIF-1. A computational approach could be very useful in the elucidation of the structure–activity relationship and in the design of new analogues with desired biological effect.

Long-Lasting Effects of Oxy- and Sulfoanalogues of l-Arginine on Enzyme Actions

Arginine residues are very important for the structure of proteins and their action. Arginine is essential for many natural processes because it has unique ionizable group under physiological conditions. Numerous mimetics of arginine were synthesized and their biological effects were evaluated, but the mechanisms of actions are still unknown. The aim of this study is to see if oxy- and sulfoanalogues of arginine can be recognized by human arginyl-tRNA synthetase (HArgS)—an enzyme responsible for coupling of L-arginine with its cognate tRNA in a two-step catalytic reaction. We make use of modeling and docking studies of adenylate kinase (ADK) to reveal the effects produced by the incorporation of the arginine mimetics on the structure of ADK and its action. Three analogues of arginine, L-canavanine (Cav), L-norcanavanine (NCav), and L-sulfoarginine (sArg), can be recognized as substrates of HArgS when incorporated in different peptide and protein sequences instead of L-arginine. Mutation in the enzyme active center by arginine mimetics leads to conformational changes, which produce a decrease the rate of the enzyme catalyzed reaction and even a loss of enzymatic action. All these observations could explain the long-lasting nature of the effects of the arginine analogues.

Oxy- and Sulfoanalogues of l -Arginine

l-Arginine (l-Arg) is a natural cationic amino acid, containing a guanidinium group. This group is positively charged at neutral pH and is involved in a variety of physiological and pathological processes. Arginine plays an important role in the normal growth and development of organisms. Arginine depletion is associated with serious cell and organ functional impairment, ultimately lethal to the body. Elevated arginine levels can influence cellular functions, too; they may cause cell death or cell proliferation. The biological role of arginine involves several aspects. Arginine in the peptide chain is important in the regulation of peptide synthesis. Usually, peptides contain several arginine residues, and this repeat signals the end of peptide chain in the biosynthesis process. This is particularly important in neuropeptides, an essential class of molecules involved in cell signalling. Neuropeptides are synthesised as long precursor molecules, containing multiple copies of the active species. They are known as “pro-peptides” and are functionally inactive. They can be conveniently stored in the cells in this inactive form, until they will be needed. The individual neuropeptide copies in the precursor molecule are separated by arginine-rich regions. These rich in arginine domains serve not only for recognising the active peptide fragments, but are a source of arginine as well. Most amino acids inside the cells are known to be stored in a bonded form, much the same as proteins. Cells unable to synthesise arginine de novo supply their free amino acids by proteolysis. Arginine is involved in many metabolic pathways in the human body. It is a precursor for the biosynthesis of peptides and proteins, but also of ornithine, polyamines, nitric oxide, proline, glutamic acid, glutamine, creatine, agmatine and dimethyl-arginines. In mammals, arginine is a substrate of the five different enzyme systems, including nitric oxide synthases, arginase, arginine:glycine amidinotransferase, arginine decarboxylase and arginine deiminase (Morris, Brit J Pharmacol 157:922–930, 2009). The latter enzyme is not expressed in mammalian cells, but takes part in arginine metabolism when expressed by pathogens. Once inside the mammalian cells, pathogens strongly affect the metabolism of arginine in the host (Fig. 5.1). Arginine is a semi-essential amino acid in mammals, because it can be synthesised from citrulline. The process is catalysed by argininosuccinate synthase, one of two enzymes, responsible for converting arginine into citrulline. The biosynthesis of arginine from citrulline is catalysed by the cytosolic fraction of arginine succinate synthase and argininosuccinate lyase.

Modeling the Relationship between Biological Activity of Delta-selective Enkephalin Analogues and Docking Results by Polynomials

