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Bioconjugate Chem. 2002, 13, 1176−1180
Radiometal-Labeled Peptide-PNA Conjugates for Targeting bcl-2 Expression: Preparation, Characterization, and in Vitro mRNA Binding Michael R. Lewis,*,†,‡ Fang Jia,† Fabio Gallazzi,§ Yi Wang,† Jiuli Zhang,† Nalini Shenoy,§ Susan Z. Lever,§,# and Mark Hannink| Department of Veterinary Medicine and Surgery, Department of Radiology, Department of Chemistry, University of Missouri Research Reactor, and Department of Biochemistry, University of MissourisColumbia, Columbia, Missouri 65211 . Received August 6, 2002
A new antisense peptide-peptide nucleic acid (peptide-PNA) conjugate, designed for targeting bcl-2 expression, has been radiolabeled, characterized, and evaluated for bcl-2 mRNA binding in a cell-free system. A PNA complementary to the first six codons of the bcl-2 gene was synthesized by standard solid-phase Fmoc chemistry and conjugated to a new derivative of 1,4,7,10-tetraazacyclododecaneN,N′,N′′,N′′′-tetraacetic acid (DOTA) that allows macrocyclic radiometal chelates to be incorporated into any sequence position of a peptide-PNA conjugate. The DOTA-PNA conjugate was then coupled to a membrane-permeating transduction peptide, PTD-4, designed for intracellular delivery of the radiolabeled PNA. The conjugate was characterized by HPLC and ESI-MS and labeled with 111In and 90Y to high specific activities (>1000 Ci/mmol) with high radiochemical purity. Northern blot analysis showed that 90Y-PTD-4-K(DOTA)-anti-bcl-2-PNA bound specifically to as little as 50 fmol of bcl-2 mRNA, a result equivalent to that obtained with the analogous 32P-labeled DNA antisense oligonucleotide. Thus, the mRNA targeting properties of 111In- and 90Y-PTD-4-K(DOTA)-anti-bcl-2-PNA demonstrate potential for diagnostic imaging and targeted radiotherapy applications in bcl-2-positive cancers.
The processes of carcinogenesis and cancer progression often involve deregulation of proto-oncogenes that confer malignant or aggressive phenotypes to tumor cells. For example, the B-cell lymphoma/leukemia-2 (bcl-2) protooncogene (1) produces an inhibitor of apoptosis (2) that is thought to provide a survival advantage to tumor cells and has been implicated in the development of radiation (3-5) and chemoresistance (6-8). Bcl-2 is overexpressed in non-Hodgkin’s lymphoma (NHL) (9-13) and many other cancers, including breast (14), lung (15, 16), colon (17, 18), prostate (19, 20), and neuroendocrine cancers (21), malignant melanoma (22), acute myeloid leukemia (23), and multiple myeloma (24). Response of NHL patients to conventional combination chemotherapy is initially good, with 30-35% achieving long-term survival at 10-15 years follow-up (25). Unfortunately, in most cases relapse is inevitable, with transformation to a more aggressive histologic phenotype and development of resistance to radiation and/or chemotherapy. In two large cohort studies of patients with aggressive NHL receiving combination chemotherapy (26, * To whom correspondence should be addressed: Michael R. Lewis, Ph.D., Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, 379 E. Campus Drive, University of Missouri-Columbia, Columbia, MO 65211. Phone: (573) 814-6000, ext. 3703. FAX: (573) 814-6551. E-mail: LewisMic@ missouri.edu. † Department of Veterinary Medicine and Surgery. ‡ Department of Radiology. § Department of Chemistry. # University of Missouri Research Reactor. | Department of Biochemistry.
27), multivariate analyses revealed that bcl-2 overexpression was the only clinical feature that correlated strongly with high relapse rate, reduced disease-free survival, and poor overall survival. Thus bcl-2 overexpression might be considered a new independent prognostic parameter in NHL, aiding in the identification of patient risk groups. Such patients might respond better to alternative treatments, such as targeted immunotherapy, radioimmunotherapy, or antisense therapy, all of which act through mechanisms that down-regulate bcl2. Therefore, development of new bcl-2-targeted diagnostic and therapeutic agents may provide a better understanding of the role this proto-oncogene plays in disease progression and treatment response or failure. The objective of the present research was to develop bcl-2 antisense agents labeled with diagnostic and therapeutic radiometals and coupled to a peptide vector for intracellular delivery. Peptide nucleic acid (PNA), developed by Nielsen et al. (28), has great potential for antisense applications. PNA is resistant to biological degradation and binds complementary mRNA with affinity, specificity, and stability exceeding those of corresponding DNA:RNA duplexes (29). However, studies of antisense properties of PNAs have been limited by their poor cellular uptake. Recent work demonstrated that PNA uptake can be increased substantially (30, 31) by conjugation to membrane-permeating peptides such as Drosophila antennapedia(43-58) (pAntp) (32) or the HIV Tat peptide (33). More recently, a synthetic protein transduction domain, PTD-4, was shown to exhibit a 33fold increase in cellular uptake, compared to the Tat sequence (34).
