Activities of poloxamer CRL8131 against Mycobacterium tuberculosis in vitro and in vivo.

A poloxamer surfactant, CRL8131, was evaluated for activity against Mycobacterium tuberculosis (Erdman) by itself and in combination with antibiotics in broth culture, in a macrophage cell line assay, and in testing with mice. In the broth culture, CRL8131 suppressed the growth of M. tuberculosis and produced synergistic effects in combination with isoniazid, rifampin, and streptomycin. It also displayed synergy with isoniazid and rifampin against two drug-resistant isolates. In the macrophage cell line assay, CRL8131 produced a synergistic effect on intracellular killing of M. tuberculosis by isoniazid, rifampin, streptomycin, pyrazinamide, thiacetazone, D-cycloserine, ethionamide, amikacin, clindamycin, and p-aminosalicylic acid. It demonstrated no synergy or antagonism with ethambutol, gentamicin, kanamycin, ciprofloxacin, or nalidixic acid. Finally, with C57BL/6 mice infected with M. tuberculosis, a combination of CRL8131 and either thiacetazone or pyrazinamide produced 100% survival at 40 days whereas the antibiotics produced only 33% survival and CRL8131 produced 0% survival when used as single agents. This improved survival rate was associated with a significant reduction in the number of organisms in the lungs and spleens of infected mice.

Design and synthesis of 14 alpha-methyl-15-aza-D-homosterols as novel antimycotics.

A novel series of 14 alpha-methyl-15-aza-D-homosterols 3-7 has been synthesized. These compounds display significant antimycotic activity in vitro (MIC = 0.8-3.1 micrograms/mL) that compares quite favorably to the activity observed for fluconazole (MIC = 0.8 micrograms/mL). Azasterols 3 and 4 were active in vivo as reflected in the increased survival time of Candida albicans infected mice. The antimycotic activity of 3-7 is hypothesized to be a consequence of the inhibition of fungal 14,15-sterol reductase.

Comparative studies of the inhibitory properties of antibiotics on human immunodeficiency virus and avian myeloblastosis virus reverse transcriptases and cellular DNA polymerases.

The inhibition of human immunodeficiency virus (HIV) reverse transcriptase by certain antibiotics and related compounds was studied in comparison with that of avian myeloblastosis virus (AMV) reverse transcriptase and cellular DNA polymerases alpha and beta. In general, compounds that inhibited HIV reverse transcriptase also inhibited AMV reverse transcriptase. For example, 10 micrograms/ml of the isoquinoline quinones used in this study inhibited approximately 80% of the activity of reverse transcriptases of HIV and AMV, but did not inhibit the activity of DNA polymerases alpha and beta even at 50 micrograms/ml. AMV enzyme was more sensitive than HIV enzyme to colistin, enduracidins A and B, janiemycin, glysperin A, and thielavins A and B. The streptonigrin alkyl esters, however, inhibited HIV reverse transcriptase only. Sakyomicin A, luzopeptins, ellagic acid and suramine inhibited the activities of reverse transcriptases and cellular DNA polymerases.

Isolation and characterization of a beta-tubulin gene from Candida albicans.

We report the isolation and nucleotide sequence determination of a beta-tubulin gene (TUB2) from the pathogenic dimorphic fungus Candida albicans. Nucleotide sequence analysis revealed that TUB2 encodes a protein of 449 amino acids (aa) with considerable sequence homology to beta-tubulins isolated from other fungal species. The nucleotide sequence of the C. albicans gene is 70% homologous to that of the Saccharomyces cerevisiae gene. The coding region for the C. albicans beta-tubulin gene is interrupted by two introns. The first intron occurs after the 4th aa and the second intron occurs after the 13th aa. A comparison with other fungal beta-tubulin genes indicates that the intron locations are highly conserved. Codon usage in the C. albicans TUB2 gene is nonrandom, as has been observed for other fungal beta-tubulin genes. The C. albicans TUB2 gene is transcribed to yield a 1.8-kb mRNA species. On the basis of genomic Southern-blot analysis, we conclude that C. albicans most likely possesses a single beta-tubulin gene.

Main properties of duck hepatitis B virus DNA polymerase: comparison with the human and woodchuck hepatitis B virus DNA polymerases.

