AZT 5'-Cholinephosphate as an anti-HIV agent: the study of biochemical properties and metabolic transformations using its 32P-labelled counterpart

Biochemical and metabolic transformations of 3'-azido-3'-deoxythymidine 5'-choline phosphate (1) were studied using its 32P-labelled counterpart for the evaluation of possible reasons for its enhanced anti-HIV activity. An effective synthesis of 32P-labelled 1 with a specific activity >1,000 Ci/mmol was developed by esterification of 32P-phosphoric acid with choline in the presence of BrCN followed by the coupling of the resulting choline phosphate with 3'-azido-3'-deoxythymidine (AZT). Chemical and enzymatic stabilities of 1 as well as the dynamics of penetration through HL-60 cell membranes were studied at the concentrations comparable to its antiviral concentrations. The products of intracellular transformations of the studied nucleotide were identified.     

Ribokinase from E. coli: expression, purification, and substrate specificity

Ribokinase (RK) was expressed in the Escherichia coli ER2566 cells harboring the constructed expression plasmid encompassing the rbsK gene, encoding ribokinase. The recombinant enzyme was purified from sonicated cells by double chromatography to afford a preparation that was ca. 90% pure and had specific activity of 75 micromol/min mg protein. Catalytic activity of RK: (i) is strongly dependent on the presence of monovalent cations (potassium>ammonium>cesium), and (ii) is cooperatively enhanced by divalent magnesium and manganese ions. Besides D-ribose and 2-deoxy-D-ribose, RK was found to catalyze the 5-O-phosphorylation of D-arabinose, D-xylose, and D-fructose in the presence of ATP, and potassium and magnesium ions; L-ribose and L-arabinose are not substrates for the recombinant enzyme. A new radiochemical method for monitoring the formation of D-pentofuranose-5-[32P]phosphates in the presence of [gamma-32P]ATP and RK is reported.     

Enzymatic synthesis of bis(5'-nucleosidyl) tetra- or triphosphates

The total fraction of aminoacyl-tRNA synthases from Escherichia coli has been shown to catalyze the synthesis of the bis(5'-nucleosidyl) oligophosphates Ap4AZT, Ap4d4T, Ap43TC, and Ap4ACV, as well as Ap3AZT and Ap3d4T, from [alpha-32P]ATP and the corresponding nucleoside-5'-tri(or di)phosphate. The resulting compounds, characterized by HPLC, are resistant to alkaline phosphatase. Ap4AZT, Ap4d4T, and Ap43TC are formed with approximately equal efficiency, whereas the efficiencies of the synthesis of Ap4ACV, Ap3AZT, and Ap3d4T are three- to fivefold lower. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.     

Sodium Channelopathies: From Molecular Physiology towards Medical Genetics

Voltage-gated sodium channels are heteromeric transmembrane proteins involved in the conduction of sodium ion currents in response to membrane depolarization. In humans, nine homologous genes, SCN1A–11A, which encode different isoforms of the voltage-gated sodium channel family, are known. Sodium channel isoforms exhibit different kinetic properties that determine different types of neurons. Mutations in different channels are described in patients with various congenital disorders, from epilepsy to congenital insensitivity to pain. This review presents an analysis of the current literature on the properties of different isoforms of voltage-gated sodium channels and associated diseases.     

Efficiency of the miRNA–mRNA Interaction Prediction Programs

miRNAs play a key role in regulation of gene expression. Nowadays it is known more than 2500 human miRNAs, while a majority of miRNA–mRNA interactions remains unidentified. The recent development of a high-throughput CLASH (crosslinking, ligation and sequencing of hybrids) technique for discerning miRNA–mRNA interactions allowed an experimental analysis of the human miRNA–mRNA interactome. Therefore, it allowed us, for the first time, make an experimental analysis of the human miRNA–mRNA interactome as a whole and an evaluation of the quality of most commonly used miRNA prediction tools (TargetScan, PicTar, PITA, RNA22 and miRanda). To estimate efficiency of the miRNA–mRNA prediction tools, we used next parameters: sensitivity, positive predicted value, predictions in different mRNA regions (3' UTR, CDS, 5' UTR), predictions for different types of interactions (5 classes), predictions of “canonical” and “nocanonical” interactions, similarity with the random generated data. The analysis revealed low efficiency of all prediction programs in comparison with the CLASH data in terms of the all examined parameters.     

Specificity and Selectivity of a Modified Radioligand Method for Estimating the Binding Activity of β1 Adrenergic Receptors in Human T Lymphocytes

Russian Journal of Bioorganic Chemistry - A radioligand-based method was proposed for quantifying the binding activity of β1-adrenergic receptors (ARs) on the surface of human T lymphocytes....     

5'-carbamoylphosphonyl-[6-3H]-AZT as a tool for studying metabolic transformations of the nonradioactive counterpart, an inhibitor of HIV replication

An effective synthesis of 5'-carbamoylphosphonyl-[6-3H]-AZT was developed from [6-3H]-AZT.For the synthesized compound, chemical and enzymatic stability were determined and its penetration across HL-60 cell membranes was studied.     

Preparation of 32P-labelled 2-methylthioadenosine di- and triphosphates

2-Methylthioadenosine 5'-mono- and diphosphates were prepared by chemical synthesis, and enzymatically phosphorylated to 5'-[beta-32P]-diphosphate and 5'-[gamma-32P]-triphosphate, respectively. They were isolated by HPLC and had activity exceeding 1000 Ci/mmol and radiochemical purity higher than 95%.     

4'-Thio-5-ethyl-2'-deoxyuridine 5'-phosphonates: synthesis and antiviral activity

A number of new 5'-phosphonate derivatives of 4'-thio-5-ethyl-2'-deoxyuridine (TEDU) were synthesized. These compounds displayed a low cytotoxicity and, except for TEDU 5'-fluorophosphate, antiherpes activity similar to that of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir) and 9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine (pencyclovir). 5'-Ethoxycarbonylphosphonate and 5'-aminocarbonylphosphonate of TEDU were also found to suppress the reproduction of herpes simplex type 1 virus, which is resistant to acyclovir.     

The synthesis and antiherpetic activity of acyclovir phosphonate esters

Alkyl esters of acyclovir phosphite, alkoxycarbonylphosphonate, ethoxycarbonylmethylphosphonate, and aminocarbonylphosphonate were synthesized. Most of them were shown to inhibit the replication of type 1 herpes simplex virus in Vero cell culture. The stability in phosphate buffer and human blood serum was studied for several of the derivatives. A correlation between the stability and antiviral activity of the synthesized compounds is discussed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.