10.1021/bc025591s CCC: $22.00 © 2002 American Chemical Society Published on Web 11/05/2002
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Figure 1. Structures of radiometal-labeled PTD-4-K(DOTA)anti-bcl-2-PNA (1a, 1b) and PTD-4-K(DOTA)-nonsense-PNA (2a, 2b).
We report here the preparation, characterization, and target binding of a PTD-4-antisense PNA conjugate, complementary to the first six codons of bcl-2 mRNA, and an analogous nonsense PNA conjugate, for which a BLAST database search revealed no homology with any known mammalian gene or DNA sequence. The conjugates were labeled with the diagnostic imaging radiometal 111In (T1/2 ) 67.4 h; EC 849 keV (100%); γ 173 keV (89%), 247 keV (94%)) and the therapeutic radiometal 90 Y (T1/2 ) 64.0 h; β- 2.27 MeV (100%)). To provide maximum flexibility in the preparation of radiometal-labeled peptide-PNA conjugates, a bifunctional derivative of 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA), which can be incorporated into any sequence position of a PNA or peptide by standard solid-phase coupling techniques, was synthesized. DOTA chelates a large number of radiometals, including 111In and 90Y, with extremely high in vivo stability. N-R-(9-Fluorenylmethoxycarbonyl)-N--[tris(tert-butyl)DOTA]-L-lysine (FmocK(DOTA)), was synthesized by activation of DOTAtris(tert-butyl ester) (Macrocyclics, Dallas, TX) with 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate (HATU, Applied Biosystems, Foster City, CA) and 1-hydroxy-7-azabenzotriazole (HOAt, Applied Biosystems) in 1-methyl-2-pyrrolidinone and reaction of the resulting active ester with N-R-Fmoc-Llysine (Advanced ChemTech, Louisville, KY). The compound was purified by solvent extraction using ethyl acetate and water, followed by concentration of the organic phase in vacuo and precipitation of the product with cold diethyl ether. Alternatively, Fmoc-K(DOTA) could be purified by semipreparative reversed-phase HPLC. Analytical reversed-phase HPLC, electrospray ionization mass spectrometry (ESI-MS), high-resolution 1 H NMR, and elemental analysis of Fmoc-K(DOTA) were consistent with the structure of the desired product. Bcl-2 antisense and nonsense PNAs (Figure 1) were synthesized manually on XAL PEG PS resin (Applied Biosystems), using Fmoc-protected PNA monomers (B ) T-OH, A(Bhoc)-OH, C(Bhoc)-OH, and G(Bhoc)-OH, Applied Biosystems) and standard solid-phase coupling techniques, followed by manual coupling of FmocK(DOTA) and Fmoc-glycine as a small spacer between the PNA conjugate and PTD-4. Then the fully protected PTD-4 sequence was added to Fmoc-G-K(DOTA)-anti-
Bioconjugate Chem., Vol. 13, No. 6, 2002 1177
Figure 2. Size exclusion HPLC of 1 (top) and 1a (bottom). The peak eluting at 10.6 min retention time corresponds to an apparent molecular weight of 5.94 kDa. Table 1.
