Developing a Peptide-Based Near-Infrared Molecular Probe for

its target, MMP-7, the fluorescence signal of the designed probe was increased by 7-fold with a Kcat/. Km of 100 000 M-1 s-1. The described synthetic ...
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Bioconjugate Chem. 2004, 15, 1403−1407

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Developing a Peptide-Based Near-Infrared Molecular Probe for Protease Sensing Wellington Pham, Yongdoo Choi, Ralph Weissleder, and Ching-Hsuan Tung* Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129. Received March 25, 2004; Revised Manuscript Received May 3, 2004

Recently near-infrared (NIR) molecular probes have become important reporter molecules for a number of types of in vivo biomedical imaging. A peptide-based NIR fluorescence probe consisting of a NIR fluorescence emitter (Cy5.5), a NIR fluorescence absorber (NIRQ820), and a protease selective peptide sequence was designed to sense protease activity. Using a MMP-7 model, we showed that NIRQ820 efficiently absorbs the emission energy of Cy5.5 resulting in a low initial signal. Upon reacting with its target, MMP-7, the fluorescence signal of the designed probe was increased by 7-fold with a Kcat/ Km of 100 000 M-1 s-1. The described synthetic strategy should have wide application for other NIR probe preparations.

INTRODUCTION

To identify promising drug candidates, reliable assays, which could truly report drug efficacy, are critical to initial screening and further evaluation. Many proteolytic assays have been reported and applied in vitro, but direct imaging of the therapeutic effect of drugs in vivo was not available until recently (1). A matrix metalloproteinase (MMP)-activatable near-infrared (NIR) fluorescence probe was developed to report inhibitor effect on proteolytic activity in animals (1). On each probe molecule, multiple NIR fluorochromes were anchored, via an MMP-selective peptide spacer. This probe is optically silent in its native (quenched) state and becomes highly fluorescent after the proteolysis of MMP substrate peptide linkers by the enzyme. Although this approach works well, it is reasonable to think of a simpler and more defined probe for in vivo assay. For detecting enzymatic activity in vivo and for the imaging of deeper tissues in vivo, the use of near-infrared (NIR) light is required because hemoglobin and water, which are the major absorbers of visible and infrared light, respectively, have their lowest absorption coefficient in the NIR region around 650-900 nm (2). Although many fluorescence enzyme substrates have been reported and widely used, for example, coumarin, p-nitrophenol, and other analogues, most of these fluorochromes are in the visible range. The challenge of moving from visible into NIR window is to find a good matching pair of fluorescence donor and acceptor. The underlying principle of designing a NIR probe relies on fluorescence resonance energy transfer (FRET) between a fluorescent donor and a fluorescent or a nonfluorescent acceptor molecule linked via a protease substrate peptide linker. We reported this type of design previously for caspase-3 enzyme detection, using a synthesized nonfluorescence azulene quencher (abs ) 750 nm) pairing with a commercial available fluorochrome (em ) 700) (3). More than * To whom correspondence should be addressed. Ching H. Tung, Ph.D., Center for Molecular Imaging Research, Massachusetts General Hospital, 149 13th St., Rm. 5406, Charlestown, MA 02129. (tel): (617) 726-5779. (fax): (617) 726-5708. e-mail: [email protected].

4-fold increase in fluorescence signal was observed when treated with its target enzyme. Lately, we found that a previously synthesized NIR fluorochrome, NIRQ820 (4), served as a better acceptor for the fluorochromes with emission around 700 nm. MMPs are known to be important in normal tissue remodeling, but also known to play critical roles in many diseases, such as lung diseases, atherosclerosis, and cancer (5-7). Elevated level of MMPs have been found in tumors and shown to correlate well with their invasive and metastatic profiles in both experimental cancer models and in human malignancies (8, 9). The presence of extracellular and membrane-bound MMPs in tumors not only aids the degradation of extracellular matrix by neoplastic cells, but also facilitates their motility and directs cell invasion (10). Thus, MMPs have long been of interest as pharmaceutical targets (11-13). Among those MMPs, MMP-7 has been an important target, because it was overexpressed in pancreatic, colon, and breast cancers (14, 15), In this report, we describe the design of a new type of well-defined low molecular weight peptidebased NIR fluorescence probe, designed primarily for sensing of tumor-associated MMP-7 activity. EXPERIMENTAL PROCEDURES

