Nucleic Acids

as Targets for Drug Action

 Nucleic Acids (NA)
 Biomacromolecules
 Universal in living things, as they are found in all cells and viruses
 Play a vital role in living organisms/things
 Carry gentetic information
 First discovered in 1871 by Friedrich Miescher
 Composed of monomeric units .i.e. Nucleotides
 DNA and RNA are natural Nucleic Acids
Discovery of NA:
 DNA was accomplished by Johann Friedrich Miescher circa 1870

 He found a substance of unknown function in the nuclei of human white blood cells (nuclein)

 Miescher separated ‘nuclein’ into protein and nucleic acid components.

 In the 1920's nucleic acids were found to be major components of chromosomes

 Elemental analysis of NA showed the presence of C, H, N, O & P

 Unlike proteins, nucleic acids contained no Sulfur (S)

 Hydrolysis of chromosomal NA gave inorganic phosphate, 2-deoxyribose and heterocyclic bases

 Chromosomal nucleic acids with unusual sugar component are called deoxyribonucleic acids (DNA)

 Analogous nucleic acids with ribose component are ribonucleic acids (RNA)

 The acidic character of the nucleic acids was attributed to the phosphoric acid moiety
Types of Nucleic Acids

Natural Nucleic Acids

 Deoxy Ribonucleic Acid (DNA)

 Ribo Nucleic Acid (RNA)

Artificial Nucleic Acids

 Peptide Nucleic Acid (PNA)

Locked Nucleic Acid (LNA)
 Glycol Nucleic Acid (GNA)
 Threose Nucleic Acid (TNA)
 Morpholino Nucleic Acids (MNA)
Constituents of Nucleic Acids

 Nucleobases

 Nucleosides

 Nucleotides

 Deoxynucleotides

Basic Components of Nucleic Acids

Phosphate Heterocylic-
ester base

Sugar
 Basic constituents of Nucleic Acids

Structures of pyrimidines and purines

 Nucleic Acid Bases:

Purines Pyrimidines

Sugar:
Phosphate:
O

HO P OH

OH
 Names of DNA Base Derivatives
Base Nucleoside 5'-Nucleotide

Adenine 2'-Deoxyadenosine 2'-Deoxyadenosine-5'-monophosphate

Cytosine 2'-Deoxycytidine 2'-Deoxycytidine-5'-monophosphate

Guanine 2'-Deoxyguanosine 2'-Deoxyguanosine-5'-monophosphate

Thymine 2'-Deoxythymidine 2'-Deoxythymidine-5'-monophosphate

 Nucleoside Base Distribution in DNA

Base Composition (mole %) Base Ratios

Organism Ratio (A+T)/(G+C)
A G T C A/T G/C
Human 30.9 19.9 29.4 19.8 1.05 1.00 1.52
Chicken 28.8 20.5 29.2 21.5 1.02 0.95 1.38
Yeast 31.3 18.7 32.9 17.1 0.95 1.09 1.79
Clostridium
36.9 14.0 36.3 12.8 1.01 1.09 2.70
perfringens
Sarcina
13.4 37.1 12.4 37.1 1.08 1.00 0.35
lutea
Base pairing

Watson-Crick H-bonding
DNA-Polymorphs
View down two successive base pairs,
showing the helical twist angle between them
Glycol Nucleic Acid (GNA)

GNA is polymer similar to DNA or RNA but differing in the composition of its "backbone"
It's backbone is composed of repeating glycerol units linked by phosphodiester bonds
Shows Watson-Crick base pairing and its more stable than its natural counter parts (DNA / RNA)
Not known to occur naturally
2,3-dihydroxypropylnucleoside analogues were first prepared by Ueda et al. (1971)
Later phosphate-linked oligomers exhibited hypochromicity in the presence of RNA and DNA in solution
It requires a high temperature to melt a duplex of GNA
It is possibly the simplest of the nucleic acids, so making it a hypothetical precursor to RNA
Locked Nucleic Acid (LNA)

