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A protease inhibitor discovery method using fluorescence correlation spectroscopy with position-specific labeled protein substrates
Analytical Biochemistry 390 (2009) 121–125
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Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
A protease inhibitor discovery method using fluorescence correlation spectroscopy with position-specific labeled protein substrates Hidetaka Nakata a,b, Takashi Ohtsuki a,*, Masahiko Sisido a a b
Department of Bioscience and Biotechnology, Okayama University, Okayama 700-8530, Japan Olympus Corporation, Tokyo 192-8512, Japan
a r t i c l e
i n f o
Article history: Received 23 January 2009 Available online 24 April 2009 Keywords: Fluorescence correlation spectroscopy Protease assay Protease substrate Four-base codon Position-specific labeling
a b s t r a c t We developed novel substrates for protease activity evaluation by fluorescence correlation spectroscopy (FCS). Substrates were labeled in a position-specific manner with a fluorophore near the N terminus and included a C-terminal, 30 kDa, highly soluble protein (elongation factor Ts [EF-Ts]). The C-terminal protein enhanced the substrate peptide solubility and increased the molecular weight, enabling sensitive detection by FCS. Using the labeled substrates, caspase-3 and matrix metalloproteinase-9 (MMP-9) activities were confirmed by FCS. To demonstrate the suitability of this FCS-based assay for high-throughput screening, we screened various chemical compounds for MMP-9 inhibitors. The screening results confirmed the inhibitory activity of one compound and also revealed another potential MMP-9 inhibitor. Thus, this combination of position-specific labeled protein substrates and FCS may serve as a useful tool for evaluating activities of various proteases and for protease inhibitor screening. Ó 2009 Elsevier Inc. All rights reserved.
Protease inhibitor discovery is an important step in drug discovery. Short peptides are generally used as model substrates for original substrate proteins. Peptides with a tryptophan or other fluorophore–quencher pair can be used to detect substrate cleavage, where cleavage is monitored by the fluorescence intensity increase following quencher release [1,2]. Although peptide substrates are inexpensive and easy to handle, they often show limited solubility and cause undesirable aggregations that may lead to incorrect conclusions. The introduction of a large-sized fluorophore–quencher pair may boost these solubility problems and possibly impede protease access. Such solubility problems can be overcome by the use of soluble protein substrates instead of short peptide substrates. In this study, we developed novel protein substrates with a fluorophore at a specific position (Fig. 1) by a cell-free translation system with an expanded genetic code [3–6]. Cleavage of the fluorescently labeled substrate by proteases generates a small, fluorophore-bearing peptide fragment. We performed protease assays to detect the small peptide fragment using fluorescence correlation spectroscopy (FCS),1 a powerful tool for studying interactions
among biomolecules at the single-molecule level in solution [7–9]. In FCS experiments, changes in the diffusion times of the labeled molecules are considered. The observed data can be interpreted by changes in the molecular weights or conformations of the biomolecules. FCS is advantageous for high-throughput screening because samples on a multiwell plate can be automatically measured by the FCS instrument. In addition, FCS requires only a small volume at a low concentration (1010–108 M) of the fluorophore-bearing substrate compared with measurements using a peptide substrate bearing a fluorophore–quencher pair (106–105 M) [2,10]. We attempted to detect the activity of caspase-3 and matrix metalloproteinase (MMP)-9 using fluorescently labeled substrates and FCS. To demonstrate the suitability of our FCS-based assay for screening, various chemical compounds were screened for MMP-9 inhibitors. Because MMPs are related to many diseases [11], particularly tumor metastasis and invasion [12,13], MMPs such as MMP-9 have attracted attention as targets for drug development. Materials and methods
* Corresponding author. Fax: +81 86 251 8219. E-mail address: [email protected] (T. Ohtsuki). 1 Abbreviations used: FCS, fluorescence correlation spectroscopy; MMP, matrix metalloproteinase; EF-Ts, elongation factor Ts; PCR, polymerase chain reaction; TAMRA-X-aminophenylalanine, 4-(6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoyl)aminophenylalanine; TAMRA–tRNA, TAMRA-X-aminophenylalanyl– tRNACCCG; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; EDTA, ethylenediaminetetraacetic acid; EGTA, ethyleneglycoltetraacetic acid; DTT, dithiothreitol; DMSO, dimethyl sulfoxide; FRET, fluorescence resonance energy transfer; FCCS, fluorescence cross-correlation spectroscopy. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.03.049
Constructing a vector expressing a caspase-3 substrate The coding sequence of the Escherichia coli elongation factor Ts (EF-Ts) gene was polymerase chain reaction (PCR)-amplified from EasyXpress–Positive Control DNA (Qiagen, Germany) using two primers: 50 -aaa ccg ggt cta atg aga cca tgg ctg aaa tta ccg cat ccc30 and 50 -aac ccc ccc cgg gag act gct tgg aca tcg cag-30 . The
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Protease inhibitor discovery method / H. Nakata et al. / Anal. Biochem. 390 (2009) 121–125
FCS measurements
Fig. 1. Design of a fluorescently labeled substrate for a protease (upper panel). Position-specific labeling of proteins with TAMRA was performed by introducing TAMRA-X-aminophenylalanine into proteins in response to a CGGG four-base codon.
