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Chap. 9 - DRUGS TARGETING NUCLEIC ACIDS DNA & RNA
Types of DRUGS ACTING on DNA (in chapter 9)
Intercalating agents
Topoisomerase poisons
Alkylating agents
Metallating agents
Chain cutters
Chain terminators
Control of gene transcription (and epigenetics)
© Oxford University Press, 2013
1
DRUGS ACTING ON DNA
Intercalating agents
Mechanism of action
• Contain planar aromatic or heteroaromatic ring systems
• Planar systems slip between the layers of nucleic acid pairs and
disrupt the shape of the helix
• A preference is often shown for either the minor or major groove
• Intercalation prevents replication and transcription
• Intercalation can inhibit topoisomerases too
© Oxford University Press, 2013
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Intercalating agents
Proflavine, also called diaminoacridine, is an acriflavine derivative,
a disinfectant bacteriostatic against many gram-positive bacteria. It
has been used in the form of the dihydrochloride and hemisulfate salts
as a topical antiseptic, and was formerly used as a urinary antiseptic.
Proflavine is toxic and carcinogenic in mammals and so it is used only
as a surface disinfectant or for treating superficial wounds.
Proflavine is also known to have a mutagenic effect on DNA by
intercalating between nucleic acid base pairs. It differs from most
other mutagenic components by causing basepair-deletions or basepairinsertions and not substitutions.
NH3 Cl
Ar
NH3 Cl
H2N
N
NH2
Planar tricyclic system
The amino substituents are protonated
Used as a topical antibacterial agent in WW II
Targets bacterial DNA
Too toxic for systemic use
NH3 HOSO3
Ar
NH3 HOSO3
as a salt proflavine
is more stable and
avoids oxidation
© Oxford University Press, 2013
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Intercalating agents: Proflavine
Proflavine
G C
AT
TA
T A
G C
G C
TA
T A
H 3N
N
NH3
sugar phosphate
backbone
T A
AT
AT
T A
C G
G C
TA
T A
T
H 3N
A
proflavine
NH3
G C
GC
GC
T A
A T
C G
O
G
C
O
TA
T A
DNA DOUBLE HELIX
van der Waals interactions
Ionic interactions
© Oxford University Press, 2013
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ethidium bromide
Br
N
NH2
H2 N
fluorescent stain for gel electrophoresis
Ethidium bromide is an intercalating agent commonly
used as a fluorescent tag (nucleic acid stain) in
molecular biology laboratories for techniques such as
agarose gel electrophoresis. When exposed to
ultraviolet light, it will fluoresce with an orange colour,
intensifying almost 20-fold after binding to DNA. It has
been commonly used since the 1950s in veterinary
medicine to treat trypanosomiasis in cattle, a disease
caused by trypanosomes. The high incidence of
antibiotic resistance makes this treatment impractical in
some areas, where the related isometamidium chloride
is used instead. Ethidium bromide may be a mutagen,
although this depends on the organism exposed and the
circumstances of exposure. Approximately 30,000
people in sub-Saharan Africa get African
trypanosomiasis each year and Chagas disease, which
causes 21,000 deaths per year mainly in Latin America.
Protozoan trypanosomes spread by the Tsetse Fly, develops over years
© Oxford University Press, 2013
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Br
N
NH
H
N
N
H2N
NH2
N
isometamidium chloride
Isometamidium is a phenanthridine aromatic amidine with a narrow therapeutic index which has
been marketed for over 30 years as both a prophylactic and a therapeutic trypanocidal agent in the
field. Isometamidium chloride is used curatively at lower dosage rates, and prophylactically at
higher dosage rates.
Indications
Intromidium is indicated for treatment and prevention of trypanosomiasis caused by Trypanosoma
spp. in cattle, goats, sheep, camels, horses and dogs. A preventive dosage ensures protection for 2 to
4 months. When clinical cases occur, the whole group should be treated.
Contra indications
Intromidium should not be administered subcutaneously.
Avoid concurrent administration of other trypanocidal drugs, particularly diminazene aceturate.
Avoid underdosing.
© Oxford University Press, 2013
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Intercalating agents - anticancer agents
Doxorubicin (also called adriamycin)
1. Intercalates via the major groove of DNA double helix.
2. Blocks the action of topoisomerase II by stabilising the DNAenzyme complex
3. Acts as a topoisomerase poison
Doxorubicin is commonly used to treat some leukemias and
Hodgkin's lymphoma, as well as cancers of the bladder, breast,
stomach, lung, ovaries, thyroid, soft tissue sarcoma, multiple
myeloma, and others Many combination therapies are used,
such as (adriamycin, cyclophosphamide),
Liposomal form
There is a pegylated (polyethylene glycol coated) liposomeencapsulated form of doxorubicin, sold as Doxil. Doxil is used
primarily for the treatment of ovarian cancer or AIDS-related
Kaposi's sarcoma where the disease has progressed or recurred
after platinum-based chemotherapy.
Cl
O
O
N
P
NH
Cyclophosphamide
Common side effects include low white blood cell
counts, loss of appetite, vomiting, hair loss, bleeding
from the bladder, an increased future risk of cancer,
infertility, allergic reactions, and pulmonary fibrosis.
Cl
Cyclophosphamide is in the alkylating agent and nitrogen
mustard family of medications. It works by interfering
with the duplication of DNA and the creation of RNA.
Planar rings
g
O
O
OH
CH2OH
OH
OMe
O
OH H
O
H
O
H
HO
Me
H
NH3
H
H
H
Doxorubicin (Adriamycin)
Extra binding to sugar phosphate
backbone by NH3
© Oxford University Press, 2013
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The biosynthesis of
Doxorubicin uses enzymes
found in fat metabolism.
This involves using acetyl
building blocks (2C) as
acetyl CoA. Such molecules
are called “kitids” (often
multiples of 2C). There are
1000s of compounds that
have been discovered from
the random synthesis of
clusters of proteins working
together. Chemists have
tried to splice in various
combinations of such genes
in microorganisms to make
novel drugs as potential lead
compounds
compounds.
a few more steps...
© Oxford University Press, 2013
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Polyethylene glycol (PEG) is a polyether compound with many
applications from industrial manufacturing to medicine. A
comparison of uricase and PEG-uricase. PEG-uricase includes 40
polymers of 10kDa PEG. PEGylation improves its solubility at
physiological pH, increases serum half-life and reduces
immunogenicity without compromising activity. Upper images
show the whole tetramer, lower images show one of the lysines
that is PEGylated.
O
O
Nu
O
O
Nu
O
etc.
O
O
OH
OH
N
O
H2N
O
H
n
OH
O
O
pH = 5
( H2O)
(-H
O
O
O
+3
Fe
H
n
OH
imine (oxime) linkage to the drug through a carbonyl group
OH
OH
O
OH
O
OH
O
O
Cl
O
H
H
O
H
O
n
Cl
phosgene or phosgene equivalent
O
O
O
O
Cl
O
O
O
O
© Oxford University Press, 2013
Intercalating agents - anticancer agents
Dactinomycin
Extra binding to sugar phosphate
backbone by cyclic peptides
planar rings
can intercallate
H
n
9
Dactinomycin
1. Intercalates via minor groove of DNA
double helix
2. Prevents unwinding of DNA double helix
3. Blocks transcription by blocking DNAdependent RNA polymerase
Dactinomycin is a chemotherapy used to treat
many cancers, including
i l di Wilms
Wil tumor,
t
rhabdomyosarcoma, Ewing's sarcoma,
trophoblastic neoplasm, testicular cancer, and
certain types of ovarian cancer. It is given by
injection into a vein. Common side effects
include bone marrow suppression, vomiting,
mouth ulcers, hair loss, liver problems,
infections, muscle pains, future cancers,
allergic reactions, and tissue death at the site of
injection. Use in pregnancy may harm the baby.
Dactinomycin. also known as actinomycin D,
is the most significant member of
actinomycines, which are a class of polypeptide
antitumor antibiotics isolated from soil bacteria
of the genus Streptomyces. It is one of the
older anticancer drugs, and has been used for
many years.
© Oxford University Press, 2013
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Many quinone methides show medicinal properties
O
O
two common
patterns
Can be extended to
naphthalene,
anthracene,
phenanthrene
ortho quinone
methide ppattern
para quinone
methide ppattern
B
resonance
OH
H
Taxodone and its oxidized rearrangement
product, taxodione, are diterpenoid quinone
methides found in Taxodium distichum (bald
cypress), Rosmarinus officinalis (rosemary),
several Salvia species and other plants, that
display anticancer, antibacterial, antioxidant,
antifungal insecticide
antifungal,
insecticide, and antifeedant activities
activities.
