Types of DRUGS ACTING on DNA (In Chapter 9) Intercalating Agents

Home , Alitretinoin, Amsacrine, Chlormethine, Dactinomycin, Mitomycins, Mitomycin C

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Chap. 9 - DRUGS TARGETING NUCLEIC ACIDS DNA & RNA Types of DRUGS ACTING on DNA (in chapter 9)

Intercalating agents

Topoisomerase poisons

Alkylating agents

Metallating agents

Chain cutters

Chain terminators

Control of gene transcription (and epigenetics)

1 © Oxford University Press, 2013

DRUGS ACTING ON DNA

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 • A preference is often shown for either the minor or major groove • Intercalation prevents replication and transcription • Intercalation can inhibit topoisomerases too

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Intercalating agents Proflavine, also called diaminoacridine, is an acriflavine derivative, a disinfectant bacteriostatic against many gram-positive bacteria. It has been used in the form of the dihydrochloride and hemisulfate salts as a topical antiseptic, and was formerly used as a urinary antiseptic. Proflavine is toxic and carcinogenic in mammals and so it is used only as a surface disinfectant or for treating superficial wounds.

Proflavine is also known to have a mutagenic effect on DNA by intercalating between nucleic acid base pairs. It differs from most other mutagenic components by causing basepair-deletions or basepair- insertions and not substitutions.

NH3 Cl Ar

NH3 Cl

H2N N NH2 HOSO Planar tricyclic system NH3 3 The amino substituents are protonated Ar Used as a topical antibacterial agent in WW II NH3 HOSO Targets bacterial DNA 3 Too toxic for systemic use as a salt proflavine is more stable and avoids oxidation

3 © Oxford University Press, 2013

Intercalating agents: Proflavine

Proflavine

GC A T TA T A H NNHN G C 3 3 G C sugar phosphate TA TA backbone TA A T TA A T T A C G G C proflavine TA H3NNH3 TA GC G C O O GC T A G C A T C G TA TA

DNA DOUBLE HELIX van der Waals interactions Ionic interactions

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ethidium bromide Ethidium bromide is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. When exposed to ultraviolet light, it will fluoresce with an orange colour, intensifying almost 20-fold after binding to DNA. It has Br N been commonly used since the 1950s in veterinary medicine to treat trypanosomiasis in cattle, a disease caused by trypanosomes. The high incidence of antibiotic resistance makes this treatment impractical in H2N NH2 some areas, where the related isometamidium chloride is used instead. Ethidium bromide may be a mutagen, although this depends on the organism exposed and the fluorescent stain for gel electrophoresis circumstances of exposure. Approximately 30,000 people in sub-Saharan Africa get African trypanosomiasis each year and Chagas disease, which causes 21,000 deaths per year mainly in Latin America.

Protozoan trypanosomes spread by the Tsetse Fly, develops over years 5 © Oxford University Press, 2013

Br N

NH H

N N NH2 H2N N

isometamidium chloride

Isometamidium is a phenanthridine aromatic amidine with a narrow therapeutic index which has been marketed for over 30 years as both a prophylactic and a therapeutic trypanocidal agent in the field. Isometamidium chloride is used curatively at lower dosage rates, and prophylactically at higher dosage rates.

Indications Intromidium is indicated for treatment and prevention of trypanosomiasis caused by Trypanosoma spp. in cattle, goats, sheep, camels, horses and dogs. A preventive dosage ensures protection for 2 to 4 months. When clinical cases occur, the whole group should be treated.

Contra indications Intromidium should not be administered subcutaneously. Avoid concurrent administration of other trypanocidal drugs, particularly diminazene aceturate. Avoid underdosing. 6 © Oxford University Press, 2013

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Intercalating agents - anticancer agents Doxorubicin (also called adriamycin) 1. Intercalates via the major groove of DNA double helix. 2. Blocks the action of topoisomerase II by stabilising the DNA- enzyme complex 3. Acts as a topoisomerase poison

Doxorubicin is commonly used to treat some leukemias and Planar rings Hodgkin's lymphoma, as well as cancers of the bladder, breast, O OH O stomach, lung, ovaries, thyroid, soft tissue sarcoma, multiple CH2OH myeloma, and others Many combination therapies are used, such as (adriamycin, cyclophosphamide), OH

Liposomal form OMe O OH H O There is a pegylated (polyethylene glycol coated) liposome- encapsulated form of doxorubicin, sold as Doxil. Doxil is used H O primarily for the treatment of ovarian cancer or AIDS-related H Me Kaposi's sarcoma where the disease has progressed or recurred H H H after platinum-based chemotherapy. HO H Cl Cyclophosphamide NH3 Common side effects include low white blood cell counts, loss of appetite, vomiting, hair loss, bleeding Doxorubicin (Adriamycin) from the bladder, an increased future risk of cancer, O N infertility, allergic reactions, and pulmonary fibrosis. Extra binding to sugar phosphate P Cl Cyclophosphamide is in the alkylating agent and nitrogen backbone by NH ONH mustard family of medications. It works by interfering 3 with the duplication of DNA and the creation of RNA. 7 © Oxford University Press, 2013

The biosynthesis of Doxorubicin uses enzymes found in fat metabolism. This involves using acetyl building blocks (2C) as acetyl CoA. Such molecules are called “kitids” (often multiples of 2C). There are 1000s of compounds that have been discovered from the random synthesis of clusters of proteins working together. Chemists have tried to splice in various combinations of such genes in microorganisms to make novel drugs as potential lead compounds.

a few more steps... 8 © Oxford University Press, 2013

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Polyethylene glycol (PEG) is a polyether compound with many applications from industrial manufacturing to medicine. A comparison of uricase and PEG-uricase. PEG-uricase includes 40 polymers of 10kDa PEG. PEGylation improves its solubility at physiological pH, increases serum half-life and reduces immunogenicity without compromising activity. Upper images show the whole tetramer, lower images show one of the lysines that is PEGylated.

O O Nu O Nu O O etc. O O

OH OH

O N O H N O H 2 O pH = 5 O H (-H2O) n OH OH n +3 O O Fe imine (oxime) linkage to the drug through a carbonyl group

OH OH OH O H O O H O O O H O O n Cl Cl O OH O Cl O O H O phosgene or phosgene equivalent O O n 9 © Oxford University Press, 2013

Intercalating agents - anticancer agents Dactinomycin 1. Intercalates via minor groove of DNA double helix 2. Prevents unwinding of DNA double helix 3. Blocks transcription by blocking DNA- dependent RNA polymerase

Dactinomycin is a chemotherapy used to treat many cancers, ildiWiltincluding Wilms tumor, rhabdomyosarcoma, Ewing's sarcoma, trophoblastic neoplasm, testicular cancer, and certain types of ovarian cancer. It is given by planar rings Dactinomycin injection into a vein. Common side effects can intercallate include bone marrow suppression, vomiting, mouth ulcers, hair loss, liver problems, infections, muscle pains, future cancers, allergic reactions, and tissue death at the site of injection. Use in pregnancy may harm the baby.