The main properties of the duck hepatitis B virus (DHBV) DNA polymerase have been studied and compared with those of the human hepatitis B virus (HBV) and of the woodchuck hepatitis virus (WHV) DNA polymerases. All 3 enzymes are active under high salt conditions in the presence of high magnesium concentration. DHBV DNA polymerase was found less sensitive to ethanol and to operate at higher optimal pH than the HBV and WHV DNA polymerases. Like the other two viral endogenous DNA polymerases, the DHBV enzyme was strongly inhibited by phosphonoformic acid but not by aphidicolin, sulfhydryl group blockers or phosphonoacetic acid. Inhibition of DHBV DNA polymerase by the triphosphate derivatives of several nucleoside analogs appeared similar to that reported for HBV or WHV endogenous polymerase. FIACTP was the most, and ACVTP the least effective inhibitor; BVdUTP was of intermediary potency; araCTP and araTTP had a greater inhibitory effect on DHBV DNA polymerase than HBV or WHV DNA polymerase. The similarities in the properties of DHBV and HBV DNA polymerase justify the use of the duck hepatitis B polymerase model for screening and evaluation of potentially active drugs against HBV infection.

Parvodicin, a novel glycopeptide from a new species, Actinomadura parvosata: discovery, taxonomy, activity and structure elucidation.

An extensive taxonomic investigation identified strain SK&F-AAJ-271 as a new species, designated Actinomadura parvosata. Fermentations of this organism produce a complex of acidic, lipophilic glycopeptide antibiotics, the parvodicins. Structures for seven of the isolated components were derived from a combination of mass spectral, high-field NMR and chemical techniques. The O-acetyl functionality present in two of the isolated components is a structural feature unique among the known members of this class of antibiotics. The parvodicins are active in vitro against a range of Gram-positive bacteria. The most active parvodicin, C1, produces high serum levels in vivo and has the potential for a long duration of action.

In vitro antiviral activity of dammar resin triterpenoids.

Nine triterpenes with antiviral activity against Herpes simplex virus types I and II in vitro were isolated from dammar resin. Each compound caused a significant reduction in viral cytopathic effect when Vero cells were exposed continuously to 1-10 micrograms/ml of compound for 48 h after viral challenge. The triterpenes were identified as dammaradienol [1], dammarenediol-II [2], hydroxydammarenone-I [3], ursonic acid [5], hydroxyhopanone [11], dammarenolic acid [15], shoreic acid [16], eichlerianic acid [17], and a novel compound, hydroxyoleanonic lactone [7], on the basis of their chromatographic, spectroscopic, and physical properties.

Effects of coordinated gold compounds on in vitro and in situ DNA replication.

Auranofin, a coordinated gold compound, inhibits in vitro DNA synthesis and displays in vivo antitumor activity. To understand the mechanisms of inhibition of DNA replication, we have examined the effects of auranofin and other gold complexes on the activities of purified cellular and herpesvirus-induced DNA polymerases, and on in situ DNA replication in permeabilized S phase KB cells. Evaluation of the data suggests the following conclusions. (1) The gold compounds varied in their abilities to inhibit DNA polymerase activities. DNA polymerase alpha was more sensitive to inhibition by gold compounds than DNA polymerase beta; (2) Inhibition of purified DNA polymerases by gold (I) compounds was noncompetitive with both DNA template and triphosphate substrates. Inhibition by SKF 101675, a gold (III) complex was competitive with DNA. (3) None of the gold compounds tested preferentially inhibited herpesvirus-induced DNA polymerases. (4) The gold complexes that inhibited in vitro DNA replication also inhibited in situ DNA synthesis. However, the potency and order of potency of the compounds varied between the in vitro and in situ systems. (5) Auranofin and other gold compounds inhibited the clonogenic capacity of KB cells in a concentration-dependent manner. The IC50 values measured in the clonogenic assay were significantly lower than those obtained from the in vitro and in situ DNA replication assays.

Distinctive properties of DNA polymerases induced by herpes simplex virus type-1 and Epstein-Barr virus.

The properties of DNA polymerases induced by two human herpesviruses, herpes simplex virus type-1 (HSV-1) and Epstein-Barr virus (EBV), have been compared. The HSV-1 and EBV polymerases can be distinguished from one another by differences in the elution profiles in phosphocellulose and single-stranded DNA cellulose columns. Although both enzymes require monovalent cations for optimum activity, the HSV-1 enzyme requires ammonium sulfate whereas the EBV enzyme activity is inhibited by it; on the other hand, the EBV polymerase requires KCl. Other reaction requirements are also different for the two viral enzymes. Thus, when the EBV DNA polymerase was assayed under conditions optimum for the HSV-1 DNA polymerase, only 15% of its activity was expressed. Differences were also noted in sensitivities of the two viral enzymes to the 5'-triphosphates of nucleoside analogs with antiherpesvirus activity such as BVdU, IVdU, ACV, FIAC and IdUrd. The HSV-1 polymerase was more sensitive than the EBV DNA polymerase to inhibition by phosphonoacetate, phosphonoformate, aphidicolin and N-ethylmaleimide. However, the EBV DNA polymerase was more sensitive than HSV-1 DNA polymerase to heat treatment at 42 degrees C. Thus, the marked differences between the two viral enzymes can be useful in identifying enzyme activities in cells producing the virus and also in studying the biochemical mechanism of action of some of the antiviral agents.

