the organic chemistry of drug design and drug action chapter 6 dna-interactive agents

The Organic Chemistry of Drug Design and Drug Action Chapter 6 DNA-Interactive Agents

DNA - another receptor Carries genetic information in cells Few differences between normal DNA and DNA from other cells. Therefore, these drugs are generally very toxic; used for life-threatening diseases, such as cancer and viral infections.

Cancer Cells Rapid, abnormal cell division Constant need for DNA and precursors Selective toxicity rapid uptake of drug molecules by cancer cells repair mechanisms too slow activation of proteins such as p53 in normal cells in response to DNA damage - leads to increased DNA repair enzymes, cell cycle arrest (to allow time for DNA repair), and programmed cell death (apoptosis)

Combination Chemotherapy In the late 1950s combination chemotherapy was introduced. Effectiveness compared to single drug: Able to fight acquired resistance Different mechanisms of action increase effectiveness Some covalent modifications can be reversed by repair enzymes, so inhibitors of DNA repair can be added

Drug Interactions Care must be given to which mechanisms of action are involved in drug combinations. For example, a renal (kidney) cytotoxic agent should not be used with a drug that requires renal elimination for excretion.

Drug Resistance 1. Increased expression of membrane glycoproteins - affects membrane permeability (blocks drug transport) 2. Increased levels of thiols (destroys electrophilic anticancer drugs) 3. Increased levels of deactivating enzymes (destroys anticancer drugs) 4. Decreased levels of prodrug-activating enzymes (prevents activation of prodrugs) 5. Increased DNA repair enzymes (repairs DNA modification) All involve gene alterations.

DNA Structure and Properties purine pyrimidine adenine cytosine guanine thymine In double-stranded DNA the ratio of A/T and G/C is always 1.

Hydrogen Bonding of Complementary Base Pairs (Watson-Crick Base Pair) 2 H-bonds

Hydrogen Bonding 3 H-bonds

FIGURE 6.1 DNA structure. Reproduced with permission from Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J.D. (1989). Molecular Biology of the Cell, 2nd ed., p. 99. Garland Publishing, New York. Copyright 1989 Garland Publishing.

The 2 glycosidic bonds that connect the base to its sugar are not directly opposite each other, giving different spacings along helix. FIGURE 6.2 Characteristic of DNA base pairs that causes formation of major and minor grooves

Duplex (double- stranded) DNA (all inside) FIGURE 6.3 Major and minor grooves of DNA. With permission from Kornberg, A. (1980); From DNA Replication by Arthur Kornberg. Copyright 1980 by W. H. Freeman and Company. Used with permission.

most stable tautomer FIGURE 6.4 Hydrogen bonding sites of the DNA bases. D, hydrogen bond donor; A, hydrogen bond acceptor Base Tautomerism

mimics thymine mimics adenine These can substitute for T and A in DNA polymerase reactions. Therefore H bonding is not essential; only need the groups to fit snugly in the binding site of DNA polymerase. FIGURE 6.5 Nonpolar nucleoside isosteres (6.4 and 6.5) of thymidine and adenosine, respectively, that base pair by non-hydrogen-bond interactions

DNA Shapes Human somatic cells - each of the 46 chromosomes consists of a single DNA duplex about 4 cm long. Therefore a total of 46 4 = 1.84 m long of DNA packed into the nucleus. Nucleus is only 5 m in diameter Done with aid of richly basic proteins called histones. Folded compact form of DNA called chromatin.

Packing of DNA into the Nucleus FIGURE 6.6 Stages in the formation of the entire metaphase chromosome starting from duplex DNA. With permission from Alberts, B., (1994). Copyright 1994 from Molecular Biology of the Cell, 3rd ed. By Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts and James D. Watson. Reproduced by permission of Routledge, Inc., part of The Taylor & Francis Group.

FIGURE 6.7 Artist rendition of the conversion of duplex DNA into chromatin fiber

Supercoiled DNA - Packing of Bacterial DNA Facilitates RNA polymerase reaction Helps in chromatin packing circular DNA (plasmid) supercoiled DNA Enzymes that interconvert supercoiled and relaxed DNA are called DNA topoisomerases. FIGURE 6.8 Conversion of duplex DNA into supercoiled DNA

DNA topoisomerases also resolve topological problems such as catenation and knotting. catenanes FIGURE 6.9 Catenane and knot catalog. Arrows indicate the orientation of the DNA primary sequence: a and b, singly linked catenanes; c and d, simplest knot, the trefoil; eh, multiply interwound torus catenanes; i, right-handed torus knot with seven nodes; j, right-handed torus catenane with eight notes; k, right-handed twist knot with seven nodes; l, 6-noded knots composed of two trefoils. Adapted with permission from Wasserman, S. A. and Cozzarelli, N. R. Biochemical topology: applications to DNA recombination and replication. Science, 1986, 232, 952. Reprinted with permission from AAAS.

