Telomeric G-Quadruplexes as TherapeuticTargets in CancerSUBMITTED TO:MA’M TAYYABA YASMINSUBMITTED                         ...
TABLE OF CONTENTS1. INTRODUCTION2. OBJECTIVES3. MEHODOLOGY & INSTRUMENTATION4. ROLE OF BIOINFORMATICS5. EXPECTED OUTCOMES6...
ABSTRACT           2
INTRODUCTION:Cancer is one of the leading health problems in Pakistan. Dr Muhammad Luqman, director of medicaleducation at...
researchers blocked telomerase activity in human breast and prostate cancer cells growing in the laboratory,prompting the ...
OBJECTIVES  To describe the mechanism of introduction and action of anti-cancerous G-   quadruplex binding molecules.  T...
METHODOLOGY:The discovery of the role of telomeres in controlling cell division has led to extensive research into its pot...
MECHANISMS OF ACTIONS:The G-quadruplex structure has four grooves of unequal width, this be important in its recognition b...
The quadruplex-binding acridine ligands BRACO-19 and RHPS4 , in common with telomestatin, induce rapidreplicative senescen...
INSTRUMENTATION:G-quadruplex structure (i.e. inter- vsintramolecular), quantity of sample, ligand-quadruplex stoichiometry...
Advantages of G-quadruplex inhibitionThe potential advantages of Stabilising G-Quadruplex DNA, a potential cancer cure, ar...
11
12
ROLE OF BIOINFORMATICSQuadruplex binding molecules are designed using Bioinfo tools .To design such molecules ,structure a...
 Compare text with pattern, if G is there in text then text, pattern and counter are incremented. If      not,reset count...
ECONOMICS/OUTCOMESThese molecules have facilitated highly selective gene silencing, and have found numerous applications i...
3. Cause a decrease in cell proliferationWhile more quadruplex-specific ligands are being identified, understanding of the...
REFERENCES(1)http://tribune.com.pk/story/333586/world-cancer-day-more-than-1-4m-cancer-patients-in-pakistan/2http://www.me...
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Telomeric G-Quadruplexes as Therapeutic Targets in Cancer

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Telomeric G-Quadruplexes as Therapeutic Targets in Cancer

  1. 1. Telomeric G-Quadruplexes as TherapeuticTargets in CancerSUBMITTED TO:MA’M TAYYABA YASMINSUBMITTED BY:HIRA BATOOL(SP10-BSB-013)MADIHA TARIQ(SP10-BSB-0145/10/2012
  2. 2. TABLE OF CONTENTS1. INTRODUCTION2. OBJECTIVES3. MEHODOLOGY & INSTRUMENTATION4. ROLE OF BIOINFORMATICS5. EXPECTED OUTCOMES6. REFERENCES 1
  3. 3. ABSTRACT 2
  4. 4. INTRODUCTION:Cancer is one of the leading health problems in Pakistan. Dr Muhammad Luqman, director of medicaleducation at Foundation University Medical College, Rawalpindi, said that the number of cancer patients inPakistan is increasing by 8 to 10 % per year[ 1].Lung cancer and Breast cancers are most frequently occurringcancers in our population. Common treatments for cancer are :  Surgery  Radiotherapy  ChemotherapyPROBLEMS WITH THESE TREATMENTS:Chemotherapyprevents or reduces the growth of cancer cells. But it can also harm healthy cells such as thosethat 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, permanentpigmentation, and scarring) in the treated area, and loss of appetite. It can cause inflammation of tissues andorgans in and around the body site radiated.Because of these and many otherside effects, scientists and researchers are trying to find new treatments forthe cancer. The majority of cytotoxic cancer chemotherapeutic agents target DNA, in a relatively unselectivemanner. The effectiveness of agents such as cis-platinum and Adriamycin is a consequence of DNA-repairdefects and high DNA topoisomerase II levels, respectively, in susceptible cancer cell types. However thesefeatures, though highly significant for the positive clinical outcomes are counter-balanced by their high toxicityand generation of resistance mechanisms. There has been little development of new cytotoxic drugs over thepast few years, contrasting remarkably with the major effort world-wide in the discovery and thedevelopment 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 andWeinberg,2000). It is now evident that this ability relates to the maintenance of telomeres, tandem repeatedDNA sequences ([TTAGGG]n in humans) at the ends of chromosomes with associated proteins.[ 4]As a cellbegins to become cancerous, it divides more often, and its telomeres become very short. If its telomeres gettoo short, the cell may die. It can escape this fate by becoming a cancer cell and activating an enzyme calledtelomerase, which prevents the telomeres from getting even shorter. If scientists can learn how to stoptelomerase, they might be able to fight cancer by making cancer cells age and die. In one experiment, 3
