Topic 5 Nucleic Acids as Drug TargetsPart 3: Section 7.3 (Drugs acting on RNA) 4.Drugs Acting On...

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




•• 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






•• 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






•• 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






•• 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


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


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


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


-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

Topic 5 Nucleic Acids as Drug TargetsPart 3: Section 7.3 (Drugs acting on RNA) 4.Drugs Acting On...

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