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Inhibitors of RNA and DNA biosynthesis
Pergamon Press
Lifa Sciaacea Vol . 15, pp " 359-369 Printed in the U .S .A .
1~IINIREVIESQ
INHIBITORS OF HNA AND DNA BIOSYNTSSSIS Shen-Ching Sung Division of Neurological Sciences, Department of Psychiatry University of British Columbia Vancouver V6T-1W5, Canada .
During the lest fifteen years our knowledge of the bioeyathesis of nucleic acids has ezpaaded greatly, due largely to the discovery and use of various inhibitors of RNA and DNA synthesis .
Among these inhibitors (1)
some are known to act by binding to template or primer DNA whereas others exert their effects directly on the enzyme polyieraee .
Some inhibitors of
RNA synthesis affect the initiation step while others block the elongation (polymerization) of the RNA chain.
Meay chemotherapeutic compounds alter
the structure and function of DNA by interaction, binding or intercalation (2) .
A wide variety of purine and pyrimidine analogs, D-arabinosyl
nucleosides, alkylating agents and several anticancer drugs have been used as inhibitors of RNA and/or DNA synthesis .
A large number of antibiotics are
inhibitors of RNA and/or DNA synthesis (3-5) .
Sore of these antibiotics
have been shown to inhibit DNA-dependent RNA polymerase by binding specifically either to DNA, to the enzyme or to one of the enzyme subunits, but no antibiotic has yet been shown to inhibit specifically the activlty of DNA-dependent DüA poly~raee.
Actinamycin, the moat thoroughly studied and comronly used antibiotic, hoe various biological effects, but its principal action ie the inhibition of
359
362
Inhibitors of RNA and DNA Biosynthesis
Vol . 15, No . 3
polymerase by a-amanitin is dependent on the amount of enzyme present but is independent of the DNA concentration, and is not competitive with the RNA precursor nucleoside triphosphates .
This substance inhibits chain elongation
and does not exert a specific effect on the initiation .
In contrast to
rifamycin, which inhibits RNA polymeraee from E . coli but not that from animal tissue, a-amanitin does not affect the former enzyme .
Eukaryotic cells possess multiple forma of DNA-dependent RNA polymerasea . RNA polymerase I is localized in the nucleolus, whereas polymerase II is found in the nucleoplasm.
The former enzyme ie largely involved in the
nucleolur synthesis of ribosomal RNA while the latter synthesizes the bulk of the nucleoplasmic RNA species (13) .
Polymerase III is also believed to be
present in the nucleoplasm but its role in cell function has not been clarified.
a-Amanitin selectively inhibits the activity of DNA-dependent RNA
polymerase II, but does not affect polymerases I and III (12,14) .
a-Amanitin
is the only example of a selective inhibitor of a RNA polymerase from eukaryotic cells and thus has become a very useful tool for the study of the poorly understood transcription mechanism in higher organisms .
Inhibitors of RNA synthesis can be used in the isolation of mutants of eukaryotic cells that contain different forms of DNA-dependent RNA polymerase . For example, mutants of Chinese hamster ovary cells that are resistant~to a-amanitin have been isolated (15) .
Some of these mutants contain an altered
form of DNA-dependent RNA polymerase that is resistant to inhibition by a-amanitin .
Such mutants may prove useful for studying the mechanism of action
of this eukaryotic enzyme .
Rifampicia is another antibiotic which inhibits bacterial enzyme not the DNA-dependent RNA polymerases from mammalian nuclei .
but
Several semi-
Vol. 15, No . 3
36 3
inhibitors of RNA and DNA Biosynthesis
synthetic derivatives of rifaapicin possessing highly hydrophobic sidechains have been made, however, which dô inhibit the mammalian enzyme (16) apparently at the initiation rather than the elongation step .
These
hydrophobic derivatives block the binding of DNA by the mammalian enzyme, whereas rifamycin does not block DNA binding by S. cola RNA polymerase .
