Telomerase and the benefits of healthy living

For reprint orders, please contact: reprints@futuremedicine.com † Author for correspondence: Gene Expression and Regulation Program, The Wistar Institute, 3601 Spruce St, Philadelphia, PA 19104, USA n Tel.: +1 215 495 6884 n Fax: n skorda@wistar.org Inappropriate activation of a single enzyme, telomerase, is associated with the uncontrollable proliferation of cells observed in as many as 90% of all of human cancers. Since the mid-1990s, when telomerase activity was detected in human tumors, scientists have eyed the enzyme as an ideal target for developing broadly effective anticancer drugs. One of the missing links in the effort to identify such therapies has been the high-resolution structure of the enzyme, a powerful tool used for the identification and development of clinical drugs. A recent structure of the catalytic subunit of teleomerase from the Skordalakes laboratory, a major advancement in the field of telomeres, has opened the door to the development of new, broadly effective cancer drugs, as well as anti-aging therapies. Here we present a brief description of telomerase biology, current efforts to identify telomerase function modulators and the potential importance of the telomerase structure in future drug development. Telomerase biology & human disease cancer cells and tumor suppression. Moreover, the fact that telomerase is highly active in carcinogenic cells but dormant in most healthy tissues suggests that any chemotherapy that targets telomerase could have limited side effects, thus making telomerase an ideal target for the identification and development of a cancer therapeutic [29] . Any organism with linear chromosomes faces a substantial obstacle in maintaining the terminal sequence of its DNA. This is referred to as the ‘end replication problem’ [1–10] . Eukaryotic cells overcome this problem and prevent loss of genetic information through the use of a specialized DNA polymerase, termed telomerase [11–15] . The minimal telomerase holoenzyme consists of a protein subunit (TERT) and an integral RNA component (TER), which contains the template TERT uses to add multiple, short, G-rich DNA repeats at the 3´-end of linear chromosomes [16– 20] . Telomere addition at the end of the chromosomes leads to the recruitment of a number of telomere-binding proteins that form a complex of at least six subunits, named shelterin. The shelterin complex in turn protects the chromosome ends from degradation and from being recognized as DNA strand breaks by the cell’s recombination and DNA-repair systems, which would otherwise lead to chromosome end-to-end fusion, genomic instability and senescence, and it also regulates telomerase function [21,22] . Telomerase is highly active in early stages of life and becomes dormant in most somatic cells during adulthood [21,22] . As a result, the telomere’s ability to provide genomic stability is diminished over time owing to natural loss of telomeric structure with every cell division, a process that has been associated with aging [23–25] . Cancer cells, however, regain the ability to activate the enzyme telomerase, which works tirelessly to maintain the short length of telomeres of rapidly dividing cancer cells [26–28] . Inhibiting telomerase activity in human tumors could lead to senescence of the TERT, the catalytic subunit of telomerase, is conserved among phylogenetic groups and consists of at least four highly conserved domains, three of which share similarities with the HIV reverse transcriptases (RTs), viral RNA polymerases and B-family DNA polymerases [15,30– 32] . TERT, however, is unique when compared with other reverse transcriptases and polymerases, in that it contains domains N-terminally to the polymerase domain that are essential for function. These include the far N-terminal domain (TEN), which is the least conserved among phylogenetic groups, but is required for appropriate human, yeast and ciliated protozoa telomerase activity in vitro and telomere maintenance in vivo [33,34] . The RNA-binding domain of telomerase, TRBD, is located between the TEN and the polymerase domains and, unlike the TEN domain, is highly conserved among phylogenetic groups and is essential for telomerase function both in vitro and in vivo [35] . The TRBD–TER interactions have been demonstrated to be important for the proper assembly and enzymatic activity of the holoenzyme in vitro [36] . Moreover, the interaction of the TRBD with the template boundary element (TBE; in organisms that contain this element) 10.2217/14796694.5.2.163 © 2009 Future Medicine Future Oncol. (2009) 5(2), 163–167 Telomerase structure & function Special Report Emmanuel Skordalakes† Future Oncology Telomerase structure paves the way for new cancer therapies Keywords aging n cancer n telomerase n telomeres ISSN 1479-6694 163 Special Report Skordalakes is thought to facilitate, in part, the faithful addition of multiple, identical telomeric repeats at the ends of chromosomes [19,35,37] . TERT structure & drug design Owing to the important role of telomerase in human cancer, tremendous effort has been made towards the identification of cancer therapies that target this enzyme. Most of the methods used for the inhibition of telomerase target either the protein or the RNA, the two main components of the enzyme, although indirect inhibition of telomerase by targeting substrates that regulate telomerase function has also been used to a lesser extent. For example, therapies tested for telomerase inhibition include both nucleoside as well as non-nucleoside inhibitors [38–48] . Vaccines consisting of small TERT peptides have also been used to induce an immune response against the catalytic subunit of telomerase, which is overexpressed in carcinogenic cells [49–56] . The use of oligonucleotides that target the RNA component of telomerase, TER, have also been tested, but with limited success owing to the challenge associated with substrate accessibility and drug stability [57–61] . Ribozymes, RNA molecules with catalytic activity, have been used to target TER degradation and telomerase inhibition, again with limited success owing to lack of specificity as well as suitable delivery systems [62–69] . Efforts to identify direct inhibitors of telomerase for the treatment of human cancer over the last decade have been met with limited success, in part due to the lack of structural information on the enzyme. Structural biology is a powerful method that has been used successfully for the identification and development of therapeutics for a wide range of diseases. Attempts to obtain the high-resolution structure of telomerase have been hampered by the complexity of the system, which has made it difficult to isolate the enzyme in sufficient quantities in a stable, active form for structure determination. We recently solved the 3D structure of the catalytic subunit, TERT, in the red flour beetle telomerase using x-ray crystallography – a technique that analyzes the diffraction patterns of x-rays beamed at crystals of a molecule [70] . The TERT structure revealed four distinct domains (the TRBD, fingers, palm and thumb domains), organized into a ring-like structure (FIGURE 1) . The arrangement of the TERT structure resembles the shape of a donut and is similar to that of retroviral RTs, viral RNA polymerases and B-family DNA polymerases, suggesting an evolutionary link between these enzyme families. 