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Topic 5 Nucleic Acids as Drug Targets Nucleic Acids-Chapter 7 Patrick and Corey 187, 188, 193-194, 198-199 1. DEOXYRIBONUCLEIC ACID (DNA) 1.2 Secondary Structure - Double Helix Thymine o 10A Me G C AT Major groove O O H2N N O O P O TA T A G C G C NH O O 5' TA T A AT AT T A C G G C TA T A G C GC GC T A A T C G TA T A DNA DOUBLE HELIX o 34A O P O O O P N O O 5' NH2 3' 3' O O P O O 5' N O O O 3' N N N T A Minor groove Adenine O N N HN 3' O P O O N N O Cytosine H2N Guanine 5' O O O P O O 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 enzyme 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 • Ribose is used instead of deoxyribose • Uracil is used rather than thymine O HOCH2 O H H OH HN H H OH OH Ribose O N H 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 protein synthesis: • Messenger RNA (mRNA) Relays the code 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) 2.3 Tertiary structure 3'p end A AMINO ACID 5'p C end C Base Pairing A G C G C mI Methylinosine G U I Inosine C G UH2 Dihydrouridine G C T Ribothymidine U U A U U UA G G C G Ps Pseudouridine A G G C C UH C G C G mGU G mG Methylguanosine C 2 U C C G G m2G Dimethylguanosine 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 ANTICODON G Yeast alanine-tRNA Anticodon - the 3 bases are specific for the attached amino acid - base pair to the complementary triplet code on mRNA (the codon) Amino acid t-RNA AC G Anticodon binding region for m-RNA 2. RIBONUCLEIC ACID (RNA) 2.5 Translation - protein synthesis PEPTIDE NH2 NH R' C H O C R C H O C HO H H Adenine HO H H O H H OH O O P O O Adenine O H H OH O O PEPTIDE P O O NH t RNA t RNA Transfer of growing peptide chain to next amino acid R C H O C NH HO H H Adenine R' C H O C OH H H O OH O P O O t RNA HO H H Adenine O H H O OH O P O O t RNA 2. RIBONUCLEIC ACID (RNA) 2.5 Translation - protein synthesis Overview mRNA Ribosome Amino acid tRNA DNA 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 – Proflavine 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 transcription • 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' Tyr DNA 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 Tyr DNA 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 5' O H H Base TopoII H H H HO O P O O Base O H Tyr H O 3' H H TopoII Tyr O O P O O Base O H H H H HO H O H H H H H O 3' H 3. DRUGS ACTING ON DNA 3.1 Intercalating agents Examples Planar rings N-Me-Gly N-Me-Gly L-Pro N-Me-L-Val D-Val O H N-Me-L-Val C Me C C H H NH C L-Pro O O C Me C C CH2OH OH O H OMe NH O OH H C N O H O O H NH2 O CH3 O OH D-Val O O O Planar rings CH3 Dactinomycin Extra binding to sugar phosphate backbone by cyclic peptide HO Me H NH3 H H H Doxorubicin (Adriamycin) Extra binding to sugar phosphate backbone by NH3 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 proteins) Example Mechlorethamine (nitrogen 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 Intrastrand cross linking Nu Nu Nu Interstrand cross linking 3. DRUGS ACTING ON DNA 3.2 Alkylating agents Mechanism of action DNA DNA O G = Guanine H N NH 2 Cl N CH3 N CH3 H N O N + N N N CH3 G Cl NH 2 N N N Cl Cl Aziridine ion Mechlorethamine N N NH N NH 2 N G N DNA O CH3 + N NH DNA H N O NH 2 NH 2 N Crosslinked DNA N N N NH 2 O O H N N N N N N NH 2 NH N O N N CH3 N N NH 2 NH O 3. DRUGS ACTING ON DNA 3.2 Alkylating agents Mechlorethamine analogues Cl Cl O N N HN Cl Aromatic ring - e withdrawing effect N is less nucleophilic Less reactive alkylating agent Selective for stronger nucleophiles (e.g. guanine) O N H Cl Uracil mustard Used vs leukemia Attached to a nucleic acid building block Concentrated in fast growing cells (tumours) Some selectivity 3. DRUGS ACTING ON DNA 3.2 Alkylating agents Cisplatin Mitomycin C Cl NH3 Pt Cl NH3 O CH2OCONH2 H2N OMe N Me 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 CH2OCONH2 H2N H2N OMe N Me NH Reduction H2N OMe N Me NH N Me O H C NH2 O OH CH2OCONH 2 H2N CH2 H2 N-DNA H2N NH-DNA H2 N-DNA N Me NH2 OH NH OH OH H O CH2OCONH 2 -MeOH O -H + Ring opening O CH2OCONH2 N Me NH2 OH Alkylating agent O Guanine N HN NH NH-DNA OH CH2 CH2 H 2N H2N -CO2 -NH3 OH NH NH-DNA OH NH2 OH O Guanine N HN N N Me N Me N N NH2 Crosslinked DNA N H 3. DRUGS ACTING ON DNA 3.3 Chain cutters CONH2 NH2 NH2 N H N O N O H2N O CH3 HN CH3 HO H N O CH3 HO N H R N O NH CH3 Bleomycin Used vs skin cancer S N S N O HO O OH O N H O OH OH BLEOMYCIN A2 BLEOMYCIN B2 R = NHCH2CH2CH2SMe2 R = NHCH2CH2CH2CH2NHC(NH2)=NH OH O OH O • • • NH2 Abstracts H from DNA to generate radicals Radicals react with oxygen resulting in chain cutting Bleomycin also inhibits repair enzymes Contents 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 rRNA Antibiotics H2N OH HO H OH C Me H O CH2OH O R O O Me Chloramphenicol (vs typhoid) H Me HO H H H H OMe O H C Me H OH Streptomycin O CH2OH H MeHN H Me H NMe2 CH3 HO O Rifamycins O OH O H3C CH3 HO OH NH2 OH Cl HO Me H3C CH3 O O CH3 O CH3 OH O Me Chlortetracycline (Aureomycin) O CH3 H NMe2 O HO O O H OH O OH OH OH H OH Me H H Me O CHCl2 NH2 H O O C CHO H OH Me HN H H H N Me HN H NH OH H HO H O2N C NH NH Erythromycin OMe 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 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 5. DRUGS ACTING ON or through RNA Small Interfering RNA-siRNA Targets specific RNA sequences for destruction si RNAds, ~21BP, 2 base overhang + phosphate “Dicer” enzyme 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 Azidothymidine (AZT) (Zidovudine;Retrovir) O CH3 HN O N O O O HO P O P O P O HO O CH3 HN OH OH O N O OH N3 N3 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 terminator 6. Drugs related to nucleic acid building blocks Examples: Antiviral agents O HN H2 N Acyclovir (Zovirax) AcO N N O HO N N Chain terminating group N OAc Famciclovir (Famvir) Notes: Same mechanisms of action as AZT Used vs herpes simplex and shingles N N NH2 Chain terminating group