Inhibition of human and woodchuck hepatitis virus DNA polymerase by the triphosphates of acyclovir, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine and E-5-(2-bromovinyl)-2'-deoxyuridine.

The triphosphates of acyclovir (ACV), 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC) and E-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) have been examined for their inhibitory effects on the endogenous DNA polymerase reactions of human hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV). All three triphosphates (ACVTP, FIACTP and BVdUTP) inhibited the HBV and WHV DNA polymerases by competing with the corresponding natural substrates. FIACTP was the most potent inhibitor of HBV and WHV DNA polymerase while ACVTP was the least effective inhibitor. The inhibitory properties of these compounds were compared with those of the 5'-triphosphates of 1-beta-arabinofuranosyl-cytosine (ara-CTP) and 1-beta-arabinofuranosylthymine (ara-TTP). The 50% inhibitory doses for HBV and WHV DNA polymerases were in the following order: FIACTP less than BVdUTP less than ara-TTP less than ACVTP less than ara-CTP. BVdUTP appeared to be an efficient alternate substrate to dTTP for HBV DNA polymerase while FIACTP was much less efficient when substituted for dCTP. ACVTP did not act as an alternate substrate to dGTP and appeared to prevent DNA chain elongation.

Effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine on several parameters of Epstein-Barr virus infection.

The selective and potent anti-herpesvirus drug, (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU), has been examined for its inhibitory effects on several parameters of Epstein-Barr virus (EBV) infection in the lymphoblastoid cell lines Raji, P3HR-1, B-95-8 and P3 hybrid cells (a human embryo oropharyngeal cell line fused with a nasopharyngeal carcinoma cell line). At a dosage of 0.03 to 0.1 mM, BVdU caused a marked inhibition of (i) spontaneous viral capsid antigen (VCA) expression in B-95-8 and P3 hybrid cells, (ii) VCA expression and DNA synthesis in B-95-8 cells induced with croton oil and n-butyrate, (iii) early antigen (EA) expression and DNA synthesis in Raji cells superinfected with EBV, and (iv) VCA expression and DNA synthesis in B-95-8 cells superinfected with EBV. In its inhibitory effects on these various parameters of EBV infection, BVdU appears to be comparable to acyclovir [9-(2-hydroxyethoxymethyl)guanine], another selective anti-herpesvirus drug which has been previously recognized as an effective inhibitor of EBV replication.

Antiviral activity of the 3'-amino derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine.

3'-NH2-BV-dUrd, the 3'-amino derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine, was found to be a potent and selective inhibitor of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) replication. 3'-NH2-BV-dUrd was about 4-12 times less potent but equally selective in its anti-herpes activity as BV-dUrd. Akin to BV-dUrd, 3'-NH2-BV-dUrd was much less inhibitory to herpes simplex virus type 2 than type 1. It was totally inactive against a thymidine kinase-deficient mutant of HSV-1. The 5'-triphosphate of 3'-NH2-BV-dUrd (3'-NH2-BV-dUTP) was evaluated for its inhibitory effects on purified herpes viral and cellular DNA polymerases. Among the DNA polymerases tested, HSV-1 DNA polymerase and DNA polymerase alpha were the most sensitive to inhibition by 3'-NH2-BV-dUTP (Ki values 0.13 and 0.10 microM, respectively). The Km/Ki ratio for DNA polymerase alpha was 47, as compared with 4.6 for HSV-1 DNA polymerase. Thus, the selectivity of 3'-NH2-BV-dUrd as an anti-herpes agent cannot be ascribed to a discriminative effect of its 5'-triphosphate at the DNA polymerase level. This selectivity most probably resides at the thymidine kinase level. 3'-NH2-BV-dUrd would be phosphorylated preferentially by the HSV-1-induced thymidine kinase (Ki 1.9 microM, as compared with greater than 200 microM for the cellular thymidine kinase), and this preferential phosphorylation would confine the further action of the compound to the virus-infected cell.

Mode of action of acyclovir triphosphate on herpesviral and cellular DNA polymerases.