O
1e-
O
1eHO
HO
HO
oxidation
lose proton
oxidation
lose proton
H
H
H
H
H
O
H
O
B
O
Taxodone
H
OH
B
O
HO
HO
H
H
N
O
nucleophiles
undergo
conjugate
addition
R
H
O
H2N
R
© Oxford University
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Intercalating agents and chain cutting - Bleomycins
CONH2
H
N
H
primary amine
Fe+2 or Fe+3
NH2
can chelate here
H NH
primary amide
O
pyrimidine N
ring
Me
H
O
HO
O
HO
S
NH
O
HN
N
H
N
O
OH
H
O
OH
O
H H H
N
N
H
Me
A variety of
possibilities.
NH
H
Me
HO
Me
imidazole
ring
Bleomycin A 2
Bleomycin B 2
OH
O
NH2
S
Bithiazole
intercalating
region
H
O
OH
O
R
N
N
H2N
primary amine
O
2
OH
R = NHCH2CH2CH2SMe2
R = NHCH2CH2CH2CH2NHC(NH2)=NH
Notes on bleomycins
y
• Used as anticancer agents
• Intercalated by means of bithiazole ring system
• Ferrous ion then chelated by nitrogens of the primary
amines, amide and pyrimidine ring
• Reaction with oxygen results in a ferric ion and reactive
oxygen species
• Results in radical formation and chain cutting
• Bleomycin prevents DNA ligase from repairing damage
© Oxford University Press, 2013
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Bleomycin is a medication used to treat cancer.
This includes Hodgkin's lymphoma, nonHodgkin's lymphoma, testicular cancer, ovarian
cancer, and cervical cancer among others.
Typically used with other cancer medications, it
can be given intravenously, by injection into a
muscle or under the skin. It may also be
administered inside the chest to help prevent the
recurrence of a fluid around the lung due to
cancer; however talc is better for this.
Common side effects include fever, weight loss,
vomiting, rash, a severe type of anaphylaxis,
possible inflammation of the lungs that can result
in lung scarring
scarring. Chest X-rays
X rays every couple of
weeks are recommended to check for this.
Bleomycin may cause harm to the baby if used
during pregnancy. It is believed to primarily work
by preventing synthesis of DNA.
13
© Oxford University Press, 2013
Free radical damage to DNA can remove bases and
DNA
DNA
DNA
O
Base
O
O
O
Base
O
H
B
Base
O
H
H
O
B
H
O
O
DNA
NH
Bleomycin
HN
Fe+3
O
HN
DNA
Fe+4
DNA
Fe+4
HN
O
O
NH
Bleomycin
Iron is believed to be the metal responsible for the creation of free
radicals (often hydroxyl) because it exists at the highest
concentration of any transition metal in most living organisms.
Free radicals can attack the deoxyribose
y
DNA backbone and
bases, potentially causing a number of lesions that can be
cytotoxic or mutagenic. Cells have developed complex and
efficient repair mechanisms to fix the lesions. In the case of free
radical attack on DNA, base-excision repair is the repair
mechanism used. Free radical reactions with the deoxyribose sugar
backbone (C1, C2, C3, C4 or C5) are initiated by hydrogen
abstraction from a deoxyribose carbon, and the predominant
consequence is eventual strand breakage and base release.
NH
Bleomycin
H
DNA
O
Base
O
O
O
DNA
H
B
DNA strands
can unravel.
© Oxford University Press, 2013
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Topoisomerase medications are split into two main classes: topoisomerase poisons, which target the
topoisomerase-DNA complex, and topoisomerase inhibitors, which disrupt catalytic turnover.
Topo II poisons
Examples of topoisomerase poisons include the following:
eukaryotic type II topoisomerase inhibitors (topo II): amsacrine, etoposide (next slide), etoposide
phosphate, teniposide and doxorubicin. These drugs are anti-cancer therapies.
b t i l type
bacterial
t
II topoisomerase
t
i
inhibitors
i hibit ((gyrase and
d ttopo IV)
IV): fluoroquinolones.
fl
i l
Th
These are
antibacterials and include such fluoroquinolones as ciprofloxacin.
Some of these poisons encourage the forward cleavage reaction (fluoroquinolones), while other poisons
prevent the re-ligation of DNA (etoposide and teniposide).
Ciprofloxacin (later slides) targets prokaryotes topo II a thousand times better than it targets eukaryotic
topo IIs.
O
Topo II inhibitors
H
N
These inhibitors target the N-terminal ATPase domain of topo II and
prevent topo II from turning over. Very few of these inhibitors.
O
N
N
O
N
An example of topoisomerase inhibitors includes :
ICRF-193. The structure of this compound bound to the
ATPase domain is known. The drug binds in a non-competitive
manner and locks down the dimerization of the ATPase domain.
H
ICRF 193
O
© Oxford University Press, 2013
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Topoisomerase poisons - non-intercalating
H
O
Me
HO
Stabilizes the complex between
DNA and topoisomerase enzymes
Used as anticancer agents
Also causes chain cutting
O
O
HO
O
O
4
O
O
O
Etoposide
MeO
4'
OMe
OH
Etoposide is used as a form of chemotherapy for cancers such as Kaposi’s sarcoma, Ewing's sarcoma,
lung cancer, testicular cancer, lymphoma, nonlymphocytic leukemia, and glioblastoma multiforme. It
is often given in combination with other drugs (such as bleomycin). It is also sometimes used in a
conditioning regimen prior to a bone marrow or blood stem cell transplant.
It is given intravenously (IV) or orally in capsule form. If the drug is given IV, it must be done slowly
over a 30- to 60-minute period because it can lower blood pressure as it is being administered. Blood
pressure is checked often during infusing, with the speed of administration adjusted accordingly.
Etoposide forms a ternary complex (3 way) with DNA and the topoisomerase II enzyme (which aids in
DNA unwinding), prevents re-ligation of the DNA strands, and by doing so causes DNA strands to
break. Cancer cells rely on this enzyme more than healthy cells, since they divide more rapidly. This
causes errors in DNA synthesis and promotes apoptosis of the cancer cell.
16
© Oxford University Press, 2013
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Topoisomerase poisons - non-intercalating
H
O
S
HO
O
O
O
HO
O
4
O
Stabilizes the complex
between DNA and
topoisomerase enzymes
Used as anticancer agents
Also causes chain cutting,
similar to etoposide.
O
O
Teniposide
MeO
OMe
4'
OH
Teniposide is a chemotherapeutic medication used in the treatment of childhood acute lymphocytic
leukemia, Hodgkin's lymphoma, certain brain tumours, and other types of cancer. It slows the growth
of cancer cells in the body (by a ternary complex).
When Teniposide is used with other chemotherapeutic agents, it results in severe bone marrow
suppression. Other common side effects include gastrointestinal toxicity, hypersensitivity reactions,
and reversible alopecia (hair loss).
Teniposide is a semisynthetic derivative of podophyllotoxin from the rhizome of the wild mandrake
(Podophyllum peltatum). More specifically, it is a glycoside of podophyllotoxin with a D-glucose
derivative. It is chemically similar to the anti-cancer drug etoposide, being distinguished only by a
thienyl where etoposide has a methyl. Both these compounds have been developed with the aim of
© Oxford University Press, 2013
creating less toxic derivatives of podophyllotoxin.
17
DRUGS ACTING ON DNA - Topoisomerase poisons - non-intercalating
Examples - Camptothecin
O
A
B
N
C
N
D
E
Me HO
O
O
Lactone ring
Notes
• Stabilizes a complex between DNA and
topoisomerase I
• Single-strand breaks accumulate in the chain
• Irreversible double-strand breaks occur during
transcription
• Semi-synthetic analogues used as anticancer
agents
Camptothecin is a cytotoxic quinoline alkaloid which inhibits the DNA enzyme
topoisomerase I (topo I). It was isolated from the bark and stem of Camptotheca
acuminata (Happy tree), a tree native to China and used as a cancer treatment in
Traditional Chinese Medicine. It showed remarkable anticancer activity in preliminary
clinical
li i l trials
i l but
b also
l low
l solubility
l bili and
d (hi
(high)
h) adverse
d
drug
d
reaction.
i Because
B
off these
h
disadvantages synthetic and medicinal chemists have developed numerous syntheses of
Camptothecin and various derivatives to increase the benefits of the chemical, with good
results. Two analogues have been approved and are used in cancer chemotherapy today,
topotecan and irinotecan.
© Oxford University Press, 2013
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Camptothecin, DNA and enzyme complex
Camptothecin binds to the topo I enzyme and
DNA complex by hydrogen bonds resulting in
a ternary complex, and thereby stabilizing it.
