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Topic 5 Nucleic Acids as DrugTargets
Nucleic Acids-Chapter 7 Patrickand Corey 187, 188, 193-194,
198-199
N
N
N
NH2N
N
NH
O
O
Me
O
O
P
O
O
OO
P
O
O
O
HN
N
N
NO
H2N
O
P
O
OO
N
N
NH2
OO
O
P
O
O
OO
P
O
O
O
O
P
O
OO
3'
3'
5'
5'
5'
5'
3'
3'
Thymine Adenine
Cytosine Guanine
1. DEOXYRIBONUCLEIC ACID (DNA)1. DEOXYRIBONUCLEIC ACID (DNA)
Base PairingBase PairingG-C base pairing involves 3 H-bondsG-C base pairing involves 3 H-bondsA-T base pairing involves 2 H-bondsA-T base pairing involves 2 H-bonds
To34A
o10A
DNA DOUBLE HELIX
TAG
C
ATC
GCTA
ATCG
AT A
T ATA
TAT A
G CC
T ATA
AG CT
CGCG
ATAT
Majorgroove
Minor groove
G
G
1.2 Secondary Structure - Double Helix1.2 Secondary Structure - Double Helix
1. DEOXYRIBONUCLEIC ACID (DNA)1. DEOXYRIBONUCLEIC ACID (DNA)1.3 Tertiary Structure1.3 Tertiary Structure
•• Double helix coils into a 3D shape - Double helix coils into a 3D shape - supercoilingsupercoiling
•• Double helix has to unravel during replicationDouble helix has to unravel during replication
•• Unravelling leads to strainUnravelling leads to strain
•• Relieved by enzyme catalysed cutting and repair of DNARelieved by enzyme catalysed cutting and repair of DNAchainchain
•• Important to the activity of the Important to the activity of the quinolone quinolone and and fluoroquinolonefluoroquinoloneantibacterial agents which act as enzyme inhibitorsantibacterial agents which act as enzyme inhibitors
2. RIBONUCLEIC ACID (RNA)2. RIBONUCLEIC ACID (RNA)2.1 Primary structure2.1 Primary structure
•• Similar to DNA with the following exceptionsSimilar to DNA with the following exceptions•• Ribose is used instead of Ribose is used instead of deoxyribosedeoxyribose•• Uracil Uracil is used rather than thymineis used rather than thymine
UracilUracil
HN
N
H
O
O
RiboseRibose
O OH
H
HH
OHOH
HOCH2
H
2. RIBONUCLEIC ACID (RNA)2. RIBONUCLEIC ACID (RNA)2.2 Secondary structure2.2 Secondary structure
•• Single strandedSingle stranded
•• Some regions of helical secondary structure exist due to baseSome regions of helical secondary structure exist due to basepairing within the same strand (see pairing within the same strand (see t-RNAt-RNA))
•• Adenine pairs to Adenine pairs to uraciluracil; guanine pairs to cytosine; guanine pairs to cytosine
2. RIBONUCLEIC ACID (RNA)2. RIBONUCLEIC ACID (RNA)
•• Three types of RNA are involved in protein synthesis:Three types of RNA are involved in protein synthesis:
•• Messenger RNA (mRNA)Messenger RNA (mRNA) Relays the code for a protein from DNA to the proteinRelays the code for a protein from DNA to the proteinproduction siteproduction site
•• Transfer RNA (Transfer RNA (tRNAtRNA)) The adapter unit linking the triplet code on mRNA to specificThe adapter unit linking the triplet code on mRNA to specificamino acidsamino acids
•• Ribosomal RNA (Ribosomal RNA (rRNArRNA)) Present in Present in ribosomes ribosomes (the production site for protein synthesis).(the production site for protein synthesis).Important both structurally and catalyticallyImportant both structurally and catalytically
2.3 Tertiary structure2.3 Tertiary structure
mI MethylinosineI InosineUH2 DihydrouridineT RibothymidinePs PseudouridinemG Methylguanosinem2G Dimethylguanosine
2. RIBONUCLEIC ACID (RNA)2. RIBONUCLEIC ACID (RNA)2.3 Tertiary structure2.3 Tertiary structure
