The Organic Chemistry of Drug Design and Drug Action Chapter 6
DNA-Interactive Agents
Slide 2
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.
Slide 3
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)
Slide 4
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
Slide 5
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.
Slide 6
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.
Slide 7
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.
Slide 8
Hydrogen Bonding of Complementary Base Pairs (Watson-Crick Base
Pair) 2 H-bonds
Slide 9
Hydrogen Bonding 3 H-bonds
Slide 10
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.
Slide 11
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
Slide 12
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.
Slide 13
most stable tautomer FIGURE 6.4 Hydrogen bonding sites of the
DNA bases. D, hydrogen bond donor; A, hydrogen bond acceptor Base
Tautomerism
Slide 14
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
Slide 15
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.
Slide 16
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.
Slide 17
FIGURE 6.7 Artist rendition of the conversion of duplex DNA
into chromatin fiber
Slide 18
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
Slide 19
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.
Slide 20
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.
Slide 21
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.
Slide 22
Table 6.1
Slide 23
Topoisomerase mechanisms FIGURE 6.11 Mechanisms of DNA
topoisomerase-catalyzed reactions. Drawings produced by Professor
Alfonso Mondragn, Department of Molecular Biosciences, Northwestern
University.
Slide 24
Topoisomerase mechanism SCHEME 6.1 DNA topoisomerase-catalyzed
strand cleavage to cleavable complexes
Slide 25
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
Slide 26
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.
Slide 27
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/
Slide 28
A- and B-DNA glycosyl bonds are always anti. anti (base in the
opposite direction as the 5-phosphate)
Slide 29
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)
Slide 30
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
Slide 31
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.
Slide 32
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.
Slide 33
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.
Slide 34
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.
Slide 35
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
Slide 36
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
Slide 37
Example of Intercalation: Ethidium bromide FIGURE 6.19
Intercalation of ethidium bromide into B-DNA
Slide 38
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.
Slide 39
Nalidixic acid binds to bacterial topoisomerase II
Slide 40
Other DNA intercalators
Slide 41
Selected Examples of DNA Intercalators
AcridinesActinomycinsAnthracyclines
Slide 42
Amsacrine - acridine analog Lead compound antibacterial Lead
modification anti-leukemia agent stabilizes cleavable complex
Slide 43
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.
Slide 44
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)
Slide 45
Bis-intercalators do not always bind as tightly as expected
FIGURE 6.22 General structure of bis-quinoxaline intercalators
Slide 46
A bis-intercalator requires the correct linker
Slide 47
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.
Slide 48
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)
Slide 49
Chemistry of Alkylating Agents Reactivity of Nu - in general:
RS - > RNH 2 > ROPO 3 = > RCOO - SCHEME 6.2 Nucleophilic
substitution mechanisms
Slide 50
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.
Slide 51
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
Slide 52
Interstrand Cross-linking of DNA by Mechlorethamine
Slide 53
Alkylation may change the preferred tautomer of the base
Slide 54
Hydrolysis of alkylated N-7 guanine leads to destruction of the
purine nucleus. SCHEME 6.4 Depurination of N-7 alkylated guanines
in DNA
Slide 55
Formation of cross-links in DNA SCHEME 6.5 Interstrand
cross-links of abasic sites in duplex DNA by reaction with
guanine
Slide 56
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
Slide 57
A naturally occurring mustard? SCHEME 6.6 Proposed mechanism
for DNA alkylation by fasicularin
Slide 58
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
Slide 59
Methanesulfonates excellent leaving group Alkylates N-7 of
guanine intrastrand cross-links
Slide 60
Cyclopropane- Containing Alkylators From Streptomyces All
contain a 4-spirocyclopropylcyclohexadienone SCHEME 6.7 Reaction of
nucleophiles with 4-spirocyclopropylcyclohexadienone
Slide 61
The nitrogen atom is conjugated with the cyclohexadienone which
lowers the reactivity. SCHEME 6.8 Stabilization of the
spirocyclopropylcyclohexadienone by nitrogen conjugation
Slide 62
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
Slide 63
Metabolically-Activated Alkylating Agents Stable compounds that
require one or more enzymes or a reducing agent to convert them
into the alkylating agent.
Slide 64
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
Slide 65
Mechanism of Action of Nitrosoureas alkylating agent
carbamoylating agent SCHEME 6.10 Decomposition of
N-methyl-N-nitrosourea
Slide 66
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
Slide 67
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
Slide 68
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.
Slide 69
Interstrand cross-link from carmustine (6.38, R = R = CH 2 CH 2
Cl) 1-[N 3 -deoxycytidyl]-2-[N-deoxyguanosinyl]ethane
Slide 70
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
Slide 71
Fotemustine also causes cross links in DNA
Slide 72
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
Slide 73
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
Slide 74
Mitomycin C SCHEME 6.15 Mechanism for the bioactivation of
mitomycin C and alkylation of DNA
Leinamycin unusual functionality Isolated from Streptomyces
Requires thiol activation for antitumor activity
Slide 78
Chemical Model Studies these intermediates were proposed for
activity SCHEME 6.18 Model reaction for the mechanism of activation
of leinamycin
Slide 79
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
Slide 80
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
Slide 81
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
Slide 82
Generation of HO from O 2 - and from 6.67 (ferric complex)
Fenton reaction SCHEME 6.22 Anthracycline semiquinone generation of
hydroxyl radicals
Slide 83
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
Slide 84
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
Slide 85
Bleomycin From Streptomyces verticellus Principal domains in
bleomycin Forms Fe II complex with O 2 Intercalates into DNA
Selective uptake by cancer cells
Slide 86
Ternary Complex of Bleomycin, Fe (II), and O 2 Active Form
Slide 87
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)
Slide 88
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.
Slide 89
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
Slide 90
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
Slide 91
Tirapazamine Kills hypoxic cells in solid tumors Damage to DNA
backbone and bases SCHEME 6.26 Mechanism for formation of hydroxyl
radicals by tirapazamine
Slide 92
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
Slide 93
Enediyne Antitumor Antibiotics
Slide 94
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
Slide 95
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
Slide 96
Dynemicin A Reductive Mechanism Intercalates into DNA Causes
DNA cleavage SCHEME 6.29 Reductive mechanism for activation of
dynemicin A
Slide 97
Dynemicin A Nucleophilic Mechanism SCHEME 6.30 Nucleophilic
mechanism for activation of dynemicin A
Slide 98
Zinostatin Activation Mechanism by thiols Intercalates into DNA
Causes DNA cleavage SCHEME 6.31 Activation of zinostatin by
thiols
Slide 99
Deactivation of zinostatin SCHEME 6.32 Polar addition reaction
to deactivate zinostatin
Slide 100
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
Slide 101
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