This prevents DNA re-ligation and causes
DNA damage which results in apoptosis. The
most important part of the structure is the
E-ring which interacts from three different
positions with the enzyme. The hydroxyl
group in position 20 forms hydrogen bond to
the side chain on aspartic acid number 533 in
the enzyme. It is critical that the configuration
of the chiral carbon is (S) because (R) is
inactive. The lactone is bonded with two
hydrogen bonds to the amino groups on
arginine 364. Toxicity of CPT is primarily a
result of conversion of single-strand breaks
into double-strand breaks during the S-phase
when the replication fork collides with the
cleavage complexes formed by DNA and CPT.
The lactone ring in CPT is highly susceptible
to hydrolysis. The open ring form is inactive
and it must therefore be closed to inhibit topo
I. The closed form is favored in acidic
condition, as it is in many cancer cells
microenvironment. CPT is transported into the
cell by passive diffusion.
19
DNA
A
B
Protein
C
D
E
Drug
R2
R1
R3
O
N
R4
N
O
There are many variations
made with different groups
at R1, R2, R3 and R4
O
OH
© Oxford University Press, 2013
Camptothecin
Topotecan
N
O
HO
O
N
N
N
N
O
O
O
O
SN-38
SN
38 is
i an antineoplastic
i
l i drug.
d
I is
It
i the
h active
i metabolite
b li off irinotecan
ii
(
(an
analog of camptothecin - a topoisomerase I inhibitor) but has 1000 times
more activity than irinotecan itself. SN-38 and its glucuronide are lost into the
bile and feces. It can cause the symptoms of diarrhoea and myelosuppression
experienced by ~25% of the patients administered irinotecan.
irinotecan
(next slide)
N
N
Topotecan (trade name Hycamtin) is a
chemotherapeutic agent that is a
topoisomerase I inhibitor. It is a synthetic,
water-soluble analog of the natural chemical
compound camptothecin. It is used in the
form of its hydrochloride salt to treat ovarian
cancer, lung cancer and other cancer types.
Topotecan was the first topoisomerase I
inhibitor for oral use. Why is it more water
soluble? Loperamide often taken with
topotecan due to severe diarhea side effect.
O
HO
O
O
N
N
O
N
N
O
O
SN-38
O
O
Loperamide
N
O
OH
N
Cl
Loperamide, sold under the brand name
Imodium among others, is a medication used
to decrease the frequency of diarrhea. It is
often used for this purpose in gastroenteritis,
inflammatory bowel disease, and short
bowel syndrome. It is not recommended for
those with blood in the stool. The
medication is taken by mouth.
© Oxford University Press, 2013
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Model of human liver cell showing blood,
bile and intestinal compartments, indicating
tissue involvement of genes in the
irinotecan pathway – eventually leads to
inhibition of DNA replication and
transcription.
Irinotecan-associated diarrhea sometimes
leads to such severe dehydration that it
requires hospitalization. This side-effect is
t t d with
treated
ith an antidiarrheal
tidi h l suchh as
loperamide.
The immune system is adversely impacted
by irinotecan, reflected by dramatically
lowered neutrophils (white blood cells).
The patient may experience a period of
neutropenia while the bone marrow
increases white cell production to
compensate.
Neutrophils
p
make up
p the majority
j y of
circulating white blood cells and serve as
the primary defense against infections by
destroying bacteria, bacterial fragments and
immunoglobulin-bound viruses in the
blood. Patients with neutropenia are more
susceptible to bacterial infections and,
without prompt medical attention, the
condition may become life-threatening
(neutropenic sepsis). Neutropenia can be
acute (temporary) or chronic (long21lasting).
© Oxford University Press, 2013
log Poct/H2O = log [neutral compound]1-octanol
[neutral compound]water
log Doct/H2O = log [all forms compound]1-octanol
[all forms compound]water
The partition-coefficient (P) or distribution-coefficient (D) is the ratio of concentrations of a compound in a mixture of two immiscible
phases at equilibrium. This ratio is a measure of the difference in solubility of the compound in these two phases. The partition-coefficient
generally refers to the concentration ratio of un-ionized species of compound whereas the distribution-coefficient refers to the concentration
ratio of all species of the compound (ionized plus un-ionized) and depends on pH. Usually one phase is water and the other is 1-octanol. The
partition coefficient measures how hydrophilic or hydrophobic a chemical substance is. Hydrophobic drugs with high octanol/water partition
coefficients are mainly distributed to hydrophobic areas such as lipid bilayers of cells, while low octanol/water partition coefficients) are found
primarily in aqueous regions such as blood serum.
solubility and partition coefficients of straight chain functiona groups in 100 g water
OH
OH
OH
OH
CH3
100%
miscible
solubility =
-0.74
log Poct/H2O =
100%
miscible
-0.38
C
solubility =
+0.25
+0.84
100%
miscible
100%
miscible
-0.54
-0.32
C7H15OH
C8H17OH
C9H19OH
0.0058 mol
0.6g
0.0008 mol
0.09g
0.0058 mol
0.6g
0.0008 mol
0.09g
O
C
100%
miscible
100%
miscible
+0.33
0 33
+0.79
0 79
+4.02
+2.97
O
O
C
C
OH C6H13
OH C5H11
OH C4H9
O
+2.62
+2.03
C
OH C3H7
O
O
O
C6H13OH
O
C
OH C2H5
C5H11OH
0.030 mol
2.6g
+1.51
O
C
OH H3C
log Poct/H2O =
0.11 mol
7.9g
O
O
H
100%
miscible
O
C
OH C7H15
OH
4.9g
1.1g
0.2g
(guess)
0.07g
+1 39
+1.39
+1 92
+1.92
+2 6
+2.6
(guess)
+3 05
+3.05
O
O
O
O
C
H
solubility =
log Poct/H2O =
H
H
H
H
H
100%
miscible
100%
miscible
100%
miscible
100%
miscible
100%
miscible
27g
+0.35
+0.45
+0.59
-0.24
+0.88
+0.29
soluble (?)
+1.3
(guess)
6g
+0.84
web site with lots of partition coef. values: https://www.nist.gov/sites/default/files/documents/srd/jpcrd367.pdf
© Oxford University Press, 2013
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log Poct/H2O = log [compound]1-octanol
[compound]water
There is good correlation
of calculated partition
coefficients with
experimental partition
coefficients
log D
1.5
1.0
What might a distribution coefficient curve
look like for the following compound?
0.5
O
pH
0.0
2
H2N
OH
4
6
8
10
12
14
-0.5
-1.0
-1.5
© Oxford University Press, 2013
23
Topoisomerase poisons - non-intercalating
Examples - Quinolones and fluoroquinilones
O
O
F
CO2H
Me
N
N
CH2CH3
Nalidixic acid
N
CO2H
N
HN
Ciprofloxacin
• Synthetic agents used as antibacterial agents
• Stabilise complex between bacterial DNA and topoisomerases
• Binding site for agents revealed once DNA strands are ‘nicked’
The quinolones are a family of synthetic broad-spectrum antibiotic drugs. They can be isolated from
natural sources (such as plants, animals and bacteria) and act as natural antimicrobials.
Quinolones exert their antibacterial effect by preventing bacterial DNA from unwinding and
duplicating. The majority of quinolones in clinical use are fluoroquinolones, which have a fluorine
atom attached to the central ring system, typically at the 6-position or C-7 position.
Fluoroquinolones are especially important for hospital-acquired infections in which resistance to
older antibacterial classes is suspected. Treatment guidelines recommend minimizing the use of
fluoroquinolones and other broad-spectrum antibiotics in less severe infections and in those in which
risk factors for multidrug resistance are not present. Side effects are severe so quinolones are
contraindicated unless no other safe alternative antibiotic exists.
© Oxford University Press, 2013
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Tertiary Structure
1.
2.
3.
4.
5.
Double helix coils into a 3D shape – supercoiling
Double helix has to unravel during replication
Unravelling leads to strain
Relieved by enzyme-catalyzed cutting and repair of DNA chain
Quinolone and fluoroquinolone antibacterial agents inhibit this enzyme
Nalidixic acid (1962) was the first of the
quinolone family of antibiotics.
Nalidixic acid (1962)
1
2
N
8
N
8a
CH3
7
3
HO
O
6
4a
4
5
O
c
N
a
N
b N
O
Ciprofloxacin (1980)
HO
F
O
Which "N" is most basic?
a, b, c
d all are similar bases
H
The quinolones are a family of synthetic broadspectrum antibiotic drugs. Quinolones, and
derivatives, have also been isolated from natural
sources (such as plants, animals and bacteria)
and can act as natural antimicrobials and/or
signalling molecules. Quinolones prevent
bacterial DNA from unwinding and duplicating.