Dactinomycin. also known as actinomycin D, is the most significant member of actinomycines, which are a class of polypeptide antitumor antibiotics isolated from soil bacteria of the genus Streptomyces. It is one of the Extra binding to sugar phosphate older anticancer drugs, and has been used for backbone by cyclic peptides many years. © Oxford University Press, 2013

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Many quinone methides show medicinal properties O two common O Taxodone and its oxidized rearrangement patterns Can be extended to product, taxodione, are diterpenoid quinone naphthalene, methides found in Taxodium distichum (bald anthracene, cypress), Rosmarinus officinalis (rosemary), phenanthrene several Salvia species and other plants, that para quinone ortho quinone display anticancer, antibacterial, antioxidant, methide pattern methide pattern B antifungal, insecticide, and antifeedant activities. resonance H OH O 1e- 1e- O HO HO HO

oxidation lose proton oxidation lose proton H H H H H H O O B Taxodone O OH H B O HO HO

nucleophiles undergo conjugate H addition N H R O H O H2N 11 © Oxford UniversityR Press, 2013

Intercalating agents and chain cutting - Bleomycins

primary amine Fe+2 or Fe+3 CONH2 NH2 can chelate here H O N H NH A variety of 2 possibilities. H primary amide R O N pyrimidine NN Me O S ring HHHH O HO N Bithiazole H2N O NH N H intercalating primary amine Me HN O region N Me HO Me S H H H HO N O OH imidazole H ring O NH O OH Bleomycin A2 R = NHCH2CH2CH2SMe2 Bleomycin B2 R = NHCH2CH2CH2CH2NHC(NH2)=NH OH Notes on bleomycins OH • Used as anticancer agents OH O O • Intercalated by means of bithiazole ring system • Ferrous ion then chelated by nitrogens of the primary NH 2 amines, amide and pyrimidine ring • Reaction with oxygen results in a ferric ion and reactive oxygen species • Results in radical formation and chain cutting • Bleomycin prevents DNA ligase from repairing damage 12 © Oxford University Press, 2013

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Bleomycin is a medication used to treat cancer. This includes Hodgkin's lymphoma, non- Hodgkin's lymphoma, testicular cancer, ovarian cancer, and cervical cancer among others. Typically used with other cancer medications, it can be given intravenously, by injection into a muscle or under the skin. It may also be administered inside the chest to help prevent the recurrence of a fluid around the lung due to cancer; however talc is better for this.

Common side effects include fever, weight loss, vomiting, rash, a severe type of anaphylaxis, possible inflammation of the lungs that can result in lung scarring. Chest X -rays every couple of weeks are recommended to check for this. Bleomycin may cause harm to the baby if used during pregnancy. It is believed to primarily work by preventing synthesis of DNA.

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Free radical damage to DNA can remove bases and DNA DNA DNA H B

O Base O Base O Base O O O H H O B H O +3 O O O O Fe HN NH DNA +4 DNA Fe+4 DNA Fe HN NH HN NH Bleomycin

Bleomycin Bleomycin H Base DNA

Iron is believed to be the metal responsible for the creation of free O O radicals (often hydroxyl) because it exists at the highest O concentration of any transition metal in most living organisms.

Free radicals can attack the deoxyribose DNA backbone and bases, potentially causing a number of lesions that can be O cytotoxic or mutagenic. Cells have developed complex and DNA H B efficient repair mechanisms to fix the lesions. In the case of free DNA strands radical attack on DNA, base-excision repair is the repair can unravel. mechanism used. Free radical reactions with the deoxyribose sugar backbone (C1, C2, C3, C4 or C5) are initiated by hydrogen abstraction from a deoxyribose carbon, and the predominant consequence is eventual strand breakage and base release. 14 © Oxford University Press, 2013

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Topoisomerase medications are split into two main classes: topoisomerase poisons, which target the topoisomerase-DNA complex, and topoisomerase inhibitors, which disrupt catalytic turnover.

Topo II poisons

Examples of topoisomerase poisons include the following: eukaryotic type II topoisomerase inhibitors (topo II): amsacrine, etoposide (next slide), etoposide phosphate, teniposide and doxorubicin. These drugs are anti-cancer therapies. btiltbacterial type II topoi somerase i nhibit ors (gyrase an d topo IV): fl uoroqu ino lones. These are antibacterials and include such fluoroquinolones as ciprofloxacin.

Some of these poisons encourage the forward cleavage reaction (fluoroquinolones), while other poisons prevent the re-ligation of DNA (etoposide and teniposide).

Ciprofloxacin (later slides) targets prokaryotes topo II a thousand times better than it targets eukaryotic topo IIs. O

Topo II inhibitors H N

These inhibitors target the N-terminal ATPase domain of topo II and O N prevent topo II from turning over. Very few of these inhibitors. N O N An example of topoisomerase inhibitors includes : H ICRF 193 ICRF-193. The structure of this compound bound to the O ATPase domain is known. The drug binds in a non-competitive manner and locks down the dimerization of the ATPase domain. 15 © Oxford University Press, 2013

Topoisomerase poisons - non-intercalating

H O Stabilizes the complex between Me O O DNA and topoisomerase enzymes HO Used as anticancer agents HO O Also causes chain cutting O 4 O O O Etoposide

MeO 4' OMe OH Etoposide is used as a form of chemotherapy for cancers such as Kaposi’s sarcoma, Ewing's sarcoma, lung cancer, testicular cancer, lymphoma, nonlymphocytic leukemia, and glioblastoma multiforme. It is often given in combination with other drugs (such as bleomycin). It is also sometimes used in a conditioning regimen prior to a bone marrow or blood stem cell transplant.

It is given intravenously (IV) or orally in capsule form. If the drug is given IV, it must be done slowly over a 30- to 60-minute period because it can lower blood pressure as it is being administered. Blood pressure is checked often during infusing, with the speed of administration adjusted accordingly.

Etoposide forms a ternary complex (3 way) with DNA and the topoisomerase II enzyme (which aids in DNA unwinding), prevents re-ligation of the DNA strands, and by doing so causes DNA strands to break. Cancer cells rely on this enzyme more than healthy cells, since they divide more rapidly. This causes errors in DNA synthesis and promotes apoptosis of the cancer cell. 16 © Oxford University Press, 2013

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Topoisomerase poisons - non-intercalating

H Stabilizes the complex O O between DNA and O HO topoisomerase enzymes S HO O Used as anticancer agents Also causes chain cutting, O 4 O similar to etoposide. O O Teniposide

MeO 4' OMe OH Teniposide is a chemotherapeutic medication used in the treatment of childhood acute lymphocytic leukemia, Hodgkin's lymphoma, certain brain tumours, and other types of cancer. It slows the growth of cancer cells in the body (by a ternary complex).

When Teniposide is used with other chemotherapeutic agents, it results in severe bone marrow suppression. Other common side effects include gastrointestinal toxicity, hypersensitivity reactions, and reversible alopecia (hair loss).

Teniposide is a semisynthetic derivative of podophyllotoxin from the rhizome of the wild mandrake (Podophyllum peltatum). More specifically, it is a glycoside of podophyllotoxin with a D-glucose derivative. It is chemically similar to the anti-cancer drug etoposide, being distinguished only by a thienyl where etoposide has a methyl. Both these compounds have been developed with the aim of 17 creating less toxic derivatives of podophyllotoxin. © Oxford University Press, 2013

DRUGS ACTING ON DNA - Topoisomerase poisons - non-intercalating

Examples - Camptothecin Notes • Stabilizes a complex between DNA and O topoisomerase I ABC N • Single-strand breaks accumulate in the chain N D • Irreversible double-strand breaks occur during E Lactone ring O transcription • Semi-synthetic analogues used as anticancer Me HO O agents

Camptothecin is a cytotoxic quinoline alkaloid which inhibits the DNA enzyme topoisomerase I (topo I). It was isolated from the bark and stem of Camptotheca acuminata (Happy tree), a tree native to China and used as a cancer treatment in Traditional Chinese Medicine. It showed remarkable anticancer activity in preliminary clini cal tri al s but a lso l ow sol ubili ty an d (high) ad verse d rug reacti on. BfhBecause of these disadvantages synthetic and medicinal chemists have developed numerous syntheses of Camptothecin and various derivatives to increase the benefits of the chemical, with good results. Two analogues have been approved and are used in cancer chemotherapy today, topotecan and irinotecan.

