Bioconjugate Chem. 2003, 14, 679−683
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PNA-Based RNA-Triggered Drug-Releasing System Zhaochun Ma and John-Stephen Taylor* Department of Chemistry, Washington University, St. Louis, Missouri 63130. Received January 30, 2003; Revised Manuscript Received March 26, 2003
A three-component sequence-specific RNA-triggered drug-releasing system is described that consists of an 8-mer PNA linked to a coumarin ester (the prodrug component) and a 14-mer PNA linked to histidine (the catalytic component) that are complementary to the C loop of E. coli 5S rRNA (the triggering component). Binding of the catalytic component to the RNA creates a prodrug-metabolizing enzyme that catalyzes a 60 000-fold acceleration in the rate of coumarin release from the prodrug compared to the rate of coumarin release from the ester subunit catalyzed by imidazole alone. RNAtriggered release of hydroxycoumarin is only slightly less efficient than that triggered by a short unfolded DNA sequence corresponding to the PNA binding sites. The lower efficiency results from a decrease in kcat and an increase in KM, presumably due to the bent nature of the RNA. The efficiency of DNA-triggered hydroxycoumarin release was found to depend on the distance between the catalytic and prodrug components.
INTRODUCTION
Chemotherapeutic approaches to cancer and infectious disease involve drugs that are selectively toxic to the diseased cell or the disease-causing virus or organism. Most conventional anticancer drugs work by interfering with replication and cell division and owe their marginal selectivity for cancer cells to the fact that cancer cells divide more rapidly than normal cells. Current approaches to increasing drug selectivity involve antibody-, gene-, and bacterial-directed enzymatic activation of prodrugs inside or in the vicinity of a cancer cell (1, 2). Other approaches are based on interfering with cancerspecific biochemical pathways through small molecule or antisense- or antigene-based drugs (3-5). All these methods depend on detailed knowledge of the biochemistry of the cancer cells and how they differ from that of normal cells. Recent DNA array-based analyses of cancer cells from patients having the same type of cancer have revealed variations in their individual pattern of gene expression (6, 7). Consequently, a drug that works well on a cancer in one patient may not work well on the same type of cancer in another patient. Another problem is that cancer cells can become resistant to drugs due to mutations in the proteins involved in the mechanism of action by the drug. Unfortunately, it would be very difficult if not impossible to tailor biochemistry-based chemotherapeutic agents for an individual patient or respond to a mutagenic event because of the time involved in developing and approving the appropriate drug. Recently, we proposed a new approach to the design of highly selective and patient-specific chemotherapeutic agents which in principle does not require any knowledge of the biochemistry of the diseased cell and could work against cancers, viruses, and other pathogenic organisms (8, 9). The idea is to use disease-specific genetic information (the signal) to trigger the release of a toxic agent within the diseased cell (signal transduction). One implementation of this idea is to use a unique or overexpressed * To whom correspondence should be addressed. Telephone: (314) 935-6721. Fax: (314) 935-4481. E-mail: taylor@ wuchem.wustl.edu.
mRNA specific to the disease state to template the association of a prodrug and a catalytic component which releases the drug (Figure 1). We have previously demonstrated the feasibility of such approach with DNAbased model systems in which the prodrug was constructed by attaching p-nitrophenolate to an oligodeoxynucleotide (ODN) that is complementary to one-half of a triggering ODN via an ester linkage (8, 9). Hydrolysis of the nitrophenyl ester would release nitrophenolate which has been used by others to release cytotoxic drugs such as fluorouracil and daunorubicin that have been appended to the ortho or para position via a carbamoylmethyl linkage (10-12). The catalytic component was made by attaching imidazole, a well-known catalyst for ester hydrolysis, to an ODN complementary to the other half of the targeting ODN. When incubated together, the nitrophenolate was released with an approximately 1000fold rate enhancement over that in the absence of the triggering ODN. The DNA-based system used to initially test the concept of nucleic acid-triggered drug release in vitro is not suitable for RNA-triggered drug release in vivo. Use of a DNA-based system could cause depletion of the RNA triggering sequence through DNA-mediated cleavage by RNaseH. DNA itself is also degraded by enzymatic action within a cell and would therefore not be suitable for sustained activity of the catalytic and prodrug components. What is also unknown from the previous studies is how well a folded RNA structure will serve as a trigger. Herein, we describe a peptide nucleic acid (PNA)-based drug-releasing system, which should not suffer from the problems inherent with a DNA-based system, and show that it can be triggered by a folded RNA molecule almost equally well as an unfolded DNA molecule. EXPERIMENTAL PROCEDURES
Materials and Methods. Reagents for automatic PNA synthesis were purchased from PerSeptive Biosystems. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP‚ HCl) was purchased from Pierce. ODNs were purchased from IDT (Coraville, IA) and purified by reversed-phase HPLC as triethylammonium salts. High-pressure liquid
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Figure 1. General concept of mRNA-triggered catalytic drug release. The catalyst and cytotoxic drug are attached to nucleic acids or analogues that are complementary to an exposed loop of an mRNA specific to the disease state (the signal). The recognition sequence of the catalytic component is made sufficiently long enough to specifically recognize and bind tightly to the triggering sequence and thereby transform the mRNA into a prodrug-metabolizing enzyme. The recognition sequence of the prodrug component is made shorter so that it binds reversibly, thereby facilitating turnover of the prodrug and release of multiple cytotoxic drugs (signal transduction) which then kill the cell. In this system, the rate of drug release catalyzed by the catalytic component in the absence of the RNA trigger is very low.