FIGURE 6.10 Visualization of trefoil DNA by electron microscopy. Reproduced with permission from Griffith J.D., Nash, H.A., Proc. Natl. Acad. Sci. USA 1985, 82, 3124.

Two Principal Types of Topoisomerases DNA topoisomerases I catalyze transient breaks of one strand of duplex DNA. DNA topoisomerases II (in bacteria called DNA gyrase) catalyze cleavage of both strands of duplex DNA.

Table 6.1

Topoisomerase mechanisms FIGURE 6.11 Mechanisms of DNA topoisomerase-catalyzed reactions. Drawings produced by Professor Alfonso Mondragn, Department of Molecular Biosciences, Northwestern University.

Topoisomerase mechanism SCHEME 6.1 DNA topoisomerase-catalyzed strand cleavage to cleavable complexes

Possible Mechanism of Topoisomerase I Reaction Conformational change to make a gap for strand to pass through Religation of the two ends Relaxed DNA is released Attack of Tyr at 5-phosphate Cleavable complex Ready for another catalytic cycle FIGURE 6.12 Artist rendition of a possible mechanism for a topoisomerase I reaction. The colored sections are the topoisomerase, and the black lines are the double-stranded DNA. With permission from Champoux, J.J. (2010). With permission from the Annual Review of Biochemistry. Volume 70 2001 by Annual Reviews. www.annualreview.org.www.annualreview.org

Mechanism for Topoisomerase I Decatenation (B) FIGURE 6.13 Artist rendition of possible mechanisms of topoisomerase IA-catalyzed relaxation of (A) supercoiled DNA and (B) decatenation of a DNA catenane. From Li, Z.; Mondragon, A.; DiGate, R. J. The mechanism of IA topoisomerase-mediated DNA topological transformations. Mol. Cell 2001, 7, 301.

DNA Conformations Right-handed helices Left-handed helix FIGURE 6.14 Computer graphics depictions of A-DNA, B-DNA, and Z-DNA. Reproduced with permission from the Jena Library of Biological Macromolecules, Institute of Molecular Biotechnology (IMB), Jena, Germany; http://jenalib.fli-leibniz.de/ Hhne R., Koch F. T., Shnel, J. A comparative view at comprehensive information resources on three-dimensional structures of biological macromolecules. Brief Funct. Genomic Proteomic 2007, 6(3), 220239.http://jenalib.fli-leibniz.de/

A- and B-DNA glycosyl bonds are always anti. anti (base in the opposite direction as the 5-phosphate)

Z-DNA glycosyl bond is anti at pyrimidines but syn at purines (responsible for zigzag appearance). syn (base in the same direction as the 5-phosphate)

Classes of DNA-Interactive Drugs Reversible binders - reversible interactions with DNA Alkylators - react covalently with DNA bases Strand breakers - generate reactive radicals that cleave polynucleotide strands

How Do Drugs Interact with DNA Packed as Chromatin? Figure 6.15AFigure 6.15B The outer surface of the DNA is accessible to small molecules.

Also, nucleosomes are in dynamic equilibrium with uncoiled DNA, so drug can bind after uncoiling. FIGURE 6.16 Schematic of how a drug could bind to DNA wrapped around histones in the nucleosome. Polach K. J. Mechanism of protein access to specific DNA sequences in chromatin: A dynamic equilibrium model for gene regulation. J. Mol Biol. 1995, 254, 130.

Reversible DNA Binders Three ways small molecules can reversibly bind to duplex DNA. FIGURE 6.17 Schematic of three types of reversible DNA binders. A, external electrostatic binder; B, groove binder; C, intercalator. In B and C, the pink bar represents the drug. Reproduced with permission from Blackburn G. M., Gait M. J., Eds. Nucleic Acids in Chemistry and Biology, 2nd ed., 1996; p. 332. By permission of Oxford University Press.