Anticodon Anticodon - the 3 bases are specific for the attached amino acid- the 3 bases are specific for the attached amino acid- base pair to the complementary triplet code on m-- base pair to the complementary triplet code on m-RNA (the codon)RNA (the codon)
Yeast Yeast alanine-tRNAalanine-tRNA
AMINO
ACID
C
G
G
C
C
G
G
C
C
G
C
G
G
C
G
C
A
U
G C
G
C G
G U
G
G C
G
Ps
C
U
mIU
CIG
UH2G
G
A
G
GC
C
U
C
m2G
UH2
C
G
G
G
A
UH2
U
A
G
C
G
A
Ps
U
T
U
mGU
A
C
C
A
C
A
U
C
C
U
3'pend
5'pend
ANTICODON
Base Pairing
ACG
Aminoacid
t-RNA
Anticodonbinding regionfor m-RNA
O
H H
HO O
Adenine
H
O P
O
t RNA
OH
O
H
CO
C HR'
NH2
C
NH
R H
CO
O
H
P O
OH
O
t RNA
O
H
Adenine
HO
HH
O
PEPTIDE
2. RIBONUCLEIC ACID (RNA)2. RIBONUCLEIC ACID (RNA)2.5 Translation - protein synthesis2.5 Translation - protein synthesis
Transfer of growing peptidechain to nextamino acid
O
H H
HO OH
Adenine
H
O P
O
t RNA
OH
O
H
NH
PEPTIDE
NH
CO
C HR
CR' H
CO
O
H
P O
OH
O
t RNA
O
H
Adenine
HO
HH
O
DNA
Nucleus
2. RIBONUCLEIC ACID (RNA)2. RIBONUCLEIC ACID (RNA)
Transcription
mRNA
mRNA
tRNA
Amino acid
Translation
2.5 Translation - protein synthesis2.5 Translation - protein synthesisOverviewOverview
Ribosome
Protein
ContentsContents
Part 2: Section 7.3 (Drugs acting on DNA)
3. Drugs acting on DNA3.1. Intercalating agents
- Topoisomerase II- Example – Proflavine
3.2. Alkylating agents3.3. Chain cutters
3. DRUGS ACTING ON DNA3. DRUGS ACTING ON DNA3.1 Intercalating agents3.1 Intercalating agents
Mechanism of actionMechanism of action
•• Contain planar aromatic or Contain planar aromatic or heteroaromatic heteroaromatic ring systemsring systems
•• Planar systems slip between the layers of nucleic acid pairs andPlanar systems slip between the layers of nucleic acid pairs anddisrupt the shape of the helixdisrupt the shape of the helix
•• Preference is often shown for the minor or major groovePreference is often shown for the minor or major groove
•• Intercalation prevents replication and transcriptionIntercalation prevents replication and transcription
•• Intercalation inhibits Intercalation inhibits topoisomerase topoisomerase II- see II- see Doxorubicin, p.198Doxorubicin, p.198CoreyCorey..
Topoisomerase IITopoisomerase II
•• Relieves the strain in the DNA helix by temporarily cleaving the DNARelieves the strain in the DNA helix by temporarily cleaving the DNAchain and crossing an intact strand through the broken strandchain and crossing an intact strand through the broken strand
3. DRUGS ACTING ON DNA3. DRUGS ACTING ON DNA
DNA
5'
3' 5'
3'
Tyr
Topo II
•• Tyrosine residues in the enzyme are involved in the chain breakingTyrosine residues in the enzyme are involved in the chain breakingprocessprocess
•• The residues form covalent bonds to DNAThe residues form covalent bonds to DNA
DNA
Tyr
Tyr
5'
3' 5'
3'
Topo II
Topoisomerase Topoisomerase IIII
•• Relieves the strain in the DNA helix by temporarily cleaving the DNARelieves the strain in the DNA helix by temporarily cleaving the DNAchain and crossing an intact strand through the broken strandchain and crossing an intact strand through the broken strand
3. DRUGS ACTING ON DNA3. DRUGS ACTING ON DNA
•• Tyrosine residues in the enzyme are involved in the chain breakingTyrosine residues in the enzyme are involved in the chain breakingprocessprocess