The effect of 5'-triphosphate of acyclovir (ACV) on DNA polymerases of two human herpes-viruses, herpes simplex virus type-1 (HSV-1) and Epstein-Barr virus (EBV) as well as human cellular DNA polymerases alpha and beta has been examined. Of the enzymes tested, HSV-1 DNA polymerase was the most sensitive to inhibition by acyclovir triphosphate (ACVTP). The EBV DNA polymerase and DNA polymerase beta were less sensitive. ACVTP inhibition was competitive with dGTP with Ki values of 0.03, 0.15, 9.8 and 11.9 microM for HSV-1 DNA polymerase, DNA polymerase alpha, EBV DNA polymerase and DNA polymerase beta, respectively. Substituting a synthetic primer template (dG) approximately 15 x (dC)n for activated DNA template did not alter the pattern of inhibition. In a time course experiment, addition of ACVTP instead of dGTP did not increase DNA synthesis and it appeared to act as a chain terminator in DNA replication catalyzed by either HSV-1 DNA polymerase or DNA polymerase alpha. Although EBV DNA polymerase was less sensitive to ACVTP inhibition, the nucleoside analog itself was inhibitory to EB virus production by P3HR1 cell line as determined by a reduction in the percentage of cells expressing virus capsid antigen (VCA). On day 4, ACV at 10 and 25 micrograms/ml reduced the cell growth by 10% and 32%, respectively, while it reduced the VCA-positive cells by 80% and 84%, respectively. These results indicate that inhibition of EBV DNA polymerase activity by ACVTP may not be the primary mechanism responsible for ACV inhibition of EBV replication.

Inhibitory effect of E-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil on herpes simplex virus replication and DNA synthesis.

The effect of E-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU) on herpes simplex virus (HSV) replication was examined and compared with that of E-5-(2-bromovinyl)-2'-deoxyuridine (BVdUrd). The 50% inhibitory dose against HSV type 1 (HSV-1) was 0.1 microgram/ml compared with 0.008 microgram/ml for BVdUrd; the antimetabolic 50% inhibitory dose of BVaraU ranged from 20 to 95 micrograms/ml. The addition of 50 micrograms of BVaraU per ml to HSV-1-infected Vero cells decreased the synthesis of viral and cellular DNA by 37 and 28%, respectively. The 5'-triphosphate (BVaraUTP) competed with dTTP in DNA synthesis by the herpes-viral and cellular DNA polymerases; the apparent Ki values of HSV-1 DNA polymerase, DNA polymerase alpha, and DNA polymerase beta were 0.14, 0.32, and 5 microM, respectively. Thus, BVaraU was a less effective antiherpesvirus agent than BVdUrd; unlike BVdUrd, it did not appear to be internally incorporated into replicating DNA in virus-infected cells.

Cellular and Epstein-Barr virus specific DNA polymerases in virus-producing Burkitt's lymphoma cell lines.

We have determined the levels of cellular DNA polymerases and Epstein-Barr virus specific DNA polymerase in three Burkitt's lymphoma cell lines producing varying amounts of EBV, one of which was induced by 12-0-tetra-decanoylphorbol-13-acetate (TPA). There was a proportional increase in the level of EBV-DNA polymerase with an increase in the percent of virus-producing cells. However, there was a reciprocal relationship between the levels of EBV-DNA polymerase and DNA polymerase alpha i.e., in cell line containing the highest level of EBV-DNA polymerase, activity of DNA polymerase alpha, but not of DNA polymerase beta, was reduced to an insignificantly low level. TPA does not have any direct effect on activities of either EBV-DNA polymerase or DNA polymerase alpha. EBV-DNA polymerases isolated from cells grown with or without TPA are indistinguishable in their properties such as elution position on phosphocellulose column, molecular weight, mono and divalent cation requirements, pH optimum, and other requirements for optimum activity. Addition of crude extracts of cells grown in presence of TPA to the purified DNA polymerase alpha did not inhibit its activity indicating that the observed loss was not due to any specific inhibitor present in TPA treated cells. Raji, a nonproducer cell line, did not contain EBV-DNA polymerase. There was no induction of EBV-DNA polymerase when Raji cells were grown in presence of TPA. The phenomenon of reduction in the levels of DNA polymerase alpha in cells induced to produce EBV may represent a mechanism by which the host DNA replication is shut off following virus infection.

Comparative effects of the 5'-triphosphates of 9-beta-(2'-azido-2'-deoxy-D-arabinofuranosyl)adenine and 9-beta-D-arabinofuranosyladenine on DNA polymerases from L1210 leukemia cells.

9-beta-(2'-Azido-2'-deoxy-D-arabiofuranosyl)adenine (arazide) causes greater and significantly more persistent inhibition of [3H]-thymidine incorporation into the DNA of neoplastic cells than the related agent 9-beta-D-arabinofuranosyladenine (araA). To elucidate the mechanism(s) responsible, we compared the effects of arazide and araA 5'-triphosphates on DNA polymerases alpha and beta of L1210 leukemia cells. Both nucleoside triphosphate analogs inhibited DNA polymerase alpha activity by competing with dATP; only araATP was inhibitory to DNA polymerase beta. Arazide triphosphate was at least four times more active than araATP as an inhibitor of DNA polymerase alpha. Preincubation of DNA polymerase alpha with either agent did not result in enzyme inactivation. The results suggest that interference with DNA polymerase alpha activity by arazide triphosphate may be in part responsible for the inhibition of DNA synthesis produced by arazide in neoplastic cells.