It’s mimic of RNA

Sometimes called as inaccessible RNA
Ribose is modified with methylene bridge between 2’,4’-positions
3’-ribose conformation is restricted/locked
Locked ribose conformation enhances base stacking and backbone pre-organization
LNA increases thermal stability of duplexes
Ideal for the detection of short RNA and DNA targets
Capable of single nucleotide discrimination
Resistant to exo- and endonucleases resulting in high stability in vivo and in vitro applications
Increased target specificity
Peptide Nucleic Acid (PNA)
PNA Composed of 3 parts
a) Backbone – Peptide bond
b) Relevant base
c) Spacer (Acetic acid derivative)

PNAS 2000, 97, 3868-3871

Sept’2009
 Morpholino Nucleic Acids

It is a molecule used to modify gene expression

These oligomers are an antisense technology used to block
access of other molecules to specific sequences within N.A.
They block small regions of the base-pairing surfaces of RNA
It won’t degrade RNA
 Nucleic Acid – Interactive Agents: Classification

Reversible Binders: interact with DNA through the reversible formation of noncovalent interactions

Alkylators: react covalently with DNA bases

Strand Breakers: generate reactive radicals that produce cleavage of the polynucleotide strands

Silverman, R.B. The Organic Chemistry of Drug Design and Drug Action, 2nd Ed. Elsevier Academic
Press, San Diego, California, 2004, 617pp
Reversible Binders

Molecules that interact with DNA through the reversible formation of noncovalent interactions

Interference with these interactions can disrupt the DNA function and sometimes structure

3 types of DNA reversible binders

External electrostatic binding

Negatively charged sugar phosphate backbone of DNA affects the structure and function
Cations and water molecules bind to DNA to stabilize secondary structure
Electrostatic interactions disrupt DNA structure
Not depend on DNA sequence
Groove Binding
Two Types: Major and Minor Grooves
Differ in electrostatic potentials, H-bonding characteristics, Steric Effects & Degree of Hydration
Major Groove
Proteins exhibit major groove binding
Peptide molecules with amide bond
Wide G-C rich regions
Minor Groove
Small molecules bind to minor groove
Ligands possess linked aromatic rings(Crescent Shaped)
Narrow A-T rich region
Molecules fit well in this groove
H-bonding between N3 of A or O2 of T with molecules
Greater electronegative potential
Cationic molecules can interact well
Unwinding of DNA occurs rarely
Eg. Antitumor agent Netropsin
Groove Binding
 These molecules bind with approximately the same
affinity to DNA as intercalators (with typical binding
affinities of 106 mole−1), but do not perturb DNA structure.

4',6-diamidino-2-phenylindole
Common structural characteristics shared by most minor-groove binding molecules:

Positive charge(s)

Linked rather than fused aromatic and/or heteroaromatic rings

An approximately crescent shape in three dimensions

Dickerson–Drew sequence: d(CGCGAATTCGCG)
and closely related sequence: d(CGCAAATTTGCG)

Complex
d(CGCAAATTTGCG) : Hoechst 33258
 Hoechst 33258

H-bonding between ligand and O2 of T or N3 of A

It is used as a DNA and Chromosomal stain

It spans 4-5 A/T base-pair binding site

Non-planar and bulky Piperdine ring forces molecule into groove

Reasons for binding of these class of compounds to AT region

Narrow cross section of groove-binding molecules

complementing groove width

The negative electrostatic potential in the AT minor groove

complementing the positive charge (s) on these molecules

Displacement of highly structured arrangement of water molecules

(Spine of Hydration)
In uncharged ligand,

Vanderwaals and hydrophobic interactions are dominant

But, H-bonding and electrostatic interactions are of less importance in groove binding
Groove binders comprises molecules with two charged amidinium groups, one at each end

X-Ray and NMR studies: It covers 3-4 A/T base pairs

H-bonding between ligand and O2 of T or N3 0f A

In the complex with Dickerson-Drew Sequence,

water molecules mediates this interaction at one end of the
Groove binders comprises molecules with two charged amidinium groups, one at each end

Flexible bis-amidinium drug

Binding pattern is similar to Berenil

It has activity against the Pneumocystis carinii pathogen,

which is responsible for the opportunistic and life threatening
pneumonia that affects the majority of AIDS patients

It binds to A/T rich region

It’s effectiveness is limited and produces a number of toxic side effects.