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Camptothecin, DNA and enzyme complex Camptothecin binds to the topo I enzyme and DNA complex by hydrogen bonds resulting in DNA a ternary complex, and thereby stabilizing it. This prevents DNA re-ligation and causes DNA damage which results in apoptosis. The most important part of the structure is the E-ring which interacts from three different Protein positions with the enzyme. The hydroxyl A B C group in position 20 forms hydrogen bond to D the side chain on aspartic acid number 533 in E the enzyme. It is critical that the configuration Drug of the chiral carbon is (S) because (R) is inactive. The lactone is bonded with two hydrogen bonds to the amino groups on arginine 364. Toxicity of CPT is primarily a result of conversion of single-strand breaks into double-strand breaks during the S-phase

R2 R1 when the replication fork collides with the cleavage complexes formed by DNA and CPT. R3 O The lactone ring in CPT is highly susceptible N to hydrolysis. The open ring form is inactive

R4 N and it must therefore be closed to inhibit topo I. The closed form is favored in acidic O There are many variations condition, as it is in many cancer cells made with different groups microenvironment. CPT is transported into the at R1, R2, R3 and R4 OH O cell by passive diffusion. 19 © Oxford University Press, 2013

Topotecan (trade name Hycamtin) is a Camptothecin N chemotherapeutic agent that is a Topotecan topoisomerase I inhibitor. It is a synthetic, water-soluble analog of the natural chemical O HO N O compound camptothecin. It is used in the N form of its hydrochloride salt to treat ovarian N cancer, lung cancer and other cancer types. N Topotecan was the first topoisomerase I O O inhibitor for oral use. Why is it more water soluble? Loperamide often taken with O O topotecan due to severe diarhea side effect. SN-38 isanantineoplastic drug. It istheactive metabolite of irinotecan (an analog of camptothecin - a topoisomerase I inhibitor) but has 1000 times irinotecan more activity than irinotecan itself. SN-38 and its glucuronide are lost into the N (next slide) bile and feces. It can cause the symptoms of diarrhoea and myelosuppression experienced by ~25% of the patients administered irinotecan. N O HO O O N N O N N

O O SN-38 O O

Loperamide, sold under the brand name Loperamide Imodium among others, is a medication used to decrease the frequency of diarrhea. It is often used for this purpose in gastroenteritis, OH N inflammatory bowel disease, and short bowel syndrome. It is not recommended for O N those with blood in the stool. The Cl medication is taken by mouth. 20 © Oxford University Press, 2013

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Model of human liver cell showing blood, bile and intestinal compartments, indicating tissue involvement of genes in the irinotecan pathway – eventually leads to inhibition of DNA replication and transcription.

Irinotecan-associated diarrhea sometimes leads to such severe dehydration that it requires hospitalization. This side-effect is treat ed with an antidi arrh eal such as loperamide.

The immune system is adversely impacted by irinotecan, reflected by dramatically lowered neutrophils (white blood cells). The patient may experience a period of neutropenia while the bone marrow increases white cell production to compensate.

Neutrophils make uppjy the majority of circulating white blood cells and serve as the primary defense against infections by destroying bacteria, bacterial fragments and immunoglobulin-bound viruses in the blood. Patients with neutropenia are more susceptible to bacterial infections and, without prompt medical attention, the condition may become life-threatening (neutropenic sepsis). Neutropenia can be acute (temporary) or chronic (long21 lasting). © Oxford University Press, 2013

[neutral compound]1-octanol [all forms compound]1-octanol log Poct/H2O = log log Doct/H2O = log [neutral compound]water [all forms compound]water The partition-coefficient (P) or distribution-coefficient (D) is the ratio of concentrations of a compound in a mixture of two immiscible phases at equilibrium. This ratio is a measure of the difference in solubility of the compound in these two phases. The partition-coefficient generally refers to the concentration ratio of un-ionized species of compound whereas the distribution-coefficient refers to the concentration ratio of all species of the compound (ionized plus un-ionized) and depends on pH. Usually one phase is water and the other is 1-octanol. The partition coefficient measures how hydrophilic or hydrophobic a chemical substance is. Hydrophobic drugs with high octanol/water partition coefficients are mainly distributed to hydrophobic areas such as lipid bilayers of cells, while low octanol/water partition coefficients) are found primarily in aqueous regions such as blood serum. solubility and partition coefficients of straight chain functiona groups in 100 g water OH OH OH OH C5H11OH C6H13OH C7H15OH C8H17OH C9H19OH CH3 0.0058 mol 0.0008 mol 0.0058 mol 0.0008 mol 100% 100% 0.11 mol 0.030 mol solubility = 100% 2.6g 0.6g 0.09g 0.6g 0.09g miscible miscible miscible 7.9g +1.51 +2.03 +2.62 +2.97 +4.02 log Poct/H2O = -0.74 -0.38 +0.25 +0.84

O O O O O O O O

C C C C C C C C C H OH C H OH C H OH H OH H3C OH C2H5 OH C3H7 OH C4H9 OH 5 11 6 13 7 15

100% 100% 100% 100% 4.9g 1.1g 0.2g 0.07g solubility = miscible miscible miscible miscible (guess)

+1.39 +1.92 +2.6 +3.05 log Poct/H2O = -0.54 -0.32 +0.33 +0.79 (guess)

O O O O O O O O

C H H H H H H 100% 100% 100% 100% 100% 27g soluble (?) 6g solubility = miscible miscible miscible miscible miscible log P = +0.35 +0.45 +0.59 -0.24 +0.88 +0.29 +1.3 +0.84 oct/H2O (guess) web site with lots of partition coef. values: https://www.nist.gov/sites/default/files/documents/srd/jpcrd367.pdf 22 © Oxford University Press, 2013

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[compound]1-octanol log Poct/H2O = log [compound]water

There is good correlation of calculated partition coefficients with experimental partition coefficients

log D

1.5

1.0

What might a distribution coefficient curve 0.5 look like for the following compound? O 0.0 pH 2 4 6 81012 14 H2N -0.5 OH -1.0

-1.5 23 © Oxford University Press, 2013

Topoisomerase poisons - non-intercalating Examples - Quinolones and fluoroquinilones

O O

CO2H F CO2H

Me NN N N

CH2CH3 HN

Nalidixic acid Ciprofloxacin • Synthetic agents used as antibacterial agents • Stabilise complex between bacterial DNA and topoisomerases • Binding site for agents revealed once DNA strands are ‘nicked’ The quinolones are a family of synthetic broad-spectrum antibiotic drugs. They can be isolated from natural sources (such as plants, animals and bacteria) and act as natural antimicrobials.

Quinolones exert their antibacterial effect by preventing bacterial DNA from unwinding and duplicating. The majority of quinolones in clinical use are fluoroquinolones, which have a fluorine atom attached to the central ring system, typically at the 6-position or C-7 position.

