Southwestern In Vivo Cellular and Molecular Imaging Program

 


 

 

Project 6
In Vivo Assessment of Anti-telomerase Peptide Nucleic Acids as a Novel Cancer Therapeutic Strategy

Investigators and Areas of Expertise:

David R. Corey, Ph.D., chemist with unequaled expertise in PNA chemistry, will synthesize all PNAs. Jerry W. Shay Ph.D., a leader in understanding telomerase and telomere biology will design protocols for testing anti-telomerase PNAs in animal tumor models. Orhan K. Öz, M.D., Ph.D., a radiologist and biophysicist specializing in the application of nuclear imaging in humans and animal models, will coordinate and interpret imaging studies. Anca Constantinescu will radiolabel the PNAs and perform animal investigations.

Hypothesis and Specific Aims:

Hypothesis: Application of imaging techniques will provide information that will improve the ability of peptide nucleic acids (PNAs) to inhibit telomerase in tumors, enhancing the ability of PNAs to block cancer cell proliferation and validating telomerase as a novel target for chemotherapy.

Specific Aims: We have demonstrated that PNAs that target telomerase cause telomere shortening and block cancer cell proliferation in vitro. We are now testing the ability of anti-telomerase PNAs to block cancer cell proliferation in vivo in a nude mouse model of human cancer, but our efforts are limited by our lack of knowledge of tissue distribution of PNAs and their uptake by tumors. Our goal is to use imaging techniques as part of a design cycle to improve the potency of anti-telomerase PNAs.

Background and Significance:

Telomerase activity has been found in most types of human tumors, but not in most adjacent normal cells. This correlation has led to the hypothesis that reactivation of telomerase is necessary for the sustained cell proliferation that characterizes cancer, and that telomerase is a promising target for a class of chemotherapeutic agents that act by a novel mechanism. Supporting this hypothesis is the observation that early stage neuroblastomas have low telomerase activity correlating with a favorable outcome, while late stage disease exhibits high telomerase activity and a poor outcome. A similar correlation between telomerase activity and poor clinical outcome has been reported for ordinary meningioma and other studies have suggested that telomerase activity is correlated with pathologic stage or tumor aggressiveness.

 Chemical structure of PNA (Figure)

To test the link between telomerase and cancer cell proliferation we have developed PNAs as telomerase inhibitors. PNAs are DNA analogs in which the ribose-phosphodiester backbone has been replaced by N-(2-aminoethyl) glycine linkages. A methylene carbonyl linker connects standard nucleotide bases to this backbone at the amino nitrogens. PNA synthesis uses standard Boc or Fmoc solid phase protocols. This ability to make PNAs easily will facilitate the labeling of PNAs with a wide array of metal chelators and tumor targeting moieties. PNAs bind to complementary sequences with high affinity binding and are resistant to degradation by nucleases and proteases.

Strengths of PNAs as inhibitors for human telomerase:

1)  Synthesis uses standard protocols derived from peptide synthesis
2)  PNAs can be readily modified at N or C termini
3)  Achiral backbone allows synthesis of homogeneous product
4)  Increased affinity for complementary sequences
5)  Increased rates of association to complementary sequences
6)  Low propensity for artifactual binding to proteins that normally bind polyanions
7)  Resistance to degradation by nucleases and proteases
8)  High mismatch discrimination
9)  The telomerase RNA template is highly accessible to PNA binding

Telomerase is an outstanding target for PNAs because it possesses an RNA domain and must be accessible to telomere binding. Thus, telomerase is highly accessible to hybridization by PNAs and we have shown that PNAs inhibit telomerase with IC50 values as low as 1 nM. We have already demonstrated that anti-telomerase PNAs can reduce the proliferation of cultured tumor cells and cause telomeres to shorten. Thus, it is clear that telomerase is a promising and novel target for chemotherapy, that it can be inhibited PNAs, and that inhibition causes cancer cell death. The challenge for this therapeutic approach is to assess telomerase inhibition in animals: efficient non-invasive imaging techniques will help us achieve this goal.

Hnatowich and coworkers demonstrated that 99mTc-labeled PNAs were able to localize to DNA oligonucleotides immobilized on beads and implanted subcutaneously in mice. They further reported that PNAs were stable, non toxic, and had favorable properties for eventual use as radiopharmaceuticals. In our initial studies, we have confirmed that our anti-telomerase PNAs are nontoxic when administered at doses as high as 20 mg/kg, over three times the therapeutic dosage of 6 mg/kg normally used for oligonucleotides. There is no information available, however, on the uptake of PNAs by tumors or their in vivo efficacy, preventing thoughtful evaluation of their potential as therapeutic leads. The research described in this proposal is significant because it will systematically examine the uptake of PNAs by tumor and other tissues, and use this knowledge to optimize the inhibition of telomerase by PNAs. The information gained by these studies will be widely useful for development of PNAs as general agents for controlling gene expression and for determining whether PNAs can act as effective anti-telomerase agents in vivo.

Scientific opportunities provided by collaboration. The neutral peptide-based chemistry of PNAs represents a dramatic departure from the chemistry of standard negatively charged oligonucleotides. There is a high probability that in vivo studies will produce novel and unanticipated results that might provide a breakthrough improvement in efficacy and pharmacokinetics. The collaboration presents a unique scientific opportunity to bring together diverse researchers in the fields of (i) oligomer synthesis and design (Corey), (ii) animal testing and telomere biology (Shay), and (iii) synthesis of radiolabeled compounds and in vivo animal testing and imaging (Oz). Alone, none of these labs could effectively pursue the aims of this proposal, but by combining their expertise, a design and development cycle will allow rapid refinement of oligomer properties leading to optimized tissue distribution and in vivo efficacy. Exciting scientific opportunities will also arise from interactions with the Kodadek laboratory's use of phage display to develop tumor targeting peptides.

 

References:

1. Corey, DR. Peptide Nucleic Acids - Expanding the options for nucleic acid recognition. Trends in Biotechnology 1997, 15, 224-229.

2. Herbert, B-S, Pitts AE, Baker SI, Hamilton SE, Wright WE, Shay JW, Corey DR. Inhibition of telomerase leads to eroded telomeres, reduced proliferation, and cell death. Proc. Natl. Acad. Sci. USA 1999, 96, 14726-14781.

3. Mayfield, LD, Corey DR. Automated synthesis of peptide nucleic acids (PNAs) and peptide nucleic acid-peptide conjugates. Anal. Bioch. 1999, 268, 401-404.

4. Norton, JC, Waggenspack JH, Varnum E, Corey DR. Targeting Peptide Nucleic Acid Protein Conjugates to Structural Features Within Duplex DNA. Bioorg. Med. Chem. 1995, 3, 437-445.

5. Norton, JC, Piatyszek MA, Wright WE, Shay JW, Corey DR. Inhibition of Human Telomerase Activity by Peptide Nucleic Acids. Nature Biotech 1996, 14, 615-620.

6. Shammas, MA, Simmons CG, Corey DR, Shmookler-Reis RJ. Telomerase Inhibition by Peptide Nucleic Acids Reverses "Immortality" of Transformed Cells. Oncogene 1999, 18, 6191-6200.

7. Smulevitch, SV, Simmons CG, Norton JC, Wise TW, Corey DR. Enhanced Strand Invasion by Oligonucleotides through Manipulation of Backbone Charge. Nature Biotechnol. 1996, 14, 1700-1705.

 


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