CHEM3407 Part 4 Stu

2/10/2015
CHEM3407/3410 & BPHM3015
Medicinal Chemistry
Nucleic acid as a drug target

Hongzhe SUN
Chemistry & Pharmacy
HKU
2nd Semester, 2015
Medicinal Chemistry
Nucleic acids as drug targets
NH2
Cytosine
(C)
BASES
3N
N
1
9
3 NH
5 3
1
Pyrimidine
7
9
N
H
Adenine
(A)
1
3
Purine
1N
3 NH
DNA
1
Uracil (U)
RNA
Thymine
(T)
Nitrogen-containing ring
Hetero-cyclic AMINES
H 3C
NH2
NH2
Guanine (G)
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7. NUCLEIC ACIDS AS DRUG TARGETS

Contents
7.1. Deoxyribonucleic Acid (DNA)
7.2. Ribonucleic Acid (RNA)
2.1. Primary & Secondary structure
2.2. Tertiary structure
2.3. Transcription
2.4. Translation - protein synthesis
7.3 Genetic Engineering
3.1 DNA Recombinant Technology
3.2 Amplification of a gene
7.4. Drugs Acting on DNA or RNA
Where is DNA found

In the cell lies the nucleus which
house the chromosomes.
Cells
Nucleus
Chromosome: For
human beings there are
22 pair of chromosomes
which determines traits
with 23rd pair
determining sex. XX female and XY - males.
Some individuals have
three or have YX
chromosomes
Genes: One-gene-oneenzyme hypothesis

Deoxyribose nucleic acid: DNA- Deoxyribose nucleic acid
houses the gene which codes for the protein which makes
each life from including you and me.
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Tidbits on DNA
DNA is responsible for all
the proteins in us.
We share 98 % of the same
genes as chimps
All the DNA in your body

unwound would stretch to
the Sun and back.
1
Nucleic Acids in Virus, Bacteria and Human Cells
bacteria
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7.1. DEOXYRIBONUCLEIC ACID (DNA)

1.1 Primary Structure
Nucleotide = Phosphate + Sugar + Base
O
NH2
N1
NH2
Base
5 7
4 9
HN1
8
5 7
4 9
H2N
N
H
N
H
Guanine
Adenine
HO3PO
O
Phosphate
Purines
H
H
Sugar
H
HO
NH2
N3
2
Deoxyadenosine
phosphate
HN3
Cytosine
2 1
N
H
CH3
N
H
Thymine
Pyrimidines

1.1 Primary Structure
NH2
5' end
N
O
O P O
OH
5'
CH2
H
H
1'
3' H H
O CH2
OH
Cytosine (C)
O
O
N
NH
N
P
OH
H
Base
O
O P O
OH
N
N
Base
O P O
NH2
O
O
5' end
Adenine (A)
O CH2
OH
Base
O
OH
H
Me
NH
O
N
P
O
O P O
Guanine (G)
NH2
O CH2
OH
Thymine (T)
3' H
Base
O
OH
O
O P O
3' End
3' End
1. Sugar phosphate backbone is invariant, bases attached in random order

2. The order of bases provides the primary structure of DNA
3. Nucleic acids are named in the 5 3 direction (No 5-5 or 3-3 linkages)
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Comparison of nucleic acids and proteins

5
Protein
Phosphate
Sugar C
5-end dpCpApT 3-end
Phosphate
d(pCAT)
Sugar A
Phosphate
Sugar T
3
Nucleic acid
Q. Any possibility to have a biopolymer with protein backbone as the

backbone and nucleic bases as the side-chain?
The peptide nucleic acids (PNAs) constitute a remarkable new class of synthetic nucleic
acid analogues, based on their peptide-like backbone. This structure gives to PNAs the
capacity to hybridize with high affinity and specificity to complementary RNA and DNA
sequences and a great resistance to nucleases and proteinases.
3-end
N-terminus
C-terminus
Protein
PNA
DNA
1
5-end
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1.2 Secondary Structure - Double Helix
o
10A
G
A T
Major
groove
TA
T A
G
C
G
TA
T
T
Minor
groove
A T
A T
T A
C G
G
o
34A
C
TA
G
T
C
G C
GC
T A
A
Sugar phosphate backbone is ionised

and faces outward (favourable
interactions with water)
Nucleic acid bases point inward and
pair up A-T or G-C
Purine pairs with pyrimidine constant diameter to helix
Base bairs are stacked (vdw
interactions between pairs)
Chains are complementary
TA
T
DNA DOUBLE HELIX