External electrostatic binders - cations that bind to anionic phosphates. Groove binders - proteins prefer major groove binding; small molecules prefer minor groove binding. Minor groove generally not as wide in A-T regions as in G-C regions. Therefore, flat aromatic, often crescent-shaped molecules (6.11) prefer A-T regions.

FIGURE 6.18 Model showing interaction of netropsin (colored ball model) with double helical DNA (colored stick model). The 2D structure of netropsin (6.8) is also shown. Image created by JanLipfert from crystallographic coordinates deposited in the Protein Data Bank, accession code 101D. Netropsin is a minor groove binder

DNA Intercalators Flat, generally aromatic or heteroaromatic molecules Insert (intercalate) and stack between base pairs Noncovalent interactions Drug is perpendicular to helix axis Sugar-phosphate backbone is distorted Energetically favorable process Does not disrupt H-bonding Destroys regular helix; unwinds DNA Therefore interferes with the action of DNA topoisomerases and DNA polymerases, which elongate DNA chain and correct mistakes in the DNA

Example of Intercalation: Ethidium bromide FIGURE 6.19 Intercalation of ethidium bromide into B-DNA

Topotecan binds to the DNA- topoisomerase I complex antitumor agent Does not appear to be a correlation between DNA intercalation and antitumor activity. It is not sufficient to intercalate without stabilization of the cleavable complex.

Nalidixic acid binds to bacterial topoisomerase II

Other DNA intercalators

Selected Examples of DNA Intercalators AcridinesActinomycinsAnthracyclines

Amsacrine - acridine analog Lead compound antibacterial Lead modification anti-leukemia agent stabilizes cleavable complex

Crystal Stucture of an Actinomycin Analog Bound to a DNA dactinomycin - antitumor from Streptomyces FIGURE 6.20 X-ray structure of a 1:2 complex of dactinomycin with d(GC). Reprinted from Journal of Molecular Biology, Vol. 68, Stereochemistry of actinomycin binding to DNA. II. Detailed molecular model of actinomycin DNA complex and its implications, pp. 2634. Copyright. 1972 Academic Press, with permission from Elsevier.

FIGURE 6.21 X-ray structure of daunorubicin intercalated into an oligonucleotide. Quigley, G. S.;Wang, A.; Ughetto, G.; Van der Marel, G.; Van Boom, J. H.; Rich, A. Molecular structure of an anticancer drug-DNA complex: Daunomycin plus d(CpGpTpApCpG). Proc. Natl. Acad. Sci. USA 1980, 77, p. 7206. Reprinted with permission from Dr. C. J. Quigley. Anthracycline Analog Complex stabilized by stacking energy and H-bonding Intercalation and topoisomerase II-induced damage anti-leukemia agent daunorubicin (daunomycin) D ring (major groove) A ring (minor groove)

Bis-intercalators do not always bind as tightly as expected FIGURE 6.22 General structure of bis-quinoxaline intercalators

A bis-intercalator requires the correct linker

DNA Alkylators Lead discovery Autopsies of soldiers killed in World War I by sulfur mustard (6.23) showed leukopenia (low white blood cells), bone marrow defects, dissolution of lymphoid tissue, ulceration of GI tract. These are all rapidly replicating cells. Nitrogen mustards sulfur mustard Suggested this may show tumor cytotoxicity too. 1931 - S mustard tried as antitumor agent, but too toxic.

Lead Modification Less toxic form of sulfur mustard sought. 1942 - first clinical trials of a nitrogen mustard Marks beginning of modern cancer chemotherapy (for advanced Hodgkins disease)

Chemistry of Alkylating Agents Reactivity of Nu - in general: RS - > RNH 2 > ROPO 3 = > RCOO - SCHEME 6.2 Nucleophilic substitution mechanisms

Purines A/G Pyrimidines T/C For DNA: N-7 of guanine > N-3 of adenine > N-7 of adenine > N-3 of guanine > N-1 of adenine > N-1 of cytosine N-3 of cytosine, the O-6 of guanine, and phosphate groups also can be alkylated.

anchimeric assistance If k 1 > k 2, S N 2 If k 2 > k 1, S N 1 Bifunctional alkylating agents DNA undergoes intrastrand and interstand cross-linking Compounds that cross-link DNA (bifunctional alkylating agents) are much more effective. SCHEME 6.3 Alkylations by nitrogen mustards

Interstrand Cross-linking of DNA by Mechlorethamine

Alkylation may change the preferred tautomer of the base

Hydrolysis of alkylated N-7 guanine leads to destruction of the purine nucleus. SCHEME 6.4 Depurination of N-7 alkylated guanines in DNA

