ABSTRACT
2,3-Dihydroxy-quinoxaline, a small molecule that promotes ATPase catalytic activity of Herpes Simplex Virus thymidine kinase (HSV-TK), was identified by virtual screening. This compound competitively inhibited HSV-TK catalyzed phosphorylation of acyclovir with Ki=250 µM (95% CI: 106-405 µM) and dose-dependently increased the rate of the ATP hydrolysis with KM=112 µM (95% CI: 28-195 µM). The kinetic scheme consistent with this experimental data is proposed.
Subject(s)
Adenosine Triphosphatases/chemistry , Quinoxalines/pharmacology , Simplexvirus/enzymology , Thymidine Kinase/antagonists & inhibitors , Acyclovir/therapeutic use , Catalysis , Humans , Kinetics , Phosphorylation/drug effects , Simplexvirus/drug effects , Thymidine Kinase/chemistryABSTRACT
In this study, we continued to study antiherpetic properties of acyclovir 5'-hydrogenphosphonate (Hp-ACV) in cell cultures and animal models. Hp-ACV was shown to inhibit the development of herpetic infection in mice induced by the HSV-1/L(2) strain. The compound suppressed replication of both ACV-sensitive HSV-1/L(2) and ACV-resistant HSV-1/L(2)/R strains in Vero cell culture. Viral population resistant to Hp-ACV (HSV-1/L(2)/R(Hp-ACV)) was developed much slower than ACV-resistant population. The analysis of Hp-ACV-resistant clones isolated from the HSV-1/L(2)/R(Hp-ACV) population demonstrated their partial cross-resistance to ACV. The mutations determining the resistance of HSV-1 clones to Hp-ACV were partly overlapped with mutations defining ACV resistance but did not always coincide. HSV-1/L(2)/R(Hp-ACV) herpes virus thymidine kinase is shortened from the C-terminus by 100 amino acid residues in length.
Subject(s)
Acyclovir/analogs & derivatives , Antiviral Agents/pharmacology , Herpesviridae/genetics , Acyclovir/chemical synthesis , Acyclovir/chemistry , Acyclovir/pharmacology , Amino Acid Sequence , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Chlorocebus aethiops , Drug Resistance, Viral , Herpesviridae/drug effects , Herpesviridae Infections/drug therapy , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Mutation , Sequence Alignment , Thymidine Kinase/genetics , Thymidine Kinase/metabolism , Vero CellsABSTRACT
The combinational use of acyclovir (ACV) phosphonate esters and alpha(2)-interferon was shown to produce a synergistic effect on inhibition of HSV-1 replication in Vero cell cultures. Unlike other acyclovir phosphonate derivatives studied earlier, ACV H-phosphonate is not an ACV prodrug. On penetrating into the cells, it may be directly converted into ACV monophosphate escaping dephosphonylation-phosphorylation steps.
Subject(s)
Acyclovir/analogs & derivatives , Antiviral Agents/pharmacology , Chemistry, Pharmaceutical/methods , Herpesviridae/metabolism , Interferon-alpha/metabolism , Organophosphonates/chemistry , Acyclovir/chemistry , Animals , Antiviral Agents/chemistry , Chlorocebus aethiops , Chromatography/methods , Drug Design , Kinetics , Models, Biological , Phosphorylation , Purine-Nucleoside Phosphorylase/chemistry , Vero CellsABSTRACT
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.
