The replication fork and anti-cancers

Replication Fork
and It’s Inhibitors
Behzad Milani
PhD Student of Biochemistry
Supervised by Prof. AmirMozaffari
May 2017

2 June 2017Replication Fork - Behzad Milani
2

Chapter 1
DNA Replication Mechanisem

Introduction
• DNA replication is the biological process of
producing two identical replicas of DNA from one
original DNA molecule.
• This process occurs in all living organisms and is
the basis for biological inheritance.
• DNA is made up of a double helix of two
complementary strands.
4

• During replication, these strands are separated.
• Each strand of the original DNA molecule then
serves as a template for the production of its
counterpart, a process referred to as
semiconservative replication.
• Cellular proofreading and error-checking
mechanisms ensure near perfect fidelity for DNA
replication.
5Introduction

6Introduction

• DNA replication begins at specific locations, or
origins of replication, in the genome.
• Unwinding of DNA at the origin and synthesis of
new strands results in replication forks growing bi-
directionally from the origin.
7Introduction

8Introduction

• A number of proteins are associated with the
replication fork to help in the initiation and
continuation of DNA synthesis.
• DNA polymerase synthesizes the new strands by
adding nucleotides that complement each
(template) strand.
• DNA replication occurs during the S-stage of
interphase.
9Introduction

10Introduction

DNA structures
• The four nucleobases adenine, cytosine, guanine,
and thymine, (A,C, G and T).
• A and G are purine bases,
• C and T are pyrimidines.
• A pairs with T (two hydrogen bonds),
• G pairs with C (stronger: three hydrogen bonds).
11

DNA structures
12

13

DNA structures
• Phosphodiester (intra-strand) bonds are stronger
than hydrogen (inter-strand) bonds.
• This allows the strands to be separated from one
another.
• The nucleotides on a single strand can therefore be
used to reconstruct nucleotides on a newly
synthesized partner strand.
14

15DNA structures

DNA structures
• DNA strands of the double helix are anti-parallel
with one being 5' to 3', and the opposite strand 3'
to 5'.
• DNA polymerase can synthesize DNA in only one
direction by adding nucleotides to the 3' end of a
DNA strand.
16

17DNA structures

18

DNA polymerase
• DNA polymerases cannot initiate synthesis of new
strands, but can only extend an existing DNA or RNA
strand paired with a template strand.
• To begin synthesis, a short fragment of RNA, called
a primer, must be created and paired with the
template DNA strand.
19

20DNA polymerase

21DNA polymerase

Replication process
• DNA replication proceeds in three enzymatically
catalyzed and coordinated steps:
22
Initiation Elongation Termination

Topoisomerase
• Relaxes the DNA from its super-coiled nature.
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24

25

26

27

28

29

DNA Gyrase
• Relieves strain of unwinding by DNA helicase; this is a
specific type of topoisomerase
30

31

Replication process - Initiation
• At particular points in the DNA, known as "origins",
• Are targeted by initiator proteins.
• In E. coli this protein is DnaA;
• In yeast, this is the origin recognition complex.
• Sequences used by initiator proteins tend to be "AT-rich"
• Initiators recruit other proteins and form the pre-
replication complex, which unzips the double-stranded
DNA.
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33

34

35

Replication Fork
• The replication fork is a structure that forms within the nucleus
during DNA replication.
• It is created by helicases, which break the hydrogen bonds holding
the two DNA strands together.
• The resulting structure has two branching "prongs", each one made
up of a single strand of DNA.
• These two strands serve as the template for the leading and
lagging strands, which will be created as DNA polymerase matches
complementary nucleotides to the templates; the templates may
be properly referred to as the leading strand template and the
lagging strand template.
36

Replication Fork
• DNA is always synthesized in the 5' to 3' direction.
• Since the leading and lagging strand templates are
oriented in opposite directions at the replication fork, a
major issue is how to achieve synthesis of nascent (new)
lagging strand DNA, whose direction of synthesis is
opposite to the direction of the growing replication fork.
37

38

39

DNA Helicase
• Also known as helix destabilizing enzyme.
• Helicase separates the two strands of DNA at the Replication Fork
behind the topoisomerase.
40

41

42

43

Single-Strand Binding (SSB) Proteins
• Bind to ssDNA and prevent the DNA double helix from re-
annealing after DNA helicase unwinds it, thus
maintaining the strand separation, and facilitating the
synthesis of the nascent strand.
44

Leading strand
• The leading strand is the strand of nascent DNA which is
being synthesized in the same direction as the growing
replication fork.
• A polymerase "reads" the leading strand template and
adds complementary nucleotides to the nascent leading
strand on a continuous basis.
45

Primase
• Provides a starting point of RNA (or DNA) for DNA
polymerase to begin synthesis of the new DNA strand.
46

