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Paper 4: Biomolecules and Their Interactions

Module 24: Interactions of small molecules with DNA

Introduction

Small molecules that bind to genomic DNA, can serve as effective anticancer, antibiotic, antiviral therapeutic agents and can affect the well being of millions of people around the world. DNA is a pharmacological target of many drugs used clinically. With the completion of human genome project in the year 2000 and developments in experimental medicine, biochemistry and biotechnology, understanding of interactions between small molecules/ligands and DNA would lead to a quantum jump in the development of DNA binding pharmaceuticals, personalized medicine and industry.

DNA has two primary functions: replication by which it makes copies of nucleotide sequence in our DNA and transcription by which it controls the synthesis of body’s vital proteins. Many small molecules can bind to different regions of replicating fork of genomic DNA (Waring 1981) and stop work of DNA polymerase. Chromomycin, echinomycin, distamycin, anthramycin, rifamicin, ethidium bromide, bleomycin and base analogs are few such examples. Although exact mechanism of action is not known, several metabolites, drugs and carcinogens can enter the cell and bind to cytosolic receptors. The complex somehow controls transcription of a specific DNA segment.

Objectives

Objective of the present module is:

a) To introduce the readers basic concepts in small molecules- DNA interactions, b) To describe experimental and theoretical techniques used, c) To describe binding of small molecules to DNA by:

External binding,

Intercalation,

Minor groove binding, Major groove binding,

Mixed mode binding,

Alkylation, and

Binding in 3-way junction. d) To introduce the reader to the most challenging field of designing sequence specific DNA

binding drugs.

24.1 Basic concepts in small molecules- DNA interaction

The small molecules can interact with DNA bases by non-bonded, electrostatic and H-bonding interactions. Alkali and alkaline earth metal ions can interact with the backbone phosphate groups. The transition metal ions can form salt bridges and glue DNA base pairs. Aromatic molecules can slide in between the base pairs and intercalate in between the base pairs. These interact with DNA bases by stacking and electrostatic interactions. Many heterocyclic aromatic molecules with cationic tails and protruding NH and H groups can interact with DNA bases in major and minor grooves by H-bonding and electrostatic interactions. Their binding to DNA can be specific (recognize DNA base sequence) or non specific. The recognition of DNA base sequence, by small molecules, aroused a great interest in the scientific community. The reason being it gives us an opportunity to control replication or transcription of a specific gene segment and subsequent biochemical events. One more reason for great outburst of

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interest in the study of ligand-DNA interactions is, ligand-DNA complex serves as an ideal prototype model system, for the study of protein–DNA interaction. Latter is important but a lot more complicated, because of inherent flexibility in the DNA structure and difficulties in co-crystalization of protein-DNA complexes. Understanding of protein-DNA recognition is still incomplete.

24.2 Use of experimental and theoretical techniques for the study of small molecules DNA interactions

Interaction of small molecules with DNA can be followed by number techniques.

X-ray fiber diffraction

Lerman (1961) gave the first proposal for intercalation of acridine orange on the basis of X-ray fiber

diffraction and hydrodynamical studies. X-ray diffraction pattern of regular helical fragments of DNA

were found to be disturbed. There was a strong meridian reflection at 3.4Å indicative of stacking. DNA

molecule was found to be lengthened. Langridge et al (1975) and Arnott et al (1980) studied fiber

diffraction patterns of many platinum antibiotics with DNA.

Spectroscopy

Majority of the small molecules which slide in between DNA base pairs, are chromophoric in nature.

Hence, their interaction with DNA can be followed by spectroscopic techniques (Sirajuddin et al 2013).

These yield both thermodynamic as well as kinetic information. When ethidium bromide or acridine

orange, form complexes with DNA, absorption spectrum is shifted towards longer wavelength side and

the molar extinction coefficient is depressed (bathochromic and hypochromic effects respectively)

(Waring 1981, Sanger 1984). Ultraviolet absorption spectra of netopsin with calf thymus DNA indicated

hypochromatic effect. There is an absorption peak beyond 320 nm. This depends upon A-T content and

disappears on heating DNA (Zimmer and Wahnert 1986).