111In
and
90Y
conjugate
radiometal
3 1 1
111In
a
111In 90Y
Labeling of Conjugates 1 and 3. labeling ratio (µCi/µg)
incorporation (%)a
specific activity (Ci/mmol)
199 201 820
98.77 ( 1.41 96.62 ( 0.29 98.61 ( 2.25
1068 1300 5397
n ) 3.
bcl-2-PNA using an automated peptide synthesizer. The PTD-4-K(DOTA)-anti-bcl-2-PNA conjugate (1) was cleaved and deprotected with trifluoroacetic acid (TFA) containing 2.5% each of triisopropylsilane, m-cresol, thioanisole, ethanedithiol, phenol, and H2O for 4 h at room temperature. Unfortunately, the fully deprotected conjugate coeluted with unmodified PNA at the solvent front when reversed-phase HPLC purification was attempted. Therefore, slightly deprotected PTD-4-K(DOTA)-anti-bcl-2-PNA was cleaved from the resin with 25% TFA, 5% triisopropylsilane, 5% m-cresol, and 5% H2O in dichloromethane for two cycles of 15 min at room temperature. Two major slightly deprotected intermediates were purified by semipreparative reversed-phase HPLC. These intermediates were collected separately and fully deprotected as described above, after which 1 was isolated by precipitation with cold diethyl ether and characterized by ESI-MS. Observed molecular ions at m/z ) 2190.0 ((M + 3H)3+), 1642.5 ((M + 4H)4+), 1314.2 ((M + 5H)5+), 1095.1 ((M + 6H)6+), and 938.9 ((M + 7H)7+) were consistent with the mass of the desired product, 6568 Da. PTD-4-K(DOTA)-nonsense-PNA (2) was synthesized and purified using the same procedure, followed by ESI-MS analysis. Molecular ions observed at m/z ) 2211.5 ((M + 3H)3+), 1659.1 ((M + 4H)4+), 1327.2 ((M + 5H)5+), 1106.4 ((M + 6H)6+), 948.5 ((M + 7H)7+), 830.2 ((M + 8H)8+), and 737.8 ((M + 9H)9+) were consistent the mass of the desired product, 6629 Da. Synthesis of PTD4-K(DOTA)-PNA conjugates has been described in brief (35a) and will be reported in detail subsequently (35b). Conjugates 1 and 2 were labeled with 111In (1a, 2a) and 90Y (1b, 2b) in 0.2 M ammonium acetate, pH 5.0, containing approximately 1 mg/mL of gentisic acid, for 30 min at 90 °C. The antisense conjugate lacking PTD4, K(DOTA)-anti-bcl-2-PNA (3) was labeled with 111In (3a) using the same conditions. Diethylenetriaminepentaacetic acid (DTPA) was added to a final concentration of 1 mM, and after 5 min at room temperature the
1178 Bioconjugate Chem., Vol. 13, No. 6, 2002
Lewis et al.
Figure 3. (Top) Northern blot analysis of bcl-2 mRNA (left) and luciferase mRNA (right), using 1b. (Bottom) Northern blot analysis of bcl-2 mRNA (left) and luciferase mRNA (right), using 32P-anti-bcl-2-DNA.
Figure 4. Northern blot analysis of bcl-2 mRNA (left) and luciferase mRNA (right), using 2b.
reaction mixtures were analyzed by normal-phase TLC and size exclusion HPLC. Both methods showed that 111 In and 90Y incorporation into 1 was nearly quantitative at specific activities >1000 Ci/mmol (Table 1), as was 111In labeling of 3. These specific activities are considerably higher than those employed previously for in vivo imaging of c-myc mRNA with 111In-labeled DNA oligonucleotides (36) and are also in the range proposed to image mRNA concentrations as low as 1 pM (37). Conjugate 2 was also labeled efficiently with 111In and 90Y by the same procedure. Analysis of 1 by size exclusion HPLC (Figure 2) showed that the conjugate had an apparent molecular weight of 5.94 kDa, determined from a calibration curve generated using a Bio-Rad (Hercules, CA) molecular weight standard. This value, lower than the calculated molecular weight, is consistent with the size of an R helix-random coil peptide-PNA conjugate (38, 39). Size exclusion HPLC of 1a (Figure 2) revealed that the radiochemical purity of the compound was 100%. The kinetic stability of 1a was evaluated by size exclusion HPLC during incubation in mouse plasma at 37 °C for 168 h (data not shown). No evidence of instability of the 111In-K(DOTA) chelate was seen, and no changes in the chromatographic profile of 1a were observed for 24 h. At later time points, a reproducible increase of approximately 0.1 min in the retention time of the 111In-labeled conjugate and peak tailing suggested that some proteolytic