Groove binders comprises molecules with two charged amidinium groups, one at each end

It is iso-structural to Beneril

Found improved biological activity.

Complex with DNA(X-ray studies): Bulky cyclohexyl groups snugly fit into the groove
 DB289
DB289 (pafuramidine maleate)

2,5-bis[4-(N-methoxyamidino)phenyl]furan monomaleate

It is a prodrug of DB75 (furamidine dihydrochloride)

Important oral dug treats Trypanosomiasis (African sleeping sickness)
In Phase-III clinical trials
It is believed to exert its action by selectively binding to A/T-rich kinetoplast DNA in trypanosomes
 It makes isohelical complex with Dickerson-Drew dodecamer

Ian Midgley et. al Drug Metabolism and Disposition 2007, DOI: 10.1124/dmd.106.013391
 Molecular structure of CGP40215A

Molecular Model

Binds minor groove A/T region

Two central NH groups of the ligand form strong

hydrogen bonds to O2 atoms of thymine bases

Ends of the ligand only make one direct DNA contact

The crystal structure of a complex with d(CGCGAATTCGCG)2

DB921

Schematic showing the hydrogen-bonding to base edges from the linear ligand DB921,
as found in the crystal structure of its DNA complex
TRIBIZ
It binds tightly, to a site of 7.5 base pairs

A view of the crystal structure (Clark et al., 1996) of the complex between the TRIBIZ molecule and
the duplex formed by d(CGCAAATTTGCG). The methoxyphenyl group of TRIBIZ is at the upper end
of the binding site in this view
Groove binding drugs netropsin and distamycin

The crystal structure of a host–guest complex involving netropsin (in

space-filling mode) and the oligonucleotide d(CTTAATTCGAATTAAG),
showing the two AATT drug-binding sites in the one sequence.
Complex or Co-crystal of D(TTGGCCAA) : Chromomycin

Mg

Chromomycin – Anti Cancer, Antibiotic

H2O
Crystal Structures of Drug-Oligonucleotide Minor-Groove Complexes
Lexitropsins:

Switching hydrogen-bond polarity at the groove floor by means of, for example,
an imidazole ring in order to hydrogen-bond to the guanine –NH2 substituent.
(a) The principles of netropsin and distamycin amide–base recognition, showing
hydrogen-bonding to the thymine of an A•T base pair.
(b) The concept of lexitropsin base recognition, with hydrogen-bonding from the
exocyclic amino substituent of a guanine to the nitrogen atom of an imidazole ring.

The crystal structure of a 2:1 complex between distamycin

(with atoms colored black) and the duplex formed by d(ICICICIC).
Detail of the hydrogen-bonding to both DNA strands in a 2:1 distamycin dimer showing
the hydrogen-bonding to bases in both strands of a five base-pair A/T sequence
The crystal structure of the polyamide molecule Im-Py-Py bound to a duplex formed by the sequence
d(CCAGATCTGG). The central part of this sequence, i.e. 5′-AGATCT, is recognized by the polyamide
A schematic view of the hydrogen-bonding to A•T and G•C base pair edges as observed in the crystal structure
(Kielkopf et al., 1998) of the complex involving the polyamide Im-Py-Py and the target sequence 5′-TCAGT
A schematic view of a hairpin polyamide, Im-Py-Py-linker-Py-Py-Py, recognizing both strands of
a G/C-containing sequence
DNA - intercalation

Intercalation is the reversible inclusion of a molecule between two DNA strands (Double helix)

Intercalation occurs with appropriate size, chemical nature fit and perpendicular to helix

Ligands are mostly polycyclic, aromatic, and planar

Used in chemotherapeutic treatment to inhibit DNA replication in rapidly growing cancer cells