Fluorescence emission of the bound drug is modified. For example fluorescence emission of ethidium

bromide belonging to phenanthriding class with core aromatic heterocyclic moiety, is enhanced, while

with amino acridines, it is quenched. This can be used for quantitative analysis following Scatchard plot

method. The ligand-DNA interaction is studied by number of other physic-chemical techniques as:

differential thermal analysis (DTA),circular dichroism (CD), linear dichroism, Infrared (IR), and Raman

spectroscopy (Sanger, 1984).

Electron Microscopy

Electron microscopy with autoradiography has been used extensively study interaction of several ligands

with DNA virus T2.

Sedimentation coefficient

Interaction of intercalating drugs with circular DNA can be followed by sedimentation coefficient

measurement discussed in module 5, to yield the unwinding angle. Virions from tumor virus SV-40

polyoma and bacteriophage X174 and lambda) have been used to study DNA unwinding.

NMR spectroscopy

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Proton NMR (nuclear magnetic resonance) has been used to study interaction of many ligands as bi-

functional antibiotics (echinomycin and tandem) with synthetic DNA fragments to yield structural

information.

Single crystal X-ray diffraction

Single crystal X-ray diffraction studies had been difficult initially, because of inherent flexibility and

heterogeneity in the DNA sequence. The studies with many planer chromophores were reported using

di-nucleotide monophosphate DNA or RNA duplexes. The interaction of daunomycin with

hexanucleotide duplex was reported by Alexander Rich (1980). Dickersen’s group reported studies on

netropsin with dodecamer CGCGAATTCGCG (Kopka et al 19845).

To date there are over 300 entries of 3D (thee dimensiona)l structures of ligand-DNA complexes using

NMR and single crystal X-ray diffraction techniques at Nucleic acid data base (NDB at Rutgers)(Berman

et al 1992) and Protein data bank (PDB) at RCSB (Research Collaboratory for structural Bioinformatics ).

DNA foot printing

Methydium propyl-EDTA (MPE) is an intercalator covalently bound by short hydrocarbon chains

together with methyl chelator. MPE together with ferrous ion efficiently produces single stranded

breaks in double helical DNA restriction fragment from PBR-322 plasmid. This mimics the behaviors of

DNA and is used in the analysis of study of DNA binding proteins. It can be also used for smaller

molecules as drugs. In the case of netropsin, it was seen that the regions which are rich in AT sequences

are highly protected when netropsin is bound to DNA. This gives a direct evidence of binding site of

netropsin on polymeric DNA. DNA foot printing had been a very important technique in understanding

sequence specific interactions of small molecules (Dervan, 1986).

Theoretical studies

Semi empirical, empirical and numerous quantum chemical methods inclusive of ab initio calculations

have been used to study interaction of small molecules with DNA. The interaction had also been

followed by computer aided molecular modeling, molecular mechanics and molecular dynamics and free

energy perturbation calculations techniques (Jayaraman et al 2011). It is beyond the scope of this

module to discuss each of these techniques in details and would be done elsewhere.

24.3 Different modes of binding small molecules with DNA

External binding

In the normal Watson-Crick hydrogen bonded double helical B-DNA, few small molecules, water and

ions (Mg2+ , Ca+2 and Na+,) can attach to the outer surface (sugar-phosphate backbone), mostly through

salt linkage, electrostatic and H-bonding interactions, leading to structural and conformational changes

in DNA. Although these interactions are of paramount importance in Biochemistry, these are usually

nonspecific (Barton J K).

Intercalation

In order to interact with DNA bases, small molecules must first be accommodated either in between the

H-bonded base pairs or in cavities (grooves) around the DNA molecule. Certain flat aromatic or hetero-

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aromatic molecules can slide in between the base pairs of DNA. This mode of binding is known as

‘intercalation’. These can stabilize the duplex without disrupting base pairing. Intercalation has the

effect of lengthening the duplex by around 3 Å per bound drug molecule. It causes unwinding of DNA,

and prevents replication by interfering with the action of topoisomerases. The degree of unwinding

depends on the structure of the intercalating molecule and the site of intercalation. The tight

ternary complex formed between the intercalated drug, DNA and the topoisomerase is lethal to

proliferating cells, so intercalators are often more toxic to cancer cells than to normal cells (wiki

intercalation).

