Effect of Rifampicin and Two of Its Derivatives on Cells Infected With Moloney Sarcoma Virus

Derivatives of rifamycin-SV with substituted cyclic-amine side chains in position 3 of the ansa ring are strong inhibitors of RNA-directed DNA and DNA-directed DNA polymerase activity of RNA tumor viruses of murine, feline, and avian origin. Among 37 3-amine derivatives of rifamycin-SV that were tested, 29 3-cyclic amine derivatives were good inhibitors of the viral polymerases. Especially active were 3-piperidyl derivatives of rifamycin-SV with cyclohexyl and cyclohexylalkyl substituents. Derivatives that were effective against the viral polymerase also blocked cell transformation by the murine sarcoma virus. A DNA-directed DNA polymerase preparation from human KB cells was less sensitive to inhibition by these derivatives than the virion polymerase.Can chemotherapy of viral diseases and cancer be based on the control of replication of specific classes of nucleic acid molecules? The search for specific inhibitors of polymerase has an excellent start, for the antibiotic rifamycin-SV and several of its semisynthetic derivatives specifically bind to and block the DNA-directed RNA polymerase of bacteria. Although these derivatives do not inhibit mammalian polymerases (1), we found that lengthening the rifampicin 3-iminomethyl4-methyl-1-piperazine side chain by substitution of benzyl for methyl yields compounds ( Fig. 1, AF/ABDMP and AB/ABP) that inhibit the RNA-directed DNA polymerase (reverse transcriptase) of RNA tumor viruses; removal of the methyl group yields N-demethyl rifampicin, a weak inhibitor (2-4).To see if even more extensive inhibition is possible, we have explored further the structural requirements for antipolymerase activity. We tested the ability of rifamycin-SV derivatives that contain 3-amine, quinoxalino, and benzoxazino substituents (see Fig. 1) to inhibit RNA-directed DNA and DNA-directed DNA polymerase. For investigation of DNA-directed DNA polymerase activity, the partially purified enzyme of the Harvey strain of murine sarcoma virus (MSV strain H) was tested with poly d(A-T) as a template. For RNA-directed DNA polymerase activity, the endogenous activity of the Moloney (M) strain of MSV was used. These polymerase activities of RNA tumor viruses are especially suitable for testing, since their true function during oncogenesis is probably DNA synthesis (2-4), and, therefore, they are a natural target for possible chemotherapy. MATERUILS AND METHODSViruses. Strains H and M of MSV, and FeLV were propagated in cultured cells and purified (2, 5, 6). AMV, kindly provided by Dr. Joseph Beard, was purified as described (7).Rifamycin Derivatives. The rifamycin-SV derivatives used in this study were generously provided by Drs

Section: -Amine Derivatives Of Rifamycin-sv Inhibit Cell Transformatmentioning

confidence: 99%

3-Cyclic Amine Derivatives of Rifamycin: Strong Inhibitors of the DNA Polymerase Activity of RNA Tumor Viruses

Green

Bragdon

Rankin

1972

“…Two rifampicin derivatives, dimethylbenzylrifampicin (DMB) and rifazacyclo-16, inhibit viral reverse transcriptase (7) and at comparable concentrations also inhibit murine sarcoma virus (MSV) transformation of mouse cells in tissue culture (10)(11)(12)(13)(14). This correlation between biological and biochemical data suggests that these drugs block transformation by inhibiting the viral induced reverse transcriptase; alternatively the drugs may inhibit other cellular enzymes required for transformation.…”

Section: Introductionmentioning

confidence: 90%

“…Previous reports (10,13,17) showed that DMB inhibited murine leukemia virus transformation of the mouse cell line UC1-B without the addition of amphotericin B, although it was subsequently shown that the inhibition was more reproducible and more complete when the amphotericin B was included (11). It was hypothesized that amphotericin B acted by altering cell membrane permeability, thus enabling the DMB to be taken up more readily by the cells.…”

mentioning

confidence: 99%

The Specificity of Dimethylbenzylrifampicin as an Inhibitor of Viral Induced Transformation

Smith¹,

Hackett²

1974

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The effect of 2',6'-dimethyl-N(4')-benzyl- Certain derivatives of rifampicin (1-7) actively inhibit oncornavirus RNA-dependent DNA polymerase (reverse transcriptase) (8,9). Two rifampicin derivatives, dimethylbenzylrifampicin (DMB) and rifazacyclo-16, inhibit viral reverse transcriptase (7) and at comparable concentrations also inhibit murine sarcoma virus (MSV) transformation of mouse cells in tissue culture (10)(11)(12)(13)(14). This correlation between biological and biochemical data suggests that these drugs block transformation by inhibiting the viral induced reverse transcriptase; alternatively the drugs may inhibit other cellular enzymes required for transformation. To further explore the latter possibility, we have compared the effect of DMB on transformation by MSV with its effect on transformation induced by simian virus 40 (SV40), a small DNA-containing virus which does not have reverse transcriptase activity. We found that SV40-induced transformation was completely normal under conditions where MSV transformation of the same cells was greatly inhibited. MATERIALS AND METHODSCell Culturing. Balb/3T3 clone A31 cells (obtained from Dr. G. Todaro) were used as the parental cell stock. The cells were grown in Dulbecco's modification of Eagle's medium (Gibco no. H21) with 10% calf serum (Colorado Serum Co.) (complete medium). To obtain a drug resistant variant, 100 Balb/3T3 cells were plated into a 60-mm dish with complete medium containing 3jug/ml of DMB and allowed to grow for 3 weeks with biweekly fluid changes. The cells were passaged one time to a Falcon flask containing complete medium plus 3 jug/ml of DMB and then passaged 4 times in the same medium containing 20;zg/ml of DMB. The cells that survived this treatment grew well in DMB and were considered resistant variants. SV40 Virus. SV40 (small plaque variant) was prepared by innoculating Vero cells in Dulbecco's medium plus 2% fetalcalf serum with 1 X 10-4 plaque-forming units per cell of SV40 and harvesting the cell supernatant 2 weeks later at the time of maximum cytopathic effect. Viral stocks were titered on TC-7 monkey cells by means of a fluorescent antibody assay. Three days after infection with various dilutions of virus, the cells (grown on coverslips) were fixed and stained for V antigen (monkey anti-V serum was kindly supplied by Dr. H. Ozer. The fluorescein-conjugated antibody to monkey globulin was obtained from Sycco Co.). SV40 Transformation. For the transformation assay, 60-mm tissue culture dishes of confluent resistant cells grown in complete medium plus DMB, 3,4g/ml, were infected by removing the medium, adding 0.5 ml of either SV40 or mockinfected cell supernatants and incubating at 370 for 3 hr with periodic shaking. The virus or mock virus was removed and fresh complete medium added for 3 hr. Cells were treated with trypsin and plated at various dilutions; cells were allowed to become confluent or colonies were obtained as described (15). In brief, the petri dishes were fixed and stained with hematoxylin (Harris-Lillie...

“…One of the rifampicin derivatives, 2',6'-dimethyl-N(4')-benzyl-N(4') [desmethyl]rifampicin, inhibited focus formation and infectious virus production in BALB/3T3 cells by Moloney Sarcoma Virus (1,2). It also inhibited Moloney leukemia virus-induced focus formation in the UC1-B cell line derived from BALB/3T3 cells (3a, 3b).…”

mentioning

confidence: 95%

Synergistic Effect of Rifamycin Derivatives and Amphotericin B on Viral Transformation of a Murine Cell Line

Hackett

Sylvester

Joss

et al. 1972

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