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3'-Amino-3'-deoxythymidine decreased the incorporation of [2-14C]thymidine into DNA of L1210 cells in vitro, and produced an accumulation of [2-14C]thymidine di- and triphosphate. The extent of these effects varied with the amount of recovery time after removal of 3'-amino-3'-deoxythymidine prior to addition of labeled thymidine. The distribution of radioactivity in the acid-soluble fraction derived from [3H]3'-amino-3'-deoxythymidine was as follows: 50% as 3'-amino-3'-deoxythymidine, 20% as the monophosphate, 10% as the diphosphate, and 20% as the triphosphate derivatives. No incorporation of [3H]3'-amino-3'-deoxythymidine into L1210 DNA could be detected. 3'-Amino-3'-deoxythymidine-5'-triphosphate is a competitive inhibitor against dTTP with a Ki of 3.3 microM, whereas the Km for dTTP was 8 microM using activated calf thymus DNA as the template and DNA polymerase-alpha. These data indicate that a major site of inhibition by 3'-amino-3'-deoxythymidine is inhibition of the DNA polymerase reaction.

Evidence was obtained which indicates that the lethal effect of 5-iodouracil (IUra) on bacteriophage T4 is not due to a mutagenic process. T4td8rII (thymine requiring, rapid lysis) double mutants were constructed. Reversion of T4td8rII to r(+) was measured. First, reversion by growth in the presence of the structural analogues chlorouracil (ClUra) and bromouracil (BrUra) did not correlate with their relative lethal effects (for mutagenesis: IUra

3'-Deoxythymidin-2'-ene (d4T) is a potent and selective inhibitor of human immunodeficiency virus replication in a variety of human cell types and is currently undergoing phase I clinical trials for the treatment of acquired immunodeficiency syndrome. As part of our ongoing studies of the cellular pharmacology of d4T, and in light of recent reports in which such nucleoside analogs as 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyadenosine were shown to permeate cells by the unusual mechanism of nonfacilitated diffusion, we have investigated the uptake of d4T in the human lymphocyte cell line H9. Several lines of evidence suggest that d4T permeation of H9 cells occurs by nonfacilitated diffusion; 1) [3H]d4T influx was linear for the first 10 sec and was nonconcentrative, reaching equilibrium with the extracellular drug concentration in 2-3 min, 2) the initial rates of influx were a linear function of concentration over the range from 1 microM to 5 mM, with no sign of uptake by a saturable mechanism, and 3) the uptake of [3H]d4T was insensitive to the nucleoside transport inhibitors nitrobenzylthioinosine and dipyridamole, as well as a large molar excess of AZT, thymidine, or adenosine. The octanol/water partition coefficient of d4T was 0.179, intermediate between those of thymidine and AZT. Thus, d4T does not appear to be a substrate for the nucleoside transport system responsible for the uptake of physiological nucleosides as well as most nucleoside analogs, and it enters the cell by nonfacilitated diffusion.

Radioactive 5'-amino-5'-deoxythymidine (5'-AdThd), an inhibitor of herpes simplex virus, was synthesized by direct amination of monotosylated thymidine with concentrated ammonium hydroxide. Incubation of 5'-AdThd with purified herpes simplex virus type 1-encoded pyrimidine deoxyribonucleoside kinase produced the diphosphate derivative. The monphosphate derivative of 5'-AdThd was not detected as a reaction product of this enzymatic phosphorylation. A purified mixture of nonviral thymidine kinase and thymidylate kinase derived from uninfected Vero cells was unable to phosphorylate 5'-AdThd under identical conditions. The rate of hydrolysis of 5'-AdThd diphosphate increased as the pH of the reaction mixture decreased, with a shoulder region appearing between pH 3 and 5. The rate of hydrolysis was markedly increased below pH 3. 5'-AdThd, but not the monophosphate derivative, was detected in the hydrolysis mixture. The hydrolysis of 5'-AdThd diphosphate pH 3 also yielded some thymine in addition to 5'-AdThd.

Pyrimidine deoxyribonucleoside kinase (thymidine kinase [TK]) was purified from two herpes simplex virus type 1 (HVS-1)-transformed TK-deficient mouse (LMTK-) cell lines and from LMTK- cells infected with HSV-1 mutant viruses coding for variant TK enzymes. These preparations exhibited normal or variant virus-induced thymidylate kinase activities correlating with their relative TK activities. Neither virus-induced activity was detected in LMTK- cells infected with an HSV-1 TK-deficient mutant. These results suggest that HSV-1 thymidylate kinase activity and TK activity are mediated by the same protein.