In this binding mode, dimension and structure of the small molecule/ligand are crucial. Planar

aromatic molecules as: acrydines, ethidium bromide and proflavin are most suitable because of their

size, which is close do H-bonded base-pair (figure 24.1 and 24.2). Latter gives a good overlap with

bases. These interact with DNA with stacking and electrostatic interaction which is nonspecific. The

binding does not necessitate any specific base sequence.

Figure 24.1 Acridines

Figure 24.2 Ethidium bromide

Figure 24.3 Classical intercalation model. Figure from wiki intercalation

Figure 24.4 Ethidium bromide with Adenine uracil duplex. Figure from

wiki ethidium bromide

There are two ways of binding small molecules by intercalation. The classical or ‘end-on model’

described by Lerman (1971), in which the long axis of the molecule is almost parallel to long axis (C6

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pyrimidine-C8 purine) of the DNA base pairs (figure 24.3), and ‘cris-cross model’ in which the long axis

of the ligand is rotated with respect to DNA long axis. Often it is closer to the ‘dyad axis’ of DNA. A

typical example is of complex of daunomycin with DNA (discussed later in this module). If only few

atoms of the ligand protrude out of the planar structure, as the aromatic ring in ethidium bromide, the

ligand rotates around its own axis so that these groups can protrude out in the major or minor groove. A

typical example (ehidium bromide with AU duplex) is shown in figure 24.4. The aromatic ring is seen to

protrude out in minor groove. (wiki Ethidium bromide).

If aromatic rings are substituted and there is a curvature it its structure, as in case of many steroids. The

molecule cannot slide in between the base pairs and may not bind by intercalation. Similarly if DNA base

is modified as in the case of 1-Me Adenosine or Ionosine, the ligand cannot fit in the structure and does

not intercalate. These observations led to the ‘steric compatibility’ as the minimum requirement for small

molecule DNA binding by intercalation.

Singles crystal X –ray diffraction studies have been reported for about 200 intercalator-DNA complexes.

In case of ethidium bromide, proflavin, acrydine orange etc with di-nucleotide monophosphate duplexes

(known as mini helix) it was observed that purines at 5’ position had C3’ endo sugar pucker, while the

pyrimidines at 3’ end was with C2’ endo sugar conformation. The glycosyl torsional angle in most cases

was in anti conformation (Sanger, 1984).

Groove recognition

Drugs such as mithramycin, chromomycin and oligomycin are specific for double stranded

DNA and recognize guanine residues. These molecules do not intercalate, in spite of normal

planarity for at least two third of their chromophores. Another class of molecules belonging to

nonintercalators are: netropsin, distamycin and hoechst 33258 (Nucleic acid drug interaction).

Figure 24.5a Recognition of DNA base sequence. Shown here are functional groups of GC, CG, AT and TA base paies in small (minor) and wide (major groove). Methyl groups in thymine and C2H in Guanine are colored green. Figure based on

introduction to protein structure by Branden and Tooze 1996.

These molecules recognize DNA base sequence and do not alter DNA structure and length. The

question arises as to how these molecules recognize DNA sequence? In normal Watson-Crick

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hydrogen bonded DNA, the only regions where the bases are visible to small molecules, are the

floors of minor and major grooves (figure 24.5a).

We show in figure 24.5b schematic representation of H-bonding donors and acceptors in major

and minor grooves of G-C, A-T, C-G and T-A base sequences.

Figure 24.5b schematic representati on of H-bonding donors and acceptors in major

and minor grooves of G-C, A-T, C-G and T-A base pairs

These molecules interact with the available nitrogen and oxygen atoms in the grooves (figure

24.5) that can make hydrogen bonds with the protruding donors (NH or H atoms) of the ligands

or protein side chains. The methyl group of thymine and corresponding hydrogen atoms of

cytosine provides additional discrimination. The donor-acceptor patterns for the AT and GC base

pairs, in the major and minor grooves are different (Brandon and Tooze 1996), so also are the sizes of

major and minor grooves. Polymorphic structural changes in DNA (A, B, C or Z) make the interactions

dependent on environmental conditions.