•• The residues form covalent bonds to DNAThe residues form covalent bonds to DNA•• The enzyme pulls the chains apart to create a gapThe enzyme pulls the chains apart to create a gap
Topo II
DNA
Tyr
Tyr
Topo II
Topo II
Topoisomerase IITopoisomerase II
•• Relieves the strain in the DNA helix by temporarily cleaving the DNARelieves the strain in the DNA helix by temporarily cleaving the DNAchain and crossing an intact strand through the broken strandchain and crossing an intact strand through the broken strand
3. DRUGS ACTING ON DNA3. DRUGS ACTING ON DNA
•• Tyrosine residues in the enzyme are involved in the chain breakingTyrosine residues in the enzyme are involved in the chain breakingprocessprocess
•• The residues form covalent bonds to DNAThe residues form covalent bonds to DNA
•• The intact strand of DNA is passed through the gapThe intact strand of DNA is passed through the gap•• The enzyme pulls the chains apart to create a gapThe enzyme pulls the chains apart to create a gap
Tyr
Tyr
5'
3' 5'
3'
Topo II
Topo II
3
Topoisomerase IITopoisomerase II
•• Relieves the strain in the DNA helix by temporarily cleaving the DNARelieves the strain in the DNA helix by temporarily cleaving the DNAchain and crossing an intact strand through the broken strandchain and crossing an intact strand through the broken strand
3. DRUGS ACTING ON DNA3. DRUGS ACTING ON DNA
•• Tyrosine residues in the enzyme are involved in the chain breakingTyrosine residues in the enzyme are involved in the chain breakingprocessprocess
•• The residues form covalent bonds to DNAThe residues form covalent bonds to DNA
•• The break is resealedThe break is resealed•• The intact strand of DNA is passed through the gapThe intact strand of DNA is passed through the gap•• The enzyme pulls the chains apart to create a gapThe enzyme pulls the chains apart to create a gap
Tyr
5'
3' 5'
3'
Topo II
4
Topoisomerase IITopoisomerase II
•• Relieves the strain in the DNA helix by temporarily cleaving the DNARelieves the strain in the DNA helix by temporarily cleaving the DNAchain and crossing an intact strand through the broken strandchain and crossing an intact strand through the broken strand
3. DRUGS ACTING ON DNA3. DRUGS ACTING ON DNA
•• Tyrosine residues in the enzyme are involved in the chain breakingTyrosine residues in the enzyme are involved in the chain breakingprocessprocess
•• The residues form covalent bonds to DNAThe residues form covalent bonds to DNA
•• The break is resealedThe break is resealed•• The intact strand of DNA is passed through the gapThe intact strand of DNA is passed through the gap•• The enzyme pulls the chains apart to create a gapThe enzyme pulls the chains apart to create a gap
Mechanism of chain cuttingMechanism of chain cutting
BaseO
HO
HH
HH
O
PO
O
O
5'
3'
BaseO
H
HH
HH
HO Tyr
TopoII
BaseO
HO
HHHH
O
PO
HO
O
5'
3'
O
BaseO
H
HHHH
Tyr
TopoII
3. DRUGS ACTING ON DNA3. DRUGS ACTING ON DNATopoisomerase IITopoisomerase II
Planar ringsPlanar rings
Planar ringsPlanar rings
Extra binding to sugarExtra binding to sugarphosphate backbone by NHphosphate backbone by NH33
DoxorubicinDoxorubicin (Adriamycin)(Adriamycin)
OMe
O
O
OH
OHH O
OH
O
CH2OH
O
NH3
H
H
H
H
Me
H
HOH
O
N
O
NH2
CH3CH3
C C
NH
O
NH
CC
D-Val
C
D-Val
L-Pro
N-Me-Gly
L-Pro
N-Me-Gly
