Table Of ContentMolecular Biology
Biochemistry and Biophysics
18
Editors:
A. Kleinzeller, Philadelphia· G. F. Springer, Evanston
H. G. Wittmann, Berlin
Advisory Editors:
C. R. Cantor, New York· F. Cramer, Gottingen . F. Egami, Tokyo
M. Eigen, Gottingen . F. Gros, Paris· H. Gutfreund, Bristol
B. Hess, Dortmund· H. Jahrmiirker, Munich· R W. Jeanloz, Boston
E. Katzir, Rehovot . B. Keil, Gif-sur-Yvette· M. Klingenberg, Munich
I. M. Klotz, Evanston· F. Lynen, MartinsriedjMunich
W. T. J. Morgan, London· K. Muhlethaler, Zurich . S. Ochoa, New York
G. Palmer, Ann Arbor· I. Pecht, Rehovot . R. R. Porter, Oxford
W. Reichardt, Tubingen . H. Tuppy, Vienna
J. Waldenstrom. Malmo
Helga Kersten· Walter Kersten
Inhibitors
of Nucleic Acid Synthesis
Biophysical and Biochemical Aspects
With 73 Figures
Springer-Verlag Berlin· Heidelberg· New York 1974
Professor Dr. HELGA KERSTEN and Professor Dr. WALTER KERSTEN
Physiologisch-Chemisches Institut
der Universitat Erlangen-Nlirnberg
D-8520 Erlangen/Fed. Rep. Germany
Wasserturmstr. 5
ISBN-13:978-3-642-S0S57-9 e-ISBN-13:97S-3-642-S0S55-5
DOl: 10.1007 /97S-3-642-S0S55-5
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ment with the publisher. ©by Springer-Verlag Berlin Heidelberg 1974.Softcover reprint of the hardcover 1s t edition 1974
Library of Congress Cataloging in Publication Data. Kersten, Helga, 1926. Inhibitors of nucleic acid synthesis.
(Molecular biology, biochemistry, and biophysics, v. 18). 1. Nucleic acid synthesis. 2. Antimetabolites. 3. Antibiotics.
I. Kersten, Walter, 1926- joint author. II. Title. III. Series. [DNLM: 1. Antibiotics - Pharmacodynamics.
2. Nucleic acids - Antagonists and inhibitors. 3. Nucleic acids - Biosynthesis. WI M0195T no. 18 I
QU58 K412i]. QP620.K47. 574.8'732. 74-11265.
The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific
statement, that such names are exempt from the relevant protective laws and regulations and therefore free for
general use.
Preface
During the last decade physical and chemical methods have improved
rapidly - a fact which allowed the mode of action of antibiotics to
be studied - and many biochemically-oriented scientists have devoted
their research to the following questions:
1. What is the metabolic pathway that is inhibited selectively, and
what are the target molecules within a sensitive cell?
2. What are the relationships between the chemical structure of an
antibiotic and the physicochemical properties of the sensitive mole
cule(s)?
3. Why and how far is the action selective?
4. Is it possible to correlate the interaction with the target mole
cule(s) with the particular biological activities observed?
This monograph deals with those antibiotics which interfere with the
biosynthesis of nucleic acids. The idea was to provide an insight
into how to investigate the preceding questions experimentally and
to solve as yet unresolved problems rather than to give a review of
the current state of knowledge. Although the biochemistry of nucleic
acid synthesis is known in general, the precise molecular mechanisms
by which deoxyribonucleic acid is replicated or transcribed has still
to be clarified. For this reason it is not yet possible to describe
the molecular mechanisms by which the inhibitors of nucleic acid and
protein synthesis exhibit their effects. The fact that the inhibitors
of nucleic acid and protein synthesis themselves served as useful tools
to obtain an insight into the mechanisms of replication, transcription
and translation was one of the most exciting discoveries in this field.
Several chapters are devoted to these aspects of antibiotic research.