Formation of cross-links in DNA SCHEME 6.5 Interstrand cross-links of abasic sites in duplex DNA by reaction with guanine

Mechlorethamine is quite unstable to hydrolysis (completely reacts within minutes of injection). Therefore, a more stable analog is needed. More stable Slows rate of aziridinium formation R = COOHtoo stable, but soluble R = (CH 2 ) 3 COOHchlorambucil

A naturally occurring mustard? SCHEME 6.6 Proposed mechanism for DNA alkylation by fasicularin

Ethylenimines Lower pK a of the aziridine N so it is not protonated at physiological pH - attach e - -withdrawing group Need at least 2 aziridines per molecule for antitumor activity

Methanesulfonates excellent leaving group Alkylates N-7 of guanine intrastrand cross-links

Cyclopropane- Containing Alkylators From Streptomyces All contain a 4-spirocyclopropylcyclohexadienone SCHEME 6.7 Reaction of nucleophiles with 4-spirocyclopropylcyclohexadienone

The nitrogen atom is conjugated with the cyclohexadienone which lowers the reactivity. SCHEME 6.8 Stabilization of the spirocyclopropylcyclohexadienone by nitrogen conjugation

Binding of these molecules to the A-T regions of DNA twists the nitrogen out of conjugation, making the cyclopropane much more reactive. N-3 of adenine reacts. SCHEME 6.9 N-3 adenine alkylation by CC-1065 and related compounds

Metabolically-Activated Alkylating Agents Stable compounds that require one or more enzymes or a reducing agent to convert them into the alkylating agent.

Nitrosoureas Lead compounds 6.38, where R = CH 3 and R = H (modest antitumor activity) (BCNU) (CCNU) Can cross blood-brain barrier for brain tumors

Mechanism of Action of Nitrosoureas alkylating agent carbamoylating agent SCHEME 6.10 Decomposition of N-methyl-N-nitrosourea

Evidence That Diazomethane (CH 2 =N + =N - ) is Not the Active Alkylating Agent, But Methyl Diazonium Is isolated If diazomethane was the actual alkylating agent, only 2 deuteriums would have been detected, but 3 deuteriums were found. SCHEME 6.11 Deuterium labeling experiment to determine mechanism of activation of nitrosoureas

Evidence That the Alkylating Agent, Not the Carbamoylating Agent, is Responsible for Activity. R = alkyl N-nitrosoamides Cannot form carbamoylating agent; still anticancer agent N-nitrosourethanes Also cannot form carbamoylating agent; still antitumor agent

However, nitrosoureas with no alkylating activity are inactive. The carbamoylating agent (O=C=NR) acylates amines in proteins and inhibits DNA polymerase and repair enzymes.

Interstrand cross-link from carmustine (6.38, R = R = CH 2 CH 2 Cl) 1-[N 3 -deoxycytidyl]-2-[N-deoxyguanosinyl]ethane

Proposed Mechanism for Cross-Linking of DNA by (2- Chloroethyl) nitrosoureas The same product is obtained when R = cyclohexyl, so 2- chloroethyldiazonium was proposed as the intermediate. Resistance: O 6 -alkylguanine-DNA alkyltransferase - repair enzyme that excises O-6 guanine adducts Resistance is evidence for this intermediate Detected by electrospray MS SCHEME 6.12 Mechanism proposed for cross-linking of DNA by (2-chloroethyl)nitrosoureas

Fotemustine also causes cross links in DNA

SCHEME 6.13 Alternative mechanism for the cross-linking of DNA by (2-chloroethyl)nitrosoureas Another Proposed Mechanism for Cross-Linking of DNA by (2-Chloroethyl) nitrosoureas

Triazene Antitumor Drugs Using [ 14 C] dacarbazine (6.52), it was shown that formaldehyde is produced and DNA is methylated at N-7 of guanine. SCHEME 6.14 Mechanism for the methylation of DNA by dacarbazine

Mitomycin C SCHEME 6.15 Mechanism for the bioactivation of mitomycin C and alkylation of DNA

SCHEME 6.16 Bioreductive monoalkylating agents

Bioreductive Bis-alkylators SCHEME 6.17 Bioreductive bis-alkylating agents

Leinamycin unusual functionality Isolated from Streptomyces Requires thiol activation for antitumor activity

Chemical Model Studies these intermediates were proposed for activity SCHEME 6.18 Model reaction for the mechanism of activation of leinamycin