10
G
Thymine
Me
A T
Major
groove
TA
T A
G
C
G
O P
O
C
TA
O
o
34A
5'
T
C
G
TA
DNA DOUBLE HELIX
O
5'
N
NH2
N
3'
O
O
N
N
HN
3'
O
P
N
O
O
O
3'
G C
GC
T A
A
C
TA
3'
A T
T A
C G
G
NH
O
O
5'
A T
O
N
T
Minor
groove
Adenine
H2N
Cytosine
H2N
Guanine
5'
O
O
O
P
Base Pairing
G-C base pairing involves 3 H-bonds
A-T base pairing involves 2 H-bonds
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Effect of base-pair composition on the denaturation

temperature of DNA
10
Too wide
Too narrow
Q: AT-rich regions melt more easily than GC-rich regions?
1. DNA Replication
New
DNA
chains
Template
DNA
Double Helix
New
DNA
chains
Template
Replication
DNA
Daughter helices
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Replication
Approach of trinucleotide
Base pairing
Enzyme-catalysed 'splicing'
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1.3 Tertiary Structure
Double helix coils into a 3D shape - supercoiling

http://www.youtube.com/watch?v=EYGrElVyHnU&feature=related
(other one is ok)
Double helix has to unravel during replication
Unravelling leads to strain
Relieved by enzyme catalysed cutting and repair of DNA

chain
Important to the activity of the quinolone and fluoroquinolone

antibacterial agents which act as Topoisomerase enzyme
inhibitors
1

1.4 Action of topoisomerase II
Relieves the strain in the DNA helix by temporarily cleaving the DNA
chain and crossing an intact strand through the broken strand
Topo II
Tyr
5'
3'
3'
DNA
5'
Tyrosine residues in the enzyme are involved in the chain breaking process
The residues form covalent bonds to DNA
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Topo II
Tyr
5'
3'
3'
Tyr
DNA
5'
Topo II
The enzyme pulls the chains apart to create a gap

Topo II
Tyr
Tyr
DNA
Topo II
The intact strand of DNA is passed through the gap
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3
Topo II
Tyr
5'
3'
3'
5'
Tyr
Topo II
The break is resealed
1

4
Topo II
Tyr
5'
3'
3'
5'
The break is resealed
1
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Mechanism of chain cutting
5'
5'
H
Base
O
H
HO
O
O P
O O
HO
Tyr
O P
O O
TopoII
Base
O
H
Tyr
Base
O
H
TopoII
Base
O
H
3'
3'
7.2. RIBONUCLEIC ACID (RNA)

2.1 Primary & Secondary Structure of RNA
Similar to DNA with the following exceptions
Ribose is used instead of deoxyribose
Uracil (U) is used rather than thymine (T)

O
HOCH2 O
H
OH
HN
H
OH
OH
Ribose
N
H
Uracil
Single stranded
Some regions of helical secondary structure exist due to base

pairing within the same strand (see t-RNA)
Adenine pairs to uracil (A-U); guanine pairs to cytosine (C-G)

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2.2 Tertiary structure
There are three type of RNA nucleic acids. They serve different
purpose.
1. Messenger RNA (mRNA):
carries the genetic info from the DNA in

the nucleus to cytoplasm
not very stable & synthesized as needed
2. Ribosomal RNA (rRNA):
This is where protein synthesis occurs also

known as the protein factory.
found in the ribosomes
small spherical bodies in the cells but outside the nuclei
large molecules
3. Transfer RNA (tRNA):
Smallest of RNA, is responsible for carrying amino acids to the

protein factory (r-RNA) and coordinating the amino acid
1
monomeric unit to make a protein..
7.2. RIBONUCLEIC ACID (mRNA)