47

Replication process - Elongation
• DNA polymerase has 5'-3' activity.
• All known DNA replication systems require a free 3'
hydroxyl group before synthesis can be initiated
• The DNA template is read in 3' to 5' direction whereas a
new strand is synthesized in the 5' to 3' direction.
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49

50

51

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• Primase adds RNA primers to the template strands.
• The leading strand receives one RNA primer while the
lagging strand receives several.
• The leading strand is continuously extended from the
primer by a DNA polymerase with high processivity.
• The lagging strand is extended discontinuously from each
primer forming Okazaki fragments.
53

• RNase removes the primer RNA fragments, and a low
processivity DNA polymerase distinct from the replicative
polymerase enters to fill the gaps.
• When this is complete, a single nick on the leading
strand and several nicks on the lagging strand can be
found.
• Ligase works to fill these nicks in, thus completing the
newly replicated DNA molecule.
54

• Multiple DNA polymerases take on different roles in the
DNA replication process.
• In E. coli, DNA Pol III is the polymerase enzyme primarily
responsible for DNA replication.
• It assembles into a replication complex at the replication
fork that exhibits extremely high processivity, remaining
intact for the entire replication cycle.
55

• In contrast, DNA Pol I is the enzyme responsible for
replacing RNA primers with DNA.
• DNA Pol I has a 5' to 3' exonuclease activity in addition to
its polymerase activity, and uses its exonuclease activity
to degrade the RNA primers ahead of it as it extends the
DNA strand behind it, in a process called nick translation.
• Pol I is much less processive than Pol III because its
primary function in DNA replication is to create many
short DNA regions rather than a few very long regions.2 June 2017Replication Fork - Behzad Milani
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• In eukaryotes, the low-processivity enzyme, Pol α, helps
to initiate replication because it forms a complex with
primase.
• In eukaryotes, leading strand synthesis is thought to be
conducted by Pol ε; however, this view has recently been
challenged, suggesting a role for Pol δ.
• Primer removal is completed Pol δ while repair of DNA
during replication is completed by Pol ε.
57

• As DNA synthesis continues, the original DNA strands
continue to unwind on each side of the bubble, forming a
replication fork with two prongs.
• In bacteria, which have a single origin of replication on
their circular chromosome, this process creates a "theta
structure" (theta: θ).
• In contrast, eukaryotes have longer linear chromosomes
and initiate replication at multiple origins within these.
58

DNA clamp
• A protein which prevents elongating DNA polymerases
from dissociating from the DNA parent strand.
59

60

DNA Polymerase
• The enzyme responsible for catalyzing the addition of
nucleotide substrates to DNA in the 5' to 3' direction
during DNA replication.
• Also performs proof-reading and error correction.
• There exist many different types of DNA Polymerase,
(such as the DNA Polymerase III) each of which perform
different functions in different types of cells.
61

DNA Polymerase
• Enzymatic hydrolysis of the resulting
pyrophosphate (from dNTPs) into inorganic
phosphate consumes a second high-energy
phosphate bond and renders the reaction
effectively irreversible.
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63

64

65DNA polymerase

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67

68

69

70

71

72

73

74DNA polymerase

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76

Lagging strand
• The lagging strand is the strand of nascent DNA whose
direction of synthesis is opposite to the direction of the
growing replication fork.
• Because of its orientation, replication of the lagging
strand is more complicated as compared to that of the
leading strand.
• As a consequence, the DNA polymerase on this strand is
seen to "lag behind" the other strand.
77

Lagging strand
• The lagging strand is synthesized in short, separated
segments.
• On the lagging strand template, a primase "reads" the
template DNA and initiates synthesis of a short
complementary RNA primer.
• A DNA polymerase extends the primed segments, forming
Okazaki fragments.
• The RNA primers are then removed and replaced with DNA,
and the fragments of DNA are joined together by DNA ligase.
78

Okazaki fragments
79

DNA Ligase
• Re-anneals the semi-conservative strands and joins
Okazaki Fragments of the lagging strand.
80

81DNA Ligase

DNA polymerase
• DNA polymerases are highly accurate, with an
intrinsic error rate of less than one mistake for
every 10,000,000 nucleotides added.
• In addition, some DNA polymerases also have
proofreading ability; they can remove nucleotides
from the end of a growing strand in order to
correct mismatched bases.
82

DNA polymerase
• Finally, post-replication mismatch repair
mechanisms monitor the DNA for errors, being
capable of distinguishing mismatches in the newly
synthesized DNA strand from the original strand
sequence.
• Together, these three discrimination steps enable
replication fidelity of less than one mistake for
every 1,000,000,000 nucleotides added.
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92