degradation of PTD-4 may have occurred. However, the retro-inverso counterpart of pAntp displays equivalent transduction activity and is much more stable to proteolysis (30). Therefore, we intend to synthesize the retro-inverso-PTD-4-K(DOTA)-anti-bcl2-PNA analogue and evaluate it in future in vitro and in vivo studies. A pcDNA3-hygro vector (Invitrogen, Carlsbad, CA) containing the open reading frame of human bcl-2 was constructed for mRNA synthesis. Bcl-2 and luciferase mRNAs were prepared by in vitro transcription, subjected to electrophoresis through a 1% agarose gel containing 2.2 M formaldehyde (40), and immobilized on nylon membranes by capillary transfer (41). RNA transfer was determined qualitatively to be complete by ethidium bromide staining of the gel before and after transfer and by methylene blue staining of the membrane. Then 50µCi aliquots of 1b and 2b (5458 Ci/mmol) were incubated with membranes in hybridization solution (CLONTECH, Palo Alto, CA), containing 5% nonfat dry milk, for 1 h at
63.2 °C, after which the membranes were washed according to the manufacturer’s instructions. Autoradiography of membranes for 1 h (Figure 3, top) showed that 1b bound specifically to as little as 5 ng (50 fmol) of the bcl-2 transcript, while no binding to luciferase mRNA was observed. The analogous anti-bcl-2 DNA oligonucleotide was end-labeled to a specific activity of 6000 Ci/mmol, using γ-[32P]ATP and T4 polynucleotide kinase, and 50 µCi of 32P-anti-bcl-2-DNA was hybridized with the immobilized mRNAs under the same conditions, except that nonfat dry milk was not added. Autoradiography with 32 P-labeled DNA was carried out for 5.25 h, such that the film was exposed to the same number of radioactive decays as for 90Y-labeled PNA. As shown in Figure 3 (bottom), 32P-anti-bcl-2-DNA gave equivalent results to those obtained with 1b. Analogous control experiments using 2b showed no binding to either transcript (Figure 4). Thus, the 90Y-labeled antisense peptide-PNA conjugate was qualitatively equal to 32P-labeled antisense DNA in terms of bcl-2 mRNA detection sensitivity and specificity. However, Northern blot analysis at elevated temperatures demonstrated that 1b bound to bcl-2 mRNA with greater thermodynamic stability than 32P-anti-bcl2-DNA (see Supporting Information). Synthetic bcl-2 and luciferase mRNAs were also spiked into a total RNA preparation from 293 human embryonic kidney cells, and the resulting mixtures were analyzed by Northern blotting with 1b after agarose gel electrophoresis. While some nonspecific binding to cellular RNA was seen (Figure 5), 10 ng (100 fmol) of bcl-2 mRNA was detected clearly in the presence of a large excess of cellular RNA, and no binding to luciferase mRNA was observed. In conclusion, a new bcl-2 antisense PNA conjugate, coupled to a membrane-permeating peptide for intracellular delivery, was prepared and labeled to high specific activities with the diagnostic imaging radiometal 111In and the therapeutic radiometal 90Y. Hybridization experiments demonstrated that 1b was able to bind to immobilized bcl-2 mRNA with high sensitivity and specificity equivalent to 32P-labeled antisense DNA, yet with greater thermodynamic stability. We are currently evaluating the bcl-2 targeting properties of 1a and 1b in cultured NHL cells and tumor-bearing animal models. In preliminary studies, uptake of 1a in Raji cells, which express high levels of bcl-2 mRNA (42), increased significantly (p < 0.05) from 6.53% of the total radioactivity added after 30 min at 37 °C to 8.04% at 4 h, while initial
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Figure 5. Northern blot analysis of total cellular RNA from 293 cells, using 1b. Lane 1: total RNA (10 µg); lane 2: total RNA (10 µg), spiked with 10 ng of synthetic bcl-2 mRNA; lane 3: total RNA (10 µg), spiked with 10 ng of luciferase mRNA. The arrow indicates the bcl-2 transcript, determined to be equal to the theoretical molecular size of 0.754 kb by ethidium bromide staining and comparison with a DNA molecular weight standard (1kb DNA Ladder, Promega, Madison, WI).