2.3 Transcription
The copying of a segment of DNA which codes for a specific protein
G
U
A
U
C
U
G
U
C
C
C
U
U
A
mRNA
G
T
A
T
C
T
G
T
C
C
C
T
T
A
DNA double helix
C
A
T
A
G
A
C
A
G
G
G
A
A
T
DNA unravelled
to reveal gene
G
T
A
T
C
T
G
T
C
C
C
T
T
A
G
U
A
U
C
U
G
U
C
C
C
U
U
A
C
A
T
A
G
A
C
A
G
G
G
A
A
T
mRNA
Transcription
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7.2. RIBONUCLEIC ACID (tRNA)
Base Pairing
3'p
end
A
AMINO
ACID
C
C
5'p
end A
G
C
G
C
G
U
C
G
G
C
U
U
A
U
U UA
G
G
C
G
A G G C C
UH2
C G C G mGU
G
C
U C C G G
G
C
T PsC
A G C G C m2G
G UH2
C
G A UH2
G
U
A
C
G
C
G
C
G
Ps
U
U
mI
I
C
G
Yeast alanine-tRNA
mI Methylinosine
I
Inosine
UH2 Dihydrouridine
T
Ribothymidine
Ps
Pseudouridine
mG Methylguanosine
m2G Dimethylguanosine
ANTICODON
Amino
acid
t-RNA
AC G
Anticodon
binding region
for m-RNA
Anticodon - the 3 bases are specific for the attached amino acid
Condon - base pair to the complementary triplet code on
m-RNA
1

2.4 Translation - protein synthesis
His
Growing
protein chain
Ribosome
His
Peptide
transfer
60S
mRNA
OH
His
GCA
P-site A-site
GCU
CGA CAU GUC
mRNA
GCU GUA
CGA CAU GUC
mRNA
GCU GUA
CGACAUGUC
40S
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2. RIBONUCLEIC ACID (RNA)

Val
OH
tRNA
OH
His
CAG
His
His
GCU
Translocation
mRNA
GCU GUA
CGACAUGUC
P-site
mRNA
GUA
CGA CAU GUC
mRNA
A-site
GUA
CGA CAU GUC

PEPTIDE
NH2
NH
R' C H
O C
R C H
O C
HO
H
H
Adenine
HO
H
H
O
H
O
H
O
OH
P O
Adenine
O
H
O
H
O
OH
O
t RNA
t RNA
Transfer of
growing peptide
chain to next
amino acid
PEPTIDE
P O
O
NH
R C H
O C
NH
HO
H
H
Adenine
R' C H
O C
OH
H
O
H
OH
O
P O
O
HO
H
H
Adenine
O
H
O
t RNA
H
OH
O
P O
O
t RNA
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Overview
mRNA
Ribosome
Amino acid
DNA
tRNA
mRNA
Translation
Transcription
Nucleus
Protein
7.3. GENETIC ENGINEERING

Introduction
(general definition): direct human manipulation of an organism's
genetic material in a way that does not occur under natural conditions
- select, isolate and copy genetic material of interest to make a construct
- introduce this construct to plasmid (circular form of DNA) of host
(bacteria or yeast or animal cell), either indirectly via a viral vector or
directly through injection techniques or physical stimulations
- wait the modified plasmid to be expressed within the host to give
genetically modified DNA or protein, then kill the host to release the
as-synthesized DNA or protein product, finally purified them by using
SDS-PAGE and affinity chromatography (~1-5 mg product / kg of host)
1
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3.1 Recombinant DNA technology
'Sticky' ends
GAATAC
C TTAAG
DNA
GAATAC
C TTAAG
Restriction
enzyme
Restriction
enzyme
G
C TTAA
G
C TTAA
AATAC
G
Annealing
GAATAC
C TTAAG
+
G AATAC
C TTAAG
AATAC
G
DNA ligase
enzyme
DNA
GAATAC
C TTAAG
isolate either one
+
G AATAC
C TTAAG
Notes:
Restriction enzymes split DNA strands at specific base pair regions
The DNA strands are split unevenly with sticky ends which allow
annealing
Ligase enzymes repair the chains
1

3.1 Recombinant DNA technology
- Segments can be added to both ends of a DNA fragment in
order to make the DNA recognisable to restriction enzymes
DNA fragment
or vector
Ligase
CCGAAT TCGG
G C C T T AAGC C
CCGAATTCGG
G C C T T AAGC C
Synthetic linker
AATTCGG
GCC
DNA fragment
or vector
CCGAAT TCGG
GC C T T AAG C C
Restriction
enzyme
CCG
DNA fragment
G C C T TAA
or vector
construct
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or construct
Plasmid split
a) Human
DNA
Restriction
enzyme
b) Ligase
Vector DNA
= plasmid
introduce back to the bacteria host
(injection or physical stimulation)
DNA/protein synthesis
gene expression
Notes:
Plasmids are circular segments of DNA transferred between bacterial cells
A gene can be inserted into a plasmid
Plasmids are introduced to bacterial cells and multiple copies are
synthesised
1

