1. DNA Interactive agents
Course: Drug Design
Course code:
Dr. Balakumar Chandrasekaran
Dr. Bilal Al-Jaidi
Assistant Professors
Faculty of Pharmacy,
Philadelphia University-Jordan

2. Learning Outcomes At the end of this lesson students will be able to
Define and explain the role of DNA in cell division.
Describe and distinguish the cancer cells Vs normal cells.
Define and describe the DNA interactive agents and
their toxicities.
Explain the concept of combination therapy.
Describe about the structure and properties of DNA.
Explain the major and minor grooves in DNA.
Describe the shape of DNA and topoisomerases.
Explain groove binders, DNA intercalators, alkylators
and DNA strand breakers.

3. DNA
Deoxyribonucleic acid (DNA), the polynucleotide that carries the genetic information in cells and this receptor is so vital to human functioning.
It is broadly defined as receptor and drugs that interact with this receptor (DNA-interactive drugs) are generally very toxic to normal cells.
The overall shape and chemical structure of DNA found in normal and abnormal cells is indistinguishable.
Therefore, these drugs are reserved only for life-threatening diseases: cancers and microbial infections.

4. Cancer cells
Cancer cells: They undergo a rapid, abnormal, and uncontrolled cell division. Genes coding for the differentiation in cancer cells appear to be shut off or inadequately expressed.
Cancer cells: As these cells are continually undergoing mitosis, there is a constant need for rapid production of DNA (and its precursors).
Because of similarities between normal and abnormal DNA, compound that reacts with a cancer cell will also react with a normal cell.

5. Cancer cells Normal cells
Quantitative differences between cancer and normal cells
Cancer cells
Normal cells
Cancer cell mitosis can be halted.
Normal cell mitosis cannot be halted due to there is sufficient time for the triggering
of repair mechanisms.
DNA damage in a cancer cell is not sensed. So they are defective in cell cycle arrest. Thus more sensitive to DNA-damaging agents.
DNA damage in a normal cell is sensed by different mechanisms involving proteins such as p53. Activation of this p53, leads to many cellular responses:
DNA repairing; Cell cycle arrest; apoptosis (programmed cell death)

6. DNA Interactive agents
The medical term for cancer is neoplasm, anticancer drugs may be referred to as antineoplastic agents.
Anticancer drugs that target DNA are most effective against malignant tumors with a large proportion of rapidly dividing cells (Ex: leukemias and lymphomas).
Unfortunately, the most common tumors are solid tumors, which have a small proportion of rapidly dividing cells.
This chapter mainly discuss about the organic chemistry of DNA-interactive drugs and the ways in which DNA damage relates to cancer chemotherapy.

7. The toxicity of DNA interactive agents
The toxicity of anticancer drugs usually observed in rapidly growing cells such as:-
bone marrow
gastrointestinal (GI) tract
mucosa
hair
The clinical effectiveness of an anticancer drug requires that
it generally be administered at doses in the toxic range so
that it kills tumor cells but allows enough normal cells in the
critical tissues thereby allowing recovery to be possible.

8. The toxicity of DNA interactive agents
Even though anticancer drugs are very cytotoxic, they must be administered repeatedly over a long period of time to be assured that all of the malignant cells have been eradicated.
The common side effects of anticancer drugs: nausea and vomiting.

9. DNA Interactive agents
Combination chemotherapy
ADVANTAGES
To avoid drug resistance.
Initial resistance to any single agent.
Initially responsive tumors rapidly acquire resistance after
drug administration.
Anticancer drugs themselves increase the rate of mutation of
cells into resistant forms.
Multiple drugs having different mechanisms of action.
Cells resistant to one drug may be sensitive to another.

10. DNA Interactive agents
Combination chemotherapy
DISADVANTAGES
Drug interaction.
Overlapping toxicities.
Example: Drugs that cause renal toxicity must be used cautiously.
never combine with other drugs that depend on renal elimination
as their primary mechanism of excretion.

11. DNA Interactive agents
DISADVANTAGES
3. The order of administration:
Example: Methotrexate should be given before 5-fluorouracil.
Methotrexate inhibits dihydrofolate reductase enzymeleads to decreased production of 5,10-methylenetetrahydrofolate, which is used as a cofactor by thymidylate synthase, a target for 5-fluorouracil.
Thus, the correct order of administration first decreases the availability of this cofactor of thymidylate synthase and then directly inactivates the enzyme itself, leading to a synergistic effect.