uptake of 3a was approximately 13-fold lower. In U937 cells, which express low levels of bcl-2 mRNA (43), uptake of 1a decreased significantly (p < 0.05) from 4.05% at 30 min to 2.07% at 4 h. At all time points, uptake of 1a was significantly greater (p < 0.05) in Raji cells than in U937 cells. We are currently developing Raji- and U937-bearing mouse models of NHL to evaluate the tumor targeting properties of 111In- and 90Y-retro-inverso-PTD-4-K(DOTA)-anti-bcl-2-PNA. ACKNOWLEDGMENT
This work was supported by NIH Grant CA86290 (W. A. Volkert, PI) and a grant from the University of Missouri Research Reactor Research Partnerships Initiative (to M.R.L.). The authors would like to acknowledge the support of the Department of Veterans Affairs and the University of Missouri Research Reactor for purchasing an Advanced ChemTech ACT Model 396 Omega Multiple Biomolecular Synthesizer used in this research. Supporting Information Available: Experimental details on purification, radiometal labeling, and analytical characterization of PTD-4-PNA conjugates, subcloning of bcl-2, and mRNA binding assays. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Tsujimoto, Y., Finger, L. R., Yunis, J., Nowell, P. C., and Croce, C. M. (1984) Cloning of the Chromosome Breakpoint of Neoplastic B Cells with the t(14; 18) Chromosome Translocation. Science 226, 1097-1099. (2) Hockenbery, D., Nun˜ez, G., Milliman, C., Schreiber, R. D., and Korsmeyer, S. J. (1990) Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348, 334-336. (3) Sentman, C. L., Shutter, J. R., Hockenbery, D., Kanagawa, O., and Korsmeyer, S. J. (1991) bcl-2 Inhibits Multiple Forms of Apoptosis but Not Negative Selection in Thymocytes. Cell 67, 879-888. (4) Strasser, A., Harris, A. W., and Cory, S. (1991) bcl-2 Transgene Inhibits T Cell Death and Perturbs Thymic SelfCensorship. Cell 67, 889-899.
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Lewis et al. (34) Ho, A., Schwarze, S. R., Mermelstein, S. J., Waksman, G., and Dowdy, S. F. (2001) Synthetic Protein Transduction Domains: Enhanced Transduction Potential in Vitro and in Vivo. Cancer Res. 61, 474-477. (35) (a) Gallazzi, F., Wang, Y., Jia, F., Shenoy, N., Lever, S. Z., and Lewis, M. R. (2002) Preparation of a radiolabeled peptide-PNA conjugate for imaging oncogene expression. 27th European Peptide Symposium, Sorrento, Italy, Abstract P E21. (b) Gallazzi, F., Wang, Y., Shenoy, N., Lever, S. Z., and Lewis, M. R., manuscript in preparation. (36) Dewanjee, M. K., Ghafouripour, A. K., Kapadvanjwala, M., Dewanjee, S., Serafini, A. N., Lopez, D. M., and Sfakianakis, G. N. (1994) Noninvasive Imaging of c-myc Oncogene Messenger RNA with Indium-111-Antisense Probes in a Mammary Tumor-Bearing Mouse Model. J. Nucl. Med. 35, 10541063. (37) Dewanjee, M. K., Haider, N., and Narula, J. (1999) Imaging with radiolabeled antisense oligonucleotides for the detection of intracellular messenger RNA and cardiovascular disease. J. Nucl. Cardiol. 6, 345-356. (38) Snyder, L. R., and Kirkland, J. J. (1979) Size-Exclusion Chromatography. Introduction to Modern Liquid Chromatography, First Edition, pp 483-540, John Wiley & Sons, Inc., New York. (39) Atkins, P. W. (1982) The structures and properties of macromolecules. Physical Chemistry, Second Edition, p 825, W. H. Freeman and Company, San Francisco. (40) Sambrook, J., and Russell, D. W. (2001) Separation of RNA According to Size: Electrophoresis of RNA through Agarose Gels Containing Formaldehyde. Molecular Cloning: A Laboratory Manual, 3rd ed., Vol. 2, pp 7.31-7.34, Cold Spring Harbor, New York. (41) Sambrook, J., and Russell, D. W. (2001) Transfer and Fixation of Denatured RNA to Membranes. Molecular Cloning: A Laboratory Manual, 3rd ed., Vol. 2, pp 7.35-7.41, Cold Spring Harbor, New York. (42) Graninger, W. B., Seto, M., Boutain, B., Goldman, P., and Korsmeyer, S. J. (1987) Expression of Bcl-2 and Bcl-2-Ig Fusion Transcripts in Normal and Neoplastic Cells. J. Clin. Invest. 80, 1512-1515. (43) Seto, M., Jaeger, U., Hockett, R. D., Graninger, W., Bennett, S., Goldman, P., and Korsmeyer, S. J. (1988) Alternative promoters and exons, somatic mutation and deregulation of the Bcl-2-Ig fusion gene in lymphoma. EMBO J. 7, 123-131.
BC025591S