This discovery that several antibiotics interact with nucleic acids,
nucleic acid polymerases or other enzymes, or cell constituents such
as ribosomes, allowed biochemists to use these "freaks of nature" as
simple model compounds, e.g. for the elucidation of nucleic acid-pro
tein interactions or nucleic aCid-carbohydrate interactions. During
the last years many physicists have become involved in biological
problems; their investigations into conformational changes occurring
during the inactivation of biologically active macromolecules will
provide us some time in the future with a more precise understanding
of how these macromolecules and suprarnolecular structures exhibit
their specific functions. The progress which .has been made in the
elucidation of the relationships between the ,chemical structure of
an antibiotic and the physicochemical properties of the target mole
cules, will probably be of help to all those chemists who are con
cerned with the production of'synthetic or semisynthetic compounds
for chemotherapeutic purposes. For the question of selectivity of
antibiotic action we turn to the virologists, who are concerned with
the relationships between the mechanism of antibiotic action and
antiviral activity. With increasing knowledge of virus structure and
virus replication, an initial picture emerges as to how antibiotics
exhibit their selective antiviral versus host activities.
VI
We have taken the risk of stressing biochemical, biophysical, chemi
cal and particular biological aspects. We are aware that we have left
many "gaps", e.g. dij;cussion of the biosynthesis of antibiotics and
of the mechanisms of antibiotic resistance. After writing this book
the authors found their feelings best expressed by EGON FRIEDEL, who
said:
"The courage to talk about relationships which are not completely
known, to report on facts which are not observed by oneself, to de
scribe processes about which nothing can be said with certainty: in
short to say something which could perhaps be wrong, this courage is
a prerequisite for all productivity."
Erlangen, October 1974 HELGA KERSTEN • WALTER KERSTEN
Acknowledgements
The authors thank Dr. ALEXIS VON AUFSESS and ANNEMARIE G5LDEL
for most valuable help in preparing the manuscript.
Contents
General Introduction ......................................... .
I. Inhibi to,rs of DNA Synthesis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . • . . 3
A. Mitomycin............................................... 4
1. Origin, Biological and Chemical Properties........... 4
2. On the Molecular Mechanism of Action................. 5
a) Interaction with DNA in Vitro. .......... .. ....... .. 6
b) Interaction with DNA within the Cell ......•....... 10
c) Effect on DNA Metabolism ..•....................•.. 10
d) Effect on RNA Metabolism .......................... 11
3. Particular Activities in Biological Systems •.......•. 12
a) Synthesis of Enzymes.............................. 12
b) Mutagenicity .•.....•.............................. 12
c) Chromosome Breakage............................... 12
d) Viruses, Phages and Episomal DNA.................. 13
e) Mitosis........................................... 14
f) Immune Response................................... 1 5
B. Streptonigrin ............ ,.............................. 15
1. Origin, Biological and Chemical Properties •........•. 15
2. On the Molecular Mechanism of Action................. 16
3. Particular Activities in Biological Systems ..•••.•.• 18
C. Sibiromycin............................................. 18
D. Phleomycin.............................................. 20
1. Origin, Biological and Chemical Properties ........•.. 20
2. On the Molecular Mechanism of Action .•........•.....• 20
a) Interaction of Phleomycin with Nucleic Acids in
Vitro. . . . • • . . . . . . . . • . • • . . . . . • . . . . . . . . • . . . . • • . • . . • . 20
b) Effect on DNA- and RNA-Dependent Processes in
Vi tro . . . • . . • . . . . . . . . . . • • . . . . . • . . . • . . . . . • . . . . • . . . . . 21
c) Effects on Nucleic Acids and Their Metabolism in
Normal and Virus-Infected Cells................... 22
d) Particular Activities in Bidlogical Systems ....... 24
E. Bleomycin............................................... 24
1. Origin, Biological and Chemical Properties ....•..•... 24
2. On the Molecular Mechanism of Action ......•.......... 27
a) Interaction with DNA in Vitro ... '. .. ... ..•.. .• .•.. •• 27
b) Effect on DNA-Dependent Processes in Vitro ..••....• 28
c) Effects on Nucleic Acids and Th~ir Metabolism
wi thin the Cell................................... 29