Gene
Phage
E. coli
Gene
E. coli
E. coli
Notes
Gene can be inserted into a bacteriophage
Bacteriophages are viruses which infect bacterial cells
Multiple copies of the bacteriophages are produced along with the gene
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Applications
mass production of insulin,
recombinant human growth
hormone (rHGH)
G-quadruplex DNA:
In addition to the familiar duplex DNA, certain DNA sequences can fold into
2nd-structures that are four-stranded;
they are made up of guanine (G) bases, such structures are called Gquadruplexes.
these structures may exist in vivo in specific regions of the genome including the
telomeric ends of chromosomes and oncogene regulatory regions.
(Trends Pharmacol Sci. 2000 21, 136)
9
7
3
7
9
1
3
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Two or more G-rich

repeats can form GG
hairpins, which in turn
dimerize to form
several types of stable
bimolecular
quadruplexes,
A tetramolecular parallel Gquadruplex from four DNA
strands each with a single
G-rich repeat.
Long G tracts can fold upon

themselves to form an
antiparallel intramolecular
quadruplex, termed an
intramolecular foldover Gquadruplex.
Structure of G-quadruplex d(GGGGTTTGGGG)
N+
H
N+
H
O
HN
O
H+
N
NH
PDBID: 1L1H
Ligand:3,6-bis-[3-pyrrolidino-propionamide] acridine (BSU6039)
Metal: K+ (Haider SM et al J. Mol. Biol. 2003, 326:117)
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7.4. DRUGS ACTING ON DNA
Intercalating agents
Alkylating agents
Metallating agents
Chain cutters
Chain terminators
Selected examples of RNA base methylation
(Chuan, Nat. Chem. Biol. 2010, 6:863-865)

1
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4.1 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
Preference is often shown for the minor or major groove
Intercalation causes the DNA replication machinery to skip

or insert additional nucleotides at the intercalated site
DNA, preventing DNA replication and transcription
Intercalation inhibits topoisomerase II
4. DRUGS ACTING ON DNA

Example - Proflavine
H2N
NH2
Proflavine
Planar tricyclic system

The amino substituents are protonated and charged
Used as an antibacterial agent in the WWII
Targets bacterial DNA
DNA breaks
stop cell cycle progression
apoptosis
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Example - Proflavine
not only ONE intercalator

per one DNA, everywhere!
Proflavine
G C
AT
TA
T A
G C
G C
H 3N
NH3
sugar phosphate
backbone
TA
T A
T A
AT
AT
T A
C
T
G
G C
H 3N
TA
T A
proflavine
NH3
G C
GC
GC
T A
A T
C G
TA
T A
DNA double helix
van der Waals interactions

Ionic interactions
transcription or replication
Proflavine
topoisomerase II
2KD4
cell death
1
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Examples- antimalarial agents
H
CH3
H
NH CH
HO
CH2CH3
(CH2)3 N
CH2CH3
MeO
Cl
Chloroquine
Quinine
pathogenic mosquitos parasite

Plasmodium falciparum
1

Examples Antibiotics with anticancer activity
N-Me-Gly
N-Me-Gly
L-Pro
N-Me-L-Val
D-Val
O
H
N-Me-L-Val
C
Me
C
C
C
Me
NH
C
C
C
CH2OH
OH
NH
OMe
OH H
NH2
O
CH3
OH
D-Val
O
O
L-Pro
O
CH3
Dactinomycin
Extra binding to sugar phosphate

backbone by cyclic peptide
HO
Me
H
NH3
Doxorubicin (Adriamycin)
Extra binding to sugar

phosphate backboneby
1 NH3
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Notes on dactinomycin
Intercalates via minor groove of DNA double helix
Prevents unwinding of DNA double helix (disables replication)
Blocks transcription by blocking DNA-dependent RNA
polymerase
Notes on doxorubicin (or adriamycin)
Intercalates via the major groove of DNA double helix
Blocks the action of topoisomerase II by stabilising the
DNA-enzyme complex (prevents the resealing of DNA double helix)
Acts as a topoisomerase poison