Fluoroquinolones are especially important for hospital-acquired infections in which resistance to older antibacterial classes is suspected. Treatment guidelines recommend minimizing the use of fluoroquinolones and other broad-spectrum antibiotics in less severe infections and in those in which risk factors for multidrug resistance are not present. Side effects are severe so quinolones are contraindicated unless no other safe alternative antibiotic exists. 24 © Oxford University Press, 2013

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Tertiary Structure 1. Double helix coils into a 3D shape – supercoiling 2. Double helix has to unravel during replication 3. Unravelling leads to strain 4. Relieved by enzyme-catalyzed cutting and repair of DNA chain 5. Quinolone and fluoroquinolone antibacterial agents inhibit this enzyme Nalidixic acid (1962) was the first of the Nalidixic acid (1962) quinolone family of antibiotics. 1 8 N 8a N CH The quinolones are a family of synthetic broad- 2 3 7 spectrum antibiotic drugs. Quinolones, and derivatives, have also been isolated from natural HO 3 6 sources (such as plants, animals and bacteria) 4a 4 5 and can act as natural antimicrobials and/or signalling molecules. Quinolones prevent O O c H bacterial DNA from unwinding and duplicating. N Ciprofloxacin (1980) isused to treat anumber a N b N of bacterial infections including bone and joint infections, intra abdominal infections, certain type of infectious diarrhea, respiratory tract HO infections, skin infections, typhoid fever, and F urinary tract infections, among others. A single O O Ciprofloxacin (1980) F at positoin 6 greatly increased both activity and cellular uptake. The piperizine ring (ionized) at Which "N" is most basic? position 7 improved pharmacokinetics. a, b, c Replacement of the N8 nitrogen reduced adverse 25 d all are similar bases reactions and increased activity© Oxford agains University S. Press,aureus 2013.

Topoisomerase poisons - non-intercalating

Examples - Quinolones and fluoroquinilones

Topoisomerase enzyme Structure of bacterial DNA gyrase complexed with DNA and two ciprofloxacin molecules (green)

Region binding to DNA R5 O O 6 Region R binding Region O binding to enzyme to enzyme R7 X8 N R1 Stacking domain Fluoroquinolones

•Drug molecules are stacked in the bound complex • Bound to DNA and enzyme by hydrogen and ionic bonds 26 © Oxford University Press, 2013

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Topoisomerases I form a covalent intermediate in which the active site tyrosine becomes attached to the 3' phosphate end of the cleaved strand rather than the 5' phosphate end.

The TOP1 protein of humans has been subdivided into four regions. The N-terminal 214 amino acids are dispensable for relaxation of supercoiling activity in vitro. The N-terminal domain is followed by a highly conserved, 421 amino acid core domain containing all of the catalytic residues exceppyt the active site tyrosine. This is followed by a ppyoorly conserved linker domain of 77 amino acids. Finally there is a 53 amino acid C- terminal domain. The active site Tyr723 is found within the C-terminal domain. Topoisomerase I After the TOP1 covalently attaches to the 3' end of the broken strand, supercoiling of the DNA is relaxed by controlled rotation of DNA about the Topoisomerase II, intact strand. Then the 5' hydroxyl end of the broken DNA strand can shows DNA too reverse the phosphotyrosyl bond, enabling the release of TOP1 and religation of the DNA. The nicking and closing reactions are fast, and about 100 cyypcles can occur per second.

One of the first inhibitors shown to target TOP1 is irinotecan. Irinotecan is an analogue of the cytotoxic natural alkaloid camptothecin, obtained from the Chinese tree Camptotheca acuminata. Irinotecan is especially effective through its metabolic product SN-38. Irinotecan and SN-38 act by trapping a subset of TOP1-DNA cleavage complexes, those with a guanine +1 in the DNA sequence. One irinotecan or SN-38 molecule stacks against the base pairs flanking the topoisomerase-induced cleavage site and poisons (inactivates) the TOP1 enzyme. 27 © Oxford University Press, 2013

DRUGS ACTING ON DNA Alkylating agents

Nucleophilic atoms on nucleic acid bases

nucleophilic atoms nucleophilic nucleophilic O NH2 atoms NH2 atoms 7 H N N 3 N 5 1 N N

N O N N H2N N nucleophilic N 1 3 atoms R R R Adenine Guanine Cytosine

electrophiles electrophiles electrophiles

O NH NH2 E 2 E E N H N N N N

N N N H2N N O N

R R R

28 https://en.wikipedia.org/wiki/Alkylating_antineoplastic_agent © Oxford University Press, 2013

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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 anticancer agents • Toxic side effects ((ge.g. alk ylation of proteins) • Can cause interstrand and intrastrand crosslinking if two electrophilic groups present • Alkylation of nucleic acid bases can result in miscoding

Crosslinking is possible for doubly electrophilic compounds

X X X X

Nu Nu Nu Nu Nu Nu Nu Nu

Intrastrand crosslinking Interstrand crosslinking

29 © Oxford University Press, 2013

Alkylating agents Normal base pairing Miscoding resulting from alkylated nucleic acid bases

Thymine Alkylated guanine Cytosine Guanine DRUG O NH2 N Me O HO N

N N HN N N R NH N N N N R R O H N 2 R O H2N

Guanine prefers Abnormal base pairing. keto tautomer Alkylated guanine prefers enol tautomer

Normally, has 2 H- How many possible tautomers are there for DNA bases? bonds with adenine Changes charge O O H H NH2 and changes shape. N 4 H 5 H 7 6 4 5 7 N N 1 N 5 N 1 5 3 3 N N 8 H 8 6 2 2 2 N 6 N O N 4 N N 2 O 9 4 N N 1 1 9 3 3 H H H 30 H H © Oxford University Press, 2013 Cytosine Thymine Adenine Guanine

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Bendamustine is a nitrogen mustard used in the treatment Alkylating agents of chronic lymphocytic leukemia and lymphomas. It belongs to the family of drugs called alkylating agents. It is also being studied for the treatment of sarcoma. It is Chlormethine (nitrogen mustard) also being investigated for the non-cancer diseases. CH Cl 3 N

Me N Cl N N

• Secretly used medicinally in 1942 (WW II), reduced Cl white blood count, many side effects, including increased cancer risk later • Causes intrastrand and interstrand cross-linking • Prevents replication • Mono-alkylation of guanine also possible • Analogues with better properties have been prepared

Chlormethine is also used to synthesize pethidine. How might this be done? In 1975 pethidine was opiod of choice of 60% of doctors for acute pain and 22% for chronic severe pain Cl O

? O N N

Cl 31 chlormethine pethidine © Oxford University Press, 2013

Alkylating agents How can DNA heal itself? Are there other possible leaving groups? How do they compare to chloride? Will Chlormethine they survive a journey through the body? Mechanism of action DNA DNA

H G = Guanine O N NH 2 H Cl ONHN N 2 N N N H3CN H C N N 3 H CN G 3 N Cl Cl Mechlorethamine Aziridine ion Cl N N NH N N NH2 2 NH N N NH G O O

DNA DNA

H O N H NH2 ONHN 2 N N N Crosslinked DNA N N H3C N N

N N N NH2 N N NH2 CH N NH N NH 3 O O 32 © Oxford University Press, 2013

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Alkylating agents O O Cl Cl Cl Nitrosoureas NN NN H H N N O O Lomustine Carmustine Mechanism Decompose in body to form an alkylating agent and a carbamoylating agent

O Protein-Lys-NH2 O C N R Protein-Lys-NH Carbamoylation Isocyanate HN R

O C N R Isocyanate O N2 + HO (carbamoylating Cl R agent) NN + Cl

N H Cl Alkylating N agent O H O N Notes OH Cl • Alkylating agent causes interstrand crosslinking DNA Cl XY XY• Crosslinking between G-G or G-C Alkylation Cross- • Carbamoylating agent reacts with Alkylating linking agent lysine residues on proteins • May inactivate DNA repair enzymes 33 DNA DNA © Oxford University Press, 2013