Unless otherwise stated, reagents and solvents were obtained from commercial sources without further purification. All moisture or air-sensitive reactions were carried out under a static argon atmosphere. Standard Fmoc-amino acids, N-hydroxybenzotriazole (HOBt), and 2-(1H-benzotriazole 1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (Applied Biosystem, Foster City, CA), Rink amide resin (100-200 mesh) (Novabiochem, San Dieago, CA), Cy5.5 mono maleimide (Amersham, Piscataway, NJ), and active human recombinant MMP-9 and MMP-7 enzymes (Calbiochem, San Diego, CA) were used. Reverse phase HPLC purification was performed on a Hitachi Model D-7000 incorporated with Diode Array Detector L-7455 using Vydac 218TP1010 C18 column (Hesperia, CA) at a flow rate of 4.0 mL/min. The elution gradient was set from 0 to 50% of buffer B (10% of A in acetonitrile) in 45 min where buffer A was 0.1% TFA in

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deionized water. Detection was monitored from 200 to 800 nm range and MALDI-TOF mass spectra were determined (Tufts Core Mass Spectrometry Facility, Boston, MA). Absorption and emission spectra were determined using Hitachi U-3000 Spectrophotometer and F-4500 Fluorescence Spectrophotometer, respectively. Probe Synthesis. MMP-7 substrate, GVPLSLTMGCNH2, with glycine and cysteine at both ends for conjugation was synthesized on an automatic synthesizer (433A Peptide Synthesizer, Applied Biosystem) by Fmoc chemistry (0.1 mmol) using Rink amide resin (0.43 mmol/g) and HBTU/HOBt as the activating reagents. After the completion of the peptide synthesis, the fluorescence quencher, NIRQ820, was coupled to the Nterminus (4). NIRQ820 (0.4 mmol, 326.16 mg) was first activated with HBTU (0.2 mmol, 75.86 mg) and HOBt (0.2 mmol, 27.02 mg) in DMF in the presence of argon for 3 h and then reacted overnight. The NIRQ820-labeled peptide 3, NIRQ820-GVPLSLTMGC-NH2, was cleaved by a cocktail of TFA/thioanisole/ethandithiol/anisole (90/ 5/3/2), and purified by C18 reversed-phase HPLC to afford green-colored product. MALDI-TOF MS calcd (M + H)+ 1774.57, found 1774.83. The fluorescence donor, Cy5.5 mono maleimide (1 mg, 0.87 µmol), was reacted with the previously purified compound 3 (1.5 µmol) in 400 µL buffer (1:1 acetonitrile/ 50 mM sodium acetate, pH ) 7.4) at room temperature in the dark with random shaking for 2 h. The final produce 4, NIRQ820-GVPLSLTMGC(Cy5.5)-NH2, was purified by reversed- phase HPLC. MALDI-TOF MS (M + H)+ calcd. 2814.76, found 2814.99. A control probe 5, NIRQ820-GSMLPVTLGC(Cy5.5)NH2, was synthesized using the same approach. Stern-Volmer Plot (16). A Stern-Volmer plot was obtained at room temperature where a fixed concentration of Cy5.5 (13 µM) was treated with an increasing amount of NIRQ820 (6.0-24 µM) in deionized water. The emission intensity was measured at 690 nm and the relative emission efficiencies of Cy5.5 in the absence and in the presence of NIRQ820 provided a straight line in the concentration range. The following equation was used to derive the Stern-Volmer quenching constant:

F0/F ) 1 + Ksv [NIRQ820] where F0 is the unquenched fluorescence intensity of Cy5.5, F is the fluorescence intensity at [NIRQ820], and Ksv is the Stern-Volmer quenching constant. In vitro probe activation. Peptide-based probes were dissolved in deionized water and the concentration was determined by Beer’s law: [C] ) A/ where A is the absorbance at 674 nm and  is the molar extinction coefficient (250 000 M-1 cm-1) of Cy5.5. The activation of the probes (10µM) was commenced with the addition of MMP-7 or MMP-9 enzymes (30 nM) in a buffer consisting of a 50 mM Tris (pH ) 7.6), 200 m NaCl, 5 mM CaCl2, and 1 µM ZnCl2. The fluorescence signal was monitored using 667 nm excitation and 690 nm emission at room temperature for 3 h by SPECTRAmax Gemini (Molecular Devices, Sunnyvale, CA). The plate was set to stir 5 s prior to measurement. Tumor Extraction. Human fibrosarcoma cell line (HT1080, ATCC, Manassas, VA) was grown in Dulbecco’s medium (DMEM, Cellgro, Mediatech, Washington, DC) containing 10% (v/v) fetal bovine serum (FBS, Cellgro) and 1% penicillin/streptomycin at 37 °C in a humidified 5% CO2 atmosphere. To induce solid tumors, 2 × 106 cells in DMEM media were injected subcutaneously into the mammary fat pad of athymic nude mice (6-7 weeks old)

Pham et al.

Figure 1. Normalized spectral overlap between the absorption of NIRQ820 (s) and emission of Cy5.5 (- - -). The spectra were obtained in water at room temperature.

(Charles River, Wilmington, MA). When the tumors were grown to 6-9 mm in diameter, the mice were anesthetized by means of intraperitoneal injection of ketamine (90 mg/kg body weight) and xylazine (10 mg/kg body weight). The tumor tissues were carefully dissected and reserved in a buffer solution (pH ) 7.4, 50 mM Tris-HCl, 0.15 M NaCl, and 10 mM CaCl2) followed by homogenization and centrifugation at 4 °C. The supernatant was collected and stored at -80 °C for future analysis. All procedures using the animals were conducted in compliance with state and federal guidelines and approved by institutional Animal Care. Casein Zymography. To confirm the presence of active MMP-7 in tumor tissue, zymography using caseinloaded SDS-polyacrylamide gel was performed as described previously (17). Briefly, a 12% SDS-polyacrylamide gel was copolymerized with bovine β-casein (final concentration: 0.5 mg/mL), and then the prepared gel was pre-run at room temperature. Tumor tissue extract (20 µL) and the control MMP-7 enzyme (6 ng) were treated with mercaptoethanol-free Laemmli buffer followed by electrophoresis at 4 °C. SDS was removed from the gel by washing twice with 50 mM Tris-HCl (pH 7.5), 2.5% Triton X-100 for 30 min, and twice more for 10 min with 50 mM Tris-HCl (pH 7.5). The degradation of β-casein in the gel was started by incubating the gel overnight at 37 °C with 50 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 10 mM CaCl2, 0.1% Triton X-100, and 0.02% NaN3. The staining was performed for 1 h at room temperature with 0.5% Coomassie brilliant blue R-250 in 10% acetic acid and destaining with 10% acetic acid. Caseinolytic activity was visualized as clear bands at 19 kDa of lysis against the dark background. Probe Activation in Tumor Extract. For the fluorescence assay using tumor extract, 50 µL of tumor extracts were added into 96-well plate and mixed with 150 µL of the buffer solutions (pH ) 7.6, 50 mM Tris, 200 m NaCl, 5 mM CaCl2, and 1 µM ZnCl2) containing probe 4 or 5 (10 µM). The solutions containing only probe 4 or probe 5 were also tested as controls. The fluorescence signal was monitored simultaneously for 3 h as described above. RESULTS AND DISCUSSION

NIRQ820, a cyclohepta polymethine fluorochrome, ex/ em ) 790/820 nm, is a water soluble NIR fluorochrome with great chemical stability, and a good NIR fluorochrome for protein labeling (4). By studying NIRQ820 with a shorter wavelength commercial available fluorochrome, Cy5.5 (ex/em ) 675/694 nm, Figure 1), it was found that NIRQ820 acted as an efficient quencher for Cy5.5. To assess the quenching efficiency of this FRET pair, a Stern-Volmer plot was obtained where the

Peptide-Based NIR Probe for Protease Sensing

Figure 2. Stern-Volmer plot for determining quenching efficiency of NIRQ820 toward Cy5.5 in water. F0 and F are fluorescence intensity of Cy5.5 (13 µM) in the absence and in the presence of NIRQ820, respectively. Values are means from two assays.

fluorescence intensity of the donor, Cy5.5, was measured with or without the acceptor, NIRQ820 (Figure 2). There was a decrease in the fluorescence intensity of Cy5.5 (1.32 × 10-5 M) with the increasing concentration of NIRQ820 (0.59 × 10-5 to 2.22 × 10-5 M). A nearly perfect linear line with a steep slope intercepting at 1 implied an effective dynamic diffusion control quenching effect (18, 19).