Minor groove binding drugs

Netropsin and distamycin

There are over 100 structures available of ligands binding in minor grooves of DNA. Netropsin and

distamycin are polyamide with antibiotic and antiviral activity. Netropsin is active against gram positive

and gram negative bacteria. Distamycin shows anticancer activity. These molecules belong to the class

of lexitropsines. These have pyrrole rings connected by peptide bonds and cationic tails (figure 24.6). The

two adjacent pyrrole rings are oriented with respect to each other at an angle ranging from 18⁰ to 20⁰.

These have a semicircular shape like a ‘necklace’.

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Figure 24.6 Distamycin and netropsin

The width and curvature of these molecules is critical for fitting in the minor groove of DNA. Both netropsin and distamycin interact with AT-rich regions of DNA, in the minor groove by

forming hydrogen bonding and hydrophobic interactions. The terminal amidine group of these molecules is basic and serves to attract the drug molecule to the negatively charged phosphodiester backbone. The binding could be reduced by incorporation of urea which shows

strong role played by electrostatic forces. Steric compatibility was found to be an important factor for their interaction with DNA.

Single crystal X-ray diffraction study on netropsin with CGCGAATTCGCG (dodecamer) was reported by Kopka et al(1985)(Figure 24.7). It was seen to bind in the minor groove of DNA displacing the water molecules in the spine of hydration. Netropsin amide NH furnish hydrogen bond donors to bridge DNA adenine N-3 and thymine O-2 atoms occurring on adjacent base pairs of opposite helix strands, exactly as with the spine of hydration. The narrowness of the groove forces the netropsin molecule to sit symmetrically in the center, with its two pyrrole rings slightly non-coplanar so that each ring is parallel to the walls of its respective region of the groove. Drug binding neither unwinds nor elongates the double helix, but it does force open the minor groove by 0.5-2.0 Å. It bends back the helix axis by 8⁰ across the region of attachment. The netropsin molecule has an intrinsic twist that favors insertion into the minor groove of B-DNA, and it is given a small additional twist upon binding. The base specificity that makes netropsin bind preferentially to AT sequencs, that runs four or more AT base pairs, is provided not by hydrogen bonding but by lose van der Waal’s contacts between adenine C-2 hydrogens and CH groups on the pyrrole rings of the drug molecule.

Distamycin can bind to DNA in a similar mode, in the minor groove of AT rich sequences by H-bonding

and hydrophobic interaction (nucleic acid drug interaction). In the case of GC rich sequences, the 2-

amino group of guanine prevents distamycin from binding to the minor groove by steric

hindrance, thus conferring AT-selectivity on the drug molecule. We show in figure 24.8 diatamycin with d(CGCGAATTCGCG) by Coll et al 1987.

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Figure 24.7 Netropsin in minor groove of DNA Figure from wiki PDB id 101D

Figure 24.8 Distamycin in minor groove of CGCGAATTCGCG.PDB ID 2DNA by Coll et al

1987

Figure 24.9 Noncovalent binding in 2:1 ratio of a ligand SG2057) (shown in blue). Figure by Hrtley and

Hochhauser (2011)UCL cancer research

Sundaralingams’s group reported structure of drug-DNA complex between distamycin A and an

alternating B-DNA octamer duplex d(ICICICIC)2. Distamycin was seen to bind DNA at 2:1 stoichiometry.

Two distamycin A molecules are bound side by side with dyad symmetry in an antiparallel orientation in

the expanded minor groove. The amides of each drug molecule are hydrogen bonded to the minor

groove base atoms of only one DNA strand. The complex not only shows binding of two drug molecules,

but the DNA duplex also exhibits striking low-high alternations in the helical twist angles, the sugar

puckering and the phosphate conformations, providing the basis for a new model for an alternating B-

DNA with a dinucleotide repeat. Another example showing 2:1 binding of sequence selective non-

covalent DNA binding agents (extended pyrrolo[2,1-c][1,4]benzodiazepine dimer SG2057) by Hartley

and Hochhauser (2011) in the minor groove of DNA is shown in figure 24.9.