Ci
Ciprofloxacin
fl
i (1980) is
i used
d to
t treat
t t a number
b
of bacterial infections including bone and joint
infections, intra abdominal infections, certain
type of infectious diarrhea, respiratory tract
infections, skin infections, typhoid fever, and
urinary tract infections, among others. A single
F at positoin 6 greatly increased both activity and
cellular uptake. The piperizine ring (ionized) at
position 7 improved pharmacokinetics.
Replacement of the N8 nitrogen reduced adverse
© Oxfordagains
University
2013
reactions and increased activity
S. Press,
aureus.
25
Topoisomerase poisons - non-intercalating
Examples - Quinolones and fluoroquinilones
Topoisomerase
enzyme
Structure of bacterial DNA gyrase
complexed with DNA and two
ciprofloxacin molecules (green)
Region binding to DNA
R5
Region
binding
to enzyme
O
O
R6
O
R7
X8
Region
binding
t enzyme
to
N
R1
Fluoroquinolones
Stacking domain
•Drug molecules are stacked in the bound complex
• Bound to DNA and enzyme by hydrogen and ionic bonds
© Oxford University Press, 2013
26
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Topoisomerases I form a covalent intermediate in which the active site
tyrosine becomes attached to the 3' phosphate end of the cleaved strand
rather than the 5' phosphate end.
The TOP1 protein of humans has been subdivided into four regions. The
N-terminal 214 amino acids are dispensable for relaxation of supercoiling
activity in vitro. The N-terminal domain is followed by a highly conserved,
421 amino acid core domain containing all of the catalytic residues
except
p the active site tyrosine.
y
This is followed byy a ppoorly
y conserved
linker domain of 77 amino acids. Finally there is a 53 amino acid Cterminal domain. The active site Tyr723 is found within the C-terminal
domain.
Topoisomerase I
After the TOP1 covalently attaches to the 3' end of the broken strand,
supercoiling of the DNA is relaxed by controlled rotation of DNA about the
intact strand. Then the 5' hydroxyl end of the broken DNA strand can
reverse the phosphotyrosyl bond, enabling the release of TOP1 and
religation of the DNA. The nicking and closing reactions are fast, and
about 100 cycles
y
can occur p
per second.
Topoisomerase II,
shows DNA too
One of the first inhibitors shown to target TOP1 is irinotecan. Irinotecan
is an analogue of the cytotoxic natural alkaloid camptothecin, obtained from
the Chinese tree Camptotheca acuminata. Irinotecan is especially effective
through its metabolic product SN-38. Irinotecan and SN-38 act by
trapping a subset of TOP1-DNA cleavage complexes, those with a guanine
+1 in the DNA sequence. One irinotecan or SN-38 molecule stacks against
the base pairs flanking the topoisomerase-induced cleavage site and poisons
(inactivates) the TOP1 enzyme.
© Oxford University Press, 2013
27
DRUGS ACTING ON DNA Alkylating agents
Nucleophilic atoms on nucleic acid bases
NH2
7
N
N
H2N
N
N
3
O
N
H
N
H2N
R
1
N
Cytosine
electrophiles
NH2
N
5
R
Guanine
electrophiles
N
O
R
R
electrophiles
NH2
E
E
N
N
N
nucleophilic
atoms
NH2
3 N
N
N
Adenine
E
nucleophilic
atoms
O
H
N
1N
nucleophilic
atoms
nucleophilic
atoms
N
R
https://en.wikipedia.org/wiki/Alkylating_antineoplastic_agent
N
O
N
R
© Oxford University Press, 2013
28
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Alkylating agents
• Contain highly electrophilic groups
• Form covalent bonds to nucleophilic groups in DNA
(e.g. 7-N of guanine)
• Prevent replication and transcription
• Useful anticancer agents
• Toxic side effects ((e.g.
g alkylation
y
of pproteins))
• Can cause interstrand and intrastrand crosslinking if two electrophilic groups present
• Alkylation of nucleic acid bases can result in miscoding
Crosslinking is possible for doubly electrophilic compounds
X
X
X
Nu
X
Nu
Nu
Nu
Nu
Nu
Nu
Nu
Intrastrand crosslinking
Interstrand crosslinking
© Oxford University Press, 2013
29
Alkylating agents
Normal base pairing
Cytosine
Miscoding resulting from
alkylated nucleic acid bases
Thymine
Guanine
Alkylated guanine
DRUG
O
NH2
N
NH
R
N
R
O
N
4
5
O
Normally, has 2 Hbonds with adenine
6
4
5
H
N
1
2
H
Cytosine
7
N
5
O
2
N
1
H
Thymine
O
9 N
4
R
H2N
Changes charge
and changes shape.
H
6
7
N1
3
N
5
N1
H
8
6
N
O
NH2
N3
N
N
Abnormal base pairing.
Alkylated guanine prefers enol
tautomer
How many possible tautomers are there for DNA bases?
H
N
N
Guanine prefers
keto tautomer
H
HO
N
H2N
O
O
N
HN
N
N
R
Me
2
N
3
H
Adenine
8
9
N
H
4
N
3
Guanine
2
N
H
© Oxford University Press, 2013
30
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Bendamustine is a nitrogen mustard used in the treatment
of chronic lymphocytic leukemia and lymphomas. It
belongs to the family of drugs called alkylating agents. It
is also being studied for the treatment of sarcoma. It is
also being investigated for the non-cancer diseases.
Alkylating agents
Chlormethine (nitrogen mustard)
CH3
Cl
Me
N
N
Cl
N
N
Cl
Cl
• Secretly used medicinally in 1942 (WW II), reduced
white blood count, many side effects, including increased cancer risk later
• Causes intrastrand and interstrand cross-linking
• Prevents replication
• Mono-alkylation of guanine also possible
• Analogues with better properties have been prepared
Chlormethine is also used to synthesize pethidine. How might this be done? In 1975 pethidine
was opiod of choice of 60% of doctors for acute pain and 22% for chronic severe pain
Cl
O
?
N
O
N
Cl
chlormethine
Alkylating agents
31
© Oxford University Press, 2013
pethidine
How can DNA heal itself? Are there other possible
leaving groups? How do they compare to chloride? Will
they survive a journey through the body?
Chlormethine
Mechanism of action
DNA
DNA
O
G = Guanine
H
N
NH2
Cl
H
N
O
N
N
H3C
N
H3C
N
N
H3C
G
Cl
NH2
N
N
N
N
Cl
Cl
Aziridine ion
Mechlorethamine
N
N
NH
N
NH2
N
G
N
DNA
DNA
NH2
N
NH2
N
Crosslinked DNA
N
N
H3C
H
N
O
H
N
NH2
NH
O
O
O
N
N
N
N
N
N
NH2
NH
N
O
N
N
CH3
N
NH2
N
NH
O
© Oxford University Press, 2013
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Alkylating agents
O
O
Cl
Nitrosoureas
Cl
N
O
Cl
N
H
N
N
O
Lomustine
N
H
N
Carmustine
Mechanism
Decompose in body to form an alkylating agent and a carbamoylating agent
O
Protein-Lys-NH2
O C N R
Isocyanate
R
N
N
N
Carbamoylation
O C N R
Isocyanate
(carbamoylating
agent)
+
O
Cl
Protein-Lys-NH
H
H
Cl
Alkylating
agent
Cl
O
N
OH
Cl
DNA
Cl
X
X
Y
Y
Crosslinking
Alkylation
Alkylating
agent
DNA
R
N2 + HO
N
O
HN
Notes
• Alkylating agent causes interstrand
crosslinking
• Crosslinking between G-G or G-C
• Carbamoylating agent reacts with
lysine residues on proteins
• May inactivate DNA repair enzymes
DNA
33
© Oxford University Press, 2013
Alkylating agents
O
H3C
Busulfan
S
different length tethers are possible
O
O
OMs
=
S
O
MsO
CH3
O
O
Synthetic agent used as anticancer agent (dimesylate). Causes interstrand crosslinking
O
O
Mechanism
MsO
OMs
O
CH3
Very stable anion
leaving group, could it
be made even better?
O
Busulfan
N
HN
N
Guanine
N
N
N
H2N
S
OMs
N
N
N
DNA
DNA
N
N
DNA
DNA
N
DNA
© Oxford University Press, 2013
34
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Alkylating agents
Dacarbazine
HN
N
H3C
N
N
• Prodrug activated by demethylation in liver
• Decomposes to form a methyldiazonium ion
• Alkylates guanine groups (methylates here)
CONH2
N
CH3
Mechanism
AIC
HN
N
HN
Cyt P-450
N
HN
-CH2O
N
HN
N
liver
N
H3C
N
N
CONH2
N
N
N
CH3
H
N
CONH2
N
H
CONH2
H2N
CONH2
H
CH3
CH3
O
NH
N
N CH3
Methyldiazonium ion
oxidation with
cytochrom P-450
diazomethane (very toxic and explosive gas), usually made as basic ether solution using Diazald.
