[CANCER RESEARCH 59, 639 – 644, February 1, 1999] Effects of Cationic Porphyrins as G-Quadruplex Interactive Agents in Human Tumor Cells 1 Elzbieta Izbicka, 2 Richard T. Wheelhouse, 3 Eric Raymond, Karen K. Davidson, Richard A. Lawrence, Daekyu Sun, Bradford E. Windle, Laurence H. Hurley, and Daniel D. Von Hoff Institute for Drug Development, San Antonio, Texas 78245 [E. I., E. R., D. S., R. A. L., B. E. W., D. D. V. H.]; University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229 [K. K. D]; and Drug Dynamics Institute, College of Pharmacy, The University of Texas, Austin, Texas 78712 [R. T. W., L. H. H.] ABSTRACT A series of cationic porphyrins has been identified as G-quadruplex interactive agents (QIAs) that stabilize telomeric G-quadruplex DNA and thereby inhibit human telomerase; 50% inhibition of telomerase activity was achieved in HeLa cell-free extract at porphyrin concentrations in the range <50 M. Cytotoxicity of the porphyrins in vitro was assessed in normal human cells (fibroblast and breast) and human tumor cells rep- resenting models selected for high telomerase activity and short telomeres (breast carcinoma, prostate, and lymphoma). In general, the cytotoxicity (EC 50 , effective concentration for 50% inhibition of cell proliferation) against normal and tumor cells was >50 M. The porphyrins were readily absorbed into tumor cell nuclei in culture. Inhibition of telomerase activity in MCF7 cells by subcytotoxic concentrations of TMPyP4 showed time and concentration dependence at 1–100 M TMPyP4 over 15 days in culture (10 population doubling times). The inhibition of telomerase ac- tivity was paralleled by a cell growth arrest in G 2 -M. These results suggest that relevant biological effects of porphyrins can be achieved at concen- trations that do not have general cytotoxic effects on cells. Moreover, the data support the concept that a rational, structure-based approach is possible to design novel telomere-interactive agents with application to a selective and specific anticancer therapy. INTRODUCTION Significant levels of telomerase activity have been detected in 85% of tumors (1). Telomerase is also present in stem and germ-line cells of normal tissues, albeit at much lower levels (2). Thus, telom- erase presents a target with potentially good selectivity for tumor over healthy tissue, and telomerase inhibition has been proposed as a new approach to cancer therapy (2– 4). The structure of the human telom- erase protein remained elusive until recently and has been shown to be closely related to other reverse transcriptases (5–7). It has been possible to inhibit telomerase activity either by antisense strategies directed toward the telomerase RNA template, for example peptide nucleic acids (8) and phosphorothioate oligonucleotides (9), or by using inhibitors of reverse transcriptases [e.g., established agents such as, 3'-azido-3'-deoxythymidine (10) and other nucleosides (11)]. In- hibition by cisplatin, possibly due to cross-linking of the telomeric repeat sequences, has also been reported (12). A novel approach toward achieving the net inhibition of telomerase is to target its substrate, the telomere. We used a rational, structure- based approach to the design of telomere interactive agents by con- sidering unique nucleic acid secondary structures associated with the telomerase reaction cycle. One such structure is the G-quadruplex formed by folding of the single-stranded G-rich overhang produced by telomerase activity. The template region of the telomerase RNA has only 1.5 copies of the complementary sequence (3'-CAAUC- CCAAUC-5'), so after each extension the end of the DNA must be translocated back to the beginning of the coding region before the next extension (13). Work by Zahler et al. (14) has shown that potassium ions stabilize the quadruplex and that high concentrations of potas- sium inhibit telomerase. Furthermore, we (15) have shown that there is an equilibrium between the DNA:RNA heteroduplex and the G- quadruplex that lies in favor of G-quadruplex formation. These two observations point to the involvement of G-quadruplex formation in dissociating the primer from the telomerase RNA template and pos- sibly providing the driving force for the translocation reaction. This led us to hypothesize that the G-quadruplex would indeed be a viable target for drug design, as first suggested by Blackburn (13), and thus, we have undertaken a study of QIAs. 4 The long-term goal of our studies is to identify an effective QIA (with significant concentration differences between telomerase inhi- bition and the cytotoxic effects), and bring it to clinical trial. Herein we report the inhibition of telomerase by TMPyP4, the related tetra- quinolyl porphine QP3, and metal complexes thereof. The cytotoxic- ity and cellular uptake of this family of porphyrins have been exam- ined in a series of human tumor and normal cell lines. We have demonstrated rapid repression of telomerase activity and cell growth arrest in intact tumor cells by subtoxic concentrations of TMPyP4. This finding suggests that the use of QIAs to directly target telomeres may be a possible therapeutic strategy. Three tumor models (breast, prostate, and lymphoma) are relevant to the future clinical develop- ment of telomerase inhibitors. The low cytotoxicity and inhibition of telomerase at low micromolar concentrations combine to make the cationic porphyrins attractive candidates for anticancer drug develop- ment. MATERIALS AND METHODS Molecular Modeling. Models were built using the Sybyl package (Tripos, Inc., St. Louis, MO). Coordinates for the DNA quadruplex (16) and TMPyP4 (15) were obtained from the Brookhaven Protein Data Bank. Hydrogen bond- ing constraints were added to the G-tetrads, and torsional constraints were set to maintain the planarity of the porphyrins. Porphyrins were inserted above and below the G-tetrads, and the complex was allowed to minimize using Kollman charges, Tripos force field, and conjugate gradient. After 100 iterations, the porphyrins were replaced, and the minimization was repeated for 500 iterations to a terminal gradient of 0.05 kcal/mol. Chemicals and Cell Lines. All porphyrins were obtained from Midcentury (Posen, IL). The experimental work with porphyrins was performed under minimum exposure to light. All human tumor cell lines and normal human breast cells Hs578Bst were purchased from the American Type Culture Col- lection. Normal human lung fibroblasts were obtained from Clonetics Corp. The cell lines were grown according to suppliers’ instructions. Received 8/6/98; accepted 12/2/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Cooperative Drug Discovery Group Grant CA67760 from the National Cancer Institute. Preliminary reports of this work were presented at the 88th Annual Meeting of the American Association for Cancer Research, San Diego, CA, in Proceedings of The American Association for Cancer Research, 38: 637, 1997. 2 To whom requests for reprints should be addressed, at the Institute for Drug Development, 7979 Wurzbach Road, Suite 337, San Antonio, TX 78229. Phone: (210) 616-5892; Fax (210) 616-5948; E-mail eizbicka@saci.org. 3 Present address: School of Pharmacy, The University of Bradford, Bradford, West Yorkshire BD7 1DP, United Kingdom. 4 The abbreviations used are: QIA, G-quadruplex interactive agent; TMPyP4, 5,10,15,20-tetra(N-methyl-4-pyridyl)porphine chloride; QP3, 5,10,15,20-tetra(N-methyl- 3-quinolyl)porphine chloride; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. 639 on July 18, 2015. © 1999 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from