Hoechst are part of family of blue fluorescent dyes. Hoechst 33258 (figure 24.10), bind to AT rich region of CGCGAATTCGCG similar to netropsin and distamycin. The structure has been explored both by linear dichroism and single crystal X-ray diffraction in Stephen Neidle’s group (Clark et al 1996).

Lexitropsines (figure 24.11) are semisynthetic ligands. Series of dimers and trimers of distamycin and netropsin have been synthesized and studied in order to increase DNA binding regions to 10 base pairs or more. These semi-synthetic compounds have been named as lexitropins. Some lexitropins also incorporate imidazole (Im) rings as well as pyrrole (Py)rings.

Lexitropsines form complex with DNA in the stoichiometry 1:1 and 2:1 in the minor groove of the

sequence 5′-AATTC with the bulky piperazine group extending over the C·G base pair. This binding is stabilized by hydrogen bonding and numerous close van der Waals contacts to the surface of the groove walls. The meta-hydroxyl group was found in two distinct orientations. Neither of them participated in

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direct hydrogen bonds to the exocyclic amino group of a guanine base. Recognition of DNA base sequence by lexitropsines aroused great interest. Their ability to recognize specific base sequence on DNA would give a very powerful technique in molecular biology and therapeutics (oligonucleotides as drugs).

Figure 24.10 Hoechst33258

Figure 24.11 Lexitropsins

Major Groove binding of Drugs:

Although major groove has number of electron donors and acceptors which can have H-bonding with small molecules, this binding mode is not much popular. The number of available structures of small molecules binding in major grooveof DNA is very small (close to 10). Methyl green is a DNA major groove binding drug (Kim and Norden (1993). The interaction with poly(dA-dT)2, poly(dA).poly(dT) and triplex poly(dA).2poly(dT) was studied with linear dichroism. A typical example of major groove binding is of a third DNA strand with the complementary H-bonding groups. Latter can wrap around DNA in the major groove and make a triple helix.

Mixed Binding Mode

Several studies were taken on interaction of bifunctional peptide antibiotics echinomycin, tandem, triostin etc using X-ray diffraction, NMR and theoretical modeling. These studies showed some interesting results. Lot of attention was focused on these structures as these can serve as a good model for protein-DNA interactions. These do not disrupt Watson-Crick base pairing, binding is in minor groove with quinoxiline ring having partial intercalation. Typical examples can be seen in figure 24.12 for triostin withd(GATATC) by Addess and Feigon (1994) (PDB ID 185D).

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Figure 24.12 Structure of triostin with d(GATATC)2 (PDB ID 185D) Figure 24.13a Daunomycin with CGTACG

PDB id 1D11

Figure 24.14 16Cis-Platin DNA structure

The noble Laureate Alexander Rich studied binding of daunomycin (figure 24.13a) with a hexanucleotide duplex dCpGpTpApCpGp). This was the first X-ray crystallographic structure of drug DNA with more than two base pairs and at a resolution 1.5Å.

Figure 24.13b Daunomycin

structure and nomenclature

Figure 24.13c Cris-cross intercalation

Figure 24.13d Sugar ring in major groove

The complex structure had been significantly different than the classical intercalator model.

A-ring of Daunomycin (figure 24.13b) intercalates in cris-cross fashion (figure 24.13c), with sugar ring in the minor groove (figure 24.13c),

The D-ring of the drug is protruding in the major groove (figure 24.13d),

O9-of daunamycin H-bonds with NH2 of guanine, O13 of daunomycin H- bonds with O2 of cytosine,

Sugar pucker belongs to C2’ endo family, the six bases have: C2’endo- C1’exo-O4’endo-C2’endo-C2’endo-C3’exo

It appears that daunomycin does not recognize any specific base sequence as N3 of Guanine and O2 of Cytosine are non specific.