Examples - Bleomycins
CONH2
H
N
H
primary amine
NH2
H
NH
O
pyrimidine N
ring
Me
H
O
H H H
N
HO
NH
HO
HN
O
N
H
N
OH
H
O
OH
O
Me
H2N
primary amine
O
2
primary amide
NH
Me
HO
Me
Bithiazole
intercalating
region
imidazole
ring
Bleomycin A2
Bleomycin B2
R = NHCH2CH2CH2SMe2
R = NHCH2CH2CH2CH2NHC(NH2)=NH
OH
OH
O
OH
NH2
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Bleomycin
A natural product and is isolated from
the Streptomyces. It can both complex a
metal centre and interact with DNA.
Small molecule-metal ion complexes that
can produce radical species capable of
cleaving DNA are known.
In the presence of Fe(II) and dioxygen or
Fe(III) and hydrogen peroxide bleomycin
can induce cleavage in both single and
double stranded DNA.
metal is bound to the
peptide section of the molecule.
How does it cleave DNA?

A ferric peroxide or possibly a high valent Fe(V)-oxo
species is formed that abstracts hydrogen atoms from
the deoxyribose rings of DNA.
The subsequent ring opening steps have been well
characterized with two pathways possible- dependent
on whether or not there is more oxygen present.
Clinical application
The first antibiotics active against cancer were discovered in the 1950s and
bleomycin was one that was later screened in response to the nephrotoxicity of
the other antibiotics already tested.
Clinical trials were effective and it was introduced commercially in 1969. It is
used against a variety of cancers.
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O2 molecule
ROS
O2 radical anion
HOO free hydroxy radical
O2- superoxide
Abstract H radical from

DNA (damage)
cell death
Tutorial1: Bisanthracycline WP631, is an antibiotic targeting on DNA.

Whats its potential binding mode? Check PDB: 1al9
O
OH
OH
OH
O
OH
H2N
H 2N
HO
O
O
OH
OH
OH
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Tutorial2: Analyze the binding of the following drugs targeting on

DNA.
Groove binding:
Groove binding: direct interaction of the bound molecule with the
edge base-pairs in either of the groove of the nucleic acids
5'-d(CGCAAATTTGCG)-3') complex with netropsin (PDB: 1dne)
+
N 9H 2
O
H2N10
N8
H
H
N7
CH3
H
HN6
H
O
N5 CH3
HN4
netropsin
O
H2N2
HN3
N 1H 2
+
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H
N
2
3
4
5
6
7
8
9
10
11
12
324
CG
GC 23
CG 22
AT 21
AT 20
AT 19
TA 18
TA 17
TA 16
GC 15
CG 14
GC 13
O
H
H3C
+
N 9H 2
O
H2N10
N8
H
H
N
N7
CH3
C'
HN6
H
'C
H
O
N5 CH3
HN4
H
O
H2N2
HN3
8.6
N 1H 2
+
netropsin
7.4
Major
groove
Deformation

4.2 Alkylating agents
(1o carbocation)
Notes
Contain highly electrophilic groups RX R+ + X 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. alkylation of proteins)
Can cause interstrand and intrastrand cross-linking if two
electrophilic groups present
normally, formation of N-alkylated DNA adduct always
compensate the energy loss due to disruption of double helix
structure maintained by intermolecular H-bonds between BPs
Alkylation of nucleic acid bases can result in miscoding
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Which one can be readily N-alkylated?

COOH3 N+
COOH3 N+
CH2
CH2
CH2
CH2
CH2
CH2
CH2
NH
NH3+
COO-
COOH3N+
H3N+
CH2
CH2
+
HN
+
HN
NH
NH2+
NH2
Discussion: Methylayion of nucleic acids

Cross linking
Nu
Nu
Nu
Nu
Nu
Intrastrand cross linking
Nu
Nu
Nu
Interstrand cross linking
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Nucleophilic groups on nucleic acid bases
nucleophilic
groups
NH2
NH2
7
nucleophilic
groups
N
3
HN
H2N
N
R
R
Adenine
Cytosine
Guanine

Miscoding resulting from

alkylated nucleic acid bases
Normal base pairing
Thymine
Cytosine
NH2
N
DRUG
Me
Abnormal base pairing.