Alkylating agents

O Busulfan different length tethers are possible O H3C S O OMs S = O MsO O CH3 O Synthetic agent used as anticancer agent (dimesylate). Causes interstrand crosslinking O Very stable anion leaving group, could it Mechanism O S CH3 MsO OMs be made even better? O OMs O Busulfan N HN N N N N N N DNA H2N N N Guanine DNA N N DNA DNA DNA

34 © Oxford University Press, 2013

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Alkylating agents

Dacarbazine HNN • Prodrug activated by demethylation in liver • Decomposes to form a methyldiazonium ion N N CONH 2 • Alkylates guanine groups (methylates here) H3C N

CH3 Mechanism AIC

HNN Cyt P-450 HNN HNN HNN -CH2O liver N N CONH2 N N CONH2 N NH CONH2 H2N CONH2 H C N N N 3 H H CH3 H O CH3 CH3 NNCH3 Methyldiazonium ion oxidation with cytochrom P-450 diazomethane (very toxic and explosive gas), usually made as basic ether solution using Diazald.

O O H H N2 + CH3 Diazald O N O S N N N OH N N N base N other carbocations CH3 H2C H C are possible CH2 acid 2 O H H H O diazomethane 35 H © Oxford University Press, 2013

DRUGS ACTING ON DNA - Alkylating agents Example - Mitomycin C O Guanine N O HN NH 2 N N O O OH NH DNA

H2N H N O CH3 2 H How? H N O N Guanine N NH H C N N 3 H3C N

O OH NH2 N Where are the DNA electrophilic sites?

• Prodrug activated in the body to form an alkylating agent • One of the most toxic anticancer drugs in clinical use

Mitomycin C is a mitomycin that is used as a chemotherapeutic agent by virtue of its antitumour activity. It is given intravenously to treat upper gastro-intestinal cancers (e.g. esophageal carcinoma), anal cancers, and breast cancers, as well as by bladder instillation for superficial bladder tumours. It causes delayed bone marrow toxicity and therefore it is usually administered at 6-weekly intervals. Prolonged use may result in permanent bone- marrow damage. It may also cause lung fibrosis and renal damage. 36 © Oxford University Press, 2013

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Coenzyme Q10, also known as ubiquinone, is a coenzyme that is ubiquitous in the bodies of most animals. It is a 1,4-benzoquinone, where Q refers to the quinone chemical group and 10 refers to the number of isoprenyl chemical subunits in its tail. This fat-soluble substance, which resembles a vitamin, is present in most eukaryotic cells, primarily in the mitochondria. It is a component of the electron transport chain and participates in aerobic cellular respiration, which generates energy in the form of ATP. Ninety-five percent of the human body's energy is generated this way. There are three redox states of CoQ10: fully oxidized (ubiquinone), semiquinone, and fully reduced (ubiquinol). The capacity of this molecule to act as a two-electron carrier (moving between the quinone and quinol form) and a one-electron carrier (moving between the semiquinone and one of these other forms) is central to its role in the electron transport chain due to the iron–sulfur clusters that can only accept one electron at a time, and as a free radical-scavenging antioxidant.

H BH H O O O

O CH3 O CH 3 O CH3 H3C H3C H3C +one electron +one electron H C +one proton +one proton 3 H3C e- H3C e- O R O R O R both directions both directions O O O H ubiquinone (CoQ10) BH ubiquinol (CoQ10) oxidized reduced semiquinone (CoQ10)

a mycolic acid

37 © Oxford University Press, 2013

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 NADH H O OH OH

O H C NH2 H O O CH OCONH OH 2 2 CH2 + H N H2N-DNA -H 2 H2N Ring H2N-DNA NH-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 OGuanine CH2 H2N H N HN N 2 NH -CO2 NH-DNA N -NH N 3 Me N Me N NH2 NH2 OH OH Crosslinked DNA 38 © Oxford University Press, 2013

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Biosynthesis of mitomycins – working on a handout (how about you?) In the bacterium Legionella pneumophila, mitomycin C induces competence for transformation. Natural transformation is a process of DNA transfer between cells, and is regarded as a form of bacterial sexual interaction. In microbiology, genetics, cell biology, and molecular biology, competence is the ability of a cell to take up extracellular ("naked") DNA from its environment in the process called transformation. Competence may be differentiated between natural competence, a genetically specified ability of bacteria which is thought to occur under natural conditions as well as in the laboratory, and induced or artificial competence, which arises when cells in laboratory cultures are treated to make them transiently permeable to DNA. In the natural world DNA usually becomes available by death and lysis of other cells,,ypy,gyggpg but in the laboratory it is provided by the researcher, often as a genetically engineered fragment or plasmid. During uptake, DNA is transported across the cell membrane(s), and the cell wall if one is present. Once the DNA is inside the cell it may be degraded to nucleotides, which are reused for DNA replication and other metabolic functions. Alternatively it may be recombined into the cell’s genome by its DNA repair enzymes. If this recombination changes the cell’s genotype the cell is said to have been transformed. O CO H 2 HO CO2H CO2H O P -2 O O OH O3PO O O O How? How? How? H H HO NH2 HO NH2 O NH -2 2 OH OH OPO3 PEP 4-phosphoerythrose OH OH aromatic intermediate phosphoenolpyruvate amino DAHP amino DHQ O

mitomycin C NH2 OH OH OH O O OH OH O

OH H2N O CH3 How? OH H How? N HO2C NH2 H C OH OH NH2 3 N NH H3C glucosamine aromatic intermediate mitosane core NH2 39 © Oxford UniversityO Press, 2013

DRUGS ACTING ON DNA • Neutral inactive molecule acting as a prodrug • Platinum covalently linked to chloro substituents Metallating agents • Ammonia molecules act as ligands (RNH2) • Activated in cells with low chloride ion concentration Cl NH3 • Chloro substituents replaced with neutral water ligands Pt Cisplatin • Produces positively charged species • Binds to DNA in regions rich in guanine units Cl NH3 • Intrastrand links rather than interstrand O • Locali sed unwi ndi ng of DNA d oubl e h eli x • Inhibits transcription O NH 3 Cisplatin, cisplatinum, platamin, neoplatin, cismaplat or cis- Pt diamminedichloridoplatinum(II) (CDDP) is a chemotherapy drug. It was the first NH member of a class of platinum-containing anti-cancer drugs, which now also O 3 includes carboplatin and oxaliplatin. These platinum complexes react in the body, O Carboplatin binding to DNA and causing the DNA strands to crosslink, which ultimately triggers cells to die in a programmed way.

Carboplatin, is a chemotherapy drug used against some forms of cancer (mainly ovarian carcinoma, lung, head and neck cancers as well as endometrial, esophageal, bladder, breast and cervical; central nervous system or germ cell tumors; osteogenic sarcoma, and as preparation for a stem cell or bone marrow transplant). It has vastly reduced side effects compared to cisplatin. Cisplatin and carboplatin belong to the group of platinum-based antineoplastic agents, and interact with DNA to interfere with DNA repair.