12. DNA Structure and Properties
Basis for the DNA structure
Watson and Crick first elucidated the DNA structure.
Todd and co-workers established the followings:
1. Four deoxyribonucleotides containing
the two purine bases—adenine (A) and guanine (G)
and the two pyrimidine bases—cytosine (C) and thymine (T).

13. DNA Structure and Properties
Structures of bases
Purine bases
Pyrimidine bases

14. DNA Structure and Properties
2. Four deoxyribonucleotides are linked by bonds joining the
5′-phosphate group of one nucleotide to a 3′-hydroxyl
group on the sugar of the adjacent nucleotide to form
3′,5′-phosphodiester linkages.
3. The phosphodiester bonds are stable because they are
negatively charged, thereby repelling nucleophilic attack.

15. DNA Structure and Properties
Watson and Crick proposed that
two strands of DNA are intertwined
into a helical duplex, which is held
together by specific hydrogen bonding
between base pairs of adenine with
thymine and guanine with cytosine.

16. DNA Structure and Properties
Hydrogen bonding between base pairs
ADENINE WITH THYMINE
GUANINE WITH CYTOSINE

17. DNA Structure and Properties
Always a complementary base pairs occur between guanines and cytosines or adenines and thymines only.
Example:
Strand -1 sequence: ′-TGCATG-3′
The complementary sequence: ′-ACGTAC-5′
Characteristic of DNA base pairs that causes formation of
major and minor grooves
The two glycosidic bonds that connect the base pair to its sugar rings are not directly opposite to each other.
The two sugar-phosphate backbones of the double helix are not equally spaced.
So, grooves that are formed between the backbones are not of equal size.

18. Major and minor grooves in DNA
Copyright 1980 by W.H. Freeman and company.

19. Differences between major and minor grooves
Major groove
Minor groove
The floor of the major groove is filled with base pair nitrogen and oxygen atoms that project inward from their sugar-phosphate backbones toward the center of the DNA
The floor of the minor groove is filled with nitrogen and oxygen atoms of base pairs that project outward from their sugar-phosphate backbones toward the outer edge of the DNA.

20. DNA Shapes DNA exists in variety of shapes and sizes.
The length of DNA varies from micrometers to centimeters in size.
The nucleus of human somatic cells contains each of
46 chromosomes.
Single DNA duplex of 4 cm length

21. DNA Shapes
If chromosomes, placed were placed end to end, the DNA would stretch about 2 m long. How??
Because of packaging of DNA into chromatin.
Copyright 1994 from Mol. Biol. Cell, 3Ed by Bruce Albert et al., Taylor and Francis group.

22. DNA Shapes
Some DNA is single stranded or triple stranded (triplex), but mostly DNA is double stranded (duplex).
Some DNA molecules are linear and other (ex: Bacteria), are in circular (plasmids).
Linear DNA can freely rotate until the ends become covalently linked to form circular DNA.
To accommodate further changes in the number of base pairs per turn of the duplex DNA, the circular DNA must twist, like when a rubber band is twisted, into supercoiled DNA.

23. DNA Shapes
Untwisting of the double helix prior to rejoining the ends in circular DNA usually leads to negative supercoiling
(left-handed direction); overtwisting results in positive
supercoiling (right-handed direction).
Virtually all duplex DNA within cells exists as chromatin in the negative supercoiled state, which is the direction opposite to that of the twist of the double helix.
Because supercoiled DNA is a higher energy state than uncoiled DNA, the cutting (called nicking) of one of the DNA strands of supercoiled DNA converts it into relaxed DNA.

24. Topoisomerases
Nicking or cutting of supercoiled DNA is catalyzed by a family of enzymes called DNA topoisomerases.
These nuclear enzymes catalyze the conversion of one topological isomer of DNA into another and also function to resolve topological problems in DNA.
Copyright 1987 by Benjamin Publishing Company.

25. Topoisomerases
The association of DNA with histones and other proteins also introduces supercoiling that requires relaxation by topoisomerases.
Topoisomerases are known that relax only negative supercoils, that relax supercoils of both signs, or that introduce either negative (bacterial DNA gyrase) or positive supercoils into the DNA (reverse gyrase).