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Previously we have demonstrated that NIRQ820 can be conveniently synthesized in large quantity and is compatible with Fmoc solid-phase peptide synthesis (SPPS) (4). We thus chose to couple NIRQ820 quencher onto a MMP-7 selective peptide on solid support and label the peptide with the fluorescence donor, Cy5.5, later in solution (Figure 3). The core specific peptide substrate, VPLSLTMG, for MMP-7 was selected from a combinatory peptide library (20). It has been shown that it is at least 10 to 100 times more selective for MMP-7 than for MMP-1, -2, -3, -9, and MT1-MMP. To the core MMP-7 substrate sequence, glycine and cysteine residues were incorporated at N- and C-termini, respectively, as handles for fluorochromes labeling. The peptide (GVPLSLTMGC) was synthesized on a Rink amide resin by Fmoc chemistry using SPPS. Upon complete synthesis of the peptide sequence, the N-terminal Fmoc group was removed and the sole active amino group was confirmed by ninhydrin test (21). NIRQ820 was preactivated by HOBt/HBTU in anhydrous DMF under the presence of argon for 2 h. The intermediate was then coupled to the peptide catalyzed by pyridine for 4 h. The green solid supports were washed with DMF, followed by methanol, before being subjected to standard cleavage conditions.

Figure 3. Synthesis of peptide-based MMP-7 probe 4. Reagents and conditions: (a) HOBt/HBTU, pyridine, DMF, 6 h; (b) TFA and scavengers of thioanisole, anisole, and ethanedithiol, 3 h; (c) sodium acetate buffer (50 mM, pH ) 7.4), 2 h.

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Figure 4. Analytical HPLC traces of probe 4, NIRQ820GVPLSLTMGC(Cy5.5)-NH2.

Figure 6. Activation of MMP-7 probe using tumor extract. (a) Casein zymography of HT1080 tumor extract. The 19 kDa bands indicate the active MMP-7 from standard (left) and from tumor extract (right). (b) Activation of NIR probes (10 µM) with tumor extract in a 50 mM Tris, 200 M NaCl, 5 mM CaCl2, and 1 µM ZnCl2 buffer or buffer only. Probe 4 plus tumor extract (solid diamond), scrambled probe 5 plus tumor extract (solid circle), probe 4 in buffer only (open diamond), scrambled probe 5 in buffer only (open circle).

Figure 5. Activation of peptide-based NIR probes with MMPs. (a) Sequence of MMP-7 probe 4 and control scramble probe 5. (b) Fluorescence signals change upon incubation with 4 alone ([), 4 + MMP-9 (1), 5 + MMP-7 (b), and 4 + MMP-7 (2) in a 50 mM Tris (pH ) 7.6), 200 M NaCl, 5 mM CaCl2, and 1 µM ZnCl2 buffer. (c) Fold increase of fluorescence signal at 100 min. Values are means of two assays.

After precipitation and washing with tert-butyl methyl ether at 4 °C, the labeled green peptide 3 was purified by HPLC and the product was stored at -78 °C for several months without trace of decomposition. Excess unreacted NIRQ820 was recovered by recrystallization in methanol/water. The thio-reactive maleimide moiety of Cy5.5 was further incorporated into the peptide sequence via the C-terminal cysteine residue in sodium acetate for 2 h following by HPLC purification to provide the MMP-7 probe 4 quantitatively (Figure 4). The enzyme selectivity of the probes was studied with different controls in an optimal buffer condition using 50 mM Tris (pH ) 7.6), 200 mM NaCl, 5 mM CaCl2, and 1 µM ZnCl2 (Figure 5). To follow the proteolytic reaction,