Several anticancer drugs are known to act at the sequential or concurrent steps involved in DNA synthesis, e.g ., the antitumr activity of FIIdR appears to result frog the inhibition of thynidylate synthesis.
Cytosine
arabinoeide (era-C) is believed to inhibit DNA synthesis through the action of its 5~-triphoaphate, ~ra-CTP, on the polys~erization of deozyribonucleotides (17,18) .
liany antibiotics are able to inhibit in vivo and in vitro
synthesis of DNA in warioue organisms (3-4), however, none of the aatibiotica studied to date appears to be a specific inhibitor of DNA synthesis .
Hydrozyurea interferes with DNA eynthesis in bacteria ae well ae in ns®alian cells but does not affect the activity of thymidine kinase or DNA-dependent DNA polymerise .
It has also been reported that hydrozyurea
inhibits a highly purified form of ribonucleoside diphosphate reductase fros B. coli (19) .
Although the bode of action of this substa~e on DNA synthesis
does appear to be connected with the conversion of ribonucleotides to deoryribonucleotides, evidence has been presented to indicate that nucleotide reductase is not the enzyme affected by hydrozyurea (20,21) .
Mito~cin, which ie known to cross-link to the DtiA molecule (22), has been shown to inhibit DNA synthesis in S . coli with little !mediate effect on RNA or protein synthesis (23) .
Mi.tomycin causes degradation (depolyeerization)
of DNA both in growing and resting cells of 8. coli and in mammalian cells in culture (24) .
The depolyoerizstion of DäA that occurs after adai.aistratian
36 4
Vol . 15, No . 3
Inhibitors of RNA and DNA Biosynthesis
of mitomycin C may be due to an activation of a latent cellular DNase (25) . Since it is difficult to measure the inhibition of DNA synthesis in the presence of DNA depolymerization, the use of mitomycin as a selective inhibitor of DNA synthesis moat by approached with caution.
Various
investigators have suggested that the mitomycin-induced breakdown of DNA may be a secondary phenomenon whereas other evidence indicates that the DNA breakdown may be the primary event and the inhibition of DNA synthesis may be a consequence of the depolymerization (26) .
Sarkomycin, an antitumor antibiotic, at certain concentrations inhibits DNA synthesis in Ehrlich ascites carcinoma cells but has little or no effect on RNA and protein synthesis .
This inhibition of DNA synthesis may be
prevented by reduced glutathione (27) .
Sarkaomycin also inhibits the
partially purified DNA polymerase from Landschultz ascites tumor cells (28) .
Multiple forma of DNA-dependent DNA polymerases are now known to exist in bacteria as well as in mammalian cells.
Specific inhibitors of the various
types of DNA-dependent DNA polymerase would certainly be eatremely valuable tools for elucidating the mechanism of DNA replication.
It is to be hoped
that their discovery is imminent .
RNA-directed DNA polymerase (reverse transcriptase) has been found in several oncogenic RNA viruses as well as in leukemic cells and tumors (29-33) .
Specific inhibitors of this enzyme would be useful for studying its
possible role in neoplasia and might become important chemotherapeutic agents for the treatment of human leukemia, cancer and viral diseases . such agents are known .
So far few
Rifampicin itself has no effect on the RNA-directed
DNA polymerase activity of RNA viruses, but removal of the 4-Nrmethyl group on the aminopiperazine side chain or substitution of a benzyl group at this
Vol. 15, No . 3
Inhibitors of IAA and DNA Biosynthesis
36 5
site converts rifampicin to derivatives which are effective inhibitors of this enzyme .
Thus 2,5-dimethyl-4-N-benayldemethylrifampicin (AF/ABDMP),
4-N-benzyldemethylrifanpicin (AF/ABP) and N-demethylrifampicin inhibit the RNA-directed DNA polymerase activity of several RNA tumor viruses, including narine sarcoma virus (MSV),
feline leukemia virus (FeLV) and avian
myeloblastosis virus (AMV) (34) .