Alkylated guanine prefers
enol tautomer
Guanine prefers
keto tautomer
Thymine
Me
Adenine
H2N
O P
O
O P
O
NH
O
O
5'
N
N
3'
3'
stronger H-bond between

NH3+ and C=O
O
5'
from A-T to A-C pair?
H2N
H2N
How about the

N-alkylated adenine?
NH
N
N
HO
HN
N
R
Alkylated guanine
Guanine
O
P
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Example
Chlormethine (nitrogen mustard)
Cl
CH3
N
Cl
Notes
Used medicinally in 1942
Causes intrastrand and interstrand cross-linking
Prevents replication
Mono-alkylation of guanine also possible
Analogues with better properties have been prepared

Mechanism of action
some bond length not in scale

DNA
DNA
O
G = Guanine
H
N
NH2
Cl
CH3
CH3
H
N
CH3
G
Cl
NH2
N
N
N
Cl
Aziridine ion
Mechlorethamine
Cl
N
N
NH
NH2
DNA
DNA
NH2
interstrand
NH2
N
Crosslinked DNA
CH3
H
N
H
N
NH2
NH
NH2
NH
N
O
CH3
NH2
NH
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urea

Example - Nitrosoureas
O
Cl
Cl
N
O
Nitroso
NH2-(C=O)-NH2
Cl
N
H
Lomustine
N
H
Nitrosyl in OM
N
Carmustine
Mechanism
Decompose in body to form an alkylating agent and a
carbamoylating agent
O C N R
Isocyanate
(carbamoylating
agent)
+
O
Cl
R
N
N2 + HO
Cl
N
Cl
Alkylating
agent
OH
or other Nu:

Example - Nitrosoureas
Cl
Cl
DNA
Alkylating
agent
DNA
DNA
Protein-Lys-NH 2
O C N R
Isocyanate
Crosslinking
Alkylation
Protein-Lys-NH
Carbamoylation
HN
Notes
Alkylating agent causes interstrand cross-linking
Cross linking between G-G or G-C
Carbamoylating agent reacts with lysine residues on proteins
May inactivate DNA repair enzymes
1
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2/10/2015

O
O
S
Me
Busulfan
Me
S
O
O
Synthetic agent used as anticancer agent

Causes inter-strand cross-linking
Mechanism
O
O
S
Me
O
O
OSO2Me
Me
S
O
O
Guanine
H2N
HN
N
-MeSO3-
N
DNA
DNA
DNA
-MeSO3-
DNA
methane sulfonate anion stable!
DNA

Example - Mitomycin C
urethane
O
1o amine
CH2OCONH2
H 2N
OMe
N
Me
O
NH
Aziridine ring
quinone ring
Notes
Prodrug activated in the body to form an alkylating agent
One of the most toxic anticancer drugs in clinical use
1
34
2/10/2015
Mechanism
O
H
OH
CH2OCONH2
H 2N
OMe
N
Me
Enzyme
H 2N
Reduction Me
NH
-MeOH
NH
Ring
opening
H2N-DNA
H2 N-DNA
NH-DNA
N
NH2
OH
Alkylating agent
NH
OH
CH2
CH2
H2N
H2N
NH
NH-DNA
NH2
OH
N
N
O Guanine
N
HN
N
Me
Me
O Guanine
HN
NH-DNA
OH
CH2
H2N
Me
NH2
OH
-CO2
-NH3
NH2
O
OH
CH2OCONH 2
Me
H2N
NH
OH
hydroquinone
-H +
Me
OH
quinone ring
CH2OCONH 2
H2N
OMe
CH2OCONH2
NH2
OH
1
Interstrand crossed-linked DNA
Cl
4.3 Metallating agents
Cl
NH3
Cisplatin
Pt
NH3
Neutral inactive molecule acting as a prodrug

Platinum covalently linked to chloro substituents
Ammonia molecules act as ligands
Activated in cells with low chloride ion concentration
Chloro substituents replaced with (by) neutral water ligands
Produces positively charged species
Cl
NH3
Pt
Cl
H2O
NH3 +
H2O
Pt
NH3
Cl
NH3
H2O
+
NH3
Pt
H2O
NH3
2+
DNA
DNA
NH3
Pt
DNA
NH3
Cisplatin
Prodrug
Drug
Platination
1
35
2/10/2015

4.3 Metallating agents
Binds to DNA in regions rich in guanine
units
Intrastrand links rather than
interstrand
Localised unwinding of DNA double
helix
Inhibits transcription
N7
Pt
N7
Discuss in details later!

