SUBMITTED BY: HIRA BATOOL(SP10-BSB-013) MADIHA TARIQ(SP10-BSB-014

5/10/2012

TABLE OF CONTENTS

1. INTRODUCTION2. OBJECTIVES3. MEHODOLOGY & INSTRUMENTATION4. ROLE OF BIOINFORMATICS5. EXPECTED OUTCOMES6. REFERENCES

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ABSTRACT

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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:Chemotherapy prevents 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 also weaken the immune system.

2 The 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 other side 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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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), bisubstituted acridines (Harrison et al., 1999), cationic porphyrins (Wheelhouse et al., 1998; Izbicka et al., 1999), a perylenetetracarboxylic diimide derivative (Fedoroff et al., 1998), indolo-quinolines (Caprio et al., 2000), and a benzonaphthofurandione tetracyclic compound (Perry et al., 1999a).

G-QUADRUPLEX STRUCTURE

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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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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 hTR template (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.

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LIGAND BINDING TO TELOMERE

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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-quadruplex stabilising molecules, 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-quadruplex ligands. 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-quadruplex topologies, 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 sequences All 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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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 natural product 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 modestAffinity. Telomestatin induces 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, telomestatin induces senescence and apoptosis in a number of different tumor cell types and exhibits less toxicity towards normal progenitor cells.[6]

The cationic porphyrin TMPyP4 is the most extensively studied to date. TMPyP4 inhibits both and Taq DNA 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-Quadruplex Stabilisation

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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INSTRUMENTATION:

G-quadruplex structure (i.e. inter- vs intramolecular), quantity of sample, ligand-quadruplex stoichiometry and the type of binding information sought. G-Quadruplex ligand binding assays to 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.

Advantages of G-quadruplex inhibition9

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 intramolecular telomeric 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 displace proteins from the shelterin complex causing telomere destabilization, a possible genotoxicity associated with many G quadruplex ligands.

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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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Compare text with pattern, if G is there in text then text, pattern and counter are incremented. If not ,reset counter to 0 and increment text.

If counter is equal to the length of pattern i.e 3,(means that 3 consequetive Gs 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

stabilizationof G-quadruplex DNAmight facilitate theregulation of geneexpression and/or theinhibition of telomeraseactivity[6]

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ECONOMICS/OUTCOMES

These molecules have facilitated highly selective gene silencing, and have found numerous applications in basic research and medicine

Small molecules capable of structureselective DNA 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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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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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 SmallMolecules retrieved from http://bioorganic-chemistry.com/quadruplex_ligand_review.pdf

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726962/?tool=pubmed [8]

chromosomes http://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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