Cl NH + 2+ DNA NH 3 H2O H2O NH3 H2O NH3 DNA 3 Pt Pt Pt + Pt DNA NH Cl NH3 Cl NH3 H2O NH3 3 Cisplatin 40 © Oxford University Press, 2013

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DRUGS ACTING ON DNA Chain cutters

CONH2 NH2

NH2 O N Bleomycin H O N R used vs skin cancer NN CH3 O H (see slides 12-13) O HO N S H2N O NH N

CH3 HN O N CH3 HO CH3 S H N HO O BLEOMYCIN A2 R = NHCH CH CH SMe OH 2 2 2 2 BLEOMYCIN B2 R = NHCH CH CH CH NHC(NH )=NH O N 2 2 2 2 2 O H OH OH Bleomycin is mostly used to treat cancer. This

OH includes Hodgkin's disease, non-Hodgkin's disease, O testicular cancer, ovarian cancer, and cervical cancer. OH It can be given intravenously, by injection into a O NH2 muscle or under the skin. It may also be administered inside the chest to help prevent the recurrence of a fluid around the lung due to cancer • Intercalating agent (shown earlier) • Abstracts H from DNA to generate radicals using oxidized iron complex • Radicals react with oxygen resulting in chain cutting • Bleomycin also inhibits repair enzymes

41 © Oxford University Press, 2013

Chain cutters • Generates DNA diradical • DNA diradical reacts with oxygen O • Results in chain cutting HO CH O NHCO Me 3 S S 2 H3C I H3C S S O H H3C I O N O Calicheamicin 1 O OMe HHO OH O Antitumour agent OMe Enedi yn e O H3C system HO H O MeO OH N H3C MeO The calicheamicins are a class of enediyne antitumor antibiotics derived from the bacterium Micromonospora echinospora, with calicheamicin g1 being the most notable. It was isolated originally in the mid-1980s from the chalky soil, or "calichi pits", located in Kerrville, Texas. It is extremely toxic to all cells . N-acetyl dimethyl hydrazide calicheamicin was developed and marketed as targeted therapy against the non-solid tumor cancer acute myeloid leukemia (AML). Calicheamicin 1 and the related enediyne esperamicin are the two of the most potent antitumor agents known. Bergman reaction (1972) D H H D H D H D ?

H D H D H D proposed D diradical H 42 intermediate © Oxford University Press, 2013

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43 © Oxford University Press, 2013

Chain cutters Mechanism

H O O HO HO O Michael HO NHCO2Me NHCO Me 2 addition NHCO2Me

S S S S R R R MeS Nu

O O HO HO Cycloaromatisation NHCO2Me NHCO2Me H DNA DNA (Diradical) S S O2 R R H Oxidative cleavage

44 © Oxford University Press, 2013

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Neocarzinostatin is a macromolecular chromoprotein enediyne antitumor antibiotic secreted by Streptomyces macromomyceticus. It consists of two parts, a labile chromophore (shown) and a 113 amino acid protein to which the chromophore is tightly and non-covalently -10 bound with high affinity (Kd ~ 10 M). The non-protein component is a very potent DNA-damaging agent; However it is extremely unstable and the role of the protein is to protect it and release it to the target DNA. Opening of the epoxide under reductive conditions present in cells creates favorable conditions for a Bergman cyclization, that leads to formation of benzyne, followed by DNA strand cleavage. As a medicine it is among the most potent known, and in Japan it is the only analog used clinically (against liver cancer). Neocarzinostatin likely evolved to kill bacteria that compete with the producing organism. Because it achieves this by causing DNA damage, however, it is capable of harming tumor cells, as well and was developed for its anticancer properties. 45 © Oxford University Press, 2013

DRUGS ACTING ON DNA Chain terminators 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 N3 Chain terminating group • Azidothymidine is a prodrug used in the treatment of HIV • AZT is phosphorylated to a triphosphate in the body • Triphosphate has two mechanisms of action - inhibits a viral enzyme (reverse transcriptase) - is added to growing DNA chain and acts as chain terminator

Azidothymidine (AZT) reduces the replication of the virus and leads to improvements in both symptoms and blood tests. It can also be used to prevent HIV transmission, such as from mother to child during the period of birth or after a needle stick injury. Used by itself in HIV-infected patients, AZT slows HIV replication in patients, but does not stop it entirely. HIV may become AZT-resistant over time, and therefore AZT is now usually used in conjunction with other anti-HIV drugs in the combination therapy called highly active antiretroviral therapy (HAART = highly active antiretroviral therapy). 46 © Oxford University Press, 2013

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O Azidothymidine (AZT) O P O Chain terminating group O H N O O 2 N OH N O O P O O P O O P O N N O NH N O O HN N N O P O O N N O O PO O NTP O O O O O P O O O NH2 O N H O O O P O B O P O N N HN O O N N O O O P O Aciclovir O H2N O O Chain terminating O PO O P O group O O Usual way of O N adding bases O P O Growing N chain HN O N Template H2N

N H B N H B H R N R N R N R N N R N H H N H 47 Azides are very reactive and lose N2 easily (best leaving group in chemistry) © Oxford University Press, 2013

Chain terminators

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

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

Acyclovir, is an antiviral medication. It is Famciclovir is a guanosine analogue primarily used for the treatment of herpes antiviral drug used for the treatment of simplex virus infections, chickenpox, and various herpesvirus infections, most shingl es. commonly for herpes zoster (shingles).

Notes: • Prodrugs used as antiviral agents • Same mechanisms of action as AZT • Used vs herpes simplex and shingles

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Chain terminators

a) Normal replication C C C C A A A A G PP G C G P C GOH 3' T A OH 3' T A A T A T

DNA Growing DNA Growing template chain b) Chain termination template chain

C C C C A A A A PP G G Chain termination P C Drug C Drug H 3' T AOH 3' T A A T A T

Mechanism of reverse transcription in class VI virus ssRNA-RT, human immunodeficiency virus (HIV). Colors mark complementary sequences (not drawn to scale). Reverse transcription occurs in the cytoplasm of host cell. In this process, viral ssRNA is transcribed by the viral reverse transcriptase protein (RT) into double stranded DNA. Reverse transcription takes place in 5'  3' direction. tRNA ("cloverleaf") hybridizes to PBS and provides -OH group for initiation of reverse transcription. Key: (U3 - promoter region),(U5 - recognition site for viral integrase); (PBS - primer binding site);(PP - polypurine section ).

1) Strong stop complementary DNA (cDNA) is formed. 2) Template in RNA:DNA hybrid is degraded by RNase H domain of reverse transcriptase 3) DNA:tRNA is transferred to the 3'-end of the template (synthesis "jumps"). 4) First strand synthesis takes place. 5) The rest of viral ssRNA is degraded by RNase H, except for PP site. 6) Synthesis of second strand of ssDNA is initiated from the 3'-end of the template. tRNA is necessary to synthesis of complementary PBS 7) tRNA is degraded 8) After another "jump", PBS from the second strand hybridizes with the complementary PBS on the first strand. 9) Synthesis of both strands is completed by the DNAP function of reverse transcriptase. Both dsDNA ends have U3-R-U5 sequences, so called long terminal repat sequences (3'LTR and 5'LTR, respectively). LTRs mediate integration of the retroviral DNA into another region of the host genome. Very error prone because no proof reading. 50 © Oxford University Press, 2013

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DRUGS ACTING ON DNA Control of gene transcription 3' 5' Notes: Me O • Design of synthetic Linker O N molecules capable of N Py controlling gene A T N O H H transcription Im N Me • Molecules capable of N N C G recogggnising and bindin g to Me H N O Py specific base pairs H N • Hairpin polyamides A T N H containing heterocyclic H Hp O N rings are capable of O Me binding to the minor Arm Me O T A H Arm groove N N Hp H O • Binding involves amide groups and heterocycles N H G C Py • Particular patterns of O N htheterocycli c ri ngs all ow H Me N TA N recognition of perticular Me N Im O base pairs • Capable of inhibiting Py H N transcription Me H • Designed to bind to H N Me N N regulatory element of a Me O gene O 51 © Oxford University Press, 2013

