This document discusses targeting telomeric G-quadruplexes as a potential therapeutic approach for cancer. It describes how G-quadruplex binding molecules can inhibit telomerase and cause cancer cell death by triggering telomere shortening. The document outlines various methodologies used to design G-quadruplex binding molecules and study their interactions with telomeric DNA, including computational modeling, biophysical assays, and structural analysis. Challenges in developing selective G-quadruplex targeting agents are also discussed.

1. Telomeric G-Quadruplexes as Therapeutic
Targets in Cancer
SUBMITTED TO:MA’M TAYYABA YASMIN
SUBMITTED BY:
HIRA BATOOL(SP10-BSB-013)
MADIHA TARIQ(SP10-BSB-014
5/10/2012

2. TABLE OF CONTENTS
1. INTRODUCTION
2. OBJECTIVES
3. MEHODOLOGY & INSTRUMENTATION
4. ROLE OF BIOINFORMATICS
5. EXPECTED OUTCOMES
6. REFERENCES
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3. ABSTRACT
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4. INTRODUCTION:
Cancer is one of the leading health problems in Pakistan. Dr Muhammad Luqman, director of medical
education at Foundation University Medical College, Rawalpindi, said that the number of cancer patients in
Pakistan is increasing by 8 to 10 % per year[ 1].Lung cancer and Breast cancers are most frequently occurring
cancers in our population. Common treatments for cancer are :
 Surgery
 Radiotherapy
 Chemotherapy
PROBLEMS WITH THESE TREATMENTS:
Chemotherapyprevents or reduces the growth of cancer cells. But it can also harm healthy cells such as those
that line mouth and intestines or cause hair damage. It alsoweaken the immune system.
2The possible side effects of radiation therapy depend on the affected location and the amount of radiation.
The most common side effects are tiredness, skin reactions (such as a rash or redness, permanent
pigmentation, and scarring) in the treated area, and loss of appetite. It can cause inflammation of tissues and
organs in and around the body site radiated.
Because of these and many otherside effects, scientists and researchers are trying to find new treatments for
the cancer. The majority of cytotoxic cancer chemotherapeutic agents target DNA, in a relatively unselective
manner. The effectiveness of agents such as cis-platinum and Adriamycin is a consequence of DNA-repair
defects and high DNA topoisomerase II levels, respectively, in susceptible cancer cell types. However these
features, though highly significant for the positive clinical outcomes are counter-balanced by their high toxicity
and generation of resistance mechanisms. There has been little development of new cytotoxic drugs over the
past few years, contrasting remarkably with the major effort world-wide in the discovery and the
development of targeted agents that can exploit the knowledge of the molecular basis of cancer.
TELOMERS AS THERAPEUTIC TARGETS:
One of the recognized acquired capabilities of cancer is a limitless replicative potential (Hanahan and
Weinberg,2000). It is now evident that this ability relates to the maintenance of telomeres, tandem repeated
DNA sequences ([TTAGGG]n in humans) at the ends of chromosomes with associated proteins.[ 4]As a cell
begins to become cancerous, it divides more often, and its telomeres become very short. If its telomeres get
too short, the cell may die. It can escape this fate by becoming a cancer cell and activating an enzyme called
telomerase, which prevents the telomeres from getting even shorter. If scientists can learn how to stop
telomerase, they might be able to fight cancer by making cancer cells age and die. In one experiment,
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5. researchers blocked telomerase activity in human breast and prostate cancer cells growing in the laboratory,
prompting the tumor cells to die.[3]
Telomeres consist of tandem repeats of guanine-rich sequences TTAGGG. In eukaryotes, telomeric DNA is
single stranded for the final few hundred bases. These single-stranded sequences can fold into a variety of
four-stranded structures (quadruplexes) held together by quartets of hydrogen-bonded guanine bases. The
reverse transcriptase enzyme telomerase is responsible for maintaining telomeric DNA length in over 85% of
cancer cells by catalyzing the synthesis of further telomeric repeats. Its substrate is the single-stranded 3'-
telomeric end. The associated activity of telomerase in the majority of tumors combined with its absence in
most adult normal tissues has generated considerable interest in targeting the enzyme and associated
telomeres in a cancer therapeutic context.G4 structures, Inhibits telomere elongation by telomerase (Zahler
et al., 1991). This has led to a rational search for small molecules that can selectively interact with and stabilize
G-quadruplexes (for reviews, see Mergny and Helene, 1998; Kerwin, 2000). a number of other compound
classes have been identified, including tricyclic anthraquinone-based G-quadruplex-interactive telomerase
inhibitors (Sun et al., 1997; Perry et al., 1998a,b), fluorenones (Perry et al., 1999b), bisubstitutedacridines
(Harrison et al., 1999), cationic porphyrins (Wheelhouse et al., 1998; Izbicka et al., 1999), a
perylenetetracarboxylicdiimide derivative (Fedoroffet al., 1998), indolo-quinolines (Caprio et al., 2000), and a
benzonaphthofurandione tetracyclic compound (Perry et al., 1999a).
