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Topic 5 Nucleic as Drug Targets

Nucleic Acids-Chapter 7 Patrick and Corey 187, 188, 193-194, 198-199 1. DEOXYRIBONUCLEIC (DNA) 1.2 Secondary Structure - Double Helix

Thymine o O 10A P Me O H2N N O G C O O A T O 3' TA P N T A O NH N G C O Major G C N N 5' groove O O TA 5' O T A O T A NH O O P Minor A T 2 N O groove 3' A T o O T A 34A C G O N O G C P O N HN 3' TA O N N T A O G C 5' 5' O H2N G C O GC O T A A T 3' O P C G O O TA O P O T A O

DNA DOUBLE HELIX Base Pairing G-C base pairing involves 3 H-bonds A-T base pairing involves 2 H-bonds 1. DEOXYRIBONUCLEIC ACID (DNA) 1.3 Tertiary Structure

• Double helix coils into a 3D shape - supercoiling

• Double helix has to unravel during replication

• Unravelling leads to strain

• Relieved by catalysed cutting and repair of DNA chain

• Important to the activity of the quinolone and fluoroquinolone antibacterial agents which act as enzyme inhibitors 2. RIBONUCLEIC ACID (RNA) 2.1 Primary structure

• Similar to DNA with the following exceptions • is used instead of • is used rather than

O HOCH2 O OH H H HN H H

OH OH O N H Ribose Uracil 2. RIBONUCLEIC ACID (RNA) 2.2 Secondary structure

• Single stranded

• Some regions of helical secondary structure exist due to base pairing within the same strand (see t-RNA)

• Adenine pairs to uracil; guanine pairs to cytosine 2. RIBONUCLEIC ACID (RNA) 2.3 Tertiary structure

• Three types of RNA are involved in synthesis:

• Messenger RNA (mRNA) Relays the for a protein from DNA to the protein production site

• Transfer RNA (tRNA) The adapter unit linking the triplet code on mRNA to specific amino acids

• Ribosomal RNA (rRNA) Present in ribosomes (the production site for protein synthesis). Important both structurally and catalytically 2. RIBONUCLEIC ACID (RNA) 3'p 2.3 Tertiary structure end

A AMINO 5'pACID C end C Base Pairing A G C mI Methylinosine G C G U I C G UH2 Dihydrouridine G C T Ribothymidine U U A U U Ps Pseudouridine G G G C U A UH U A G G C C mG Methylguanosine 2 C G C G mG G Amino C acid U C C G G C m2G Dimethylguanosine G A G C G C m2G C T Ps G UH2 C G UH2 A G U A t-RNA C G C G C G Yeast alanine-tRNA ACG U Ps Anticodon U mI binding region I C G ANTICODON for m-RNA Anticodon - the 3 bases are specific for the attached - to the complementary triplet code on m- RNA (the codon) 2. RIBONUCLEIC ACID (RNA) 2.5 - protein synthesis

PEPTIDE

NH NH2 R C H R' C H O C O C HO O HO O H H H H H H H H OH OH O O P O Adenine O O P O Adenine O O NH t RNA t RNA R C H O C Transfer of NH growing peptide chain to next R' C H amino acid HO OH O C H H H H HO O OH H H H H Adenine O O P O OH O Adenine O O P O t RNA O t RNA 2. RIBONUCLEIC ACID (RNA)

2.5 Translation - protein synthesis mRNA Overview Ribosome

DNA Amino acid tRNA mRNA

Translation

Transcription

Nucleus

Protein Contents

Part 2: Section 7.3 (Drugs acting on DNA)

3. Drugs acting on DNA 3.1. Intercalating agents - Topoisomerase II - Example – 3.2. Alkylating agents 3.3. Chain cutters 3. DRUGS ACTING ON DNA 3.1 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 prevents replication and • Intercalation inhibits topoisomerase II- see Doxorubicin, p.198 Corey. 3. DRUGS ACTING ON DNA 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' 5' DNA

