Strong Inhibitory Properties Research Articles

The herpes simplex virus type I DNA polymerase

Polyclonal antibodies responding specifically to the N-terminal, central and C-terminal polypeptide domains of the herpes simplex virus type I (HSV-1) DNA polymerase of strain Angelotti were generated. Each of the five different rabbit antisera reacted specifically with a viral 132 +/- 5-kDa polypeptide as shown by immunoblot analysis. Enzyme binding and inhibition studies revealed that antibodies raised to the central and the C-terminal domains of the protein inhibited the polymerizing activity by 70-90%, respectively, which is well in line with the proposed site of the catalytic center of the enzyme and with the possible involvement of these polypeptide chains in DNA-protein interactions. In agreement with this, antibodies directed towards the N-terminal domain bound to the enzyme without effecting the enzymatic activity. The strong binding but low inhibitory properties of antibodies directed to the polypeptide region between residues 1072 and 1146 confirms previous suggestions that these C-terminal sequences, which share no homology to the Epstein-Barr virus DNA polymerase, are less likely involved in the building of the polymerase catalytic site. Antibodies, raised to the very C terminus of the polymerase (EX3), were successfully used to identify a single 132 +/- 5-kDa polypeptide, which coeluted with the HSV DNA polymerase activity during DEAE-cellulose chromatography, and were further shown to precipitate a major viral polypeptide of identical size. From the presented data it can be concluded that the native enzyme consists of a single polypeptide with a size predicted from the long open reading frame of the HSV-1 DNA polymerase gene.

Quinoneimines as substrates for quinone reductase (NAD(P)H : (Quinone-acceptor)oxidoreductase) and the effect of dicumarol on their cytotoxicity

Several quinoneimines have been shown to be substrates for partly purified rat liver cytosolic quinone reductase with either NADH or NADPH as cofactor. K m and V max values with NADH as cofactor for N-acetyl- p-benzoquinoneimine were 54.9 μM and 278 μmol/min/mg; for 2-amino-1, 4-naphthoquinoneimine, 2.8 μM and 38 μmol/min/mg; for N, N-dimethylindoaniline, 1.7 μM and 22 μmol/min/mg; and 2-acetamido- N, N-dimethylindoaniline, 0.4 μM and 9 μmol/min/mg. All the quinoneimines showed substrate inhibition at high concentrations. At 30 μM dicumarol, an inhibitor of quinone reductase, potentiated the acute toxicity of quinoneimines to cultured phenobarbital-induced rat hepatocytes by 0.7- to 2.9-fold. Dicumarol was toxic to cultured non-induced rat hepatocytes and produced little or no increase in quinoneimine toxicity. Dicumarol potentiated the toxicity of 2-methyl-1, 4-naphthoquinone (menadione) to cultured non-induced, as well as phenobarbital-induced, hepatocytes. Levels of quinone reductase in both types of hepatocytes were similar. Quinoneimines exhibited strong growth inhibitory properties with Chinese hamster ovary (CHO) cells and A204 human rhabdomyosarcoma cells. Dicumarol, 0.1 mM, potentiated growth inhibition by N, N-dimethylindoaniline and 2-acetamido- N, N-dimethylindoaniline in A204 but not in CHO cells. Growth inhibition by 2-amino-1,4-naphthoquinoneimine was inhibited by dicumarol in both cell lines. Dicumarol potentiated growth inhibition by 2-methyl-1,4-naphthoquinone in A204 and CHO cells. Quinone reductase activity in A204 cells was 48% and in CHO cells 1% of the activity in cultured hepatocytes. The lack of a correlation between the effects of dicumarol on quinoneimine and quinone growth inhibition and levels of cellular quinone reductase suggests that dicumarol has effects in cells in addition to, or other than, inhibition of quinone reductase. It is concluded that quinone reductase may protect cells against quinoneimine toxicity under certain conditions, as with phenobarbital-induced hepatocytes, but does not appear to play a major role in modifying quinoneimine toxicity in non-induced hepatocytes, or growth inhibition in CHO cells or A204 cells.

In vitro screen of inhibitors of rat brain serotonin synthesis.

Over 700 compounds were screened at 10−4 M concentration as inhibitors of the conversion of L-tryptophan-14C to serotonin-14C in crude rat brain homogenates. Most of the compounds had little or no inhibitory effect. Those with strong inhibitory properties were tested as inhibitors of 5-hydroxytryptophan decarboxylase and, if active on the decarboxylase, were assayed as tryptophan hydroxylase inhibitors. Except for a few oxidizing and complexing agents and for some substituted p-phenylenediamines, the compounds found to inhibit tryptophan hydroxylase by >50% belonged to the three types of inhibitors already known, i.e. catechols, phenylalanine and ring-substituted phenylalanines, and 6-substituted tryptophans. The numerous data in this screen make possible some comments as to the structural requirements for activity within each class. A comparison of the results on tryptophan hydroxylase with data on tyrosine hydroxylase inhibition in similar homogenates makes it clear that two separate, if somewhat similar, enzymes are involved.

IN VITRO SCREEN OF INHIBITORS OF RAT BRAIN TYROSINE HYDROXYLASE

Over 600 compounds were tested at 10−4 M concentration as inhibitors of tyrosine hydroxylase activity in crude rat-brain homogenate. Most of the compounds had little or no inhibitory effect and those with strong inhibitory properties were, except for a very few oxidizing and complexing agents, all close structural analogues of tyrosine or of its catechol metabolites. Data obtained in this screen are generally in accord with previous data reported in the literature. For high inhibitory activity in the tyrosine series, side-chain substitution is critical. N-Substitution is particularly undesirable. For high inhibitory activity in the catechol series, the ring hydroxy groups should be 3,4 in relation to a C—C—N side chain. Further ring substitution is undesirable, but some side-chain substitution is permissible.