G-QUADRUPLEX STRUCTURE
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6. OBJECTIVES
 To describe the mechanism of introduction and action of anti-cancerous G-
quadruplex binding molecules.
 To study relationships between telomeric G-quadruplex structures and
chromosomal end capping activities.
 To discuss approaches, current progress, and the mechanistic issues posed by
quadruplex targeting.
 To understand the kinetic, thermodynamic and mechanical properties of
binding molecules.
 To discuss the available data on these compounds.
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7. METHODOLOGY:
The discovery of the role of telomeres in controlling cell division has led to extensive research into its potential
application to treat cancer. Telomeres and the telomerase activity provides a vast variety of potential
therapeutic targets.The inhibition of telomerase has been proposed to be used in stopping the growth of
cancerous cells by triggering telomere shortening and cell death.Thus laboratories and research centres
around the world are working to devise molecular therapeutic strategies focusing on the inhibition of
telomerase.
The design of molecules that inhibit telomerase
As Telomerase is a complex enzyme; hence the design of the molecule that inhibit telomerase can target any
one of the following mentioned features of the enzyme. These include the following:
 Telomerase Genes.
 Interacting proteins such as Pot1 (Protection of telomeres protein 1 is a protein that in humans is
encoded by the POT1 gene), TRF1 and TRF2(negative regulator of telomere length).
 The hTRtemplate (human telomerase RNA template).
 The active site of hTERT(hTERT, or "human Telomerase Reverse Transcriptase," is a ribonucleoprotein
that maintains telomere ends by addition of the telomere repeat sequence TTAGGG)
 Telomere strand interaction - Stabilising G-Quadruplex DNA
All these approaches are the subject of current investigation.The latest and most promising of these is the
stabilisation of G-Quadruplex DNA which is also the topic under discussion.
Z
LIGAND BINDING TO TELOMERE
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8. MECHANISMS OF ACTIONS:
The G-quadruplex structure has four grooves of unequal width, this be important in its recognition by small
artificial molecules. Initially it was thought that these molecules would bind within the grooves (intercalate) in
a manner similar to that of DNA intercalators.
In order to allow intercalation of the stabilising molecule into the cavity between the tetrads, distortion of the
quadruplex must occur. The G-quadruplex is an extremely stable and rigid structure and therefore this
distortion is not favoured. An alternative model for the binding of these G-quadruplexstabilisingmolecules, is
the stacking of the molecule on the outside of the tetrads. This avoids the need for any distortion of the
structure and is far more favourable.