Examples
doxorubicin (adriamycin) & daunorubicin - for treatment of Hodgkin's lymphoma
dactinomycin - used in Wilm's tumour, Ewing's Sarcoma, rhabdomyosarcoma
Driving forces for intercalation
 Stability
 Charge-transfer interactions
 Hydrogen bonding (for stabilization)
 Electrostatic forces (for stabilization)

1st described in 1961 by Lerman

 It occurs perpendicular to the helix axis

 Energetically favorable process

 Intercalation energy 7-13 kcal/mol

 It is a result of hydrophobic effect &

removal of drug from aq. medium

Vander Waal forces are more for

base pairs with intercalated molecules
Neighbor exclusion principle:

Intercalators can bind @ alternate base pair sites on DNA

Negative co-operativity

Binding in one site causes a conformational

change in the adjacent site which prevents
binding of intercalator in that adjacent site
Structural influences

Intercalation doesn’t disrupt Watson-Crick H-bonding

But, it does destroy the regular helical structure

Helical structural Interferes with action of

Intercalation variation DNA-binding enzymes Or
Interference of Topoisomerases or Polymersases

Topoisomerages: Alters the degree of super coiling DNA

Polymerases: inhibits the elongation of DNA & prevents the correction of mistakes
in the DNA by inhibiting the clipping out of mismatched residues in the terminus

Most intercalators display no sequence preferences on their binding

But show slight G-C preference
Intercalators binds less base pairs compared to groove binders
Steps involved in intercalation and Topoisomerase-induced DNA Damage:

Step 1:
Intercalator interacts with negatively charged DNA sugar-phospate backbone

Step 2:
Diffuses along the surface of the helix, until it encounters the gap (Cavity creation)

Step 3:
Topoisomerases involves i.e. interacts with DNA-Drug complex

Step 4:
Cleavable complex formation (Breakage-rejoining of DNA )
 DNA-Toposiomerases Unwinding of DNA occurs during replication
Scope for formation of tangling structures i.e. supercoils or catenanes
Major role of topoisomerases is to prevent DNA tangling

Topoisomerase-I Topoisomerase-II

structure of supercoils
Structure of the Topo-I/DNA complex.  PDB ID = 1A36
 Topoisomerase-I

It produces transient single-strand breaks in DNA

Type-I enzyme removes supercoils from DNA one at a time
It doesn’t require energy from ATP hydrolysis
DNA-Toposimerase-I is target for anticancer agent
Topotycan Hydro chloride (Hycamtin)

Topoisomerase-II

It produces transient double-strand breaks

Type II enzyme removes supercoils two at a time
It requires energy from ATP hydrolysis
DNA-Topoisomerase-II is target for anticancer drugs
Classes: Anthracyclins, anthracenediones, acridine, actinomycines

(a) To remove supercoils

(b) To remove catenanes  
Mechanism is unclear

DNA DNA

Topoisomerase
Drug

DNA -Drug Topoisomerase - DNA

Topoisomerase Drug

Topoisomerase - DNA – Drug Ternary Complex

 Mechanism of TAS-103

DNA

Drug

DNA -Drug
Antineoplastic agent

Topoisomerase

Topoisomerase - DNA – Drug

The chemical structure of TAS-103 (6-[[2-(dimethylamino)

ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-one
dihydrochloride)
Correlation between intercalation and anti tumor activity

Potency of intercalation
Strong correlation
Little correlation Cleavable complex formation

Anti tumor activity

•Some strong intercalators do not induce cleavable complexes

•Some non-intercalating molecules are DNA topoisomerase II poisons

eg. etoposide

Anti cancer drug

Intercalators:

Acridines

Actinomycins

Anthracyclins

Amsacrine
 Mechanism

Intercalation was first proposed by Leonard Lerman in 1961.

