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 Adenine 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' Cytosine Guanine 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 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 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 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) 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 Inosine 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 amino acid - base pair to the complementary triplet code on m- RNA (the codon) 2. RIBONUCLEIC ACID (RNA) 2.5 Translation - 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 PEPTIDE 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 – 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' 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 sugar Extra binding to sugar phosphate 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 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 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.