Telomerase
• Lengthens telomeric DNA by adding repetitive nucleotide
sequences to the ends of eukaryotic chromosomes.
• This allows germ cells and stem cells to avoid the
Hayflick limit on cell division.
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94

Termination
• Telomerase can become mistakenly active in somatic
cells, sometimes leading to cancer formation.
• Increased telomerase activity is one of the hallmarks of
cancer.
95

Termination
• Termination requires that the progress of the DNA
replication fork must stop or be blocked.
• Termination at a specific locus, involves the interaction
between two components:
1. a termination site sequence in the DNA, and
2. a protein which binds to this sequence to physically stop DNA replication.
In various bacterial species, this is named the DNA replication terminus
site-binding protein, or Ter protein.
96

Termination
• Because bacteria have circular chromosomes, termination of
replication occurs when the two replication forks meet each
other on the opposite end of the parental chromosome.
• E. coli regulates this process through the use of termination
sequences that, when bound by the Tus protein, enable only
one direction of replication fork to pass through.
• As a result, the replication forks are constrained to always
meet within the termination region of the chromosome.
97

Chapter 2
DNA Replication Inhibitors

DNA replication inhibitors categories
1.Alkylating antineoplastic agents
2.Nitrogen mustards
3.Topoisomerase inhibitors
99

2.Nitrogen mustards
100

DNA replication inhibitors categories:
Alkylating antineoplastic agents
• An alkylating antineoplastic agent is an alkylating agent
used in cancer treatment that attaches an alkyl group
(CnH2n+1) to DNA.
• The alkyl group is attached to the guanine base of DNA,
at the number 7 nitrogen atom of the purine ring.
• Since cancer cells, in general, proliferate faster and with
less error-correcting than healthy cells, cancer cells are
more sensitive to DNA damage — such as being alkylated.
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• Alkylating agents are used to treat several cancers.
• However, they are also toxic to normal cells (cytotoxic),
particularly cells that divide frequently, such as those in
the gastrointestinal tract, bone marrow, testicles and
ovaries, which can cause loss of fertility.
• Most of the alkylating agents are also carcinogenic.
• Hyperthermia is especially effective at enhancing the
effects of alkylating agents.
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• Some of the substances require conversion into active
substances in vivo (e.g., Cyclophosphamide is one of the
most potent immunosuppressive substances).
• In small dosages, it is very efficient in the therapy of
systemic lupus erythematosus, autoimmune hemolytic
anemias, granulomatosis with polyangiitis, and other
autoimmune diseases.
• High dosages cause pancytopenia and hemorrhagic
cystitis.
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• Dialkylating agents can react with two different 7-N-
guanine residues, and, if these are in different strands of
DNA, the result is cross-linkage of the DNA strands, which
prevents uncoiling of the DNA double helix.
• Busulfan is an example of a dialkylating agent: it is the
methanesulfonate diester of 1,4-butanediol.
Methanesulfonate can be eliminated as a leaving group.
• Both ends of the molecule can be attacked by DNA bases,
producing a butylene crosslink between two different
bases. 2 June 2017Replication Fork - Behzad Milani
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• Monoalkylating agents can react only with one 7-N of
guanine.
• Limpet attachment and monoalkylation do not prevent
the separation of the two DNA strands of the double helix
but do prevent vital DNA-processing enzymes from
accessing the DNA.
• The final result is inhibition of cell growth or stimulation
of apoptosis, cell suicide.
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106
Classical alkylating agents
Alkylating-like
Nonclassical

These include true alkyl
groups, and have been
known for a longer time
than some of the other
alkylating agents.
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• Melphalan
• Chlorambucil
• Ifosfamide
• Bendamustine
• Nitrosoureas
• Carmustine
• Lomustine
• Streptozocin
• Alkyl sulfonates
• Busulfan
• Nitrogen mustards
• Cyclophosphamide
• Mechlorethamine or mustine (HN2) (trade name Mustargen)
• Uramustine or uracil mustard

108
Alkylating-like
Nonclassical

Platinum-based chemotherapeutic
drugs (termed platinum analogues) act
in a similar manner.
These agents do not have an alkyl
group, but nevertheless damage DNA.
They permanently coordinate to DNA
to interfere with DNA repair, so they
are sometimes described as
"alkylating-like".
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• Platinum
• Cisplatin
• Carboplatin
• Nedaplatin
• Oxaliplatin
• Satraplatin
• Triplatin tetranitrate
These agents also bind at N7 of guanine.