Proposed mechanism of intercalation:

 Cationic intercalator is attracted electrostatically to the polyanionic DNA in

aqueous isotonic solution.
 The ligand displaces a Na and/or Mg cation that always surround DNA (to balance
its charge) and forms a weak electrostatic bond with the outer surface of DNA.
 From this position, the ligand may then slide into the hydrophobic environment found
between the base pairs and away from the hydrophilic outer environment surrounding the DNA.
 The base pairs transiently form such openings due to energy absorbed during collisions with
solvent molecules.
Acridine Analogues

By-products of aniline-dye manufacture

Proflavine
Early 19th century: 1st clinical medicine against malaria
During 1st World War: Proflavine was antibacterial agent
After 2nd World war: Aminacrine was principal antibacterial agent
During 1960-70: Many acridine derivatives were tested for anti tumor activity
3,4-diamino analogue has potent activity, but unstable to air oxidation
SAR studies: Donor is necessary on aniline ring

It is equally potent as 3,4-diamino analogue

This was used a lead compound
Amsacrine, Acridine analogue Binds in
minor groove

Amsacrine was most potent

It stabilises the cleavable complex
Small structural changes in 9-amino acridines results
large difference in potency
Log P values close to amsacrine were most potent
Correlation exists between electronic properties on p-aniline and acridine pKa Interacts with
Steric effects playing a major role over lipophilic and electronic effects regulatory proteins

Its now used in leukemia

Amsacrine lacks broad spectrum of clinical activity to formulate

because of 1) low aqueous solubility
2) high pKa

New potent drug:

Aqueous solubility is relatively higher

Low pKa
High DNA binding affinity
Dactinomycin

Also known as Actinomycin D

1st family of chromopeptide antibiotics
In 1940: Isolated from Streptomyces strain
In 1952: found anti tumor activity clinically
Phenoxazone Chromophore intercalates between base pairs (X-ray)
It binds to double-stranded DNA (Guanine rich region)
2-NH2 group is crucial in intercalation
Inhibits DNA-directed RNA synthesis or DNA synthesis
RNA chain initiation is not prevented rather chain elongation is blocked
Cyclic peptide lie in minor groove region
Cyclic peptide side chains: strong H-bonding and hydrophobic interactions with DNA

H-bonds between:
NH of D-Val with C=O of neighboring D-Val
Guanine NH2 and C=O of L-Threonine
Guanine N-3 ring ‘N’ and NH of L-Threonine

Stacking forces - responsible for recognition and preferential binding of Guanine

Dactinomycin
•Biological activity depends on slow rate of Drug-DNA interaction
i.e. inter molecular interactions

H-bonding and planar interactions between purin and chromophore

van der Waal interactions between Polypeptide side chains and DNA

•Peptide substituents block the progression of RNA polymerase along the DNA

•Intercalators can cause cytotoxicity by interfering with DNA normal preocesses

Doxorubicin and Daunorubicin
Bis-intercalators
DNA-Alkylators
DNA Alkylators

Mustards

Nitrogen mustard
Sulfur Mustard
 SN1

SN2

SN1-reaction

SN2-reaction
Nucleophilic Sites

Nucleic Acid Bases:

Purines Pyrimidines

Relative reactivity
N-7, G > N-3, A > N-7, A > N-3, G > N-1, A > N-1, C

•N-3, C; O-6, G; Phospate groups also can be alkylated

 Reactivity of Nucleobases is controlled by

Steric,

Electronic

H-bonding effects in DNA

•Order of nucleophilicity is also depends on chemical structure of alkylating agent

Mechlorethamine

Bi-functional alkylating agent

Inter-strand cross-link Adduct

Miscoding

T G

•It’s G-T instead of G-C

Lead Modification
Ethylenimines
Methanesulfonates
(+)-CC-1065 & Duocarmycin

Natural products
Isolated from Streptomcin strains
Sequence-selective DNA-alkylating agents
Common feature
Resonance stabilization
Metabolically Activated Ureas

Nitrosoureas

Triazene antitumor drugs

Mytocin C

Leinamycin
Nitrosoureas
Nitrosoamide Nitrosourethane
Inter-strand cross-link

C G

Nitrosourea drug