Moreover, Green et al . (35) have
demonstrated that of the 37 rifamycin-SV derivatives possessing 3-amine subatituents studied, 29 are good inhibitors of both the RNA-directed and DNA-directed polymerase activities of MSV, FeLV and AMV, and that the 3-piperidyl derivatives of rifarycin-SV possessing cyclohezyl and cyclohezylalkyl subetituents are especially effective .
Green et al . also found that
the DNA-directed DNA polymerase preparation from human RB cells vas leas sensitive to inhibition by these derivatives than the viral polymerase ; 5-10 times higher concentrations were required for the same percent inhibition of the DNA polymerase of RB cells than with viral polynerase .
Caution ie
indicatied in the use of the rifampicin derivatives as a diagnostic teat for the presence of reverse tranecriptase because these drugs are relatively uaetable in solution and also lack complete specificity, although they are more effective inhibitors of RNA-directed DNA polymerase of RNA tumor viruses than of DNA-dependent DLiA polyoerase from mammalian cells or from E. cola .
The streptovaricine codes, a known inhibitor of bacterial but not of mammalian DNA-dependent RNA polymerase, is another agent with an eztremely potent inhibitory effect on the RNA-dependent DdiA polymerase activity associated with oncogenic RNA viruses (36) .
An alkaloid extract of a
medicinal plant Sacred Lily (Narcissus tarzetta L .) has been shown to inhibit the purified DNA polymerase from avian myeloblastosis virus, possibly by binding to the polymerase enzyme rather than to the RNA template (37) . It appears to possess equal inhibitory potency when either d(AT) or 70S RNA
366
Inhibitors of RNA and DNA Hiosyntheais
is used ae the template .
Vol . 15, No . 3
Rous sarcoma virus reverse transcriptase is
inhibited by low concentrations of distamycin A (an inhibitor of DNAdependent RNA polymerase which acts by intercalating with DNA molecules), as well as of its derivative compound XIII (which possesses five pyrrole groups in the molecule instead of the three present in diatamycin A and contains the same side chains) (38) .
The reverse transcriptase activity of
Rauscher marine leukemia virus is strongly and specificallq inhibited by nontemplate single-stranded polyribonucleotides, apparently ss a result of the binding of these inhibitory homopolymers to the template site of the polymerase (39) .
The degree of inhibition obtained with single-stranded
polyribonucleotides is clearly dependent on the base composition of the homopolymer;
poly U and poly G are bound with much greater affinity than
are poly A or poly C .
Among the other potential inhibitors of RNA-dependent
DNA polymerase so far studied, adriamycin and daunomycin appear to be the most potent but they lack specificity .
Both of these antibiotics are known
inhibitors of DNA and RNA synthesis (40,41) .
DNA co~lementary to viral RNA (i .e ., DNA (-) ) can be synthesized preferentially in the presence of high concentrations of actinomycin D (e .g ., 100 Ug/ml) with either purified reverse traaecriptase or disrupted virions ; the DNA-instructed synthesis of the DNA (+) strands is severely inhibited under these conditions (42) .
This is the basis of methods used to detect
reverse transcriptase as well as to prepare highly purified DNA transcripts of various RNA template molecules .
The biological function of reverse tranecriptase ie not yet fully understood .
It has been hypothesized that RNA-dependent DNA synthesis is
involved in gene amplification (43), and recent evidence that 2~,5~-dimethyl-4~-N-benzyldemethylrifampicin preferentially inhibits the DNA
Vol. 15, No . 3
Inhibitors of RNA and DNA Hioayathesis
36 7
synthesis responsible for ribosooal gene asplification ie in accord with this hypothesis .
The use of specific inhibitors for reverse transcriptase
may provide more information about the function of this enzyme in neoplastic transformation .
The direct correlation found, for ezasple, between the
antiviral activity and the inhibition of reverse traascriptase by rifamycin derivatives suggests that reverse tranecriptase is necessary for neoplastic transformation by RNA ts~or viruses (44,45) ;
although this conclusion still
remains open to question (46) .