The antiherpesvirus agent (E)-5-(2-bromovinyl)-2'-deoxyuridine caused marked alterations in the synthesis and processing of several herpes simplex virus type 1 (HSV-1)-infected-cell polypeptides. Analogous to other thymidine analogs, there was a dose-dependent decrease in several beta and gamma polypeptides and an accumulation of HSV-1 thymidine kinase. In contrast to the action of other thymidine analogs, there were alterations in alpha polypeptides, including an increase in the synthesis and phosphorylation of infected-cell polypeptide 4b and a decrease in the synthesis of infected-cell polypeptide 27. The phosphorylation of several other HSV-1 phosphoproteins was mildly inhibited. (E)-5-(2-Bromovinyl)-2'-deoxyuridine inhibited the glycosylation of the major HSV-1 glycoproteins, and this activity appeared to be independent of the incorporation of the drug into the viral DNA. Thus, the alterations in HSV-1 polypeptide expression appear to be due to the presence of the drug in a low-molecular-weight form as well as its presence in the viral DNA. This suggests that this analog or a phosphorylated derivative might act as an inhibitor of an enzyme(s) responsible for posttranslational modification of polypeptides.

Vero cells treated for 24 h with a concentration of 2-deoxy-D-glucose (2dGlc) that inhibited the production of infectious herpes simplex virus type 1 grew at the same rate as untreated cells. Longer exposures to 2dGlc inhibited the growth of Vero cells in a dose-dependent manner, but without any loss of viability, and could be reversed by replating the cells in the absence of drug. To exhibit antiviral activity, 2dGlc had to be present during the replication cycle of herpes simplex virus type 1. Treatment of Vero cells, even with a cytotoxic dose of 2dGlc, was without effect on the yield of infectious virus, provided the drug was removed before infection. Thus the antiviral effects of 2dGlc were not the result of, and appeared to be independent of, persistent host cell toxicity.

Methyl daunosamine inhibited the replication of herpes simplex virus type 1 in a dose-dependent manner. The growth of the host Vero cells was not affected by daunosamine levels that had significant antiviral activity (2.5 mM) but was inhibited by concentrations of 5 mM or greater. Methyl daunosamine appears to be unique among the sugars with antiviral activity because at antiviral concentrations it did not inhibit the glycosylation of macromolecules.

E-5-(2-Bromovinyl)-2'-deoxyuridine (BrvdUrd) produced a dose-dependent shift in the density of herpes simplex virus type 1 (HSV-1) DNA at concentrations which yielded potent inhibition of virus replication in cultured Vero cells. Although the density of cellular DNA was not altered by these concentrations of BrvdUrd, incorporation of this analogue into cellular DNA of HSV-1-infected cells has been previously observed in this laboratory. The degree of inhibition correlated with the amount of BrvdUrd substituted for thymidine in HSV-1 DNA. BrvdUrd-substituted DNA was more labile as determined by a dose-dependent increase in single strand breaks when examined by centrifugation in alkaline sucrose gradients. Thus, the potent antiviral action of BrvdUrd observed in cell culture correlates not only with its incorporation into HSV-1 DNA but also with an altered stability of this DNA.

-Compound 1, the carbocyclic analogue of Ara-T, was synthesized by a four-step synthesis from (+)-(lα,2α,3β,5β)-3-amino-5-(hydroxymethyl)-l,2,-cyclopemane-diol (2), which was prepared from cyclopentadiene via a eight-step route as a potential antiviral agent.

Fractionation of desiccated defatted hog stomach through the use of isoelectric and ammonium sulfate precipitation technics, and guided by assays in patients with pernicious anemia in relapse given daily oral doses of 5 µg. of vitamin B12, has led to the concentration of intrinsic factor activity in an amount of protein equivalent to 0.03 per cent of the original weight of crude material. The electrophoretic pattern of this active fraction indicated a lack of homogeneity. The vitamin B12-binding activities of three fractions obtained by precipitation with ammonium sulfate were investigated by microbial inhibition and dialysis methods. There was no correlation between the ability of the fractions to bind vitamin B12 and to exert intrinsic factor activity. Among these fractions, that with the highest intrinsic factor activity in patients with pernicious anemia in relapse bound the least amount of vitamin B12.