26. Types of topoisomerases
Type I
Topoisomerases
Type II
Topoisomerases
Removes positive and negative supercoils by catalyzing a transient break of one strand
of duplex DNA and allowing the unbroken, complementary strand to pass through the
enzyme-linked strand.
It catalyzes the transient
break of both strands of
duplex DNA.
Supercoiling of the DNA
in negative direction.
DNA relaxation by one positive turn.
Subfamilies: 1A, 1B, 1C
Subfamilies: 1IA, 1IB

27. DNA Interactive agents
Three classes of DNA interactive agents
1. Reversible binders:
They interact with DNA through the reversible formation
of non-covalent interactions.
2. Alkylators:
They react covalently with DNA bases.
3. DNA strand breakers:
They generate reactive radicals that produce cleavage
of the polynucleotide strands.

28. Reversible DNA binders
Three ways of reversible binding
1. Electrostatic binding
They occurs along the exterior of the helix.
2. Groove binding
They interact with the edges of the base pairs.
either major or minor groove.
3. Intercalation between the base pairs

29. Reversible DNA binders
Three ways of reversible binding: Schematic representation
Pink colour: Drug
Copyright 1996 by Oxford University Press.

30. Electrostatic binding
Duplex DNA contains negatively charged sugar phosphate backbone.
Cations and water molecules bind to DNA and alters the structure of DNA.
Upon binding of cations, the phosphate groups released which provide both favourable and unfavourable contribution to the overall free energy.
This finally leads to disruption of the structure of DNA.

31. Groove binding Major and minor grooves are different in terms of
Electrostatic potentials
Hydrogen bonding characteristics
Steric effects
Degree of hydration
Proteins prefer the major groove interaction.
Small molecules prefer the minor groove binding.
The small molecules generally have aromatic rings
connected via single bonds enabling for tortional rotation
to fit to the helical curvature of the groove with the
displacement of water molecules.

32. Groove binding Proteins prefer the major groove interaction.
Small molecules prefer the minor groove binding.
The small molecules generally have aromatic rings
connected via single bonds enabling for tortional rotation
to fit to the helical curvature of the groove with the
displacement of water molecules.
As the A-T regions are narrow, it amenable to flat aromatic
molecules into the minor groove and lead to van der Waals
interactions with the DNA functional groups.
Hydrogen bonding from the C-2 carbonyl oxygen of T or
the N-3 nitrogen of A to minor groove binders is very
important.

33. Groove binding
In case of G-C base pair, the amino group of G sterically
hinders the hydrogen bonding thereby placing the G-C
base pairs in the minor groove.
Due to the greater negative electrostatic potential in the
A–T regions of the minor groove, there is a higher
selectivity for cationic molecules in A–T regions.
Molecules that bind in the A–T regions of the minor groove
typically are crescent shaped with hydrogen bonding NH
groups on the interior of the crescent.
Cationic groups undergo electrostatic interactions with the
negative electrostatic potential in the minor groove.
Example: Netropsin (Anticancer, antibacterial and antiviral)

34. Netropsin Netropsin displaces the water molecule and placed in the
centre of AATT region of the
minor groove.
It forms three good bifurcated
hydrogen bonds with
N-3 of adenine and the
C-2 carbonyl oxygen of thymine
along the floor of the groove.
pyrrole rings of netropsin are
packed against the C-2 positions
of adenines.

35. Netropsin Netropsin binding causes a widening of the minor
groove (0.5–2.0 Å) in the AATT region and a
bending of the helix axis (8°) away from the
site of binding.
The result of this widening is to interfere with the
interaction of topoisomerase II leading to the
DNA damage.

36. Intercalation and Topoisomerase- Induced DNA Damage
Aromatic and heteroaromatic molecules bind to
DNA by inserting (i.e., intercalating) and stacking
between the base pairs of the double helix.
The main driving forces for intercalation are stacking
and charge transfer interactions, but hydrogen bonding
and electrostatic forces also play a role in stabilization.
Intercalation is a non-covalent interaction in which the
drug is held rigidly perpendicular to the helix axis.
This causes the base pairs to separate vertically,
thereby distorting the sugar-phosphate backbone and
decreasing the pitch of the helix.