110
Alkylating-like
Nonclassical

There is not a perfect
consensus on which items
are included in this
category, but, in general,
they include:
111
• procarbazine
• altretamine

Limitations
• Their functionality has been found to be limited when in
the presence of the DNA-repair enzyme O-6-
methylguanine-DNA methyltransferase (MGMT).
• If the MGMT promoter region is methylated, the cells no
longer produce MGMT, and are therefore more responsive
to alkylating agents.
• Methylation of the MGMT promoter in gliomas is a useful
predictor of the responsiveness of tumors to alkylating
agents.
112

2.Nitrogen mustards
113

Nitrogen mustards
• The nitrogen mustards are cytotoxic chemotherapy
agents similar to mustard gas.
• Although their common use is medicinal, in principle
these compounds can also be deployed as chemical
warfare agents.
• Nitrogen mustards are nonspecific DNA alkylating agents.
114

Nitrogen mustards
• Nitrogen mustard gas was stockpiled by several nations
during the Second World War, but it was never used in
combat.
• As with all types of mustard gas, nitrogen mustards are
powerful and persistent blister agents and the main
examples (HN1, HN2, HN3, see below) are therefore
classified as Schedule 1 substances within the Chemical
Weapons Convention.
• Production and use is therefore strongly restricted.
115

Nitrogen mustards Examples:
• The original nitrogen mustard drug, mustine (HN2), is no
longer commonly in use because of excessive toxicity.
• Other nitrogen mustards developed as treatments
include cyclophosphamide, chlorambucil, uramustine,
ifosfamide, melphalan, and bendamustine.
• Bendamustine has recently re-emerged as a viable
chemotherapeutic treatment.
• Nitrogen mustards that can be used for chemical warfare
purposes are tightly regulated. 2 June 2017Replication Fork - Behzad Milani
116

Nitrogen mustards Examples:
• Their weapon designations are:
• HN1: Bis(2-chloroethyl)ethylamine
• HN2: Bis(2-chloroethyl)methylamine
• HN3: Tris(2-chloroethyl)amine
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Nitrogen mustards Mechanism of action:
• Nitrogen mustards (NMs) form cyclic aminium ions
(aziridinium rings) by intramolecular displacement of the
chloride by the amine nitrogen.
• This aziridinium group then alkylates DNA once it is
attacked by the N-7 nucleophilic center on the guanine
base.
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• A second attack after the displacement of the second chlorine
forms the second alkylation step that results in the formation of
interstrand cross-links (ICLs) as it was shown in the early 1960s.
• At that time it was proposed that the ICLs were formed between
N-7 atom of guanine residue in a 5’-d(GC) sequence.
• These kinds of lesions are effective at forcing the cell to undergo
apoptosis via p53, a protein which scans the genome for defects.
• Note that the alkylating damage itself is not cytotoxic and does
not directly cause cell death.
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• Later it was clearly demonstrated that NMs form a 1,3
ICL in the 5’-d(GNC) sequence.
• The strong cytotoxic effect caused by the formation of
ICLs is what makes NMs an effective chemotherapeutic
agent.
• Other compounds used in cancer chemotherapy that
have the ability to form ICLs are cisplatin, mitomycin C,
carmustine, and psoralen.
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2.Nitrogen mustards
121

Topoisomerase inhibitors: Anthracyclines
• Anthracyclines are a class of drugs used in cancer
chemotherapy extracted from
1. Streptomyces bacterium
2. Streptomyces peucetius var. caesius.
• These compounds are used to treat many cancers,
including leukemias, lymphomas, breast, stomach,
uterine, ovarian, bladder cancer, and lung cancers.
122

• The anthracyclines are among the most effective
anticancer treatments ever developed and are effective
against more types of cancer than any other class of
chemotherapeutic agents.
• Their main adverse effect is cardiotoxicity, which
considerably limits their usefulness.
• Use of anthracyclines has also been shown to be
significantly associated with cycle 1 severe or febrile
neutropenia.
123

• The first anthracycline discovered was daunorubicin
(trade name Daunomycin), which is produced naturally
by Streptomyces peucetius, a species of actinobacteria.
• Doxorubicin (trade name Adriamycin) was developed
shortly after, and many other related compounds have
followed, although few are in clinical use.
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125

Topoisomerase inhibitors: Medical use
• Anthracyclines are used to treat various cancers and as
of 2012 were among the most commonly used
chemotherapeutic agents.
• Doxorubicin and its derivative, epirubicin, are used in
breast cancer, childhood solid tumors, soft tissue
sarcomas, and aggressive lymphomas.
• Daunorubicin is used to treat acute lymphoblastic or
myeloblastic leukemias, and its derivative, idarubicin is
used in multiple myeloma, non-Hodgkin's lymphomas,
and breast cancer.
126

Topoisomerase inhibitors: Medical use
• Other anthracycline derivates include nemorubicin, used
for treatment of hepatocellular carcinoma, pixantrone,
used as a second-line treatment of non-Hodgkin's
lymphomas, sabarubicin, used for non-small cell lung
cancer, hormone refractory metastatic prostate cancer,
and platinum- or taxane-resistant ovarian cancer, and
valrubicin, which is used for the topical treatment of
bladder cancer.
127