DRUGS ACTING ON rRNA

NH Antibiotics – attack bacteria NH H HN C NH2 H2N C NH H OH H HO OH OH Me H H H OH HO H Me N H O 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 H H CH2OH Chloramphenicol H MeHN H (vs typhoid) H H H H Me OMe O Me H OH OH H C NMe O CH3 2 HO Rifamycins O H C 3 CH3 OH O OH O O HO O OH HO O CH3

NH2 H3C CH3 O CH O 3 OH O H CH HO Me 3 Cl NMe2 O OH Chlortetracycline Me OMe (Aureomycin) Erythromycin 52 © Oxford University Press, 2013

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HO H

O2N CH2OH HN H

C O CHCl2 Chloramphenicol (vs typhoid)

Chloramphenicol is an antibiotic useful for the treatment of a number of bacterial infections. This includes as an eye ointment to treat conjunctivitis. By mouth or by injection into a vein it is used to treat meningitis, plague, cholera, and typhoid fever (largely drug resistant now). Monitoring of blood levels of the medication and blood cell levels every two days is recommended during treatment. Common side effects include bone marrow suppression, nausea, and diarrhea. The bone marrow suppression may result in death. To reduce the risk of side effects treatment duration should be as short as possible. It has excellent blood-brain barrier penetration. Chloramphenicol is active against the three main bacterial causes of meningitis: Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. Chloramphenicol is extremely lipid-soluble; it remains relatively unbound to protein and is a small molecule. It penetrates effectively into all tissues of the body, including the brain. Chloramphenicol increases the absorption of iron.

The rifamycins are a group of antibiotics that are synthesized either naturally by the bacterium Amycolatopsis rifamycinica or artificially. They are a OH OH Me subclass of the larger family of ansamycins. Rifamycins are particularly H effective against mycobacteria, and are therefore used to treat Me N tuberculosis, leprosy, and mycobacterium avium complex (MAC) O infections. O R H Me OH Me Isoniazid is a prodrug and must be activated by a bacterial catalase- O OH peroxidase enzyme in Mycobacterium tuberculosis called KatG, which O Me Me H catalyzes the formation of the isonicotinic acyl radical, which spontaneously H H couples with NADH to form form the nicotinoyl -NAD adduct. This complex binds H tightly to the enoyl-acyl carrier protein reductase known as InhA, thereby H H Me blocking the natural enoyl-AcpM substrate and the action of fatty acid OMe O Me H OH C synthase. This process inhibits the synthesis of mycolic acids, which are O required components of the mycobacterial cell wall. A range of radicals are produced by KatG activation of isoniazid, including nitric oxide, Rifamycins Rifamycins have been used for the treatment of many diseases, the most important one being HIV-related tuberculosis.

H O N bacterial O H NH O N R 2 catalase- N R peroxidase O O N R enzyme +4 Fe

N N Isoniazid N N Forma a complex binds to protein important in mycolic fatty acid metabolism and blocks fatty acids used in cell wall of terberculosis bacteria.54 © Oxford University Press, 2013

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NH NH

H HN C NH2 H2N C NH H OH H H HO H OH H O H O CHO Me H OH Strep tomyc in H O HO O CH2OH H MeHN H H OH H Streptomycin is an antibiotic used to treat a number of bacterial infections. This includes tuberculosis, Mycobacterium avium complex, endocarditis, brucellosis, Burkholderia infection, plague, tularemia, and rat bite fever. For active tuberculosis it is often given together with isoniazid, rifampicin, and pyrazinamide. Common side effects include feeling like the world is spinning, vomiting, numbness of the face, fever, and rash.

Streptomycin is a protein synthesis inhibitor. It binds to the small 16S rRNA of the 30S subunit of the bacterial ribosome, interfering with the binding of formyl-methionyl-tRNA to the 30S subunit. This leads to codon misreading, eventual inhibition of protein synthesis and ultimately death of microbial cells through mechanisms that are still not understood. Speculation on this mechanism indicates that the binding of the molecule to the 30S subunit interferes with 50S subunit association with the mRNA strand. This results in an unstable ribosomal-mRNA complex, leading to a frameshift mutation and defective protein synthesis; leading to cell death. Humans have ribosomes which are structurally different from those in bacteria, so the drug does not have this effect in human cells. Streptomycin is an antibiotic that inhibits both Gram-positive 55 and Gram-negative bacteria, and is therefore a useful broad-spectrum antibiotic.© Oxford University Press, 2013

Chlortetracycline (trade name Aureomycin, Lederle) is a tetracycline antibiotic, the first tetracycline to be OH O OH O O identified. It was discovered in 1945. Duggar identified OH the antibiotic as the product of an actinomycete In NH2 veterinary medicine, chlortetracycline is commonly used to treat conjunctivitis in cats and other animals. OH H HO Me Chlortetracycline may increase the anticoagulant Cl NMe2 activities . The risk or severity of adverse effects can be Chlortetracycline increased when chlortetracycline is combined with (Aureomycin) acitretin, adapalene, or alitretinoin.

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NMe CH3 2 Erythromycin is an antibiotic useful for the treatment of a HO O number of bacterial infections, including respiratory tract H C 3 CH3 infections, skin infections, chlamydia infections, pelvic HO O HO O CH3 inflammatory disease, and syphilis. It may also be used during pregnancy to prevent Group B streptococcal infection in the H3C CH3 O CH newborn. An eye ointment is routinely recommended after O 3 O delivery to prevent eye infections in the newborn. Common CH 3 side effects include abdominal crampp,s, vomitin g, and O OH diarrhea. More serious side effects may include Clostridium Me OMe Erythromycin difficile colitis, liver problems, prolonged QT, and allergic reactions. It is generally safe in those who are allergic to penicillin

Erythromycin displays bacteriostatic activity or inhibits growth of bacteria. By binding to the 50s subunit of the bacterial rRNA complex, Erythromycin interferes with aminoacyl translocation, preventing the transfer of the tRNA bound at the A site of the rRNA complex to the P site of the rRNA complex. Without this translocation, the A site remains occupied, thus the addition of an incoming tRNA and its attached amino acid to the nascent polypeptide chain is inhibited. This interferes with the production of functionally useful proteins, which is the basis of this antimicrobial action.