DNA-repair fails! (shielded by HMG binding)
http://www.youtube.com/watch?v=Wq_up2uQRDo&NR=1
1

4.4 Chain cutters
trisulfide chain
HO
CH3
O
H3C
OMe
H3C
NHCO2Me
S
S
H3C
O N
HHO
OH
Calicheamicin g1
Antitumour agent
O
O
OMe
H3C
HO
MeO
O
OH
H3C
H
N
MeO
enediyne
system
Generates DNA diradical

DNA diradical reacts with oxygen
Results in chain cutting (prevent DNA ligase from repairing the damage)
36
2/10/2015

4.4 Chain cutters
Mechanism
H
HO
NHCO2Me
S
S
HO
- Nu-S-SMe
HO
Michael
addition
NHCO2Me
H
NHCO2Me
MeS
Nu
Cycloaromatisation
HO
HO
NHCO2Me
H
NHCO2Me
DNA
DNA
(Diradical)
O2
R
H
Oxidative
cleavage
(formation of
benzene ring)
Oxidative Cleavage of DNA by OH radicals (sugar)

abstraction of one H of deoxyribose initiates
two chains break away

base
O
O
N
HN
H
H 2N
N
dR
Guanine (lower oxidation potential)
HN
+ OH
OH
H 2N
N
dR
8-hydroxyguanosine
37
2/10/2015
Oxidative Damage of DNA by OH radicals (base)
tautomerize
free bond rotation
cell death or
induce mutation?
1
miscoded G-A base
pairing

4.5 Chain terminators
Basic requirements
- must be recognized by the bases in the DNA template
- must have triphosphate group so that they can undergo the same
enzyme-catalysed reaction as the normal substrate does
- must contain non-hydroxy group so that addition of new nucleotide
to the growing DNA chain is not achievable
Mimicking nucleotide !
38
2/10/2015

False substrate (nucleotide)
Thymine (T)
Azidothymidine (AZT)
(Zidovudine;Retrovir)
O
HN
O
HO P O P O P O
HO
CH3
HN
CH3
OH
OH
N
O
OH
N3
N3
dR mimick
(functionalised)
Chain terminating
group
Notes:
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
Glossary
Prodrug a molecule that is inactive in itself, but which is
converted to the active drug in the body, normally by an
enzymatic reaction.
The purpose of using it is to avoid problems related to the
pharmacokinetics of the active drug and for targeting. (discuss in
next section!)
39
2/10/2015
2-aminopurine
(not 6-aminopurine (A))

Guanine(G)
O
HN
H2 N
Aciclovir
(Zovirax)
AcO
N
O
HO
Chain
terminating
group
OAc
Famciclovir
(Famvir)
N
N
NH2
Chain
terminating
group
Notes:
Prodrugs used as antiviral agents
Same mechanisms of action as AZT
Used for herpes () simplex and shingles
1
Aciclovir (AVORAX) Chain Terminator
40
2/10/2015

a) Normal replication
C
DNA polymerase
DNA
template
Free OH
A
A
P
3'
OH
Growing
chain
DNA
template
3'
OH
Growing
chain

b) Chain termination
C
G
C
Drug
DNA
template
No free OH
P
3'
OH
Growing
chain
Chain termination
Drug
DNA
template
Growing
chain
41
2/10/2015
7.5. DRUGS ACTING ON rRNA

- antibiotics (antibacterial agent derived from a natural source)
- binds rRNA or proteins of ribosomes, resulting in inhibition
of translation process in protein synthesis)
7.5. DRUGS ACTING ON rRNA