F. Neocarcinostatin........................................ 30
1. Origin, Biological and Chemical Properties ....••..... 30
2. On the Mechanism of Action ..................•........ 31
G. Edeine.................................................. 31
H. Nalidixic Acid.......................................... 33
VIII
1. Origin, Biological and Chemical Properties......... 33
2. Mechanism of Action and Biological Activities...... 34
a) Effect on DNA and Nucleic Acid Metabolism....... 34
b) Mutagenicity.................................... 38
c) Recombination................................... 38
d) Hemolysin Production............................ 38
II. Inhibitors of RNA Synthesis that Interact with the DNA
Template .................... '" ........ .... ..... . . .. ... . . 40
A. Actinomycin........................................... 40
1. Origin, Biological and Chemical Properties......... 40
2. On the Molecular Mechanism of Action............... 42
a) ~nte;action with DNA, Poly- and Mononucleotides
'l-n V'l-tro......................................... 42
b) Interaction with Deoxyguanosine................. 44
c) Proposed Models for the Actinomycin-DNA Complex. 50
d) Inhibition of DNA Dependent RNA Synthesis in
Vitro........................................... 61
e) Effect on RNA Metabolism........................ 62
3. Particular Activities in Biological Systems........ 64
a) Synthesis of Enzymes............................ 64
b) Phages and Viruses.............................. 66
c) Immune Response................................. 67
B. Anthracyclines........................................ 67
1. Origin, Biological and Chemical Properties... ...... 67
2. On the Molecular Mechanism of Action............... 71
a) Interaction with DNA in Vitro.................... 71
b) Interaction of Planar Dyes with DNA and tRNA in
Vitro ................. , ..... , .. . ... ... ... .. . ... . 74
c) Effect on DNA-Dependent Processes in Vitro ....... 81
d) Effect on Nucleic Acid Metabolism............... 82
3. Particular Acti vi ties in Biological Systems........ 84
a) Viruses and Phages.... .......................... 84
b) Mutagenicity of Intercalating Dyes.............. 85
c) Chemotherapeutic Aspects........................ 85
C. Chromomycin, Olivomycin and Mithramycin........ ....... 86
1. Origin, Biological and Chemical Properties......... 86
2. On the Molecular Mechanism of Action............... 89
a) Interaction wi th DNA in Vitro ................... 89
b) Effects on DNA-Dependent Processes in Vitro ..... 94
c) Effects on Nucleic Acid Metabolism......... .. . . . 94
3. Particular Activities against Viruses.. ... ... ...... 96
D. Kanchanomycin......................................... 96
1. Origin, Biological and Chemical Properties......... 96
2. On the Mechanism of Action......................... 96
E. Distamycin and Netropsin ......... ~ . . . . . . . . . . . . . . . . . . . . 98
1. Origin, Biological and Chemical Properties.. ....... 98
2. On the Molecular Mechanism of Action............... 99
a) Interaction with DNA in Vitro ................... 99
b) Effect on DNA-Dependent Processes in Vitro....... 102
F. Anthramycin........................................... 103
1. Origin, Biological and Chemical Properties......... 103
2. On the Molecular Mechanism of Action............... 104
IX
a) Interaction with DNA in Vitro •• ..•••..•.••••.•..• 104
b) Inhibition of DNA-Dependent Processes in Vitro
and within the Cello •....•........•....•.......• 105
c) Chemosterilant Action of Anthramycin ............ 106
III. Inhibitors of RNA Synthesis Interacting with RNA Poly-
merases .......... " ...•.....•............................ 107
A. Rifamycins ... .' ..•.....•............................... 108
1. Origin, Biological and Chemical Properties •.•..•... 108
2. On the Molecular Mechanism of Action ..•............ 108
a) Interaction with RNA Polymerase in Vitro •..••••• 110
b) On the Mechanism of Inhibition of RNA Polymerase
Reaction. . . . . . . . . . . • . . . . . . • . . • . . . . . . . . . . . . . . . . •• 112
c) Effect on RNA in Intact Cells or Organelles •.... 113
3. Particular Activities in Biological Systems ........ 115
a) Bacteriophageq, Viruses and Episomes ............ 115
b) Trachoma Agent.................................. 117
B. Streptovaricin ..•••............•............••........ 117
1. Origin, Biological and Chemical Properties ......... 117
2. On the Molecular Mechanism of Action ......•....•... 119
C. Streptolydigin ...•.......•.........•......•.•......... 120
1. Origin, Biological and Chemical Properties ......... 120