DRUGS ACTING ON mRNA Antisense Therapy

antisense molecule Protein synthesis

m-RNA

antisense molecule AGUCUACGUU

GAAGUU A A UCAGAUGCAA m-RNA

Antisense therapy is a form of treatment for genetic disorders or infections. When the genetic sequence of a particular gene is known to be causative of a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (mRNA) produced by that gene and inactivate it, effectively turning that gene "off". This is because mRNA has to be single stranded for it to be translated. Alternatively, the strand might be targeted to bind a splicing site on pre-mRNA and modify the exon content of an mRNA. This synthesized nucleic acid is termed an "anti-sense" oligonucleotide (ASO) because its base sequence is complementary to the gene's messenger RNA (mRNA), which is called the "sense" sequence (so that a sense segment of mRNA " 5'-AAGGUC-3' 58 " would be blocked by the anti-sense mRNA segment " 3'-UUCCAG-5' "). © Oxford University Press, 2013

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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

•‘Exppgosed’ sections of mRNA must be targeted • Instability and polarity of oligonucleotides (pharmacokinetics) • Short lifetime of oligonucleotides and poor absorption across cell membranes

DRUGS ACTING ON mRNA Micro-RNA (miRNA)

• Short segments of double stranded RNA • Recognised by enzyme complex RISC to produce single stranded RNA – small interfering or small inhibitory RNA (siRNA) • Binds to complementary region of mRNA • mRNA is cleaved byyy enzyme comp lex

The RNA-induced silencing complex, or RISC, is a multiprotein complex, specifically a ribonucleoprotein, which incorporates one strand of a single-stranded RNA (ssRNA) fragment, such as microRNA (miRNA), or double stranded small interfering RNA (siRNA). The single strand acts as a template for RISC to recognize complementary messenger RNA (mRNA) transcript. Once found, one of the proteins in RISC, called Argonaute, activates and cleaves the mRNA. This process is called RNA interference (RNAi) and it is found in many eukaryotes; it is a key process in gene silencing and defense against viral infections.

miRNA

mRNA miRNA siRNA

RISC RISC RISC RISC

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Micro-RNA (miRNA)

Advantages • siRNAs have potential to be used in gene therapy • Greater efficiency in silencing mRNA than conventional antisense therapy • One siRNA could lead to cleavage of several mRNA molecules

Problems • siRNAs need to be metabolically stable • Need to reach target cells • Need to enter target cells

Epigenetics – a new frontier: DNA methylation, methylcytosine, histone acylation

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Five major families of histones exist: H1/H5, H2A, H2B, H3 and H4. Histones H2A, H2B, H3 and H4 are known as the core histones, while Histones H1 and H5 are known as the linker histones.

Histones make five types of interactions with DNA:

1. Helix-dipoles form alpha-helixes in H2B, H3, and H4 cause a net positive charge to accumulate at the point of interaction with negatively charged phosphate groups on DNA 2. HdHydrogen b on ds bet ween th e DNA b ackb one and th e amid e group on th e mai n ch ai n of hi st one prot ei ns 3. Nonpolar interactions between the histone and deoxyribose sugars on DNA 4. Salt bridges and hydrogen bonds between side chains of basic amino acids (especially lysine and arginine) and phosphate oxygens on DNA 5. Non-specific minor groove insertions of the H3 and H2B N-terminal tails into two minor grooves each on the DNA molecule

Histones are highly basic facilitating DNA-histone interactions and contribute to their water solubility.

Histones are subject to post translational modification by enzymes primarily on their N -terminal tails, but also in their globular domains. Such modifications include methylation, citrullination, acetylation, phosphorylation, SUMOylation, ubiquitination, and ADP-ribosylation. This affects their function of gene regulation.

In general, genes that are active have less bound histone, while inactive genes are highly associated with histones during interphase. Histones are evolutionarily conserved, and any deleterious mutations appear to be severely maladaptive. All histones have a highly positively charged N-terminus with many lysine and arginine residues. 63 © Oxford University Press, 2013

Methylations in biochemistry

CH3

H H H2N N N H2N N N 1 8 2 7 6 N3 4 5 N 5 H N 9 H N

10 O H N O CH3 N H Ar H Ar THF 5 (tetrahydrofolate) N methyl-THF Me-vit B12 Me-vit B-12 vit B-12

CH H CH 3 3 H N N 2 S S S O N N ATP NH3 N NH3 NH3 OH H H H OH O2C O2C O2C homocysteine methionine S-adenosyl methionine (SAM) NH 2 NH2

H H3C N N SAM

N O N O

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Citrullination or deimination is the conversion of the amino acid arginine in a protein into the amino acid citrulline. Enzymes called peptidylarginine deiminases (PADs) replace the primary ketimine group (=NH) by a ketone group (=O).

Citrullination controls the expression of genes, particularly in the developing embryo. The immune system often attacks citrullinated proteins, leading to autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.

Citrulline is not one of the 20 standard amino acids encoded by DNA in the genetic code. Instead, it is the result of a post-translational modification.

Citrullination is distinct from the formation of the free amino acid citrulline as part of the urea cycle.

Arginine is positively charged at a neutral pH, whereas citrulline is uncharged.Thisincreases the hydrophobicity of the protein, leading to changes in protein folding. Therefore, citrullination can change the structure and function of proteins.

Proteins that normally contain citrulline residues include myelin basic protein (MBP), filaggrin, and several histone proteins, while other proteins, like fibrin and vimentin, can get citrullinated during cell-death and tissue inflammation.

Fibrin and fibrinogen may be favored sites for arginine deimination within rheumatoid joints. Test for presence of anti- citrullinated protein (ACP) antibodies are highly specific (88-96%) for rheumatoid arthritis (RA), about as sensitive as rheumatoid factor (70-78%) for diagnosis of RA, and are detectable from even before the onset of clinical disease.

NH2 H H2N NH2 B H N H2N O H 2 O H O NH NH H NH + PAD + NH3 (enzyme) etc.

H H H

N N N N N N

Acylations in biochemistry Histone proteins are acetylated and deacetylated on lysine residues in the N-terminal tail as part of gene regulation. The regulation of transcription factors, effector proteins, molecular chaperones, and cytoskeletal proteins by acetylation and deacetylation is a significant post-translational regulatory mechanism. These regulatory mechanisms are analogous to phosphorylation and dephosphorylation by the action of kinases and phosphatases. Not only can the acetylation state of a protein modify its activity but there has been recent suggestion that this post-translational modification may also crosstalk with phosphorylation, methylation, ubiquitination, sumoylation, and others for dynamic control of cellular signaling .

H O H O H O N N N N N N H H lysine H lysine lysine Co-A-SH O O Co-A Co-A HN S N BH N O S H H H O Glu-Enz O acetyl Co-A O HO Glu-Enz glutamate aa acylated glutamate aa lysine

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Small Ubiquitin-like Modifier (or SUMO) proteins are a family of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. SUMOylation is a post-translational modification involved in various cellular processes, such as Ubiquitin is a small (8.5 kDa) regulatory protein that has nuclear-cytosolic transport, transcriptional regulation, been found in almost all tissues (ubiquitously) of apoptosis, protein stability, response to stress, and eukaryotic organisms. There are four genes in the human progression through the cell cycle. SUMO proteins are genome that produce ubiquitin: UBB, UBC, UBA52 and similar to ubiquitin, and SUMOylation is directed by an RPS27A. Ubiquitination can affect proteins in many enzymatic cascade analogous to that involved in ways: it can signal for their degradation via the ubiquitination. In contrast to ubiquitin, SUMO is not used proteasome, alter their cellular location , affect their activity , to tag proteins for degradation. Mature SUMO is and promote or prevent protein interactions. Ubiquitination produced when the last four amino acids of the C-terminus is carried out in three main steps: activation, conjugation, have been cleaved off to allow formation of an isopeptide and ligation, performed by ubiquitin bond between the C-terminal glycine residue of SUMO and an acceptor lysine on the target protein.

ADP-ribosylation is the addition of one or more ADP-ribose moieties to a protein. It is a reversible post-translational modification that is involved in many cellular processes, including cell signaling, DNA repair, gene regulation and apoptosis. Improper ADP-ribosylation has been implicated in some forms of cancer. It is also the basis for the toxicity of bacterial compounds such as cholera toxin, diphtheria toxin, and others.

H N O 2 O ADP H2N O N

H2N N Arg H O O N N H N N O P O P O N H OH OH O O O O O

O Glu

H N OH OH NAD+ OH OH 2

Arg H OH N N O ADP H O

O Glu

OH OH