Detailed structural analyses of G-quadruplex- ligand complexes by X-ray crystallography have demonstrated at
least two types of binding sites for G-quadruplexligands. The most common is co facial end-stacking or ‘hemi
intercalation’ of the ligand onto one or both of the terminal G-tetrads. Other binding sites are defined by the
surface features of the grooves and/or loop regions. In both cases, subtle variations of G-
quadruplextopologies, groove widths, and loop sequences can facilitate selective binding interactions even
between closely related G-quadruplex structures.[6]
[7]Small molecules binding to G-quadruplexes have been largely based on polycyclic planar aromatic
compounds with at least one substituent terminating in a cationic group. The original rationale for the planar
moiety was that this would stack effectively onto planar G-quartets, which has been subsequently visualised in
a number of crystallographic studies of G-quadruplex-ligand complexes. Structural studies are unanimous in
showing that such planar ligands stack onto a terminal G-quartet. The cationic charge requirement has led to
the dogma that these groups reside in quadruplex grooves and directly contact phosphate groups. However
the crystallographic evidence to date indicates that these electrostatic contacts are rarely direct but are often
mediated by bridging water molecules, with the space in the grooves containing structured water networks,
analogous to those around duplex DNA sequencesAll quadruplex structures are very distinctive from duplex
nucleic acids, offering considerable potential for differential molecular recognition, and thus have enabled a
number of small molecules to be developed that have much higher quadruplex compared to duplex affinity.
The classic model of telomerase inhibition and consequent telomere attrition leading to senescence and
apoptosis requires that cells with a mean telomere length of 5 kb, a 24 h cell-doubling time and a subsequent
loss of ∼ 100 nucleotides per round of replication would reach critical telomere shortening in ∼ 40–50 days.
COMMON G-QUADRUPLEX BINDING MOLECULES:
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9. The quadruplex-binding acridine ligands BRACO-19 and RHPS4 , in common with telomestatin, induce rapid
replicative senescence in cancer cells and activate the same DNA damage response that follows DNA double-
strand breaks. This involves in particular p16INK4a kinase and p53 pathways which can be visualized by a
significant population of cells undergoing end-to-end fusions in metaphase.. Q-FISH studies have shown that
telomestatin is localized at telomeres during replication and importantly, that telomere replication is
unaffected in mouse embryonic fibroblast (i.e. untransformed) cell lines .
Telomestatin, a naturalproduct isolated from Streptomyces anulatus 3533-SV is one of the strongest and most
specific inhibitors of telomerase reported to date. Telomestatin has molecular dimensions similar to those of
G-tetrad DNA and can bind to various G-quadruplexes with modest
Affinity.Telomestatininduces telomere shortening in treated cells more rapidly than is expected for a single
mechanism involving telomerase inhibition.Recent studies have shown that telomere uncapping and the loss
of telomeric DNA is related to the competition between telomestatin and POT1 – a shelterin protein that
binds to the 3’ single-stranded overhang.While it is unknown if this type of activity might be cancer-selective,
telomestatininduces senescence and apoptosis in a number of different tumor cell types and exhibits less
toxicity towards normal progenitor cells.[6]
The cationic porphyrinTMPyP4 is the most extensively studied to date. TMPyP4 inhibits both and TaqDNA
polymerase.X-ray crystallography studies have shown that TMPyP4 can bind to G-quadruplex DNA at many
different positions, including the terminal G-tetrads,[98] and the loops, grooves, and phosphodiester
backbone.[99],[6]
Anthraquinones and G-QuadruplexStabilisation
The first demonstration of telomerase inhibition by a G-quadruplex-interactive molecule in living cells was
using an anthraquinone.This molecule has moderate preference for binding to quadruplex DNA(four strand
structure) over duplex DNA (two stranded structure). This is a vital characteristic in order to avoid toxicity in
the body. Experiments have also shown the molecule
In the presence of anthraquinone, significant inhibition of more than 50% was observed for telomeres that
were 5 or more repeat units long and thus capable of forming a quadruplex structure. For telomeres of shorter
lengths, the presence of anthraquinone had little effect on the inhibition, since quadruplexes could not be
formed
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10. INSTRUMENTATION:
G-quadruplex structure (i.e. inter- vsintramolecular), quantity of sample, ligand-quadruplex stoichiometry and
the type of binding information sought.G-Quadruplex ligand binding assaysto study the interactions between
this type of structure and it’s ligands.Selection of assay will depend on a number of factors such as the ligands
to be tested,
Calorimetric techniques:
ITC (Isothermal titration calorimetry) and DSC (Differential scanning calorimetry) provide means of
quantifying the thermodynamic properties and processes of G-Quadruplex ligand systems.