Mechanism Proposed for Leinamycin This reacts by an additional mechanism Isolated, but does not directly alkylate DNA; in equilibrium with 6.64 SCHEME 6.19 Mechanism for DNA alkylation by leinamycin

Another Mechanism for How Leinamycin Damages DNA Causes strand breakage SCHEME 6.20 Mechanism for hydrodisulfide activation of molecular oxygen to cause oxidative DNA damage

Strand Breakers Anthracycline Radical Formation superoxide O 2 - and anthracycline semiquinone can generate HO HO Cleaves DNA SCHEME 6.21 Electron transfer mechanism for DNA damage by anthracyclines

Generation of HO from O 2 - and from 6.67 (ferric complex) Fenton reaction SCHEME 6.22 Anthracycline semiquinone generation of hydroxyl radicals

Third Possible Mechanism of DNA Damage by Anthracyclines Ferric complex This could react with O 2 - to give O 2 + Fe(II) Fenton reaction of Fe(II) with H 2 O 2 gives HO

The cardiotoxicity of doxorubicin can be prevented by iron chelators SCHEME 6.23 Conversion of iron chelator prodrug 6.70 into iron chelator 6.71

Bleomycin From Streptomyces verticellus Principal domains in bleomycin Forms Fe II complex with O 2 Intercalates into DNA Selective uptake by cancer cells

Ternary Complex of Bleomycin, Fe (II), and O 2 Active Form

Activation of Bleomycin From another ternary complex or from NADPH-cytochrome P450 reductase SCHEME 6.24 Cycle of events involved in DNA cleavage by bleomycin (BLM)

Possible Mechanisms for Activation of Bleomycin All three mechanisms involve generation of free radicals that can abstract H from DNA, leading to DNA strand scission.

Proposed Mechanism for the Reaction of Activated BLM with DNA DNA fragments (2 major products isolated) 3-phosphoglycolate nucleic base propenals SCHEME 6.25 Alternative mechanisms for base propenal formation and DNA strand scission by activated bleomycin: (A) Modified Criegee mechanism

Proposed Alternative Mechanism for the Reaction of Activated BLM with DNA SCHEME 6.25 Alternative mechanisms for base propenal formation and DNA strand scission by activated bleomycin: (B) Grob fragmentation mechanism

Tirapazamine Kills hypoxic cells in solid tumors Damage to DNA backbone and bases SCHEME 6.26 Mechanism for formation of hydroxyl radicals by tirapazamine

Tirapazamine also reacts with DNA radicals under hypoxic conditions, acting as a surrogate O 2. SCHEME 6.27 Mechanism for DNA-strand cleavage by tirapazamine

Enediyne Antitumor Antibiotics

Common Structural Features of Enediyne Antitumor Antibiotics Macrocyclic ring with at least one double bond and two triple bonds. (ene) (diyne) Common modes of action: intercalation into minor groove reaction (activation) with either a thiol of NADPH - generates radical radical cleavage of DNA

Mechanism for Esperamicins/Calicheamicins Intercalates into DNA Trisulfide reduction initiates the activation Responsible for DNA strand scission SCHEME 6.28 Activation of esperamicins and calicheamicins

Dynemicin A Reductive Mechanism Intercalates into DNA Causes DNA cleavage SCHEME 6.29 Reductive mechanism for activation of dynemicin A

Dynemicin A Nucleophilic Mechanism SCHEME 6.30 Nucleophilic mechanism for activation of dynemicin A

Zinostatin Activation Mechanism by thiols Intercalates into DNA Causes DNA cleavage SCHEME 6.31 Activation of zinostatin by thiols

Deactivation of zinostatin SCHEME 6.32 Polar addition reaction to deactivate zinostatin

SCHEME 6.33 DNA-strand scission by activated zinostatin and other members of the enediyne antibiotics. NCS, neocarzinostatin (Zinostatin) Two mechanisms for DNA cleavage by any of the biradicals generated in the presence of O 2 under reducing conditions Strand scission Major No Criegee rearrangement because under reducing conditions

SCHEME 6.34 Catalytic antibody-catalyzed conversion of an enediyne into a quinone via oxygenation of the corresponding benzene biradical Enediynes can also form quinones

the organic chemistry of drug design and drug action chapter 6 dna-interactive agents

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normal dna

dna structure

dna damage

dna replication

dna bases

inhibitors of dna repair

doublestranded dna

minor grooves of dna