164 Future Oncol. (2009) 5(2) The overall organization of the TERT domains appears to facilitate elongation complex formation and has significant implications for nucleic acid binding and telomere synthesis. In particular, the arrangement of the TERT domains creates a hole in the interior of the particle whose depth, width and geometry resemble to a remarkable degree the shape of double-stranded nucleic acid. To test this hypothesis we modeled an RNA/ DNA heteroduplex in the interior of the TERT ring using the HIV RT-DNA structure [71] .The model reveals a perfect fit between the protein and the nucleic acid substrate, and positioned the 3´-end of the DNA primer at the active site of the enzyme, and the 5´-end of TER at the RNAbinding site of TRBD, thus providing evidence for the formation of a telomerase elongation complex. The TERT–RNA–DNA model is further supported by the fact that several of the motifs, identified as contact points with the nucleic acid, are formed partly by positively charged residues (mostly lysines), the side chains of which extend towards the center of the ring and are poised for direct contact with the backbone of the DNA substrate. Structural comparisons of TERT with viral HIV RTs bound to ATP [72] places the nucleotide-binding pocket of telomerase at a shallow cavity located at the interface of the fingers and palm subdomains and where the active site of the enzyme is located. The structure presents a unique opportunity in our effort to identify direct inhibitors of telomerase that target the catalytic subunit of the enzyme for the treatment of human cancer. TERT contains a number of sites required for the full activity of the enzyme. These include the RNA-binding site, a well-defined and RNA DNA-binding site 3´ 5´ DNA Palm Thumb Active site TRBD Fingers RNA-binding site Figure 1. Partial telomerase model in complex with DNA. TERT sites that can be utilized for the development of cancer therapies are shown. future science group Telomerase structure paves the way for new cancer therapies surface-exposed pocket required for the assembly of TERT with the RNA component of telomerase, TER – the active site of the enzyme required for nucleotide binding and catalysis of the phosphodiester bond formation between nucleotides that form the telomeric repeats – and several DNA-binding sites at various parts of the molecule. All of these sites are essential for telomerase function and disruption of any of them could interfere with telomere synthesis, which in turn could lead to senescence of cancer cells and tumor suppression. The structure of telomerase opens the door to the creation of new, broadly effective cancer drugs, as well as anti-aging therapies. However, the identification and development of therapies for the treatment of human disease usually requires the concerted effort from a wide range of disciplines to insure the resulting drug is effective with limited side effects, a process that can take up to several years to complete. Efforts for the identification and development of such therapies using the TERT structure as a tool are already under way. Future perspective Special Report the above issues would be a major breakthrough in our effort to combat cancer. Telomerase appears to be an appealing target for drug design because there is emerging evidence that supports the fact that the enzyme is active in almost all human tumors, but inactive in most healthy tissues. This could mean that a drug that affects telomerase would likely work against all cancers, with few side effects. The progress that has been made in the last few years in our effort to understand the role of telomerase in cancer biology and to identify compounds that target this enzyme has been impressive. A concerted effort by many academic institutions and the pharmaceutical industry has produced significant information on telomerase biology, which in turn has assisted in our search for compounds that modulate telomerase function. However, many gaps remain in our understanding of telomerase function and telomere biology and its link to human disease. Understanding the link between telomere integrity and cancer will require dissecting structure/function relationships of the proteins that act on them. Financial & competing interests disclosure Over the years, significant advances have been made in the effort to identify useful therapies for the treatment of human cancer. Despite this progress, current chemotherapies have significant drawbacks in that they have major side effects, target only specific cancers and they usually work only on patients identified with early stages of the disease. For this reason, identifying new cancer therapies that overcome any or all of The author acknowledges financial support by the Pennsylvania Department of Health and The Ellison Medical Foundation. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. Executive summary n n n n The enzyme that replicates telomeres – telomerase – has been found to be highly active in approximately 90% of human tumors when it is dormant in most healthy tissues. The important role of telomerase in human disease makes it an ideal target for the identification and development of therapies that impact on cancer and age-related diseases. Efforts to identify cancer therapeutics that target telomerase have been hampered, in part, due to the lack of structural data on the enzyme. In a major breakthrough in the telomere field, the Skordalakes laboratory recently deciphered the high-resolution structure of the catalytic subunit of telomerase. The structure presents a unique opportunity in our search for small molecules that modulate telomerase function, thus enhancing our effort to identify and develop therapies to combat human disease. chromosome ends. Nature 250(5466), 467–470 (1974). Bibliography Papers of special note have been highlighted as: n of interest nn of considerable interest 1. n 2. Blackburn EH: The molecular structure of centromeres and telomeres. Annu. Rev. Biochem. 53, 163–194 (1984). 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Special Report Affiliation n Emmanuel Skordalakes Gene Expression & Regulation Program, The Wistar Institute, 3601 Spruce St, Philadelphia, PA 19104, USA Tel.: +1 215 495 6884 skorda@wistar.org Yokoyama Y, Wan X, Shinohara A, Takahashi Y, Tamaya T: Hammerhead ribozymes to modulate telomerase activity of endometrial carcinoma cells. Hum. Cell. 14(3), 223–231 (2001). www.futuremedicine.com 167