Polymerase Chain Reaction assays
SPR (Surface plasmon resonance) assay
SPR is a fast and sensitive technique useful in screening libraries of small ligands and is ideally used to
characterise interactions between ligands and macromolecules.
FRET (Fluorescence Resonance Energy Transfer) melting assay
A FRET melting assay can determine the ‘affinity’ and ‘selectivity’ of ligands by measuring the increase in
melting temperature of a quadruplex induced by the linkage of ligands to G4 Dna
TRE is a new assay which utilises SPR and does not require PCR amplification.
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11. Advantages of G-quadruplex inhibition
The potential advantages of Stabilising G-Quadruplex DNA, a potential cancer cure, are summarized below:
Unlike direct inhibition of telomerase, this method has bought about a more generalized approach
that can be applied to telomerase positive cells .
Although both the G-quadruplex inhibitors and the direct-acting telomerase inhibitors have been
shown to inhibit cell growth and induce senescence, a great contrast was that G-quadruplex
inhibitors caused no toxicity. The toxicity of this method is low due to the selectivity of the
molecules for binding to quadruplex DNA over duplex DNA.
One of the main advantages of this method over conventional telomerase inhibition is that G-
quadruplex inhibitors might not require an extended period of time before any significant effect
takes hold.
senescence is relatively rapid in comparison to telomerase inhibitors.
Challenging Problems:
A major challenge is to distinguish sequence, structural and biophysical properties of the human
intramoleculartelomeric G-quadruplex to facilitate the design of ligands selective for the human telomere,[8]
3Measuring telomerase may be a new way to detect cancer.. But there are risks. Blocking telomerase could
impair fertility, wound healing, and production of blood cells and immune system cells.
.Chromosomal ends are associated with a wide variety of proteins that bind to telomeric DNA. The ‘shelterin’
protein complex maintains the structural integrity of telomeres in vivo. Ligands displaceproteins from the
shelterin complex causing telomere destabilization, a possible genotoxicity associated with many G quadruplex
ligands.
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13. 12

14. ROLE OF BIOINFORMATICS
Quadruplex binding molecules are designed using Bioinfo tools .To design such molecules ,structure and topology of G-
quadruplex should be known.
Bioinformatics provides following sources of knowledge to discover new cancer therapies:
Information on the 20,000 to 40,000 genes that comprise the human genome, the proteins they
encode, and the variation in these genes and proteins that occur in disease.
Genome-wide analysis of cancer cells and tissues leads to the identification of new drug targets
and the design of new therapeutic interventions.
Bioinformatics deals with the storage and analysis of large amounts of diverse information on
genetic variation, gene and protein functions, and interactions in regulatory processes and
biochemical pathways.
Cancer bioinformatics deals with organizing and analyzing the data so that:
o Specific gene and protein targets on which cancer cells depend can be identified.
o Therapeutic agents directed against these targets can then be developed and
evaluated.
o Molecular and genetic variation within a population may become the basis of
individualized treatment.
Occurrences of quadruplexes within the human and other genomes have been mapped by bioinformatics
surveys, which have revealed over-representations in promoter regions, especially of genes involved in
replication, such as oncogenes, as well as in 5′ UTR regions.