• Tyrosine residues in the enzyme are involved in the chain breaking process • The residues form covalent bonds to DNA 3. DRUGS ACTING ON DNA 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' 5' DNA Tyr

Topo II

• Tyrosine residues in the enzyme are involved in the chain breaking process • The residues form covalent bonds to DNA • The enzyme pulls the chains apart to create a gap 3. DRUGS ACTING ON DNA 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

DNA Tyr

Topo II • Tyrosine residues in the enzyme are involved in the chain breaking process • The residues form covalent bonds to DNA • The enzyme pulls the chains apart to create a gap • The intact strand of DNA is passed through the gap 3. DRUGS ACTING ON DNA Topoisomerase II • Relieves the strain in the DNA helix by temporarily cleaving the DNA chain and crossing an intact strand through the broken strand

3

Topo II Tyr 5' 3' 3' 5' Tyr Topo II • Tyrosine residues in the enzyme are involved in the chain breaking process • The residues form covalent bonds to DNA • The enzyme pulls the chains apart to create a gap • The intact strand of DNA is passed through the gap • The break is resealed 3. DRUGS ACTING ON DNA Topoisomerase II • Relieves the strain in the DNA helix by temporarily cleaving the DNA chain and crossing an intact strand through the broken strand

4

Topo II Tyr

5' 3' 3' 5'

• Tyrosine residues in the enzyme are involved in the chain breaking process • The residues form covalent bonds to DNA • The enzyme pulls the chains apart to create a gap • The intact strand of DNA is passed through the gap • The break is resealed 3. DRUGS ACTING ON DNA Topoisomerase II Mechanism of chain cutting

5' Base O H H 5' Base O H H H H TopoII HO H H H TopoII HO Tyr Tyr O H O O P O P O Base O O Base O O O H H H H H H H H O H O H

3' 3' 3. DRUGS ACTING ON DNA 3.1 Intercalating agents Examples

Planar rings

N-Me-Gly N-Me-Gly O OH O N-Me-L-Val L-Pro N-Me-L-Val L-Pro CH2OH D-Val D-Val O O OH C C C O C O H C C H H H Me Me OMe O OH H O NH NH C O O C H O N NH2 H Me H H H HO O O Planar rings H NH3 CH3 CH3

Dactinomycin Doxorubicin (Adriamycin) Extra binding to Extra binding to sugar phosphate backbone by NH3 backbone by cyclic peptide 3. DRUGS ACTING ON DNA 3.2 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 anti-tumour agents • Toxic side effects (e.g. alkylation of )

Example Mechlorethamine ( mustard) Cl

CH3 N

Cl 3. DRUGS ACTING ON DNA 3.2 Alkylating agents Cross linking

X X X X

Nu Nu Nu Nu Nu Nu Nu Nu

Intrastrand cross linking Interstrand cross linking 3. DRUGS ACTING ON DNA 3.2 Alkylating agents Mechanism of action

DNA DNA

H G = Guanine O N NH2 Cl H O N NH2 N + N N N N CH3 N CH3 N CH3 N N G Cl Cl Mechlorethamine Aziridine ion Cl N N NH2 N N NH2

N NH N NH G O O

DNA DNA

H O N H NH2 O N NH2 N N N Crosslinked DNA N N CH N 3 + N

N N N NH2 N N NH2 CH N NH N NH 3 O O 3. DRUGS ACTING ON DNA 3.2 Alkylating agents Mechlorethamine analogues

Cl Cl O N N HN Cl Cl O N H

Aromatic ring - e withdrawing effect Uracil mustard N is less nucleophilic Used vs leukemia Less reactive alkylating agent Attached to a nucleic acid building Selective for stronger nucleophiles block (e.g. guanine) Concentrated in fast growing cells (tumours) Some selectivity 3. DRUGS ACTING ON DNA 3.2 Alkylating agents