Antibiotics
NH
NH
H2N
NH
HN
NH2
H
OH
HO H
OH
OH H
HO
H
Me
H
N
Me
O2N
H
O
CH2OH
CHO
H
OH
Me
C
O
HN H
Chloramphenicol
(vs typhoid)
Me
Me
HO
H
H
H
OMe
O
C
Streptomycin
OH
H
OH
Me
Me
CHCl2
O
CH2OH
H MeHN
H
OH
H
NMe2
CH3
HO
Rifamycins
O
OH
H3C
CH3
HO
OH
NH2
Cl
HO
Me
H3C
Chlortetracycline
(Aureomycin)
CH3
CH3
CH3
O
CH3
H
NMe2
HO
OH
Me
Me H OH
OH
OH
H
OH
O
Me
Erythromycin
OMe
42
2/10/2015
Interaction of Rifamycins with RNA polymerase
(Campbell et al, Cell 2001, 104, 901)
7.4. DRUGS ACTING ON mRNA

Antisense Therapy
- design and synthesis of antisense molecules (oligonucleotides)
which will bind to specific regions of mRNA and prevent the mRNA
acting as a code for protein synthesis
43
2/10/2015

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 sections of mRNA must be targeted
Instability and polarity of oligonucleotides (pharmacokinetics)
Short lifetime of oligonucleotides and poor absorption across cell membranes

Antisense Therapy
block translation
antisense
molecule
(oligonucleotide)
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 duplex
- treat some genetic disease, i.e. muscular dystrophy ()
1
44

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CHEM3407/3410 & BPHM3015

Nucleic acid as a drug target

Nucleic acids as drug targets

7. NUCLEIC ACIDS AS DRUG TARGETS

7.4. Drugs Acting on DNA or RNA

Where is DNA found

Genes: One-gene-oneenzyme hypothesis

All the DNA in your body

Nucleic Acids in Virus, Bacteria and Human Cells

7.1. DEOXYRIBONUCLEIC ACID (DNA)

7.1. DEOXYRIBONUCLEIC ACID (DNA)

1. Sugar phosphate backbone is invariant, bases attached in random order

Comparison of nucleic acids and proteins

Q. Any possibility to have a biopolymer with protein backbone as the

7.1. DEOXYRIBONUCLEIC ACID (DNA)

Sugar phosphate backbone is ionised

DNA DOUBLE HELIX

7.1. DEOXYRIBONUCLEIC ACID (DNA)

DNA DOUBLE HELIX

Effect of base-pair composition on the denaturation

Q: AT-rich regions melt more easily than GC-rich regions?

7.1. DEOXYRIBONUCLEIC ACID (DNA)

7.1. DEOXYRIBONUCLEIC ACID (DNA)

Double helix coils into a 3D shape - supercoiling

(other one is ok)

Double helix has to unravel during replication

Unravelling leads to strain

Relieved by enzyme catalysed cutting and repair of DNA

Important to the activity of the quinolone and fluoroquinolone

7.1. DEOXYRIBONUCLEIC ACID (DNA)

7.1. DEOXYRIBONUCLEIC ACID (DNA)

7.1. DEOXYRIBONUCLEIC ACID (DNA)

7.1. DEOXYRIBONUCLEIC ACID (DNA)

7.1. DEOXYRIBONUCLEIC ACID (DNA)

7.1. DEOXYRIBONUCLEIC ACID (DNA)

7.2. RIBONUCLEIC ACID (RNA)

Ribose is used instead of deoxyribose

Uracil (U) is used rather than thymine (T)

Some regions of helical secondary structure exist due to base

Adenine pairs to uracil (A-U); guanine pairs to cytosine (C-G)

7.2. RIBONUCLEIC ACID (RNA)

carries the genetic info from the DNA in

This is where protein synthesis occurs also

Smallest of RNA, is responsible for carrying amino acids to the

7.2. RIBONUCLEIC ACID (mRNA)

DNA double helix

7.2. RIBONUCLEIC ACID (tRNA)

7.2. RIBONUCLEIC ACID (RNA)

2. RIBONUCLEIC ACID (RNA)

7.2. RIBONUCLEIC ACID (RNA)

7.2. RIBONUCLEIC ACID (RNA)

7.3. GENETIC ENGINEERING

7.3. GENETIC ENGINEERING

isolate either one

7.3. GENETIC ENGINEERING

7.3. GENETIC ENGINEERING

7.3. GENETIC ENGINEERING

7.3. GENETIC ENGINEERING

Two or more G-rich

Long G tracts can fold upon

Structure of G-quadruplex d(GGGGTTTGGGG)

7.4. DRUGS ACTING ON DNA

Selected examples of RNA base methylation