BIOINFORMATICS TOOLS TO FIND G-QUADRUPLEXES:
There are many online tools to predict whether a particular sequence can form quadruplex or not. These tools
simply take an input sequence and give output in form of region that can form quadruplex. Generally, a simple
pattern match is used for searching for possible quadruplex forming sequences:
G3+N1-7G3+N1-7G3+N1-7G3+ where N is any base (including G).For a quadruplex forming sequence there must
be 4 G’s triplets with 1-7 N nucleotides between them.
A simple pseudocode to find a quadruplex in an input sequence is:
 Input telomere sequence as text and GGG as the pattern.
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15.  Compare text with pattern, if G is there in text then text, pattern and counter are incremented. If
not,reset counter to 0 and incrementtext.
 If counter is equal to the length of pattern i.e 3,(means that 3 consequetiveGs has been found) store
the start and end position of consecutive 3Gs.
 Repeat the whole process until 4 or more G triplets are found.
 Also make sure that intervening nucleotides between G triplets should be beween 1 to 7.
Bioinformatics supports therapeutic molecules development by combining information science, biostatistics,
simulation and modeling techniques. Research is performed in silico and provide informatics and statistical
support for the design and analysis of laboratory experimentation
Ligandmediated
stabilization
of G-quadruplex DNA
might facilitate the
regulation of gene
expression and/or the
inhibition of telomerase
activity[6]
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16. ECONOMICS/OUTCOMES
These molecules have facilitated highly selective gene silencing, and have found numerous applications in
basic research and medicine
Small molecules capable of structureselectiveDNA binding may provide an exciting new avenue for the
development of anti-cancer agents and molecular probes
. As novel therapeutics, they have the potential to extend and improve the lives of those suffering from one of
the most devastating and common causes of premature death
). G-quadruplex-specific antibodies generated by in vitro evolution may provide key tools for target
validation,[75,119] while the design and synthesis of new high affinity G-quadruplex ligands will provide new
drug candidates and molecular probes. These molecules will provide researchers with new tools for studying
the potential relationships between DNA folding and gene expression, chromosome stability, viral integration,
and recombination.[6]
Of greater practical importance is that future G-quadruplex ligands are developed with regard to their ability
to be used as drugs, so that they have: [5]
(a) effective and selective tumour uptake and penetration,
[5]http://onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2009.07463.x/full
The development of small molecules that specifically bind to a particular DNA secondary structure may
improve cancer-specific targeting and decrease the side effects associated with chemotherapeutic treatments.
More studies are also expected to come out on the mechanism and clinical potential of quadruplex ligands,
especially in view of the frequency of potential quadruplexes in the human genome.
. The addition of the inhibitor should;
1. Reduce telomerase activity.
2. Lead to the eventual shortening of the telomeres being observed.
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17. 3. Cause a decrease in cell proliferation
While more quadruplex-specific ligands are being identified, understanding of their binding is currently limited
as few structures are available. More structural studies on drug-G-quadruplex complexes are anticipated to
address this need. In addition, a better understanding of the biological roles of G-quadruplexes and G-
quadruplex-interactive proteins should emerge from research targeting these issues. We also expect to see
more reports on quadruplexes formed in RNAs.
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18. REFERENCES
(1)http://tribune.com.pk/story/333586/world-cancer-day-more-than-1-4m-cancer-patients-in-pakistan/
2http://www.medicinenet.com/radiation_therapy/article.htm
3http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726962/
4http://molpharm.aspetjournals.org/content/61/5/1154.full.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726962/?tool=pubmed[7]
[6]Nathan W. Luedtke (2009) Targeting G-Quadruplex DNA with Small
Molecules retrieved from http://bioorganic-chemistry.com/quadruplex_ligand_review.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726962/?tool=pubmed[8]
chromosomeshttp://www.ncbi.nlm.nih.gov/pubmed/11745111
http://exploreable.wordpress.com/tag/replicative-senescence/
http://www.ncbi.nlm.nih.gov/pubmed/18855731
http://www.quadruplex.org/?view=reviews
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