Topoisomerase inhibitors: Mechanism of action
• Anthracyclines have four mechanisms of action:
1. Inhibition of DNA and RNA synthesis by intercalating
between base pairs of the DNA/RNA strand, thus
preventing the replication of rapidly growing cancer
cells.
128

2. Inhibition of topoisomerase II enzyme, preventing the relaxing of
supercoiled DNA and thus blocking DNA transcription and
replication. Some sources say that topoisomerase II inhibitors
prevent topoisomerase II turning over which is needed for
dissociation of topoisomerase II from its nucleic acid substrate.
In other words, topoisomerase II inhibitors stabilise the
topoisomerase II complex after it has broken the DNA chain. This
leads to topoisomerase II mediated DNA-cleavage, producing
DNA breaks.
129

3. Iron-mediated generation of free oxygen radicals that
damage the DNA, proteins and cell membranes.
130

4. Induction of histone eviction from chromatin that
deregulates DNA damage response, epigenome and
transcriptome.
131

Topoisomerase inhibitors: Cardiotoxicity
• This cardiotoxicity may be caused by many factors,
which may include inhibition and/or poisoning of
topoisomerase-IIB in cardiomyocytes, interference with
the ryanodine receptors of the sarcoplasmic reticulum,
free radical formation in the heart, or from buildup of
metabolic products of the anthracycline in the heart.
• The cardiotoxicity often presents as ECG changes and
arrhythmias, or as a cardiomyopathy leading to heart
failure.
132

• This cardiotoxicity is related to a patient's cumulative
lifetime dose.
• A patient's lifetime dose is calculated during treatment,
and anthracycline treatment is usually stopped (or at
least re-evaluated by the oncologist) upon reaching the
maximum cumulative dose of the particular
anthracycline.
133

• Dexrazoxane is a cardioprotectant that is sometimes used to
reduce the risk of cardiotoxicity; it has been found to reduce the
risk of anthracycline cardiotoxicity by about two-thirds, without
affecting response to chemotherapy or overall survival.
• The liposomal formulations of daunorubicin and doxorubicin are
less toxic to cardiac tissue than the non-liposomal form because a
lower proportion of drug administered in the liposome form is
delivered to the heart.
• Longer infusion rates will result in a reduced plasma level and a
much lower left ventricular peak concentration.
134

Topoisomerase inhibitors: Neurotoxicity
• At least one study which found lower verbal memory
performance on tests of immediate and delayed recall
suggests that anthracycline may increase the risk for
developing "chemobrain".
135

Topoisomerase inhibitors: Examples
•Aclarubicin •Acridine carboxamide •Amarogentin
•Belotecan •Biosynthesis of doxorubicin •Camptothecin
•Daunorubicin •Doxorubicin •Epirubicin
•Etoposide •Exatecan •Fisetin
•Idarubicin •Irinotecan •Liposomal daunorubicin
•Losoxantrone •Lurtotecan •Mitoxantrone
•Nalidixic acid •Novobiocin •Pirarubicin
•Pixantrone •Rebeccamycin •Rubitecan
•SN-38 •Teniposide •Topoisomerase inhibitor
•Topotecan •Valrubicin •Vosaroxin
•Zorubicin
136

Some DNA replication inhibitors Examples
Acyclovir
• Viral DNA polymerase inhibitor
137
Alternative Names: Aciclovir, Acycloguanosine
Chemical Name:
2-Amino-1,9-dihydro-9-[(2-hydroxyethoxy)methyl]-6H-purin-6-one
Biological Activity
Antiviral agent, active against herpes simplex viruses HSV-1 and
HSV-2 (EC50 values are 0.85 and 0.86 μM respectively). Interferes
with viral DNA polymerization through competitive inhibition with
guanosine triphosphate. Induces apoptosis in cells transfected
with HSV-TK (suicidal gene therapy).

AM-TS23
• DNA polymerase λ and β inhibitor
138
Chemical Name: (5Z)-5-[[(4-[(2-Methylphenyl)thio]-3-
nitrophenyl]methylene]-2-thioxo-4-thiazolidinone
Biological Activity
DNA polymerase λ and β inhibitor (IC50 values are 3.9 and
18.2 μM, respectively). Sensitizes human colorectal cancer
cells to hydrogen peroxide and temozolomide (Cat. No.
2706) in vitro.

