Devron Averett

A Steady-state and Pre-steady-state Kinetic Analysis of the NTPase Activity Associated with the Hepatitis C Virus NS3 Helicase Domain

The helicase domain of hepatitis C virus NS3 (genotype 1b) was expressed in Escherichia coli and purified to homogeneity. The purified protein catalyzed the hydrolysis of nucleoside triphosphates (NTP) and the unwinding of duplex RNA in the presence of divalent metal ion. The enzyme was not selective for the NTP substrate. For example, UTP and acyclovir triphosphate were hydrolyzed efficiently by the enzyme. The rate of NTP hydrolysis was stimulated up to 27-fold by oligomeric nucleic acids (NA). Furthermore, NA bound to the enzyme with concomitant quenching of the intrinsic protein fluorescence. The dissociation constants of the enzyme for selected NA in the absence of NTP were between 10 and 35 μM at pH 7.0 and 25°C. The enzyme had maximal affinity for NA with 12 or more nucleotides. A detailed steady-state and pre-steady-state kinetic analysis of ATP hydrolysis was made with (dU)18 as the effector. The kcat values for ATP hydrolysis in the presence and absence of (dU)18 were 80 s−1 and 2.7 s−1, respectively. The association (dissociation) rate constants for the enzyme and (dU)18 in the presence and absence of ATP were 5.7 μM−1 s−1 (3.9 s−1) and 290 μM−1 s−1 (2.27 s−1), respectively. The association (dissociation) rate constants for the enzyme and ATP in the presence and absence of (dU)18 were 0.4 μM−1 s−1 (<0.5 s−1) and 0.9 μM−1 s−1 (<10−1 s−1), respectively. These data were consistent with a random kinetic mechanism.

Anti-human immunodeficiency virus synergism by zidovudine (3'-azidothymidine) and didanosine (dideoxyinosine) contrasts with their additive inhibition of normal human marrow progenitor cells.

The anti-human immunodeficiency virus (HIV) activity and hemopoietic toxicity of zidovudine (AZT) and didanosine (dideoxyinosine;ddI), alone and in combination, were assessed in a variety of cell types. AZT was more potent than ddI as an inhibitor of HIV in vitro. Synergistic inhibition of HIV by the combination of these agents was observed in MT4 cells, peripheral blood lymphocytes, and macrophages. Toxicity assessment in vitro by using progenitor (erythroid and granulocyte-macrophage) colony-forming assays with normal human bone marrow showed ddI to be less toxic than AZT. Addition of inhibitory concentrations of ddI to AZT resulted in additive inhibition of progenitor CFUs. These in vitro findings suggest that combinations of ddI and AZT at appropriately modified doses may provide an enhanced degree of selectivity in anti-HIV chemotherapy.

A simple method to purify ribonucleotide reductase

Assays of ribonucleotide reductase in extracts of Detroit 98 (human) cells were found to be complicated by the rapid depletion of the substrate (CDP) by nucleoside diphosphate kinase. Assays of either 100,000g supernatants or ammonium sulfate-fractionated extracts resulted in the conversion of greater than 90% of the substrate to CTP within 2 min. It was therefore desirable to separate nucleoside diphosphate kinase from ribonucleotide reductase. Chromatography of the fractionated extract on an ATP-agarose column resulted in the delivery of nondissociated ribonucleotide reductase in the void volume and the retention of greater than 99.9% of the nucleoside diphosphate kinase. The kinase could be eluted by 2 mM ATP. The ribonucleotide reductase was recovered from this commercially available gel with an apparent yield of greater than 200%. It could be accurately assayed with only minimal extraneous depletion of substrate. Furthermore, it was stable to storage at -80 degrees C. Tris-HCl was found to inhibit the enzyme. When HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)-Na buffer was used in place of Tris-HCl, the rate of CDP reduction was increased by 2.5-fold. Since the above procedure selectively removes nucleoside diphosphate kinase from crude preparations of ribonucleotide reductase, it should have general applicability for purifying ribonucleotide reductase from other sources.

Anabolic pathway of 6-methoxypurine arabinoside in cells infected with varicella-zoster virus.

6-Methoxypurine arabinoside (ara-M) exhibits potent activity against varicella-zoster virus (VZV) as a result of ara-Ms anabolism to the triphosphate of adenine arabinoside (ara-ATP) in VZV-infected cells. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) enhanced the formation of ara-ATP by inhibiting ara-M demethoxylation. In contrast, deoxycoformycin and coformycin, inhibitors of both adenosine deaminase and AMP deaminase, blocked the formation of ara-ATP and reversed the anti-VZV activity of ara-M. These results indicate that after the initial phosphorylation of ara-M by the VZV-coded thymidine kinase, the monophosphate is demethoxylated by AMP deaminase to form ara-IMP, which is converted to ara-ATP by the sequential actions of the cellular adenylosuccinate synthetase, adenylosuccinate lyase, and nucleotide kinases.

Purine Arabinosides as Inhibitors of Human Haemopoietic Progenitor Cells

Four purine arabinosides that inhibit varicella-zoster virus (VZV) replication in vitro were tested as inhibitors of colony formation by progenitor cells from normal human bone marrow. In general, erythroid burst forming cells (BFU-E) were more sensitive to inhibition by these compounds than were either erythroid colony forming cells (CFU-E) or granulocyte/macrophage colony forming cells (CFU-GM). A 50% reduction in colony formation (IC50) was observed for BFU-E in the presence of 8 μM 6-methoxypurine arabinoside. Adenine arabinoside and hypoxanthine arabinoside had IC50 values of 1 μM and 4 μM respectively, whereas 6-ethoxypurine arabinoside was not inhibitory (IC50 > 50 μM). Enzyme studies showed that both 6-methoxypurine arabinoside and adenine arabinoside were converted to hypoxanthine arabinoside by adenosine deaminase. 6-Ethoxypurine arabinoside was a much less efficient substrate. When the BFU-E assays were performed in the presence of an inhibitor of adenosine deaminase, 6-methoxypurine arabinoside became non-inhibitory. In contrast, adenine arabinoside became much more inhibitory (IC50 = 0.03 μM). The potency of hypoxanthine arabinoside was unaffected. Thus, incubation of 6-methoxypurine arabinoside and adenine arabinoside under conditions appropriate for the BFU-E assay resulted in the in situ conversion of these compounds to hypoxanthine arabinoside. Biotransformation of compounds must be considered in the assessment of toxicity in vitro.

ACYCLOVIR'S ANTIHERPETIC ACTIVITY IS POTENTIATED BY INHIBITION OF RIBONUCLEOTIDE REDUCTASE: 205

Compound A723U, a 2-acetylpyridine thiosemicarbazone, was found to inactivate herpes simplex virus (HSV) ribonucleotide reductase. Inactivation occurred only while the enzyme was catalyzing the reduction of substrate and at a maximum rate of 17 per hr. A723U inhibited virus replication (ED50 = 3-5 μM) at concentrations that were not toxic to the confluent host cells. It also exhibited mutually potentiated anti-HSV activity with acyclovir (ACV). Sub-inhibitory concentrations of either compound significantly decreased the ED50 of the other compound. As expected, A723U caused the dGTP pool to decrease. However, it also caused the level of ACV-triphosphate to markedly increase. The consequence is a facilitation of the binding of ACV-triphosphate to its target enzyme, HSV DNA polymerase.