O
O
H
H
Diazald
N
S
N2 + CH3
O
O
N
N
N
OH
H2C
CH2
H
H
O
N
N
N
CH3
H
O
H
N
base
acid
other carbocations
are possible
H2C
diazomethane
© Oxford University Press, 2013
35
DRUGS ACTING ON DNA - Alkylating agents
Example - Mitomycin C
O
Guanine
N
O
HN
NH2
O
N
N
O
H2N
O
OH
CH3
How?
DNA
NH
H2 N
H
N
H
N
O
Guanine
N
H3 C
O
NH
N
H3 C
OH
Where are the
electrophilic sites?
N
NH2
N
N
DNA
• Prodrug activated in the body to form an alkylating agent
• One of the most toxic anticancer drugs in clinical use
Mitomycin C is a mitomycin that is used as a chemotherapeutic agent by virtue of its
antitumour activity. It is given intravenously to treat upper gastro-intestinal cancers (e.g.
esophageal carcinoma), anal cancers, and breast cancers, as well as by bladder instillation
for superficial bladder tumours. It causes delayed bone marrow toxicity and therefore it is
usually administered at 6-weekly intervals. Prolonged use may result in permanent bonemarrow damage. It may also cause lung fibrosis and renal damage.
© Oxford University Press, 2013
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Coenzyme Q10, also known as ubiquinone, is a coenzyme that is ubiquitous in the bodies of most animals. It is a
1,4-benzoquinone, where Q refers to the quinone chemical group and 10 refers to the number of isoprenyl chemical
subunits in its tail. This fat-soluble substance, which resembles a vitamin, is present in most eukaryotic cells,
primarily in the mitochondria. It is a component of the electron transport chain and participates in aerobic
cellular respiration, which generates energy in the form of ATP. Ninety-five percent of the human body's
energy is generated this way. There are three redox states of CoQ10: fully oxidized (ubiquinone), semiquinone,
and fully reduced (ubiquinol). The capacity of this molecule to act as a two-electron carrier (moving between the
quinone and quinol form) and a one-electron carrier (moving between the semiquinone and one of these other
forms) is central to its role in the electron transport chain due to the iron–sulfur clusters that can only accept one
electron at a time, and as a free radical-scavenging antioxidant.
H
B
H
H
O
O
O
CH3
O
CH3
O
H3C
eR
O
CH3
O
H3C
+one electron
+one proton
H3C
H3C
H3 C
R
O
both directions
+one electron
+one proton
e-
O
H3C
R
O
both directions
O
O
ubiquinone (CoQ10)
oxidized
B
H
ubiquinol (CoQ10)
reduced
H
semiquinone (CoQ10)
a mycolic acid
37
© Oxford University Press, 2013
H
O
OH
CH2OCONH2
H2N
H2N
OMe
N
Me
NH
-MeOH
N
NH
N
Me
O
H
H
C
H
H2N
-H +
Ring
opening
Me
NH2
O
OH
CH2OCONH2
CH2
H2N-DNA
H2N
H2N-DNA
N
NH2
OH
NH
OH
OH
O
CH2OCONH2
H2N
OMe
Reduction
NADH Me
O
O
CH2OCONH2
NH-DNA
N
Me
NH2
OH
Alkylating agent
O Guanine
N
HN
NH
NH-DNA
OH
CH2
OH
NH2
OH
N
HN
N
N
Me
N
N
O Guanine
NH
NH-DNA
Me
N
CH2
H2N
H2N
-CO2
-NH3
OH
N
NH2
Crosslinked DNA
© Oxford University Press, 2013
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Biosynthesis of mitomycins – working on a handout (how about you?)
In the bacterium Legionella pneumophila, mitomycin C induces competence for transformation. Natural transformation
is a process of DNA transfer between cells, and is regarded as a form of bacterial sexual interaction. In microbiology,
genetics, cell biology, and molecular biology, competence is the ability of a cell to take up extracellular ("naked") DNA
from its environment in the process called transformation. Competence may be differentiated between natural
competence, a genetically specified ability of bacteria which is thought to occur under natural conditions as well as in
the laboratory, and induced or artificial competence, which arises when cells in laboratory cultures are treated to make
them transiently permeable to DNA. In the natural world DNA usually becomes available by death and lysis of other
p
byy the researcher,, often as a genetically
g
y engineered
g
fragment
g
or plasmid.
p
Duringg
cells,, but in the laboratoryy it is provided
uptake, DNA is transported across the cell membrane(s), and the cell wall if one is present. Once the DNA is inside the
cell it may be degraded to nucleotides, which are reused for DNA replication and other metabolic functions.
Alternatively it may be recombined into the cell’s genome by its DNA repair enzymes. If this recombination changes the
cell’s genotype the cell is said to have been transformed.
O
CO2H
O
HO
CO2H
P
O
O
-2
OH
O3PO
CO2H
O
O
O
How?
How?
H
How?
H
HO
OPO3-2
OH
OH
PEP
phosphoenolpyruvate
4-phosphoerythrose
HO
NH2
NH2
O
OH
OH
amino DHQ
amino DAHP
NH2
aromatic intermediate
O
mitomycin C
OH
OH
OH
OH
OH
O
O
OH
How?
H2N
OH
H
OH
OH
HO2C
NH2
NH2
CH3
NH
NH2
© Oxford University
O
Press, 2013
NH3
Cisplatin
Pt
NH3
O
O
NH3
Pt
O
NH3
Carboplatin
39
• Neutral inactive molecule acting as a prodrug
• Platinum covalently linked to chloro substituents
• Ammonia molecules act as ligands (RNH2)
• Activated in cells with low chloride ion concentration
• Chloro substituents replaced with neutral water ligands
• Produces positively charged species
• Binds to DNA in regions rich in guanine units
• Intrastrand links rather than interstrand
• Localised
L li d unwinding
i di off DNA double
d bl helix
h li
• Inhibits transcription
Metallating agents
O
N
H3C
mitosane core
DRUGS ACTING ON DNA
Cl
O
How?
N
H3C
aromatic intermediate
glucosamine
Cl
NH2
O
Cisplatin, cisplatinum, platamin, neoplatin, cismaplat or cisdiamminedichloridoplatinum(II) (CDDP) is a chemotherapy drug. It was the first
member of a class of platinum-containing anti-cancer drugs, which now also
includes carboplatin and oxaliplatin. These platinum complexes react in the body,
binding to DNA and causing the DNA strands to crosslink, which ultimately
triggers cells to die in a programmed way.
Carboplatin, is a chemotherapy drug used against some forms of cancer (mainly ovarian carcinoma, lung, head and
neck cancers as well as endometrial, esophageal, bladder, breast and cervical; central nervous system or germ cell
tumors; osteogenic sarcoma, and as preparation for a stem cell or bone marrow transplant). It has vastly reduced
side effects compared to cisplatin. Cisplatin and carboplatin belong to the group of platinum-based antineoplastic
agents, and interact with DNA to interfere with DNA repair.
Cl
NH3
Pt
Cl
H2O
NH3 +
H2O
Pt
NH3
Cl
NH3
H2O
+
NH3
Pt
H2O
NH3
2+
DNA
NH3
DNA
Pt
DNA
NH3
Cisplatin
© Oxford University Press, 2013
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DRUGS ACTING ON DNA Chain cutters
NH2
CONH2
NH2
N
H
N
O
CH3
N
O
HO
CH3 HN
NH
S
N
O
N
H
CH3 HO
CH3
S
N
O
HO
R
N
O
H
N
O
H2N
Bleomycin
used vs skin cancer
(see slides 12-13)
O
BLEOMYCIN A2
BLEOMYCIN B2
OH
O
N
H
O
OH
Bleomycin is mostly used to treat cancer. This
includes Hodgkin's disease, non-Hodgkin's disease,
testicular cancer, ovarian cancer, and cervical cancer.