fluorescence changes of Cy5.5 at 690 nm (excitation at 667 nm) were accessed by a fluorescence plate reader. Within 100 min, the Cy5.5 fluorescence signal of probe 4 increased 7-fold with MMP-7 as compared to the control enzyme, MMP-9, or when the probe 4 was incubated without MMP-7 (Figure 5). To further prove the specificity of this probe, a scrambled control probe 5, SMLPVTLG, was synthesized (Figure 5a). There was no increase in the fluorescence intensity when MMP-7 was incubated with scrambled probe. The enzyme kinetics of probe 4 with MMP-7 was determined using the Michaelis-Menten equation. To obtain accurate initial rate of substrate cleavage, the inner filter effect was corrected according to a previously reported method (22). The determined kcat/Km was 100 000 M-1 s-1. This result is consistent with the kinetics of the originally reported peptide sequence, kcat/Km ) 120 000 M-1 s-1 (20). It suggests that chemical attachment of the fluorescence donor and acceptor did not significantly alter enzymatic reactivity and selectivity. To evaluate the usage of probe 4 in detecting MMP-7 activity in biological systems, a HT1080 human fibrosarcoma tumor model was selected. It has been demonstrated previously that HT1080 has the overexpression of MMP-7 enzyme (23). The tumors were dissected when they were about 6-9 mm in size, and the tumorassociated enzymes were extracted by gradient centrifugation. The presence of active 19 kDa MMP-7 in the tumor extract was first confirmed using casein zymography (Figure 6). The fluorescence signal of probe 4 increased by 4-fold after incubating with the tumor extract in 3 h; however, the control probe 5 also gave a 2-fold increase in signal. In the previous experiment, it has been demonstrated that the control probe 5 could not be activated by recombinant MMP-7 (Figure 5C), thus the signal increase has to be caused by other proteases which were extracted from the whole tumor. This result suggests that nonspecific activation of the probe could be expected in the future in vivo imaging of MMP-7 activity.

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Peptide-Based NIR Probe for Protease Sensing

In summary, we described a rational design of a peptide-based NIR probe. Efficient quenching effect between NIR fluorochrome at 700 nm and NIRQ820 was demonstrated. In the MMP-7 model, the probe is able to release fluorescence signal by 7-fold after enzymatic degradation. This probe design could be applied to assay enzyme activity in vitro, for example high-throughput screening of various inhibitors, and potentially can be used for in vivo imaging of tumor-associated enzyme activity. ACKNOWLEDGMENT

This research was supported by NIH RO1-CA99385, DOD DAMD17-02-1-0693, and NSF BES-0119382. LITERATURE CITED (1) Bremer, C., Tung, C. H., and Weissleder, R. (2001) In vivo molecular target assessment of matrix metalloproteinase inhibition. Nat. Med. 7, 743-8. (2) Weissleder, R., and Ntziachristos, V. (2003) Shedding light onto live molecular targets. Nat Med. 9, 123-8. (3) Pham, W., Weissleder, R., and Tung, C. H. (2002) An azulene dimer as a near-infrared quencher. Angew Chem., Int. Ed. 41, 3659-3662. (4) Pham, W., Lai, W. F., Weissleder, R., and Tung, C. H. (2003) High efficiency synthesis of a bioconjugatable near-infrared fluorochrome. Bioconjugate Chem. 14, 1048-51. (5) Ohbayashi, H. (2002) Matrix metalloproteinases in lung diseases. Curr. Protein Pept. Sci. 3, 409-21. (6) Thompson, R. W., and Baxter, B. T. (1999) MMP inhibition in abdominal aortic aneurysms. Rationale for a prospective randomized clinical trial. Ann. N. Y. Acad. Sci. 878, 159-78. (7) McCawley, L. J., and Matrisian, L. M. (2000) Matrix metalloproteinases: multifunctional contributors to tumor progression. Mol. Med. Today 6, 149-56. (8) Moses, M. A., Wiederschain, D., Loughlin, K. R., Zurakowski, D., Lamb, C. C., and Freeman, M. R. (1998) Increased incidence of matrix metalloproteinases in urine of cancer patients. Cancer Res. 58, 1395-9. (9) Stearns, M. E., and Wang, M. (1993) Type IV collagenase (M(r) 72, 000) expression in human prostate: benign and malignant tissue. Cancer Res. 53, 878-83. (10) Brooks, P. C., Stromblad, S., Sanders, L. C., von Schalscha, T. L., Aimes, R. T., and Stetler-Stevenson, W. G., et al. (1996) Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3. Cell 85, 683-93.

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