  5. 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 issingle stranded for the final few hundred bases. These single-stranded sequences can fold into a variety offour-stranded structures (quadruplexes) held together by quartets of hydrogen-bonded guanine bases. Thereverse transcriptase enzyme telomerase is responsible for maintaining telomeric DNA length in over 85% ofcancer 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 inmost adult normal tissues has generated considerable interest in targeting the enzyme and associatedtelomeres in a cancer therapeutic context.G4 structures, Inhibits telomere elongation by telomerase (Zahleret al., 1991). This has led to a rational search for small molecules that can selectively interact with and stabilizeG-quadruplexes (for reviews, see Mergny and Helene, 1998; Kerwin, 2000). a number of other compoundclasses have been identified, including tricyclic anthraquinone-based G-quadruplex-interactive telomeraseinhibitors (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), aperylenetetracarboxylicdiimide derivative (Fedoroffet al., 1998), indolo-quinolines (Caprio et al., 2000), and abenzonaphthofurandione tetracyclic compound (Perry et al., 1999a).G-QUADRUPLEX STRUCTURE 4
  6. 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. 5
  7. 7. METHODOLOGY:The discovery of the role of telomeres in controlling cell division has led to extensive research into its potentialapplication to treat cancer. Telomeres and the telomerase activity provides a vast variety of potentialtherapeutic targets.The inhibition of telomerase has been proposed to be used in stopping the growth ofcancerous cells by triggering telomere shortening and cell death.Thus laboratories and research centresaround the world are working to devise molecular therapeutic strategies focusing on the inhibition oftelomerase.The design of molecules that inhibit telomeraseAs Telomerase is a complex enzyme; hence the design of the molecule that inhibit telomerase can target anyone 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 DNAAll these approaches are the subject of current investigation.The latest and most promising of these is thestabilisation of G-Quadruplex DNA which is also the topic under discussion. Z LIGAND BINDING TO TELOMERE 6
  8. 8. MECHANISMS OF ACTIONS:The G-quadruplex structure has four grooves of unequal width, this be important in its recognition by smallartificial molecules. Initially it was thought that these molecules would bind within the grooves (intercalate) ina manner similar to that of DNA intercalators.In order to allow intercalation of the stabilising molecule into the cavity between the tetrads, distortion of thequadruplex must occur. The G-quadruplex is an extremely stable and rigid structure and therefore thisdistortion is not favoured. An alternative model for the binding of these G-quadruplexstabilisingmolecules, isthe stacking of the molecule on the outside of the tetrads. This avoids the need for any distortion of thestructure and is far more favourable.Detailed structural analyses of G-quadruplex- ligand complexes by X-ray crystallography have demonstrated atleast two types of binding sites for G-quadruplexligands. The most common is co facial end-stacking or ‘hemiintercalation’ of the ligand onto one or both of the terminal G-tetrads. Other binding sites are defined by thesurface 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 evenbetween closely related G-quadruplex structures.[6][7]Small molecules binding to G-quadruplexes have been largely based on polycyclic planar aromaticcompounds with at least one substituent terminating in a cationic group. The original rationale for the planarmoiety was that this would stack effectively onto planar G-quartets, which has been subsequently visualised ina number of crystallographic studies of G-quadruplex-ligand complexes. Structural studies are unanimous inshowing that such planar ligands stack onto a terminal G-quartet. The cationic charge requirement has led tothe dogma that these groups reside in quadruplex grooves and directly contact phosphate groups. Howeverthe crystallographic evidence to date indicates that these electrostatic contacts are rarely direct but are oftenmediated 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 duplexnucleic acids, offering considerable potential for differential molecular recognition, and thus have enabled anumber 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 andapoptosis requires that cells with a mean telomere length of 5 kb, a 24 h cell-doubling time and a subsequentloss of ∼ 100 nucleotides per round of replication would reach critical telomere shortening in ∼ 40–50 days.COMMON G-QUADRUPLEX BINDING MOLECULES: 7
  9. 9. The quadruplex-binding acridine ligands BRACO-19 and RHPS4 , in common with telomestatin, induce rapidreplicative 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 asignificant population of cells undergoing end-to-end fusions in metaphase.. Q-FISH studies have shown thattelomestatin is localized at telomeres during replication and importantly, that telomere replication isunaffected in mouse embryonic fibroblast (i.e. untransformed) cell lines .Telomestatin, a naturalproduct isolated from Streptomyces anulatus 3533-SV is one of the strongest and mostspecific inhibitors of telomerase reported to date. Telomestatin has molecular dimensions similar to those ofG-tetrad DNA and can bind to various G-quadruplexes with modestAffinity.Telomestatininduces telomere shortening in treated cells more rapidly than is expected for a singlemechanism involving telomerase inhibition.Recent studies have shown that telomere uncapping and the lossof telomeric DNA is related to the competition between telomestatin and POT1 – a shelterin protein thatbinds 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 lesstoxicity towards normal progenitor cells.