It can be given intravenously, by injection into a
muscle or under the skin. It may also be
administered inside the chest to help prevent the
recurrence of a fluid around the lung due to cancer
OH
OH
O
OH
NH2
O
R = NHCH2CH2CH2SMe2
R = NHCH2CH2CH2CH2NHC(NH2)=NH
• Intercalating agent (shown earlier)
• Abstracts H from DNA to generate radicals using oxidized iron complex
• Radicals react with oxygen resulting in chain cutting
• Bleomycin also inhibits repair enzymes
41
© Oxford University Press, 2013
• Generates DNA diradical
• DNA diradical reacts with oxygen
• Results in chain cutting
Chain cutters
CH3
O
H3 C
I
S
O
OMe
O
H3 C
S
H
O
Enediyne
ed y e
system
O
OH
H3 C
O
H
N
Calicheamicin 1I
Antitumour agent
O
OMe
H3 C
HO
MeO
O
NHCO2Me
S
S
H3 C
O N
HHO
OH
HO
MeO
The calicheamicins are a class of enediyne antitumor antibiotics derived from the bacterium
Micromonospora echinospora, with calicheamicin g1 being the most notable. It was isolated originally
in the mid-1980s from the chalky soil, or "calichi pits", located in Kerrville, Texas. It is extremely
toxic to all cells . N-acetyl dimethyl hydrazide calicheamicin was developed and marketed as targeted
therapy against the non-solid tumor cancer acute myeloid leukemia (AML). Calicheamicin 1 and the
related enediyne esperamicin are the two of the most potent antitumor agents known.
Bergman reaction (1972)
D
H
H
H
D
H
D
H
D
D
?
H
H
D
proposed
diradical
intermediate
D
H
© Oxford University Press, 2013
D
42
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© Oxford University Press, 2013
43
Chain cutters
Mechanism
H
O
HO
O
HO
NHCO2Me
S
NHCO2Me
S
S
R
O
HO
Michael
addition
NHCO2Me
S
R
R
MeS
Nu
Cycloaromatisation
O
O
HO
HO
NHCO2Me
H
NHCO2Me
DNA
DNA
(Diradical)
S
R
O2
S
R
H
Oxidative
cleavage
© Oxford University Press, 2013
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Neocarzinostatin is a macromolecular
chromoprotein enediyne antitumor antibiotic
secreted by Streptomyces
macromomyceticus. It consists of two
parts, a labile chromophore (shown) and a
113 amino acid protein to which the
chromophore is tightly and non-covalently
bound with high affinity (Kd ~ 10-10 M). The
non-protein
non
protein component is a very potent
DNA-damaging agent; However it is
extremely unstable and the role of the
protein is to protect it and release it to the
target DNA. Opening of the epoxide under
reductive conditions present in cells creates
favorable conditions for a Bergman
cyclization, that leads to formation of
benzyne, followed by DNA strand cleavage.
As a medicine it is among the most potent
known, and in Japan it is the only analog
used clinically (against liver cancer).
Neocarzinostatin likely evolved to kill
bacteria that compete with the producing
organism. Because it achieves this by
causing DNA damage, however, it is capable
of harming tumor cells, as well and was
developed for its anticancer properties.
© Oxford University Press, 2013
45
DRUGS ACTING ON DNA Chain terminators
O
Azidothymidine (AZT)
(Zidovudine;Retrovir)
O
HN
N
O
O
N
O
O
O
HO P O P O P O
HO
O
CH3
HN
CH3
OH
OH
O
OH
N3
N3
Chain terminating group
• Azidothymidine is a prodrug used in the treatment of HIV
• AZT is phosphorylated to a triphosphate in the body
• Triphosphate has two mechanisms of action
- inhibits a viral enzyme (reverse transcriptase)
- is added to growing DNA chain and acts as chain terminator
Azidothymidine (AZT) reduces the replication of the virus and leads to improvements in both
symptoms and blood tests. It can also be used to prevent HIV transmission, such as from mother to
child during the period of birth or after a needle stick injury. Used by itself in HIV-infected patients,
AZT slows HIV replication in patients, but does not stop it entirely. HIV may become AZT-resistant
over time, and therefore AZT is now usually used in conjunction with other anti-HIV drugs in the
combination therapy called highly active antiretroviral therapy (HAART = highly active
antiretroviral therapy).
© Oxford University Press, 2013
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O
O
O
O
P
H2N
N
P
O
OH
O
O
O
O
O
P
NTP
O
O
N
O
P
O
O
N
HN
O
N
O
N
N
N
N
O
P
O
N
O
O
NH
Azidothymidine (AZT)
Chain
terminating
group
O
P
O
O
O
O
O
O
P
O
O
O
NH2
O
P
O
N
O
B
O
N
HN
O
H2N
O
N
N
O
O
P
P
O
O
Growing
chain
O
O
Aciclovir
Chain
terminating
group
O
Usual way of
adding bases
O
O
O
O
P
O
O
N
N
HN
Template
P
O
O
N
H2N
N
H
N
H
R
P
O
N
O
O
H
O
O
R
N
N
R
N
H
R
B
H
B
H
N
N
R
N
N
H
Azides are very reactive and lose N2 easily (best leaving group in chemistry)
© Oxford University Press, 2013
47
Chain terminators
O
N
N
HN
AcO
H2 N
O
HO
Acyclovir
(Zovirax)
N
N
N
Chain
terminating
group
Acyclovir, is an antiviral medication. It is
primarily used for the treatment of herpes
simplex virus infections, chickenpox, and
shingles.
hi l
OAc
Famciclovir
(Famvir)
N
N
NH2
Chain
terminating
group
Famciclovir is a guanosine analogue
antiviral drug used for the treatment of
various herpesvirus infections, most
commonly for herpes zoster (shingles).
Notes:
• Prodrugs used as antiviral agents
• Same mechanisms of action as AZT
• Used vs herpes simplex and shingles
© Oxford University Press, 2013
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Chain terminators
a) Normal replication
C
C
C
C
A
A
A
A
G
C
G
T
A
A
T
DNA
template
b) Chain termination
P
G
P
P
3'
OH
Growing
chain
C
C
C
A
A
A
A
T
3'
3
OH
Growing
chain
A
P
G
C
Drug
T
A
A
T
DNA
template
G
T
DNA
template
C
A
C
P
3'
OH
Growing
chain
P
Chain termination
G
C Drug
T
A
A
T
DNA
template
3'
H
Growing
© chain
Oxford University Press, 2013
49
Mechanism of reverse transcription in class VI virus ssRNA-RT,
human immunodeficiency virus (HIV). Colors mark complementary
sequences (not drawn to scale). Reverse transcription occurs in the
cytoplasm of host cell. In this process, viral ssRNA is transcribed
by the viral reverse transcriptase protein (RT) into double
stranded DNA. Reverse transcription takes place in 5'  3'
direction. tRNA ("cloverleaf") hybridizes to PBS and provides -OH
group for initiation of reverse transcription. Key: (U3 - promoter
region),(U5 - recognition site for viral integrase); (PBS - primer
binding site);(PP - polypurine section ).
1) Strong stop complementary DNA (cDNA) is formed.
2) Template in RNA:DNA hybrid is degraded by RNase H domain of
reverse transcriptase
3) DNA:tRNA is transferred to the 3'-end of the template (synthesis
"jumps").
4) First strand synthesis takes place.
5) The rest of viral ssRNA is degraded by RNase H, except for PP
site.
6) Synthesis of second strand of ssDNA is initiated from the 3'-end of
the template. tRNA is necessary to synthesis of complementary PBS
7) tRNA is degraded
8) After another "jump", PBS from the second strand hybridizes with
the complementary PBS on the first strand.
9) Synthesis of both strands is completed by the DNAP function of
reverse transcriptase.
Both dsDNA ends have U3-R-U5 sequences, so called long terminal
repat sequences (3'LTR and 5'LTR, respectively).
LTRs mediate integration of the retroviral DNA into another region
of the host genome. Very error prone because no proof reading.