References 1.
S .C . SUNG, in ^Metabolic Inhibitors^, Dol . III, ads. R.K. Hochstet, K. Rates, J.H . Qusetel, pp . 175-204, Academic Press, New York London (1972) .
2.
B .A . NEWTON, Advan. Pharmacol. Chemother. 8 149-184 (1970) .
3.
D. GORTLIEB and P .D . SHAW, Eds. ^Antibiotics ^, Vol . I, Springer-Verlag, Berlin - New York (1967) .
4.
LH. GOLDBERG and P .A . FRISDMAN, Arm. Rev. Biochem.
5.
R.K . HAROLD, is ^1Ketabolic Inhibtitvre ^, Vol. III, Eds. R.K . Hochstet,
40 775-510 (1971) .
K. Rates, and J.H . Quastel, pp . 305-360, Academic Press, New York London (1972) . 6.
R.P . PERKY, &ep . CeLZ Res. 29 400-406 (1963) .
7.
L.D . HAKILTON, W. FULLER, aad E . REICH, Asturs 198 53g-540 (1963) .
8.
J.P . RICHARDSON, J. IläZ. Biol . 78 703-714 (1973) .
9.
D. RABIISSAY and W. ZILLIG, FBBS Letters 5 104-106 (1969) .
10 . W. ZILLIG, R. ZECHEL, D. BABUSSAY, M. SCHACHHER, V.S . SETHI, P . PAL!!, A. HELL, and W. SEIFERT,
Cold Spring Barbor Symp .
Quant. BioZ .
35
47-58 (1970) . 11 . R.D . KOSTELLER and C . YANOFSRY, J. Ab Z. Biol . 48 525-531 (1970) . 12 . C. REDII~iGER, K. GN T~~~T , J.L . KANDEL, Jr ., F. GISSINGER, and P. CHAMBON,
inhibitors of RNA and DNA Biosynthesis
368
Vol . 15, No . 3
Bioahem . Biophys. Res . Cormnae. 38 165-171 (1970) . 13 . R. G. ROEDSR and W.J . RUTTER, Proc . AatZ . Acad . Sci. U.S. 65 675-682 (1970) . 14 . T .J . LINDELL, F . WEINBERG . P .W . MORRIS, R.G . ROSDER, and W .J . RiT1TER, Science,
170 447-449 (1970) .
15 . V.L . CHAN, G.F . WHITMORE, and L. SIMINOVITCH, Fror . NatZ . Accrd. Sci. U.S. 69 3119-3123 (1972) . 16 . M. MIELHAC, Z . TYSPER, and P . CHAMBON, Eur. J. Biochem. 28 291-300 (1972) . 17 . S .S . COHEN, Frog PucZeic Acid Res. and Mb Z . BioZ . 5 1-88 (1966) . 18 . R.J . SUHADOLNIK, "Aueleoside ANTIBIOTICS", Wiley (Interscience), New York (1970) . 19 . I .H . KRAROFF, N.C . BROWN, and P. REICHARD, Cancer Res . 28 1559-1565 (1968) . 20 . H .S . ROSENKRANZ, H.S . CARR, and R.D . POLLAK, Biochim . Biophys. Aeta, 149 228-245 (1967) . 21 . J .W. YARBRO, Career Res . 28 1082-1087 (1968) . 22 . W. SZYBALSR.I and V .N . IYER, in "Antibiotics"
Vol . I, Eds . D. Gottlieb and
P .D . Shaw, pp . 211-245, Springer-Verlag, Berlin - New York (1967) . 23 . S . SHIBA, A. TERAWARI, T . TAGIICHI, and J. RAWAMATA, Aatia~e 183 1056-1057 (1959) . 24 . A.J . SHATKIN, E. REICH, R. M. FRANKLIN, and E.L . TATUM, Biochim. Biophys. Aeta . 55 277-289 (1962) . 25 . H . BERSTEN, Biothun. Biophys. Acta, 55 558-560 (1962) . 26 . E. REICH, A.J . SHATRIN, and E.L . TATDM, Biochim. Biophys . Acta, 53 132-149 (1961) . 27 . S .C . SONG and J.H . QUASTEL, Cancer Res. 23 1549-1554 (1963) . 28 . H.M. KEIR and J.B . SHEPHERD, Biochem J. 95 483-489 (1965) . 29 . D . BALTIMORE, Asture, 226 1209-1211 (1970) . 30 . H .M . TSMIN and S. MIZIITANI, Aatu~,
226 1211-1213 (1970) .