[6]The cationic porphyrinTMPyP4 is the most extensively studied to date. TMPyP4 inhibits both and TaqDNApolymerase.X-ray crystallography studies have shown that TMPyP4 can bind to G-quadruplex DNA at manydifferent positions, including the terminal G-tetrads,[98] and the loops, grooves, and phosphodiesterbackbone.[99],[6]Anthraquinones and G-QuadruplexStabilisationThe first demonstration of telomerase inhibition by a G-quadruplex-interactive molecule in living cells wasusing an anthraquinone.This molecule has moderate preference for binding to quadruplex DNA(four strandstructure) over duplex DNA (two stranded structure). This is a vital characteristic in order to avoid toxicity inthe body. Experiments have also shown the moleculeIn the presence of anthraquinone, significant inhibition of more than 50% was observed for telomeres thatwere 5 or more repeat units long and thus capable of forming a quadruplex structure. For telomeres of shorterlengths, the presence of anthraquinone had little effect on the inhibition, since quadruplexes could not beformed 8
  10. 10. INSTRUMENTATION:G-quadruplex structure (i.e. inter- vsintramolecular), quantity of sample, ligand-quadruplex stoichiometry andthe type of binding information sought.G-Quadruplex ligand binding assaysto study the interactions betweenthis type of structure and it’s ligands.Selection of assay will depend on a number of factors such as the ligandsto be tested,Calorimetric techniques:ITC (Isothermal titration calorimetry) and DSC (Differential scanning calorimetry) provide means ofquantifying the thermodynamic properties and processes of G-Quadruplex ligand systems.Polymerase Chain Reaction assaysSPR (Surface plasmon resonance) assaySPR is a fast and sensitive technique useful in screening libraries of small ligands and is ideally used tocharacterise interactions between ligands and macromolecules.FRET (Fluorescence Resonance Energy Transfer) melting assayA FRET melting assay can determine the ‘affinity’ and ‘selectivity’ of ligands by measuring the increase inmelting temperature of a quadruplex induced by the linkage of ligands to G4 DnaTRE is a new assay which utilises SPR and does not require PCR amplification. 9
  11. 11. Advantages of G-quadruplex inhibitionThe 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 humanintramoleculartelomeric 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 couldimpair 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 theshelterin complex causing telomere destabilization, a possible genotoxicity associated with many G quadruplexligands. 10
  12. 12. 11
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  14. 14. ROLE OF BIOINFORMATICSQuadruplex 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 bioinformaticssurveys, which have revealed over-representations in promoter regions, especially of genes involved inreplication, 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 toolssimply take an input sequence and give output in form of region that can form quadruplex. Generally, a simplepattern 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. 13
  15. 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 statisticalsupport for the design and analysis of laboratory experimentationLigandmediatedstabilizationof G-quadruplex DNAmight facilitate theregulation of geneexpression and/or theinhibition of telomeraseactivity[6] 14
  16. 16. ECONOMICS/OUTCOMESThese molecules have facilitated highly selective gene silencing, and have found numerous applications inbasic research and medicineSmall molecules capable of structureselectiveDNA binding may provide an exciting new avenue for thedevelopment 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 ofthe most devastating and common causes of premature death). G-quadruplex-specific antibodies generated by in vitro evolution may provide key tools for targetvalidation,[75,119] while the design and synthesis of new high affinity G-quadruplex ligands will provide newdrug candidates and molecular probes. These molecules will provide researchers with new tools for studyingthe 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 abilityto 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/fullThe development of small molecules that specifically bind to a particular DNA secondary structure mayimprove 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. 15
  17. 17. 3. Cause a decrease in cell proliferationWhile more quadruplex-specific ligands are being identified, understanding of their binding is currently limitedas few structures are available. More structural studies on drug-G-quadruplex complexes are anticipated toaddress 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 seemore reports on quadruplexes formed in RNAs. 16
  18. 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.htm3http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726962/4http://molpharm.aspetjournals.org/content/61/5/1154.full.pdfhttp://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.pdfhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726962/?tool=pubmed[8]chromosomeshttp://www.ncbi.nlm.nih.gov/pubmed/11745111http://exploreable.wordpress.com/tag/replicative-senescence/http://www.ncbi.nlm.nih.gov/pubmed/18855731http://www.quadruplex.org/?view=reviews 17

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