DNA Alkylation

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This mode of binding involves forming H-bonds directly with DNA bases (figure 24.14). This mode is used

by cis-platin drugs, used commonly in cancer therapy. Original cis-platin was discoved in mid sixty’s and

put in clinical use in seventy’s. Three further drugs carboplatin, nedaplatin and oxaliplatin are in clinical

use and bind by alkylation. Calf Thymus + ethyleneamine (2,2’ bipyridine) platinum (II) DNA double helix

is unwound into a ladder structure with DNA base pair separation 3.4 Å. The Drug intercalates in

alternate base pairs. One of the bases is in syn conformation. The diffraction pattern is similar to Z-DNA

and not A and B form of DNA (Arnott et 1980)

Drug binding in a three-way junction

The scientists have developed a synthetic drug agent that targets and binds to the centre of a 3-way junction in the DNA. These 3-way junction structures are formed where three double-helical regions join together (figure 24.15). This is particularly exciting, as three way junctions have been found to be present in diseases, such as: Huntingtons disease, myotonic dystrophy and during cancer growth. A totally new mode of binding was given by Dr. Mike Hannon (University of Birmigham) and Miquel Coll at Spanish research centre at Barcelona. The ligand fits perfectly into the centre of a three way junction. These molecules recognize DNA triplex and quadruplex structures. The recognition mode of these molecules is similar to that in DNA duplexes. There are several noncovalent forces that hold the drug in three way junction. The authors have shown how a cylinder of six phenyl rings forms almost a perfect pi-stacking interaction with the six DNA bases that are placed at the junction of three double

stranded arms that come together.

Figure24.15 Recognition of 3 way junction

Figure 24.16.Face to face stacking (Figure 24.15 and 24.16 from Mike Hannon)

The two phenyl rings from a strand of the cylinder, stack with the two bases of one of the strands of the DNA. This is shown in the figure 24.16. This stacking is almost too good to be believed and leads

to the cylinder fitting into the junction just like a hand into a glove.

24.5 Designing DNA binding drugs

The objective behind designing sequence specific DNA binding drugs is to stop replication or transcription of a particular DNA segment. The designing strategy is based on understanding the mechanism of recognition of DNA base sequence by small molecules Gibson D. For example Zimmer has found that number of methyl pyrrole groups increases stability. The melting temperature Tm shows

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that binding is predominantly electrostatic. Non electrostatic forces as H-bonding and stacking also contribute.

Isohelical analysis was done by Dickerson suggested that a long flexible molecules with proper curvatures can bind to DNA. He also observed that substitution of one or more pyrroles by imidazole in netropsin could permit recognition of GC base pairs as well and lead to a class of synthetic ‘lexitropsins,’ capable of reading any desired short sequence of DNA base pairs.

Simple oligomeric compounds like distamycin and netropsin do not have the ideal crescent shape to wrap around the minor groove of DNA, and they fail to recognize longer stretches of DNA. Dervan took this approach further in synthesizing ‘isolexins’ a series of oligomeric ‘hairpin’ polyamide molecules containing pyrrole and imidazole ring systems that are able to bind side-by-side in the minor groove of DNA with high affinity and in a sequence-specific manner.

Summary

Primary objective behind study of small molecule-DNA interactions is to understand mechanism of recognition of DNA base sequence by small molecules. This would enable us to design ligands which can recognize DNA base sequence and stop replication of a specific gene. In the present module have introduced the reader to the basic concepts in small molecules DNA interactions and described experimental and theoretical methods used in order to study their interactions. We have elaborated on small molecules DNA binding and recognition by: external binding, interacalation, minor and major groove binding, mixed mode binding, alkylation and binding in a three way junction. We discussed specific examples as: binding of ethidium bromide, netropsin, distamycin, hoest33258, lexitrosins, echinomycin, daunomycin, cis platin compounds. A new synthetic molecule binding at three way DNA junction is also described. Lastly we discussed strategies used for designing small

molecules which can recognize DNA base sequence.