PCR-amplified sequence was inserted between the NcoI and SmaI sites of a pROX–FL vector, which was included in an In vitro Pinpoint Fluorescence Labeling Kit 543 (Olympus, Japan), according to the insertion protocol of the QuickChange Site-Directed Mutagenesis Kit (Stratagene, USA). The sequence 50 -gac gaa gtt gac-30 , encoding the caspase-3 cleavage site (amino acid sequence DEVD [14]), was inserted between a CGGG four-base codon and the coding sequence of the E. coli EF-Ts gene. The CGGG codon was used to encode TAMRA-X-aminophenylalanine, (4-(6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoyl)aminophenylalanine), as described below. The vector included a C-terminal His tag and an N-terminal tag (MSKQIEVNXSNET, where X = TAMRA-labeled amino acid) containing a CGGG four-base codon. Constructing a vector expressing an MMP-9 substrate The coding sequence of the E. coli EF-Ts gene was PCR-amplified from EasyXpress–Positive Control DNA by using two primers, 50 gcg agc tca tgg ctg aaa tta ccg cat ccc-30 and 50 -aac ccc ccc cgg gag act gct tgg aca tcg cag-30 , and cloned into the SacI and SmaI sites of a pROX–FL vector. The sequence 50 -ccg ctg ggt atg tgg tct cgt-30 , encoding the MMP-9 cleavage site (amino acid sequence PLGMWSR [1]), was cloned into the NdeI and XhoI sites.
FCS measurements were performed at 25 °C with an MF20 instrument (Olympus) using a UAPO 40/NA 1.15 water immersion objective (Olympus). Sample solutions were distributed into the wells of a 384-well glass-bottomed microplate with a flat thickness of 0.175 ± 0.02 mm, and measurements were carried out in each well containing 30 ll of sample solution. To detect TAMRA-labeled molecules, an He–Ne laser (543 nm) and an HQ590/60 filter were used. Repeated FCS measurements showed that the fluorescence of TAMRA remained constant (data not shown). To ensure the stable output (100 lW) of the laser beam for excitation, the laser was warmed up for more than 2 h before performing the measurements. All experiments were performed under identical conditions, with a data acquisition time of 10 s for each measurement, and the measurements were repeated five times at each well. To detect cleavage of the caspase-3 substrate, 1 nM labeled substrate was incubated with 1 U/ll of caspase-3 (Calbiochem, Germany) at 37 °C for 1 h in 20 mM Hepes-K (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethyleneglycoltetraacetic acid (EGTA), and 10 mM dithiothreitol (DTT) [15] in the presence or absence of 1 mM Ac-DEVDCHO (Biomol, USA), a caspase-3 inhibitor. The reaction mixtures were used for FCS measurements. To detect cleavage of the MMP-9 substrate, 1 nM labeled substrate was incubated with 0.3 lg/ml of MMP-9 (Calbiochem) at 25 °C for 1 h in 50 mM Tris–HCl (pH 7.5), 175 mM NaCl, 10 mM CaCl2, 0.05% Brij-35, 4.75% methanol, and 0.5% dimethyl sulfoxide (DMSO) [16]. Solutions (10 lM) of each compound from SCADS inhibitor kits I, II, and III [17,18] were used as candidates for MMP-9 inhibitors. The kits were kindly provided by the Screening Committee of Anticancer Drugs (Japan) and contained a total of 282 chemical compounds. After heat inactivation of MMP-9 at 95 °C for 5 min, FCS measurements were performed. SDS–PAGE analysis of caspase-3 substrate cleavage The reaction mixture (10 ll) contained 0.1 pmol of caspase-3 substrate and 100 U of caspase-3 in 20 mM Hepes-K (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 10 mM DTT. The reaction mixture was incubated at 37 °C for 1 h in the presence or absence of 1 mM Ac-DEVD-CHO and was then analyzed by 20% SDS–PAGE. The gel was visualized with an FMBIOIII fluorescence imager (Hitachi Software Engineering, Japan) with excitation at 532 nm and emission at 580 nm.
Preparation of fluorescently labeled protease substrates
Results
The TAMRA-labeled substrates were synthesized using an RTS 100 E. coli HY Kit (Roche Diagnostics, Switzerland) and the TAMRA–tRNA (TAMRA-X-aminophenylalanyl–tRNACCCG) included in the In vitro Pin-point Fluorescence Labeling Kit 543. The reaction mixture for in vitro translation (150 ll) contained 15 ll of the plasmid DNA (100 ng/ll), 15 ll of TAMRA–tRNA, 36 ll of amino acids, 3 ll of methionine, 30 ll of reaction mix, and 36 ll of E. coli lysate. Amino acids, methionine, reaction mix, and E. coli lysate were included in the RTS 100 E. coli HY Kit. The reaction mixture was incubated at 30 °C for 2 h. Expressed TAMRA-labeled substrates were purified using His Spin Trap Columns (GE Healthcare, UK) according to the supplier’s instructions. Substrates were concentrated with UltraFree-0.5 centrifugal devices (Millipore, USA), and the substrate sizes and concentrations were confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and FCS.