Vol . 15, No . 3
Inhibitors of RNA and DNA Hiosynthesia
369
31 . M . GREEN, M . BORUTANM, R . FUJINAGA, R .R . BAY, H . RORiAQTANDA, and C . GURGO, Pros . Aatl . Acad . Soi . U. S. 67 385-393 (1970) . 32 . S . SPIEGSLMAN, A. BURNY, M .R . DRS, J . REYDAR, J . 3CHLOM, M . TRAVNICB&, and R . WATSON, Rature, 227 563-567 (1970) . 33 . R .C . GALLO, S .S . YANG, and R .C . TING, Rature, 228 927-929 (1970) . 34 . C . GURGO, B .R . BAY, L . THIRY, and M . GRBBri, Rature Rem Biology, 229 111-114 (1971) . 35 . M . GREEN, J . BBAGDON, and A . RADRIN, Pros . Aatl . Aaad . Scti . U. S. 6 9 1294-1298 (1972) . 36 . W .W . BROCRMAN, W.A . CABTSR, L .H . LI, F . REQSSSR, and F.R . NICHOL, Rature, 230 249-250 (1971) . 37 . T .S . PAPAS, L . 3ANDHAUS, M .A . CHIRIG03, and E . FORIIBAWA, Biochem. Btophys. Res Canmat . 52 88-92 (1973) .
38 . M . ROTLER and Y . BECRER, Rature Rem Biology 234 212-214 (1971) . 39 . F .W . TOOMINSIQ and F .T . RS[aQEY, Pros . AatZ . Road. Bai. U.S . 6 8 2198-2202 (1971) . 40 . H .B . BOBMANN and D . RESBEL, FSBS Lattera 15 273-276 (1971) . 41 . W .E .G . MIR.LER, R.R . ZAHN and H .J . SEIDEL, Rature Aem Biology 232 143-145 (1971) . 42 . R.M. RUPRECHT, N .C . GOODMAN, and S . SPIEGELMAN, Bioohim. Biaphys. Acts 294 192-203 (1973) . 43 . M. CRIPPA and G .P . TOCCHINI-VALENTINI, Pros. AatZ . Aaad. Sci. U.S. 6 8 2769-2773 (1973) . 44 . R.C . TING, S .S . YANG, and R .C . GAid.O, Aatta~e Rem Biology 236 163-166 (1972) . 45 . A .M . WQ, R .C .Y . TING and R .C . GALIA, Pros. AatZ . Road. Sci. U. S. 70 1298-1302 (1973) . 46 . W. LEVINSON, A . FARAS, B . WOODSON, J . JACASON, aad J .M. BISHOP, Prop . AatZ . Acad. Sci. U .S .7 0 164-168 (1973) .
Lifa Sciaacea Vol . 15, pp " 359-369 Printed in the U .S .A .
1~IINIREVIESQ
INHIBITORS OF HNA AND DNA BIOSYNTSSSIS Shen-Ching Sung Division of Neurological Sciences, Department of Psychiatry University of British Columbia Vancouver V6T-1W5, Canada .
During the lest fifteen years our knowledge of the bioeyathesis of nucleic acids has ezpaaded greatly, due largely to the discovery and use of various inhibitors of RNA and DNA synthesis .
Among these inhibitors (1)
some are known to act by binding to template or primer DNA whereas others exert their effects directly on the enzyme polyieraee .