Aphidicolin
• DNA polymerase α, δ and ε inhibitor
139
Chemical Name: (3R,4R,4aR,6aS,8R,9R,11aS,11bS)-
Tetradecahydro-3,9-dihydroxy-4,11b-dimethyl-8,11a-
methano-11aH-cyclohepta[a]naphthalene-4,9-dimethanol
Biological Activity
DNA polymerase α, δ and ε inhibitor. Exhibits selectivity
over DNA polymerase β and γ. Antimitotic, antibiotic and
antiviral.

BIBR 1532
• Selective telomerase inhibitor
140
Chemical Name: 2-[[(2E)-3-(2-Naphthalenyl)-1-oxo-2-
butenyl1-yl]amino]benzoic acid
Biological Activity
Selective telomerase inhibitor (IC50 values are 93, >
100000 and > 100000 nM for human telomerase, human RNA
polymerase I and human RNA polymerase II + III
respectively). Causes telomere shortening in exponentially
growing NCI-H460 lung carcinoma cells and eventual growth
arrest.

BRACO 19 trihydrochloride
• Telomerase inhibitor
141
Chemical Name: N,N'-[9[[4-(Dimethylamino)phenyl]amino]-
3,6-acridinediyl]bis-1-pyrrolidinepropanamide
trihydrochloride
Biological Activity
Telomerase inhibitor (IC50 = 115 nM). Inhibits expression of
human telomerase reverse transcriptase (hTERT), induces
cellular senescence and inhibits growth of uterine cancer
cells in vitro. Inhibits growth of uterine tumor xenografts in
mice.

Capecitabine
• Prodrug of 5-Fluorouracil (Cat. No. 3257). Inhibits DNA synthesis
142
Alternative Name: Ro 09-1978
Chemical Name: 5'-Deoxy-5-fluoro-N-
[(pentyloxy)carbonyl]cytidine
Biological Activity
Prodrug of 5-Fluorouracil (5-FU) (Cat. No. 3257).
Selectively activated in tumor cells by thymidine
phosphorylase; inhibits DNA synthesis upon conversion to 5-
FU. Orally available.

Carboplatin
• Inhibitor of DNA synthesis
143
Alternative Names: NSC 241240, Paraplatin, JM 8
Chemical Name: cis-Diammine(1,1-
cyclobutanedicarboxylato)platinum(II)
Biological Activity
Antitumor agent that forms platinum-DNA adducts. Causes
intra- and interstrand DNA crosslinks blocking DNA
replication and transcription. Enhances radiation-induced
single-strand DNA breakage and displays lower
nephrotoxicity than analog cisplatin (Cat. No. 2251).

Costunolide
• Inhibitor of human telomerase activity
144
Chemical Name: (3aS,6E,10E,11aR)-3a,4,5,8,9,11a-
Hexahydro-6,10-dimethyl-3-methylene-cyclodeca[b]furan-
2(3H)-one
Biological Activity
Inhibitor of human telomerase activity (IC50 = 65 μM in
MCF-7 breast cancer cells). Suppresses proliferation and
induces apoptosis in a variety of human tumor cell lines.
Selectively blocks endothelial cell proliferation induced by
VEGF. Inhibits expression of iNOS and IL-1β and disrupts NF-
κB activation. Displays anti-inflammatory, antifungal and
antiviral properties.

Cytarabine
• Nucleoside analog; inhibits DNA replication
145
Alternative Name: Cytosine b-D-arabinofuranoside
Chemical Name: 4-Amino-1-β-D-arabinofuranosyl-2(1H)-
pyrimidinone
Biological Activity
Nucleoside analog of deoxycytidine; inhibits DNA
replication by incorporating into DNA (IC50 = 0.04 μM in
L1210 and CEM cell lines). Displays no inhibitory effects on
RNA synthesis. Causes S phase cell cycle arrest in ML-1 cell
lines; cytotoxic in L5817Y leukemia cells. Antineoplastic
and antileukemic agent.

Daptomycin
• Antibiotic; inhibits
protein, DNA and
RNA synthesis in
gram-positive
bacteria
146

147
Alternative Name: LY 146032
Chemical Name: N-(1-Oxodecyl)-L-tryptophyl-D-asparaginyl-L-α-aspartyl-
L-threonylglycyl-L-ornithinyl-L-α-aspartyl-D-alanyl-L-α-aspartylglycyl-D-
seryl-(3R)-3-methyl-L-α-glutamyl-α,2-diamino-γ-oxo-benzene butanoic
acid (13-4) lactone
Biological Activity
Lipopeptide, calcium-dependent antibiotic. Exhibits potent bacteriocidal
activity against most gram-positive bacteria in vitro and in vivo, including
antibiotic-resistant strains such as MRSA and VRE. Disrupts plasma
membrane function; activity results in membrane depolarization leading
to inhibition of protein, DNA and RNA synthesis.