2. On the Molecular Mechanism of Action ...........•.•• 121
D. Amanitins ....................••...........•........... 122
1. Origin, Biological and Chemical Properties ....•••.. 122
2. On the Molecular Mechanism of Action ..........•.•.. 122
a) Interaction of Amanitins with Eukaryotic RNA
Polymerases ..................................... 122
b) Effects on RNA Metabolism ....................... 124
IV. Inhibitors Interferring at the Precursor Level or with
Regulatory Processes of Nucleic Acid Synthesis ........... 127
A. Nucleoside Antibiotics................................ 127
a) Toyocamycin, Tubercidin, Sangivamycin ........... 127
b) Cordycepin...................................... 129
c) 3'-Amino-3'-Deoxyadenosine ....•................. 131
d) Formycin ........•.............•................. 131
e) 6(p-Hydroxyphenylazo)-Uracil (HPUra) .........•.. 131
B. Mycophenolic Acid ....................................• 132
C. Amino Acid Analogs.................................... 133
a) Azaserine and DON .•............•..............•. 133
b) Azotomycin ...............•...•......•.••........ 133
c) Hadacidin .•....•..•........... ',' ...•............ 134
D. Quinone Antibiotics ......•.........•. '. . . • . . . . . . . . . . . .. 135
Granaticin, Mitomycin Derivatives, Synthetic
Quinones. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . . . . . . • •. 135
1. Origin, Biological and Chemical Properties ..••..... 135
2. Mechanism of Action................................ 135
References. . . . . . . • . . . . . . . . . . . . . . . . . . • • . • . • . . • . . . . . • . . . . . . • . . •• 140
Subject Index ....................................•••...•...... 183
General Introduction
With the continual discoveries of new antibiotics, the number of
compounds that are known to interfere with nucleic acid or protein
biosynthesis is steadily increasing. No one"knows why different mi
croorganisms produce a variety of substances with complex chemical
structures, which are not involved in any metabolic process of the
microorganisms and which are extremely toxic. Are they produced:
1. as a chemotherapeutic aid to physicians; 2. to provide physicists
with highly sophisticated structures to be resolved; 3. to show
chemists by their biosynthetic pathway how to develop new synthetic
compounds; or 4. by their specific inhibitory effects, to help bio
chemists in elucidating as yet unknown aspects of cell metabolism?
With respect to the progress in research on DNA, RNA and protein
synthesis, e.g. replication, transcription and translation, which
has characterized the past 20 years of work in the field of Molecular
Biology, the aid provided by antibiotics discussed in the following
chapters should be acknowledged.
In this monograph the antibiotics and a few synthetic compounds
inhibiting nucleic acid synthesis have been subdivided into four
categories and are described in Chapters I - IV:
I. Inhibitors which preferentially affect DNA synthesis
II. Inhibitors which preferentially affect RNA synthesis through
interaction with the DNA template
III. Inhibitors which affect RNA synthesis through interaction with
RNA polymerases
IV. Inhibitors interfering at the precursor level or with the re
gulatory processes of nucleic acid synthesis.
For each antibiotic a brief survey on its "origin, chemical and
biological properties" is presented, followed by a more detailed
description dealing with the interaction of the antibiotic with its
target molecule in vitro. In this section, "on the molecular mecha
nism of antibiotic action", the physicochemical methods which can be
applied to an investigation of antibiotic nucleic acid or protein
interactions are introduced. The techniques available for these
binding studies can be divided into two broad categories: 1. non
spectroscopic methods; 2. spectroscopic methods. The nonspectroscopic
methods include: equilibrium dialysis, with the tar
cosedi~entation
get molecule on sucrose gradients or in the analytical ultracentri
fuge, gel filtration, electrophoresis, viscosimetry, buoyant density
measurements, polarography. Spectroscopic methods include examples
of UV and visible absorption, fluorescence spectroscopy, optical
rotatory dispersion and circular dichroism, NMR spectroscopy, elec
tron spin resonance, light sca.ttering and X-ray diffraction. A clear
comprehensive review ef these methods, their principles and their
theories has been published by CHIGNELL (1971). Although the methods
are discussed with respect to drug-protein interactions, they can
in principle be applied to studies of antibiotic nucleic acid or