chromatography was carried out on a Beckman HPLC system consisting of two 110B pumps, a 406 Analogue Interface, and a 166 variable wavelength detector, under control of an IBM PS/2 and System Gold software. PNA and ODNs were quantified by spectrophotometric A260 values. 5S rRNA was purchased from Sigma and renatured before use according to a literature procedure (13). UV spectral data were acquired on a Bausch and Lomb Spectronic 1001 spectrophotometer or Varian Cary 1E UV-vis spectrophotometer. Fluorescence measurements were carried out on a SPEX Fluoromax instrument. MALDI mass spectra of oligonucleotides and PNAs were acquired on a PerSeptive Voyager RP MALDI-TOF mass spectrometer using sinapinic acid as a matrix and calibrated versus insulin (average [M + H+] ) 5734.5) that was present as an internal standard. Synthesis of the 8-mer PNA and Catalytic Component. The PNA-peptide conjugates NH2-LysCTGGGGTA-Cys-H and NH2-His-CGGCTTGAGTCCLys-H were synthesized on a 2 µmol scale by standard Fmoc off chemistry on an Applied Biosystems (Foster City, CA) Expedite 8909 Synthesizer using trityl-histidine, Boc-lysine, trityl-cysteine, and Bhoc-protected PNA building blocks and the universal support XAL-PEG-PS (Applied Biosytems) using a standard protocol. The PNAs were purified by reversed phase HPLC on a Microsorb C18 column (300 Å pore, 5 µm particle size, 4.6 × 250 mm) using 1 mL/min linear gradients of solvent B [0.1% TFA in acetonitrile] in A [0.1% TFA in water]. The effluent was monitored by absorbance at 260 nm, and the major peaks were collected, concentrated to dryness in vacuo, and analyzed by MALDI-TOF mass spectrometry. NH2-His-CGGCTTGAGTCTTC-Lys-H was purified using a 5% to 70% linear gradient of solvent B in A over 65 min to yield 180 nmol of purified product eluting at 22 min: calcd avg (M + H+) 4059.9, found 4061.5. NH2-
Ma and Taylor
Lys-CTGGGGTA-Cys-H was purified with a 5 min 5% solvent B in A, followed by a linear gradient of 5% B 35% B in A over 30 min to yield 488 nmol of purified product eluting at 22 min: avg calcd (M + H+) 2473.4, found 2469.2. Synthesis of the Prodrug Component. The maleoyl coumarin ester was linked to the 8-mer PNA according to a general procedure (14). Thus, the PNA-peptide conjugate NH2-Lys-CTGGGGTA-Cys-H (24.4 nmol) was treated with TCEP (110 nmol) in 500 µL of 0.1 M sodium phosphate buffer, pH 7.0, for 2 h at room temperature under argon. N-Maleoyl-D-valine coumarin ester (9) (293 nmol in 10 µL of acetonitrile) was then added and allowed to stir for an additional 1 h. The PNA conjugate was purified by reversed phase HPLC as described above with a linear gradient of 5% to 70% solvent B in solvent A in 65 min. The desired fraction eluting at 29 min was collected (9.7 nmol, 40% yield), concentrated to dryness in vacuo, and analyzed by MALDI-TOF mass spectrometry, calcd avg (M + H+) 2814.74, found 2812.9. Kinetics of Hydroxycoumarin Release. A fluorescence calibration curve was obtained by plotting fluorescence intensity (cps) as a function of 7-hydroxycoumarin concentration in the buffer used for the kinetic experiments (λex ) 350 nm, λem ) 452 nm). An equation relating concentration of 7-hydroxycoumarin to the observed fluorescence intensity was obtained by linear regression analysis of the calibration curve. The catalytic component (1 µM) was incubated with the trigger (1 µM complementary DNA or 5S rRNA) and a given concentration of the prodrug component and the release of 7-hydroxycoumarin was monitored by fluorescence spectroscopy for 1 h. Michaelis Menten kcat and KM parameters were obtained by measuring the rate of 7-hydroxycoumarin release as a function of prodrug concentration (0.2, 0.4, 0.8, 2.0, 5.0, and 20 µM). The initial rates of hydroxycoumarin release by linear regression and corrected for background hydrolysis. The initial rates were plotted against the prodrug concentration and fitted to the MichaelisMenten equation using KaleidaGraph software. RESULTS AND DISCUSSION