50
© Oxford University Press, 2013
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DRUGS ACTING ON DNA Control of gene transcription
5'
3'
Notes:
• Design of synthetic
molecules capable of
controlling gene
transcription
• Molecules capable of
recognising
g
g and binding
g to
specific base pairs
• Hairpin polyamides
containing heterocyclic
rings are capable of
binding to the minor
groove
• Binding involves amide
groups and heterocycles
• Particular patterns of
h t
heterocyclic
li rings
i
allow
ll
recognition of perticular
base pairs
• Capable of inhibiting
transcription
• Designed to bind to
regulatory element of a
gene
O
Me
N
Linker
O
N
Py
A
T
N
H
O
H
N
C
H
Me
N
Me
Im N
N
G
O
Py
H
A
N
N
T
H
H
Hp
O
O
N
Me
Arm
Me
T
O
N
Hp
A
H
H
N
G
C
O
Py
N
O
T
N
Me
Arm
N
H
M
Me
H
A
N
N Im
Me
H
N
N
H
O
Py
H
N
Me
N
O
Me
O
© Oxford University Press, 2013
51
DRUGS ACTING ON rRNA
Antibiotics – attack bacteria
NH
NH
H2N
C
NH
HN
H
C
NH2
H
OH
HO H
OH H
HO
H
Me
OH
H
H
N
Me
O2N
H
O
CH2OH
R
O
HN H
O
Chloramphenicol
(vs typhoid)
H
Me
H
H
H
OMe
O
C
H
O
O
HO
CH3
HO
OH
Me
O
HO
NH2
HO
NMe2
CH3
OH
Cl
H
O
H3C
OH
H
OH
Rifamycins
O
O
Me
Me H OH
O
OH
O
CH2OH
H MeHN
HO
H
H
Streptomycin
OH
H
OH
Me
H
Me
Me
O
CHCl2
O
O
CHO
H
OH
Me
C
OH
H
O
H3C
O
CH3
O
CH3
CH3
O
O
H
CH3
NMe2
Chlortetracycline
(Aureomycin)
OH
O
Me
Erythromycin
OMe
© Oxford University Press, 2013
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HO H
O2N
CH2OH
HN H
C
CHCl2
O
Chloramphenicol
(vs typhoid)
Chloramphenicol is an antibiotic useful for the treatment of a number of bacterial infections. This includes as an eye
ointment to treat conjunctivitis. By mouth or by injection into a vein it is used to treat meningitis, plague, cholera, and
typhoid fever (largely drug resistant now). Monitoring of blood levels of the medication and blood cell levels every
two days is recommended during treatment. Common side effects include bone marrow suppression, nausea, and
diarrhea. The bone marrow suppression may result in death. To reduce the risk of side effects treatment duration should
be as short as possible. It has excellent blood-brain barrier penetration. Chloramphenicol is active against the three
main bacterial causes of meningitis: Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae.
Chloramphenicol is extremely lipid-soluble; it remains relatively unbound to protein and is a small molecule. It
penetrates effectively into all tissues of the body, including the brain. Chloramphenicol increases the absorption of iron.
© Oxford University Press, 2013
OH
The rifamycins are a group of antibiotics that are synthesized either naturally
by the bacterium Amycolatopsis rifamycinica or artificially. They are a
subclass of the larger family of ansamycins. Rifamycins are particularly
effective against mycobacteria, and are therefore used to treat
tuberculosis, leprosy, and mycobacterium avium complex (MAC)
infections.
Me
OH
H
N
Me
O
R
O
H
OH
Me
Me
O
O
OH
Me
H
Me
H
H
H
H
H
OMe
O
C
53
Me
Me H OH
O
Isoniazid is a prodrug and must be activated by a bacterial catalaseperoxidase enzyme in Mycobacterium tuberculosis called KatG, which
catalyzes the formation of the isonicotinic acyl radical, which spontaneously
couples with NADH to form the nicotinoyl-NAD
nicotinoyl NAD adduct
adduct. This complex binds
tightly to the enoyl-acyl carrier protein reductase known as InhA, thereby
blocking the natural enoyl-AcpM substrate and the action of fatty acid
synthase. This process inhibits the synthesis of mycolic acids, which are
required components of the mycobacterial cell wall. A range of radicals are
produced by KatG activation of isoniazid, including nitric oxide,
Rifamycins
Rifamycins have been used for the treatment of many diseases, the most
important one being HIV-related tuberculosis.
H
N
O
NH2
N
Isoniazid
H
bacterial O
catalaseperoxidase
enzyme
N
R
O
N
R
O
O
N
R
+4
Fe
N
N
N
Forma a complex binds to protein important in mycolic fatty acid
metabolism and blocks fatty acids used in cell wall of terberculosis bacteria.
© Oxford University Press, 2013
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NH
NH
H2N
C
NH
H
HN
C
NH2
H
OH H
HO
H
H
H
OH
H
O
O
CHO
H
Me
St t
Streptomycin
i
OH
H
HO
O
CH2OH
H MeHN
H
O
H
OH
H
Streptomycin is an antibiotic used to treat a number of bacterial infections. This includes tuberculosis,
Mycobacterium avium complex, endocarditis, brucellosis, Burkholderia infection, plague, tularemia, and rat bite
fever. For active tuberculosis it is often given together with isoniazid, rifampicin, and pyrazinamide.
Common side effects include feeling like the world is spinning, vomiting, numbness of the face, fever, and rash.
Streptomycin is a protein synthesis inhibitor. It binds to the small 16S rRNA of the 30S subunit of the
bacterial ribosome, interfering with the binding of formyl-methionyl-tRNA to the 30S subunit. This leads to
codon misreading, eventual inhibition of protein synthesis and ultimately death of microbial cells through
mechanisms that are still not understood. Speculation on this mechanism indicates that the binding of the
molecule to the 30S subunit interferes with 50S subunit association with the mRNA strand. This results in an
unstable ribosomal-mRNA complex, leading to a frameshift mutation and defective protein synthesis;
leading to cell death. Humans have ribosomes which are structurally different from those in bacteria, so the
drug does not have this effect in human cells. Streptomycin is an antibiotic that inhibits both Gram-positive
55
and Gram-negative bacteria, and is therefore a useful broad-spectrum antibiotic.© Oxford University Press, 2013
OH
O
OH
O
O
OH
NH2
OH
Cl
HO
Me
H
NMe2
Chlortetracycline
(Aureomycin)
Chlortetracycline (trade name Aureomycin, Lederle)
is a tetracycline antibiotic, the first tetracycline to be
identified. It was discovered in 1945. Duggar identified
the antibiotic as the product of an actinomycete In
veterinary medicine, chlortetracycline is commonly
used to treat conjunctivitis in cats and other animals.
Chlortetracycline may increase the anticoagulant
activities . The risk or severity of adverse effects can be
increased when chlortetracycline is combined with
acitretin, adapalene, or alitretinoin.
© Oxford University Press, 2013
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NMe2
CH3
HO
O
H3C
CH3
O
HO
HO
H3C
O
CH3
O
CH3
CH3
O
O
CH3
OH
O
Me
Erythromycin
OMe
Erythromycin is an antibiotic useful for the treatment of a
number of bacterial infections, including respiratory tract
infections, skin infections, chlamydia infections, pelvic
inflammatory disease, and syphilis. It may also be used during
pregnancy to prevent Group B streptococcal infection in the
newborn. An eye ointment is routinely recommended after
delivery to prevent eye infections in the newborn. Common
side effects include abdominal cramps,
p , vomiting,
g, and
diarrhea. More serious side effects may include Clostridium
difficile colitis, liver problems, prolonged QT, and allergic
reactions. It is generally safe in those who are allergic to
penicillin
Erythromycin displays bacteriostatic activity or inhibits
growth of bacteria. By binding to the 50s subunit of the
bacterial rRNA complex, Erythromycin interferes with
aminoacyl translocation, preventing the transfer of the tRNA
bound at the A site of the rRNA complex to the P site of the
rRNA complex. Without this translocation, the A site remains
occupied, thus the addition of an incoming tRNA and its
attached amino acid to the nascent polypeptide chain is
inhibited. This interferes with the production of functionally
useful proteins, which is the basis of this antimicrobial action.
© Oxford University Press, 2013
57
DRUGS ACTING ON mRNA Antisense Therapy
antisense
molecule
Protein synthesis
m-RNA
A G U C U A C G U U
antisense
molecule
G U A A U C A G A U G C A A A A G U
m-RNA
Antisense therapy is a form of treatment for genetic disorders or infections. When the genetic sequence
of a particular gene is known to be causative of a particular disease, it is possible to synthesize a strand
of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (mRNA)
produced by that gene and inactivate it, effectively turning that gene "off". This is because mRNA has to
be single stranded for it to be translated. Alternatively, the strand might be targeted to bind a splicing site
on pre-mRNA and modify the exon content of an mRNA. This synthesized nucleic acid is termed an
"anti-sense" oligonucleotide (ASO) because its base sequence is complementary to the gene's messenger
RNA (mRNA), which is called the "sense" sequence (so that a sense segment of mRNA " 5'-AAGGUC-3'
58
" would be blocked by the anti-sense mRNA segment " 3'-UUCCAG-5' "). © Oxford University Press, 2013
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DRUGS ACTING ON mRNA
Antisense Therapy
Advantages
• Same effect as an enzyme inhibitor or receptor antagonist
• Highly specific where the oligonucleotide is 17 nucleotides or more
• Smaller dose levels required compared to inhibitors or antagonists
• Potentially less side effects
Disadvantages
• ‘Exposed’
p
sections of mRNA must be targeted
g
• Instability and polarity of oligonucleotides (pharmacokinetics)
• Short lifetime of oligonucleotides and poor absorption across cell
membranes
© Oxford University Press, 2013
59
DRUGS ACTING ON mRNA Micro-RNA (miRNA)
• Short segments of double stranded RNA
• Recognised by enzyme complex RISC to produce single stranded RNA – small
interfering or small inhibitory RNA (siRNA)
• Binds to complementary region of mRNA
• mRNA is cleaved byy enzyme
y complex
p
The RNA-induced silencing complex, or RISC, is a multiprotein complex, specifically a
ribonucleoprotein, which incorporates one strand of a single-stranded RNA (ssRNA) fragment, such
as microRNA (miRNA), or double stranded small interfering RNA (siRNA). The single strand acts
as a template for RISC to recognize complementary messenger RNA (mRNA) transcript. Once
found, one of the proteins in RISC, called Argonaute, activates and cleaves the mRNA. This process
is called RNA interference (RNAi) and it is found in many eukaryotes; it is a key process in gene
silencing and defense against viral infections.