C
O
N-Me-L-ValN-Me-L-Val
C
O
Me MeH H
CO O
H H
O
DactinomycinDactinomycin
Extra binding to sugar phosphateExtra binding to sugar phosphatebackbone by cyclic peptidebackbone by cyclic peptide
3. DRUGS ACTING ON DNA3. DRUGS ACTING ON DNA3.1 Intercalating agents3.1 Intercalating agentsExamplesExamples
3.2 Alkylating agents3.2 Alkylating agents
•• Contain highly electrophilic groupsContain highly electrophilic groups•• Form covalent bonds to nucleophilic groups in DNA Form covalent bonds to nucleophilic groups in DNA
(e.g. 7-N of guanine)(e.g. 7-N of guanine)•• Prevent replication and transcriptionPrevent replication and transcription•• Useful anti-tumour agentsUseful anti-tumour agents•• Toxic side effects (e.g. alkylation of proteins)Toxic side effects (e.g. alkylation of proteins)
ExampleExampleMechlorethamine (nitrogen mustard)Mechlorethamine (nitrogen mustard)
CH3 N
Cl
Cl
3. DRUGS ACTING ON DNA3. DRUGS ACTING ON DNA
Interstrand cross linkingInterstrand cross linkingIntrastrand cross linkingIntrastrand cross linking
Cross linkingCross linking
Nu
Nu
X X
Nu
Nu Nu
Nu
X X
Nu
Nu
3.2 Alkylating agents3.2 Alkylating agents3. DRUGS ACTING ON DNA3. DRUGS ACTING ON DNA
CH3 N
Cl
Cl
Mechlorethamine
+
Cl
NCH3
Aziridine ion
G = Guanine
DNA
G
N
H
N
N
N
N
NNH
N
O
O
NH2
NH2
G
DNA
N
N
N
N
N
H
N
NH
N
O
NH2
O NH2
CH3 N
Cl
+
DNA
N
N
N
NNH
N
O
NH2
N
H
NO NH2
NCH3
DNA
CH3
N
N
N
N
N
N
H
N
NH
N
O NH2
O
NH2
Crosslinked DNA
3.2 Alkylating agents3.2 Alkylating agents3. DRUGS ACTING ON DNA3. DRUGS ACTING ON DNA
Mechanism of actionMechanism of action
Aromatic ring - e withdrawing effectN is less nucleophilicLess reactive alkylating agentSelective for stronger nucleophiles (e.g. guanine)
Mechlorethamine analoguesMechlorethamine analogues
Uracil mustardUsed vs leukemiaAttached to a nucleic acid buildingblockConcentrated in fast growing cells(tumours)Some selectivity
N
Cl
Cl
N
Cl
Cl
HN
N
H
O
O
3.2 Alkylating agents3.2 Alkylating agents3. DRUGS ACTING ON DNA3. DRUGS ACTING ON DNA
Binds to DNA in regions rich in guanineunitsIntrastrand links rather than interstrandInhibits transcription
Converted to alkylating agent in thebody
Cisplatin Cisplatin
Pt
NH3Cl
NH3Cl
Mitomycin C Mitomycin C O
O
N
H2N
Me
CH2OCONH2
NH
OMe
3.2 Alkylating agents3.2 Alkylating agents3. DRUGS ACTING ON DNA3. DRUGS ACTING ON DNA
-MeOH
O
OH
N
H2N
Me
CH2OCONH2
NH
H
H
Ringopening
-H +
O
OH
N
H2N
Me
CH2OCONH2
NH2
Alkylating agent
H
H2N-DNA
OH
OH
N
H2N
Me
CH2
NH2
NH-DNA
O
C
O
NH2
H2N-DNA
H
-CO2-NH3
OH
OH
N
H2N
Me
CH2
NH2
NH-DNA
NH-DNA
Crosslinked DNA
O
O
N
H2N
Me
CH2OCONH2
NH
OMe
Reduction
OH
OH
N
H2N
Me
CH2OCONH2
NH
OMe
OH
OH
N
H2N
Me
CH2
NH2
NH
NH
HN
N N
N
O
HN
N N
N
O Guanine
Guanine
•• Abstracts H from DNA to generate radicalsAbstracts H from DNA to generate radicals•• Radicals react with oxygen resulting in chain cuttingRadicals react with oxygen resulting in chain cutting•• Bleomycin also inhibits repair enzymesBleomycin also inhibits repair enzymes
BLEOMYCIN A2 R = NHCH2CH2CH2SMe2BLEOMYCIN B2 R = NHCH2CH2CH2CH2NHC(NH2)=NH
BleomycinUsed vs skin cancer
N N
N
H
NH2CONH2
NH2
H2N
CH3 HN
O
N
H
O HO
CH3
CH3
O
CH3HO
H
N
NH N
S
S
N
O
O
N
H
N
O
HO
OH
OH
OH
OH
OH
O
O NH2
O
RO
O
3.3 Chain cutters3.3 Chain cutters3. DRUGS ACTING ON DNA3. DRUGS ACTING ON DNA
ContentsContents
Part 3: Section 7.3 (Drugs acting on RNA)