PNA has a number of properties that make it ideal for a RNA-triggered drug-releasing system: (1) it binds to RNA with higher affinity than natural ODNs and can invade duplex regions (15), (2) it can bind RNA in vivo as judged by its ability to sequence-specifically inhibit translation (16-20), (3) it has much higher biological stability than ODNs in serum and in cells (21), (4) unlike ODNs it does not stimulate RNA degradation by RNaseH upon duplex formation with RNA (22), (5) it can be efficiently delivered into cells by conjugation with protein transduction domains (PTDs) such as HIV-I TAT protein (23, 24), and (6) it can be easily synthesized together with amino acids by solid-phase peptide synthesis on commercially available DNA/PNA or peptide synthesizers (25, 26). Design and Synthesis of the PNA Components. The three-component PNA-based RNA-triggered drugreleasing system used in this study is shown in Figure 2. The RNA trigger was chosen to be the 120 nt 5S rRNA of E. coli because its folded structure has been well established by a variety of methods (27, 28) and because its C region has been previously been shown to bind an antisense ODN (29). The catalytic component consists of a 14-mer PNA complementary to the C region that is linked on its carboxy end to histidine, which bears the imidazole functionality that was successfully used to
PNA-Based RNA-Triggered Drug-Releasing System
Figure 2. PNA-based drug-releasing system using E. coli 5S rRNA and ODNs as triggering sequences. The prodrug component is in italics, whereas the catalytic component is underlined. The 25-mer ODN corresponds to the 25-mer section of the 5S rRNA containing the C-loop used as the triggering sequence. The R-25-mer ODN is the same sequence but in reverse, and the 25An-mer ODNs have n As between the binding sites for the prodrug and catalytic components. The carboxy terminal amide is indicated by NH2 and the amino terminal amino group by H (see Figure 3 for a more detailed structure).
catalyze ester hydrolysis in the ODN-based systems (8, 9). The prodrug component consists of an 8-mer PNA complementary to the adjacent site on the C region that is linked to 7-hydroxy coumarin through an ester linkage which renders the hydroxycoumarin nonfluorescent. Coumarin has been previously used to monitor the stability of esterase-sensitive prodrugs of amines, peptides, and peptidomimetics (30). Both components are linked on their other ends to lysine to increase water solubility and facilitate HPLC purification. The required PNA-peptide hybrids, NH2-His-CGGCTTGAGTCC-Lys-H and NH2Lys-CTGGGGTA-Cys-H, were synthesized by standard automated Fmoc-based PNA synthesis using tritylprotected histidine, Boc-lysine, trityl-cysteine, and Bhocprotected PNA building blocks. The prodrug component was synthesized by coupling N-maleoyl-D-valine coumarin ester (9) at neutral pH to the sulfhydryl of NH2Lys-CTGGGGTA-Cys-H by a general procedure (14). Effect of Orientation and Spacing on DNA-Triggered Hydroxycoumarin Release. We first examined the ability of a 25-mer ODN corresponding to the C loop region of 5S rRNA (nt 30-54) to trigger release of 7-hydroxycoumarin from the PNA-based system in 10 mM phosphate, 0.1 M NaCl, pH 7, at 37 °C (1 µM in all three substrates). In accord with the known orientation preference (31), coumarin was found to be released at a significantly higher rate in the presence of the antiparallel than the parallel 25-mer (taking the amino terminal end of the PNA to be equivalent to the 5′-end of DNA), which was no better than the background rate for the prodrug alone (7.1:1.0:0.8 × 10-5 µM/s respectively). The background rate is much higher than previously seen with the N-maleoyl-D-valine coumarin ester used to form the prodrug component (9) and may be due to lactam formation with the free amino group of the terminal cysteine to which it is attached (Figure 3). As expected, the rate of 7-hydroxycoumarin release also depended on the distance between prodrug and catalytic component as evidenced by the decreasing rate of release upon
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Figure 3. Synthetic scheme for the preparation of the catalytic and prodrug components.