One of the areas of bioinformatics is to develop a fast and reliable method for predicting the biological activity of compounds. This will abbreviate the way for design of new compounds and reduce costs. The process of creating the selective ligands of delta opioid receptor (DOR) was directed towards the synthesis of enkephalin analogues. Their biological activity was determined using the in vivo and in vitro methods, allowing establishing the relationship between structure and biological activity. The application of computational methods in the design of this type of compounds reduces the stages of synthesis and biological tests. The relationship of the efficacy with the values of the so-called ChemScore scoring function from GOLD 5.2 and the values of total energy of ligand-receptor complex was modeled with first- to third-degree polynomials and surface fitted method. The polynomial surface of the third degree has the best fit, assessed by least squares method. In our previous study with theoretical model of DOR (PDBid:1ozc) was established the relationship of the efficacy with the values of the GoldScore scoring function and the values of total energy of ligand-receptor complex. This relationship was modeled with third degree of polynomial in Matlab. The GoldScore scoring function is used for the prediction of ligand binding positions and it takes into account factors such as H-bonding energy, van der Waals energy, metal interaction and ligand torsion strain. In contrast to it the Chemscore scoring function incorporates a protein-ligand atom clash term and an internal energy term. It takes account of hydrophobic-hydrophobic contact area, hydrogen bonding, ligand flexibility and metal interaction. Therefore, the aim of presented work is to find an optimal fitting polynomial function by which to model the relationship between quantitative parameters of {\it in vitro} bioassay (efficacy, affinity and potency) and the values obtained from molecular docking with crystal structure of DOR (PDBid:4ej4). The finding, established in this study, suggests that the third degree polynomial could be successfully used for modeling of the relationship between the efficacy of delta-selective enkephalin analogues and the results from the docking experiments. It is described by a polynomial surface of the third degree. This function could serve to predict the biological activity of new analogues and to be very useful in the design of new delta-selective analogues. Acknowledgments: This work is partially supported by the project of the Bulgarian National Science Fund, entitled: “Bioinformatics research: protein folding, docking and prediction of biological activity”, code NSF I02/16, 12.12.14.

Molecular Docking of Amino Acid Analogues of Rimantidine

M2 channel is a 97-residue single-pass membrane protein with its N-and C-termini directed toward the outside and inside of the virion, re-spectively; it is a homotetramer in its native state. The four TM helicesform a channel in which His37 is the pH sensor and Trp41, the gate. Theadamantane-based drugs, amantadine and rimantadine, which target theM2 channel, have been used as rst-choice antiviral drugs against commu-nity outbreaks of inuenza A viruses for many years, but resistance to theadamantanes has recently become widespread. To overcome this dierentanalogues of rimantidine have been synthesized. The aim of this study isto predict the biological activity of amino acid analogues of rimantidinewith a help of docking studies in order to synthesize only promising can-didates. Twenty analogues of rimantidine with natural amino acids wereused. Docking was performed with the M2 channel as it is very impor-tant in the replicative cycle of inuenza A virus. Crystal structure of thechannel was obtained from RCSB (PDB id: 2rlf). Docking was performedwith GOLD 5.2 using GoldScore tness function. The complexes of riman-tidine analogues with M2 channel were analyzed and their total energieswere calculated in Molegro Molecular Viewer. Total energy of the com-plex rimantidine/M2 channel is -29.437 kJ/mole. All complexes of aminoacid analogues of rimantidine with the channel have lower energies, butthe lowest are the energies of the complexes of Asn-Rim/M2 channel andHis-Rim/M2 channel with -72.475 and -76.440 kJ/mole, respectively. Fromthe energetically point of view complexes will be more stables. This meansthat all rimantidine analogues will block the M2 channel thus will aectthe viral replication cycle. Docking studies are an useful tool for predictionof biological eect of dierent type of compounds and could be applied inshortening the drug design process.

Conformational Analysis and HF ab initio Geometry Optimization of Kyotorphine and Its Sulfo-Analogues Norsulfoarginine-Tyrosine and Tyrosine- Norsulfoarginine

Molecular mechanics (MM) conformational search has been performed for the molecules of the endogenous dipeptide kyotorphin (kyo) and its synthetic sulfo-analogues norsulfoarginine-tyrosine (NsArg-Tyr) and tyrosine-norsulfoarginine (Tyr-NsArg). The MM-found minimum-energy conformations were further optimized at HF ab initio level (3-21G* basis set) in gas phase and in water medium. The non-ionic and the zwitter-ionic forms and the E- and Z-diastereomers of the dipeptides were considered. In all cases, all the lowest-energy conformations adopt a specific scorpion-like conformation with close proximity between the guanidino and phenolic residues. The relative enegies of the different forms, the geometric parameters and the role of the intramolecular hydrogen bonding in stabilizing the structures are discussed.

Comparative Evolution of four Scoring Functions with Three Models of Delta Opioid Receptor Using Molecular Docking

The present study was performed in order to find the most appropriate scoring functions and the model for docking of enkephalin analogues with delta-opioid receptor (DOR) that correlated well with the results obtained from in vitro tests. The capabilities of the four scoring functions embedded in GOLD were explored with three different models of DOR: a theoretical model published in ePDB (id: 1ozc), a model obtained by as with homology modeling, and a crystal structure of DOR published in PDB (id: 4ej4). Eleven enkephalin analogues were consistently docked with each of the models with each of the four scoring function. The analysis of the obtained results shows that after the docking with our modeled DOR values of scoring functions correlate with the data from in vitro tests at the highest degree. Furthermore, the use of the scoring functions ASP (Astex Statistical Potential) and GoldScore enable more precise docking of the test ligands as correlation coefficients were....