Some inhibitors of
RNA synthesis affect the initiation step while others block the elongation (polymerization) of the RNA chain.
Meay chemotherapeutic compounds alter
the structure and function of DNA by interaction, binding or intercalation (2) .
A wide variety of purine and pyrimidine analogs, D-arabinosyl
nucleosides, alkylating agents and several anticancer drugs have been used as inhibitors of RNA and/or DNA synthesis .
A large number of antibiotics are
inhibitors of RNA and/or DNA synthesis (3-5) .
Sore of these antibiotics
have been shown to inhibit DNA-dependent RNA polymerase by binding specifically either to DNA, to the enzyme or to one of the enzyme subunits, but no antibiotic has yet been shown to inhibit specifically the activlty of DNA-dependent DüA poly~raee.
Actinamycin, the moat thoroughly studied and comronly used antibiotic, hoe various biological effects, but its principal action ie the inhibition of
359
362
Inhibitors of RNA and DNA Biosynthesis
Vol . 15, No . 3
polymerase by a-amanitin is dependent on the amount of enzyme present but is independent of the DNA concentration, and is not competitive with the RNA precursor nucleoside triphosphates .
This substance inhibits chain elongation
and does not exert a specific effect on the initiation .
In contrast to
rifamycin, which inhibits RNA polymeraee from E . coli but not that from animal tissue, a-amanitin does not affect the former enzyme .
Eukaryotic cells possess multiple forma of DNA-dependent RNA polymerasea . RNA polymerase I is localized in the nucleolus, whereas polymerase II is found in the nucleoplasm.
The former enzyme ie largely involved in the
nucleolur synthesis of ribosomal RNA while the latter synthesizes the bulk of the nucleoplasmic RNA species (13) .
Polymerase III is also believed to be
present in the nucleoplasm but its role in cell function has not been clarified.
a-Amanitin selectively inhibits the activity of DNA-dependent RNA
polymerase II, but does not affect polymerases I and III (12,14) .
a-Amanitin
is the only example of a selective inhibitor of a RNA polymerase from eukaryotic cells and thus has become a very useful tool for the study of the poorly understood transcription mechanism in higher organisms .
Inhibitors of RNA synthesis can be used in the isolation of mutants of eukaryotic cells that contain different forms of DNA-dependent RNA polymerase . For example, mutants of Chinese hamster ovary cells that are resistant~to a-amanitin have been isolated (15) .
Some of these mutants contain an altered
form of DNA-dependent RNA polymerase that is resistant to inhibition by a-amanitin .
Such mutants may prove useful for studying the mechanism of action
of this eukaryotic enzyme .
Rifampicia is another antibiotic which inhibits bacterial enzyme not the DNA-dependent RNA polymerases from mammalian nuclei .
but
Several semi-
Vol. 15, No . 3
36 3
inhibitors of RNA and DNA Biosynthesis
synthetic derivatives of rifaapicin possessing highly hydrophobic sidechains have been made, however, which dô inhibit the mammalian enzyme (16) apparently at the initiation rather than the elongation step .
These
hydrophobic derivatives block the binding of DNA by the mammalian enzyme, whereas rifamycin does not block DNA binding by S. cola RNA polymerase .
Several anticancer drugs are known to act at the sequential or concurrent steps involved in DNA synthesis, e.g ., the antitumr activity of FIIdR appears to result frog the inhibition of thynidylate synthesis.
Cytosine
arabinoeide (era-C) is believed to inhibit DNA synthesis through the action of its 5~-triphoaphate, ~ra-CTP, on the polys~erization of deozyribonucleotides (17,18) .
liany antibiotics are able to inhibit in vivo and in vitro
synthesis of DNA in warioue organisms (3-4), however, none of the aatibiotica studied to date appears to be a specific inhibitor of DNA synthesis .
Hydrozyurea interferes with DNA eynthesis in bacteria ae well ae in ns®alian cells but does not affect the activity of thymidine kinase or DNA-dependent DNA polymerise .