Figure 4. Effect of spacing between the prodrug and catalytic components on 7-hydroxycoumarin release. The prodrug component (1 µM) was incubated with 1 µM of catalytic component (+His) and 1 µM of the triggering ODN in 10 mM phosphate, 0.1 M NaCl, pH 7, at 37 °C. Also shown is the effect of removing the histidine from the catalytic component (-His).
increasing the number of A's between the two binding sites (1-3 A’s: 5.7:2.0:1.3 × 10-5 µM/s respectively) (Figure 4). This decrease in rate with increasing distance contrasts with a recent publication describing distanceindependent rates of DNA-templated chemical reactions (32). RNA-Triggered Release of Hydroxycoumarin. The rate of hydroxycoumarin release from the 5S rRNA was slightly slower (4.0 × 10-5 µM/s) than that from the 25mer ODN and is intermediate between the one and two nucleotide-gapped DNA triggers (5.7 and 2.0 × 10-5 µM/ s, respectively) (Figure 5). The lower rate could be due to reduced binding affinity of the drug component for a bent RNA sequence and/or to reduced catalytic efficiency due to a change in geometry. To gain further insight into the origin of the difference in rate between the ODN- and RNA-triggered reactions, drug release was examined as
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much of the large increase in rate acceleration on going from the DNA-based system to the PNA-based system. The significantly lower KM for the PNA system may be due in part to the higher binding affinity of PNA for DNA (31) and in part to elimination of unfavorable electrostatic interactions between the catalytic and prodrug components in the DNA-based system. The RNA-triggered system has a slightly lower kcat of 6.2 ( 0.3 × 10-5 s-1 and a slightly higher KM of 0.91 ( 0.14 µM, which is consistent with the idea that both the binding affinity and catalytic efficiency are reduced because of binding to a bent RNA sequence. In this case the apparent rate acceleration is 60 000. CONCLUSIONS
Figure 5. Kinetics of RNA-triggered 7-hydroxycoumarin release. The prodrug component (1 µM) was incubated in the presence of 1 µM of the catalytic component (+His) and 1 µM of the 5S rRNA in 10 mM phosphate, 0.1 M NaCl, pH 7, at 37 °C. Hydroxycoumarin release was also measured with the catalytic component lacking the histidine (-His) and on the reversed DNA template (R25-mer).
We have shown that a folded RNA molecule is capable of triggering the release of coumarin from a PNA-based drug-releasing system with a high rate of acceleration over free imidazole that can be attributed to the high binding affinity of the PNA-based prodrug component. We also show that the rate of folded RNA-triggered drug release is only slightly less efficient than the corresponding linear DNA template and is due to a drop in kcat and a rise in KM, probably as a result of the bent nature of the RNA trigger. Unlike a previous report, we have shown that the rate of nucleic acid-templated chemical reactions are distance dependent as would be expected. We are now trying to determine whether this PNA-based drug-releasing system can be made to work inside human cells. ACKNOWLEDGMENT
Supported by NIH (RO1-CA92477) and a Wheeler Fellowship for Z. Ma. The assistance of the Washington University Mass Spectrometry Resource, a NIH Research Resource (Grant No. P41RR0954), is also acknowledged. Supporting Information Available: HPLC traces and ESI spectra of the catalytic componenet, the 8-mer PNA, and the prodrug component. This material is available free of charge via the Internet at http://pubs.acs.org/BC. LITERATURE CITED Figure 6. Michaelis-Menten behavior of RNA- and ODNtriggered hydroxycoumarin release. Initial rate of 7-hydroxycoumarin release corrected for background hydrolysis plotted against the concentration of the prodrug component in the presence of 1 µM catalytic component and 1 µM 25-mer ODN or 5S rRNA in 10 mM phosphate, 0.1 M NaCl, pH 7, at 37 °C.
a function of the concentration of the prodrug component (Figure 6). In both cases, hydroxycoumarin release showed saturation kinetics as expected for the Michaelis-Menten mechanism which had previously been shown to operate for the ODN-based drug-releasing system (8, 9). The ODN-triggered system has a kcat of 8.4 ( 0.6 × 10-5 s-1 and a KM of 0.46 ( 0.12 µM, which corresponds to an effective rate acceleration of 160 000 over free imidazole (kIm ) 1.13 ×10-3 M-1 s-1) as calculated from (kcat/KM)/kIm. The value of kcat is almost twice that of 4.4 × 10-5 s-1 previously observed for hydroxycoumarin release from a corresponding DNA-based system (9) and may be due to the more constrained nature of the linkers tethering the hydroxycoumarin ester and imidazole to the PNA. The value of KM is almost 100-times lower than that of 50 µM observed for a DNA-based 8-mer prodrug in a different sequence context and is responsible for
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