miRNA
miRNA
RISC
RISC
mRNA
siRNA
RISC
RISC
© Oxford University Press, 2013
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Micro-RNA (miRNA)
Advantages
• siRNAs have potential to be used in gene therapy
• Greater efficiency in silencing mRNA than conventional
antisense therapy
• One siRNA could lead to cleavage of several mRNA
molecules
Problems
• siRNAs need to be metabolically stable
• Need to reach target cells
• Need to enter target cells
© Oxford University Press, 2013
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Epigenetics – a new frontier: DNA methylation, methylcytosine, histone acylation
© Oxford University Press, 2013
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Five major families of histones exist: H1/H5, H2A, H2B, H3 and H4.
Histones H2A, H2B, H3 and H4 are known as the core histones, while
Histones H1 and H5 are known as the linker histones.
Histones make five types of interactions with DNA:
1. Helix-dipoles form alpha-helixes in H2B, H3, and H4 cause a net positive charge to accumulate at the
point of interaction with negatively charged phosphate groups on DNA
2 Hydrogen
2.
H d
bonds
b d between
b t
the
th DNA backbone
b kb
andd the
th amide
id group on the
th main
i chain
h i off histone
hi t
proteins
t i
3. Nonpolar interactions between the histone and deoxyribose sugars on DNA
4. Salt bridges and hydrogen bonds between side chains of basic amino acids (especially lysine and arginine)
and phosphate oxygens on DNA
5. Non-specific minor groove insertions of the H3 and H2B N-terminal tails into two minor grooves each
on the DNA molecule
Histones are highly basic facilitating DNA-histone interactions and contribute to their water solubility.
Histones are subject to post translational modification by enzymes primarily on their N
N-terminal
terminal tails, but
also in their globular domains. Such modifications include methylation, citrullination, acetylation,
phosphorylation, SUMOylation, ubiquitination, and ADP-ribosylation. This affects their function of gene
regulation.
In general, genes that are active have less bound histone, while inactive genes are highly associated with
histones during interphase. Histones are evolutionarily conserved, and any deleterious mutations appear to
be severely maladaptive. All histones have a highly positively charged N-terminus with many lysine and
63
arginine residues.
© Oxford University Press, 2013
Methylations in biochemistry
CH3
H2 N
H
N
8
N
1
2
N3 4
5
N
H
O
H
N
N
H2N
7
6
H
THF
(tetrahydrofolate)
N
N
H
5
N
H
9
10
O
CH3
N
H
Ar
Ar
N5 methyl-THF
Me-vit B-12
Me-vit B12
vit B-12
H
CH3
S
CH3
N
H2 N
S
S
O
N
ATP
NH3
O2C
O2C
NH3
N
NH3
H
homocysteine
N
OH
OH
H
O2 C
H
S-adenosyl methionine (SAM)
methionine
NH2
NH2
H
H3 C
N
N
SAM
N
DNA
cytosine
O
N
O
DNA
5-Methylcytosine
© Oxford University Press, 2013
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Citrullination or deimination is the conversion of the amino acid arginine in a protein into the amino acid citrulline.
Enzymes called peptidylarginine deiminases (PADs) replace the primary ketimine group (=NH) by a ketone group
(=O).
Citrullination controls the expression of genes, particularly in the developing embryo. The immune system often
attacks citrullinated proteins, leading to autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.
Citrulline is not one of the 20 standard amino acids encoded by DNA in the genetic code. Instead, it is the result of a
post-translational modification.
Citrullination is distinct from the formation of the free amino acid citrulline as part of the urea cycle.
Arginine is positively charged at a neutral pH, whereas citrulline is uncharged. This increases the hydrophobicity of
the protein, leading to changes in protein folding. Therefore, citrullination can change the structure and function of
proteins.
Proteins that normally contain citrulline residues include myelin basic protein (MBP), filaggrin, and several histone
proteins, while other proteins, like fibrin and vimentin, can get citrullinated during cell-death and tissue inflammation.
Fibrin and fibrinogen may be favored sites for arginine deimination within rheumatoid joints. Test for presence of anticitrullinated protein (ACP) antibodies are highly specific (88-96%) for rheumatoid arthritis (RA), about as sensitive as
rheumatoid factor (70-78%) for diagnosis of RA, and are detectable from even before the onset of clinical disease.
H2N
B
NH2
NH2
H2N
H
O
NH
+
NH3
etc.
H
H
N
N
N
H
O
NH
PAD
(enzyme)
H
N
H2N
H
NH
+
H
O
H
O
H
H
O
arginine
N
N
O
© Oxford University Press, 2013
citrulline
65
Acylations in biochemistry
Histone proteins are acetylated and deacetylated on lysine residues in the N-terminal tail as part of gene regulation.
The regulation of transcription factors, effector proteins, molecular chaperones, and cytoskeletal proteins by acetylation
and deacetylation is a significant post-translational regulatory mechanism. These regulatory mechanisms are analogous
to phosphorylation and dephosphorylation by the action of kinases and phosphatases. Not only can the acetylation
state of a protein modify its activity but there has been recent suggestion that this post-translational modification may
also crosstalk with phosphorylation, methylation, ubiquitination, sumoylation, and others for dynamic control of
cellular signaling.
signaling
H
O
H
N
O
N
N
N
lysine
H
O
N
N
H
lysine
H
lysine
H
Co-A-SH
O
Co-A
acetyl Co-A
Co-A
O
HN
S
B
H
H
S
N
O
N
H
H
O
Glu-Enz
glutamate aa
O
HO
Glu-Enz
glutamate aa
O
acylated
lysine
© Oxford University Press, 2013
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Small Ubiquitin-like Modifier (or SUMO) proteins are a
family of small proteins that are covalently attached to and
detached from other proteins in cells to modify their
function. SUMOylation is a post-translational
modification involved in various cellular processes, such as
nuclear-cytosolic transport, transcriptional regulation,
apoptosis, protein stability, response to stress, and
progression through the cell cycle. SUMO proteins are
similar to ubiquitin, and SUMOylation is directed by an
enzymatic cascade analogous to that involved in
ubiquitination In contrast to ubiquitin
ubiquitination.
ubiquitin, SUMO is not used
to tag proteins for degradation. Mature SUMO is
produced when the last four amino acids of the C-terminus
have been cleaved off to allow formation of an isopeptide
bond between the C-terminal glycine residue of SUMO and
an acceptor lysine on the target protein.
Ubiquitin is a small (8.5 kDa) regulatory protein that has
been found in almost all tissues (ubiquitously) of
eukaryotic organisms. There are four genes in the human
genome that produce ubiquitin: UBB, UBC, UBA52 and
RPS27A. Ubiquitination can affect proteins in many
ways: it can signal for their degradation via the
proteasome alter their cellular location,
proteasome,
location affect their activity,
activity
and promote or prevent protein interactions. Ubiquitination
is carried out in three main steps: activation, conjugation,
and ligation, performed by ubiquitin
67
© Oxford University Press, 2013
ADP-ribosylation is the addition of one or more ADP-ribose moieties to a protein. It is a reversible
post-translational modification that is involved in many cellular processes, including cell signaling,
DNA repair, gene regulation and apoptosis. Improper ADP-ribosylation has been implicated in some
forms of cancer. It is also the basis for the toxicity of bacterial compounds such as cholera toxin,
diphtheria toxin, and others.
H2N
O
O
ADP
O
H2N
N
H2N
N
O
O
P
O
P
O
O
N
N
H
N
N
H
Arg
O
H
N
O
OH
O
O
OH
OH
OH
O
O
NAD+
OH
Glu
H2N
OH
H
Arg
N
H
N
O
OH
ADP
O
O
OH
Glu
OH
© Oxford University Press, 2013
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