It has also been reported that hydrozyurea
inhibits a highly purified form of ribonucleoside diphosphate reductase fros B. coli (19) .
Although the bode of action of this substa~e on DNA synthesis
does appear to be connected with the conversion of ribonucleotides to deoryribonucleotides, evidence has been presented to indicate that nucleotide reductase is not the enzyme affected by hydrozyurea (20,21) .
Mito~cin, which ie known to cross-link to the DtiA molecule (22), has been shown to inhibit DNA synthesis in S . coli with little !mediate effect on RNA or protein synthesis (23) .
Mi.tomycin causes degradation (depolyeerization)
of DNA both in growing and resting cells of 8. coli and in mammalian cells in culture (24) .
The depolyoerizstion of DäA that occurs after adai.aistratian
36 4
Vol . 15, No . 3
Inhibitors of RNA and DNA Biosynthesis
of mitomycin C may be due to an activation of a latent cellular DNase (25) . Since it is difficult to measure the inhibition of DNA synthesis in the presence of DNA depolymerization, the use of mitomycin as a selective inhibitor of DNA synthesis moat by approached with caution.
Various
investigators have suggested that the mitomycin-induced breakdown of DNA may be a secondary phenomenon whereas other evidence indicates that the DNA breakdown may be the primary event and the inhibition of DNA synthesis may be a consequence of the depolymerization (26) .
Sarkomycin, an antitumor antibiotic, at certain concentrations inhibits DNA synthesis in Ehrlich ascites carcinoma cells but has little or no effect on RNA and protein synthesis .
This inhibition of DNA synthesis may be
prevented by reduced glutathione (27) .
Sarkaomycin also inhibits the
partially purified DNA polymerase from Landschultz ascites tumor cells (28) .
Multiple forma of DNA-dependent DNA polymerases are now known to exist in bacteria as well as in mammalian cells.
Specific inhibitors of the various
types of DNA-dependent DNA polymerase would certainly be eatremely valuable tools for elucidating the mechanism of DNA replication.
It is to be hoped
that their discovery is imminent .
RNA-directed DNA polymerase (reverse transcriptase) has been found in several oncogenic RNA viruses as well as in leukemic cells and tumors (29-33) .
Specific inhibitors of this enzyme would be useful for studying its
possible role in neoplasia and might become important chemotherapeutic agents for the treatment of human leukemia, cancer and viral diseases . such agents are known .
So far few
Rifampicin itself has no effect on the RNA-directed
DNA polymerase activity of RNA viruses, but removal of the 4-Nrmethyl group on the aminopiperazine side chain or substitution of a benzyl group at this
Vol. 15, No . 3
Inhibitors of IAA and DNA Biosynthesis
36 5
site converts rifampicin to derivatives which are effective inhibitors of this enzyme .
Thus 2,5-dimethyl-4-N-benayldemethylrifampicin (AF/ABDMP),
4-N-benzyldemethylrifanpicin (AF/ABP) and N-demethylrifampicin inhibit the RNA-directed DNA polymerase activity of several RNA tumor viruses, including narine sarcoma virus (MSV),
feline leukemia virus (FeLV) and avian
myeloblastosis virus (AMV) (34) .
Moreover, Green et al . (35) have
demonstrated that of the 37 rifamycin-SV derivatives possessing 3-amine subatituents studied, 29 are good inhibitors of both the RNA-directed and DNA-directed polymerase activities of MSV, FeLV and AMV, and that the 3-piperidyl derivatives of rifarycin-SV possessing cyclohezyl and cyclohezylalkyl subetituents are especially effective .
Green et al . also found that
the DNA-directed DNA polymerase preparation from human RB cells vas leas sensitive to inhibition by these derivatives than the viral polymerase ; 5-10 times higher concentrations were required for the same percent inhibition of the DNA polymerase of RB cells than with viral polynerase .