4. Drugs Acting On rRNA- Antibiotics
5. Drugs Acting On mRNA- Antisense Therapy- siRNA
6. Drugs related to nucleic acid building blocks- Examples: Antiviral agents- Examples
4. DRUGS ACTING ON 4. DRUGS ACTING ON rRNArRNAAntibiotics Antibiotics
Rifamycins
Chloramphenicol(vs typhoid)
Streptomycin
Chlortetracycline(Aureomycin)
Erythromycin
O2N
CH2OH
HO
HN
H
H
C
O CHCl2
R
Me
O
H
NMe
Me
OH
O
OHOH
OH
O
Me
O
O
C
Me Me
OMe O Me
Me
OH
H
H
H
H
H
H
H
H
H2N C
NH
NH
O
HN C
NH
NH2
O
OO
H
OH
H HO
H
H
OHH
H
CHO
OH
H
Me
CH2OH
H
HO
H
OH
H
H
MeHN
H
H
OOHOOH
Cl
OH
NMe2
O
HO
NH2
MeH
OHHO
O
O
O
H3C
CH3
CH3
O
CH3
CH3
HO
H3C
O
O
O
CH3
CH3
Me
NMe2
HO
OH
OMe
Antisense Antisense RNA Therapy RNA Therapy
5. DRUGS ACTING ON mRNA5. DRUGS ACTING ON mRNA
AdvantagesAdvantages•• Same effect as an enzyme inhibitor or receptor antagonistSame effect as an enzyme inhibitor or receptor antagonist•• Highly specific where the oligonucleotide is 17 nucleotides orHighly specific where the oligonucleotide is 17 nucleotides or
moremore•• Smaller dose levels required compared to inhibitors orSmaller dose levels required compared to inhibitors or
antagonistsantagonists•• Potentially less side effectsPotentially less side effects
DisadvantagesDisadvantages•• ‘‘ExposedExposed’’ sections of mRNA must be targeted sections of mRNA must be targeted•• Instability and polarity of oligonucleotides (pharmacokinetics)Instability and polarity of oligonucleotides (pharmacokinetics)•• Short lifetime of oligonucleotides and poor absorption acrossShort lifetime of oligonucleotides and poor absorption across
cell membranescell membranes
Antisense Therapy Antisense Therapy
5. DRUGS ACTING ON mRNA5. DRUGS ACTING ON mRNA
Small Interfering Small Interfering RNA-siRNA RNA-siRNA
5. DRUGS ACTING ON 5. DRUGS ACTING ON or throughor through RNA RNA“Dicer” enzyme
si RNA-ds, ~21BP, 2base overhang +phosphate
Targets specific RNAsequences for destruction
Small Interfering Small Interfering RNA-siRNA RNA-siRNA
5. DRUGS ACTING ON 5. DRUGS ACTING ON or throughor through RNA RNA
http://www.rnaiweb.com/RNAi/RNAi_Web_Resources/RNAi_Therapy___Clinical_Trials/
6. Drugs related to nucleic acid building blocks6. Drugs related to nucleic acid building blocks
Examples: Examples: Antiviral agents Antiviral agents
Chain terminating group
••Enzyme inhibitorEnzyme inhibitor••AZT is phosphorylated to a triphosphate in the bodyAZT is phosphorylated to a triphosphate in the body••Triphosphate has two mechanisms of actionTriphosphate has two mechanisms of action
- inhibits a viral enzyme (reverse transcriptase)- inhibits a viral enzyme (reverse transcriptase)- added to growing DNA chain and acts as chain terminator- added to growing DNA chain and acts as chain terminator
Azidothymidine (AZT) (Zidovudine;Retrovir) HN
N
O
CH3
O
OHO
N3
HN
N
O
CH3
O
OO
N3
P
O
OH
OP
O
OH
OP
O
OH
HO
ChainChainterminating terminating groupgroup
6. Drugs related to nucleic acid building blocks6. Drugs related to nucleic acid building blocks
Examples: Examples: Antiviral agents Antiviral agents
HN
N
O
H2NN
N
O
HO
Acyclovir(Zovirax)
ChainChainterminating terminating groupgroup
NotesNotes::Same mechanisms of action as AZTSame mechanisms of action as AZTUsed vs herpes simplex and shinglesUsed vs herpes simplex and shingles
Famciclovir(Famvir)
N
NN
NNH2
AcO
OAc