37. Intercalation of ethidium bromide into B-DNA
Copyright 1987 by Benjamin Publishing Company.

38. Intercalation and Topoisomerase- Induced DNA Damage
Intercalation is an energetically favorable process.
The van der Waals forces that hold the intercalated
molecules to the base pairs are stronger than those
found between the stacked base pairs.
Intercalation occurs preferentially (by 7–13 kcal/mol)
into pyrimidine-3′,5′-purine sequences.
Intercalators do not bind between each base pair.
Intercalation destroy the regular helical structure,
interferes with the action of topoisomerases which
alters the degree of supercoiling of DNA.
Most of the intercalators prefer G-C base pair.

39. Intercalation Steps Two steps for cationic intercalators:-
Step-1: Cation interacts with the negatively charged
DNA sugar-phosphate backbone.
Step-2: Diffusion of intercalator along the surface of the
helix creating a cavity for intercalation.
DNA intercalation is the first step for the DNA damage
followed by topoisomerase inhibition.
DNA-topoisomerase 1 complex is the target for the drug Topotecan hydrochloride.
DNA-topoisomerase I1 complex is the target for drugs:-
Acridines, actinomycin and anthracyclines.

40. DNA Intercalators 1. Acridine compounds Acridine Amsacrine
The DNA intercalators (reversible inhibitors) react with the DNA (enzyme) with non-covalent interactions.
1. Acridine compounds
They are by-products of aniline dye manufacture.
After 2nd world war, Aminacrine was used as an antibacterial.
Later another acridine-derivative (amsacrine) is mainly
used in the treatment of leukemia.
Acridine
Amsacrine

41. DNA Intercalators 2. Actinomycin compounds
Actinomycin D also known as Dactinomycin is an
anticancer agent used in clinics.
The phenoxazone moeity intercalates with DNA by
binding towards guanine.
3. Anthracyclines
1. Doxorubicin (active against leukemia and other
solid tumours)
2. Daunorubicin (active against leukemia)

42. DNA Alkylators The DNA alkylators (irreversible inhibitors) react with
the DNA (enzyme) to form covalent bonds.
Examples of DNA alkylators:-
1. Nitrogen mustards.
2. Ethylenimines.
3. Methanesulfonates.
4. (+)-CC-1065 and Duocarmycins.
5. Metabolically activated alkylating agents.

43. Nitrogen mustards
The alkylating agent is a compound that can replace a
hydrogen atom with an alkyl group under physiological conditions (pH 7.4, 37°C, aqueous solution).
Mechlorethamine
The alkylation reaction
Substitution reaction by nucleophiles

44. DNA Strand breakers
They initially intercalate into DNA and generate radicals.
These radicals abstract hydrogen atoms from the
DNA sugar-phosphate backbone or from the DNA bases
leading to DNA strand scission.
Therefore, these DNA-interactive compounds are
metabolically activated radical generators.
Examples:
Anthracyclines (antitumour-antibiotic)
Bleomycin
Tirapazamine
Enediyne (antitumour-antibiotic).

45. Electron transfer mechanism for DNA damage
DNA Strand breakers
Electron transfer mechanism for DNA damage
by Anthracyclines.

46. Bleomycin It is an anticancer drug isolated from fungus
Streptomyces verticellus.
There are three domains of bleomycin
interacts to DNA
A domain targets cancer cells.
A domain intercalates into cancer cell DNA.
A domain damages cancer cell DNA.

47. Bleomycin
Cycle of events involved in DNA cleavage by Bleomycin

48. Tirapazamine It is an anticancer drug that selectively kills
oxygen-poor (hypoxic) cells in solid tumors.
One-electron reduction, possibly by enzymes such as
NADPH-cytochrome P450 reductase or xanthine
oxidase, produces the key radical intermediate, which
can undergo homolytic cleavage and hydroxyl radicals,
highly reactive radicals that readily degrade DNA.
It reacts with these DNA radicals and converts them
into strand breaks.

49. Recommended Books
The organic chemistry of drug design by Richard B. Silverman. Second edition, Elsevier, 2004.
An introduction to Medicinal Chemistry by Graham L. Patrick. Fourth edition, Oxford, 2009.