Caution ie
indicatied in the use of the rifampicin derivatives as a diagnostic teat for the presence of reverse tranecriptase because these drugs are relatively uaetable in solution and also lack complete specificity, although they are more effective inhibitors of RNA-directed DNA polymerase of RNA tumor viruses than of DNA-dependent DLiA polyoerase from mammalian cells or from E. cola .
The streptovaricine codes, a known inhibitor of bacterial but not of mammalian DNA-dependent RNA polymerase, is another agent with an eztremely potent inhibitory effect on the RNA-dependent DdiA polymerase activity associated with oncogenic RNA viruses (36) .
An alkaloid extract of a
medicinal plant Sacred Lily (Narcissus tarzetta L .) has been shown to inhibit the purified DNA polymerase from avian myeloblastosis virus, possibly by binding to the polymerase enzyme rather than to the RNA template (37) . It appears to possess equal inhibitory potency when either d(AT) or 70S RNA
366
Inhibitors of RNA and DNA Hiosyntheais
is used ae the template .
Vol . 15, No . 3
Rous sarcoma virus reverse transcriptase is
inhibited by low concentrations of distamycin A (an inhibitor of DNAdependent RNA polymerase which acts by intercalating with DNA molecules), as well as of its derivative compound XIII (which possesses five pyrrole groups in the molecule instead of the three present in diatamycin A and contains the same side chains) (38) .
The reverse transcriptase activity of
Rauscher marine leukemia virus is strongly and specificallq inhibited by nontemplate single-stranded polyribonucleotides, apparently ss a result of the binding of these inhibitory homopolymers to the template site of the polymerase (39) .
The degree of inhibition obtained with single-stranded
polyribonucleotides is clearly dependent on the base composition of the homopolymer;
poly U and poly G are bound with much greater affinity than
are poly A or poly C .
Among the other potential inhibitors of RNA-dependent
DNA polymerase so far studied, adriamycin and daunomycin appear to be the most potent but they lack specificity .
Both of these antibiotics are known
inhibitors of DNA and RNA synthesis (40,41) .
DNA co~lementary to viral RNA (i .e ., DNA (-) ) can be synthesized preferentially in the presence of high concentrations of actinomycin D (e .g ., 100 Ug/ml) with either purified reverse traaecriptase or disrupted virions ; the DNA-instructed synthesis of the DNA (+) strands is severely inhibited under these conditions (42) .
This is the basis of methods used to detect
reverse transcriptase as well as to prepare highly purified DNA transcripts of various RNA template molecules .
The biological function of reverse tranecriptase ie not yet fully understood .
It has been hypothesized that RNA-dependent DNA synthesis is
involved in gene amplification (43), and recent evidence that 2~,5~-dimethyl-4~-N-benzyldemethylrifampicin preferentially inhibits the DNA
Vol. 15, No . 3
Inhibitors of RNA and DNA Hioayathesis
36 7
synthesis responsible for ribosooal gene asplification ie in accord with this hypothesis .
The use of specific inhibitors for reverse transcriptase
may provide more information about the function of this enzyme in neoplastic transformation .
The direct correlation found, for ezasple, between the
antiviral activity and the inhibition of reverse traascriptase by rifamycin derivatives suggests that reverse tranecriptase is necessary for neoplastic transformation by RNA ts~or viruses (44,45) ;
although this conclusion still
remains open to question (46) .
References 1.
S .C . SUNG, in ^Metabolic Inhibitors^, Dol . III, ads. R.K. Hochstet, K. Rates, J.H . Qusetel, pp . 175-204, Academic Press, New York London (1972) .
2.
B .A . NEWTON, Advan. Pharmacol. Chemother. 8 149-184 (1970) .
3.
D. GORTLIEB and P .D . SHAW, Eds. ^Antibiotics ^, Vol . I, Springer-Verlag, Berlin - New York (1967) .
4.
LH. GOLDBERG and P .A . FRISDMAN, Arm. Rev. Biochem.
5.
R.K . HAROLD, is ^1Ketabolic Inhibtitvre ^, Vol. III, Eds. R.K . Hochstet,
40 775-510 (1971) .
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