Dexrazoxane hydrochloride
• Topoisomerase II inhibitor
148
Alternative Name: ICRF-187
Chemical Name: 4-[(2S)-2-(3,5-Dioxopiperazin-1-
yl)propyl]piperazine-2,6-dione hydrochloride
Biological Activity
Topoisomerase II inhibitor and intracellular ion chelator.
Bridges and stabilizes an interface between two ATPase
promoters to inhibit topoisomerase II activity.
Cardioprotective when co-administered with doxorubicin;
decreases formation of reactive oxygen species (ROS) and
activates the PI3K/Akt survival pathway.

Epirubicin hydrochloride
• Inhibits DNA synthesis and function.
• Inhibits DNA topoisomerase II
149
Alternative Name: 4'-Epidoxorubicin
Chemical Name: (8S,10S)-10-[(3-Amino-2,3,6-trideoxy-α-L-arabino-
hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-
methoxy-5,12-naphthacenedione hydrochloride
Biological Activity
Antibiotic antitumor agent. Inhibits the synthesis and function of DNA (IC50 = 62.7
μM in rat glioblastoma cell lines) and inhibits the relaxing property of
topoisomerase II.

Floxuridine
• Disrupts DNA replication; inhibits thymidylate synthetase
150
Alternative Name: FdUrd
Chemical Name: 5-Fluoro-2'-deoxyuridine
Biological Activity
Antineoplastic antimetabolite. Exhibits antiproliferative
activity; inhibits thymidylate synthetase and disrupts DNA
replication in human cells. Induces double-strand DNA
breaks; activates ATR and ATM signaling pathways. Induces
phosphorylation of Chk1 and Chk2 in OVCAR-8 and SKOV3ip
ovarian cancer cell lines.

Fludarabine
• Purine analog; inhibits DNA synthesis
151
Chemical Name: 9-β-D-Arabinofuranosyl-2-fluoro-9H-purin-
6-amine
Biological Activity
Purine analog that inhibits DNA synthesis. Exhibits
antiproliferative activity (IC50 = 1.54 μM in RPMI cells) and
triggers apoptosis through increasing Bax and decreasing
Bid, XIAP and survivin expression. Inhibits cytokine-induced
activation of STAT1 and STAT1-dependent gene
transcription in lymphocytes. Also displays anticancer
activity against hematological malignancies in vivo.

5-Fluorouracil
• Inhibits RNA and DNA synthesis
152
Alternative Name: 5-FU
Chemical Name: 5-Fluoro-2,4-(1H,3H)-pyrimidinedione
Biological Activity
Anticancer agent. Metabolized to form fluorodeoxyuridine
monophosphate (FdUMP), fluorodeoxyuridine triphosphate
(FdUTP) and fluorouridine (FUTP). FdUMP inhibits
thymidylate synthase, causing a reduction in dTMP
synthesis. FUTP and FdUTP are misincorporated into RNA
and DNA respectively.

Gatifloxacin
• Antibiotic; inhibits bacterial type II topoisomerase
153
Alternative Name: AM 1155
Chemical Name: 1-Cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methyl-1-
piperazinyl)-4-oxo-3-quinolinecarboxylic acid
Biological Activity
Fluoroquinolone antibiotic. Inhibits bacterial type II topoisomerases (IC50 values are
0.109 and 13.8 μg/ml for E.coli DNA gyrase and S.aureus topoisomerase IV
respectively). Displays potent activity against gram-positive and gram-negative
bacteria. Stimulates short-term self-renewal in both human and mouse embryonic
stem cells in vitro.

Gemcitabine hydrochloride
• DNA synthesis inhibitor
154
Chemical Name: (+)-2'-Deoxy-2',2'-difluorocytidine
hydrochloride
Biological Activity
Deoxycytidine analog that inhibits DNA synthesis.
Metabolized to form gemcitabine triphosphate (dFdCTP)
and gemcitabine diphosphate (dFdCDP). dFdCTD inhibits
ribonucleotide reductase causing a reduction in cellular
nucleotides. dFdCTP is incorporated in DNA resulting in DNA
strand termination. Displays antitumor activity in vitro and
in vivo.

6-Hydroxy-DL-DOPA
• Allosteric inhibitor of RAD52; also APE1 inhibitor
155
Chemical Name: 2,5-Dihydroxy-DL-tyrosine
Biological Activity
Allosteric inhibitor of RAD52; inhibits RAD52 binding to
single strand DNA binding domains (IC50 = 1.1 μM).
Selectively inhibits proliferation of BRCA-deficient cancer
cells in vitro. Also inhibits APE1.

6-Mercaptopurine
• Purine analog; inhibits DNA and RNA synthesis
156
Chemical Name: Purine-6(1H)-thione
Biological Activity
Inhibitor of de novo purine synthesis through interference
with DNA and RNA synthesis. Immunosuppressive and
antileukemic drug; reduces the anticoagulation elicited by
warfarin. Active metabolite of azathioprine (Cat. No.
4099).