Cisplatin Mitomycin C

O CH2OCONH2 Cl NH3 H2N Pt OMe Cl NH3 Me N NH O

Binds to DNA in regions rich in guanine Converted to alkylating agent in the units body Intrastrand links rather than interstrand Inhibits transcription H O OH O CH2OCONH2 CH2OCONH2 CH2OCONH2 H N H2N OMe H2N OMe 2

-MeOH Reduction N NH Me N NH Me N NH Me H O OH OH

O H C NH2 H O O CH OCONH OH 2 2 CH2 H N H N-DNA -H + 2 H2N 2 NH-DNA Ring H2N-DNA opening N Me Me N NH2 NH OH OH 2 Alkylating agent O Guanine

HN N N NH N NH-DNA OH OH CH2 O CH2 Guanine H2N H N HN N 2 NH NH-DNA -CO N N 2 N -NH Me 3 Me N NH2 NH2 OH OH Crosslinked DNA 3. DRUGS ACTING ON DNA 3.3 Chain cutters

CONH2 NH2

NH2 O N Bleomycin H O Used vs skin cancer N R N N CH3 O H O HO N S H2N O NH N

CH3 HN O N CH3 HO CH3 S H

HO O N OH O N O H BLEOMYCIN A2 R = NHCH2CH2CH2SMe2 BLEOMYCIN B2 R = NHCH CH CH CH NHC(NH )=NH OH OH 2 2 2 2 2

OH O OH

O NH2

• Abstracts H from DNA to generate radicals • Radicals react with resulting in chain cutting • Bleomycin also inhibits repair Contents

Part 3: Section 7.3 (Drugs acting on RNA)

4. Drugs Acting On rRNA - Antibiotics 5. Drugs Acting On mRNA - - siRNA 6. Drugs related to nucleic acid building blocks - Examples: Antiviral agents - Examples 4. DRUGS ACTING ON rRNA

NH Antibiotics NH H HN C NH2 H2N C NH H OH H H HO OH OH Me H H OH HO H Me N O H O2N H O CH2OH O CHO O R H HN H Me H Me OH Me OH Streptomycin C O H O CHCl OH O 2 HO O O Me Me H CH OH H H 2 Chloramphenicol H MeHN H (vs typhoid) H H H H Me OMe O Me H OH OH H C NMe CH3 2 O O HO

Rifamycins H C 3 CH3 HO O OH O OH O O HO O CH3 OH

NH 2 H3C CH3 O CH O 3 O OH CH H 3 HO Me Cl NMe2 O OH Me OMe Chlortetracycline Erythromycin (Aureomycin) 5. DRUGS ACTING ON mRNA Antisense RNA Therapy 5. DRUGS ACTING ON mRNA Antisense Therapy

Advantages • Same effect as an enzyme inhibitor or receptor antagonist • Highly specific where the is 17 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 (pharmacokinetics) • Short lifetime of oligonucleotides and poor absorption across membranes 5. DRUGS ACTING ON or through RNA “Dicer” enzyme Small Interfering RNA-siRNA

Targets specific RNA sequences for destruction

si RNA- ds, ~21BP, 2 base overhang + phosphate 5. DRUGS ACTING ON or through RNA Small Interfering RNA-siRNA

http://www.rnaiweb.com/RNAi/RNAi_Web_Resources/RNAi_Therapy___Clinical_Trials/ 6. Drugs related to nucleic acid building blocks Examples: Antiviral agents

O O CH Azidothymidine (AZT) HN 3 CH3 (Zidovudine;Retrovir) HN O N O N O O O HO P O P O P O O HO O OH OH OH

N3 N 3 Chain terminating group •Enzyme inhibitor •AZT is phosphorylated to a triphosphate in the body •Triphosphate has two mechanisms of action - inhibits a viral enzyme (reverse transcriptase) - added to growing DNA chain and acts as chain 6. Drugs related to nucleic acid building blocks Examples: Antiviral agents

O N N HN N AcO N N N NH2 H2N N

O Chain Chain OAc HO terminating terminating group group Acyclovir Famciclovir (Zovirax) (Famvir)

Notes: Same mechanisms of action as AZT Used vs herpes simplex and shingles