Mithramycin A
• Inhibitor of DNA and
RNA polymerase
157

158
Chemical Name: (1S)-5-Deoxy-1-C-[(2S,3S)-7-[[2,6-dideoxy-3-O-(2,6-dideoxy-β-D-arabino-
hexopyranosyl)-β-D-arabino-hexopyranosyl]oxy]-3-[(O-2,6-dideoxy-3-C-methyl-β-D-ribo-
hexopyranosyl-(1.fwdarw.3)-O-2,6-dideoxy-β-D-lyxo-hexopyranosyl-(1.fwdarw.3)-2,6-dideoxy-
β-D-arabino-hexopyranosyl)oxy]-1,2,3,4-tetrahydro-5,10-dihydroxy-6-methyl-4-oxo-2-
anthracenyl]-1-O-methyl-D-threo-2-pentulose
Biological Activity
Anticancer antibiotic that selectively binds to G-C-rich DNA in the presence of Mg2+ or Zn2+,
inhibiting RNA and DNA polymerase action. Inhibits c-myc expression and induces myeloid
differentiation of HL-60 promyelocytic leukemia cells.

Mitomycin C
159
Alternative Name: Ametycine
Chemical Name: [1aS-(1aα,8β,8aα,8bα)]-6-Amino-8-
[[(aminocarbonyl)oxy]methyl]-1,1a,2,8,8a,8b-hexahydro-
8a-methoxy-5-methylazirino[2',3':3,4]pyrrolo[1,2-a]indole-
4,7-dione
Biological Activity
Antibiotic and antitumor agent. Covalently binds DNA
forming intra- and interstrand crosslinks. Inhibits DNA
synthesis. Also used for MEF/STO feeder layer preparation
in stem cell culture.

Oxaliplatin
160
Alternative Name: Eloxatin
Chemical Name: Oxalato[(1R-trans)-1,2-
cyclohexanediamine]platinum(II)
Biological Activity
Antitumor agent that forms platinum-DNA adducts. Causes
intra- and interstrand DNA crosslinks blocking DNA
replication and transcription. Displays higher cytotoxicity
and lower nephrotoxicity than analog cisplatin (Cat. No.
2251) and shows antitumor activity in cell lines with
acquired cisplatin resistance.

Ribavirin
• Antiviral guanosine analog; blocks eIF4E activity
161
Chemical Name: 1-β-D-Ribofuranosyl-1H-1,2,4-triazole-3-
carboxamide
Biological Activity
Antiviral guanosine ribonucleoside analog; misincorporated
into mRNA by viral-dependent RNA polymerases. Binds to
and redistributes mammalian eIF4E from the nucleus to the
cytoplasm (Ki ~ 0.3 μM for the active metabolite, ribavirin
triphosphate). Represses colony formation of primary AML-
M5 progenitor cells (IC50 ~ 1 μM); reduces disease severity
in acute myeloid leukemia (AML). Orally available.

Trovafloxacin mesylate
• Antibiotic; inhibits bacterial DNA synthesis
162

163
Alternative Name: CP 99219
Chemical Name: 7-[(1α,5α,6α)-6-Amino-3-azabicyclo[3.1.0]hex-3-yl]-1-(2,4-difluorophenyl)-
6-fluoro-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylic acid mesylate
Biological Activity
Fluoroquinolone antibiotic. Inhibits bacterial DNA topoisomerase IV and DNA gyrase and
forms a stable quinolone-DNA complex with these enzymes which reversibly inhibits DNA
synthesis. Displays potent activity against gram-positive and gram-negative bacteria.
Increases the production of mitochondrial NO in immortalized hepatocytes; also increases
mitochondrial Ca2+. Inhibits Panx-1 (IC50 ~ 4μM).

Viomycin
• Antibiotic; inhibits bacterial DNA synthesis
164

165
Chemical Name: (S)-3,6-Diamino-N-((3S,9S,12S,15S,Z)3((2R,4S)-6-amino-4-hydroxy-1,2,3,4-
tetrahydropyridin-2-yl)-9,12-bis(hydroxymethyl)-2,5,8,11,14-pentaoxo-6-(ureidomethylene)-
1,4,7,10,13-pentaazacyclohexadecan-15-yl)hexanamide disulfate
Biological Activity
Member of the tuberactinomycin family of antibiotics. Inhibits group I intron splicing and
prokaryotic protein synthesis. Freezes bacterial ribosomes in either the pre- or post-
translational state. Facilitates trans-cleavage of the Neurospora VS ribozyme.

The End

The replication fork and anti-cancers

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