FCS analysis of the protease reaction and its inhibition The diffusion time of the fluorescently labeled caspase-3 substrate was measured by FCS after substrate cleavage with caspase-3 in either the presence or absence of the caspase-3 inhibitor Ac-DEVD-CHO (Fig. 2). Two-component fit analysis of the separation of the substrate and the free TAMRA amino acids showed that the substrate was almost completely free of TAMRA amino acids. The remaining free TAMRA amino acids represented 3% of the total TAMRA dye in the substrate. The diffusion time of the substrate was 551 ls, a reasonable value considering the substrate size (34 kDa). When the substrate was cleaved with caspase3, the diffusion time was significantly decreased (264 ls). The diffusion time of the small fragment (2 kDa) generated by cleavage with caspase-3 was estimated to be 244 ls by performing threecomponent fit analysis of the separation of uncleaved substrate,
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Fig. 2. FCS analysis of the cleavage of a caspase-3 substrate. FCS analysis was performed after incubation of 1 nM labeled substrate at 37 °C for 1 h in the presence or absence of 1 U/ll of caspase-3 and 1 mM Ac-DEVD-CHO (inhibitor). Values represent means ± SD of five measurements.
cleaved substrate, and free TAMRA amino acid. Cleavage of caspase-3 substrate was confirmed by SDS–PAGE (72% of the substrate was cleaved [Fig. 3]). However, the diffusion time was not different from that of the intact substrate when the substrate cleavage reaction was successfully inhibited (as confirmed by SDS–PAGE [Fig. 3]) by Ac-DEVD-CHO (Fig. 2). These results indicate that the FCS-based assay is useful for detecting protease activity and applicable for compound screening for protease inhibitors. Screening compounds by FCS to identify MMP-9 inhibitors Substrate cleavage with MMP-9 was also detected by FCS (Fig. 4A). The diffusion time of the substrate (35 kDa) was 535 ls, and the diffusion time significantly decreased (286 ls) when the substrate was cleaved with MMP-9. The diffusion time of the small fragment (3 kDa) generated by cleavage with MMP-9 was estimated to be 276 ls by three-component fit analysis. To demonstrate that the FCS-based assay was suitable for screening, we screened 278 chemical compounds (see Supplementary Table 1
Fig. 3. SDS–PAGE analysis of caspase-3 substrate cleavage. The excitation and emission wavelengths for visualization were 532 and 580 nm, respectively.
in supplementary material) from the SCADS inhibitor kits for MMP-9 inhibitors. Three compounds from the kits could not be used for FCS measurement due to their high autofluorescence, and one compound could not be used because of its low concentration. After the cleavage reaction was performed in microtubes, the reaction solution was transferred onto a multiwell plate and the plate was set in the FCS instrument for high-throughput measurement. Fig. 4B shows the diffusion times of the fluorescently labeled substrate after incubation with MMP-9 in the presence of the chemical compounds. The largest SD value among those measured using the MMP-9 substrate was 38 ls (see Supplementary Tables 1 and 2). In general, a value larger than standard value + 3 SD value is considered to be significantly larger than the standard value. Thus, compounds that exhibited diffusion times longer than 400 ls (286 + 3 38) can be considered to have inhibitory activity. We confirmed that GM6001, a known MMP-9 inhibitor [19],
Fig. 4. Screening of 278 chemical compounds by FCS to identify MMP-9 inhibitors. (A) Diffusion times of the substrate only and of the substrate with MMP-9 without the inhibitor candidate. (B) Diffusion times of the substrate with MMP-9 and with inhibitor candidates. Chemical compounds as candidates for MMP-9 inhibitors are numbered as shown in Supplementary Table 1 (see Supplementary material). The number under each bar corresponds to that of each compound. Each value represents the mean of five measurements.
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are very important. In this study, considerable changes in the molecular size of the substrate through its cleavage were achieved by fusion of a 30-kDa protein (EF-Ts) to the substrate. As examples, the substrates for caspase-3 and MMP-9 were synthesized and cleaved with these proteases. The diffusion times of the substrate decreased significantly after the cleavage reaction (Figs. 2 and 4A). The EF-Ts protein successfully enhanced the molecular weight. In the screening of compounds by FCS to identify MMP-9 inhibitors, we found some possible inhibitors (Fig. 4B), indicating that the FCS-based protease assay is suitable for inhibitor screening. The developed fluorescently labeled substrates had a single fluorophore near the N terminus that was proteolytically cleaved from the C-terminal region, including the EF-Ts protein. In fluorescence analysis using a fluorophore–quencher pair or fluorescence resonance energy transfer (FRET) analysis using two fluorophores, incomplete substrate labeling can lead to a narrow dynamic range. Protease activity analyses can also be performed using fluorescence cross-correlation spectroscopy (FCCS), an extension of FCS [15,21]. FCCS does not depend on molecular size changes but does require two fluorescent proteins or fluorophores. FCS analysis using a single fluorophore does not have the problem of incomplete labeling because the unlabeled substrate is ignored by the FCS analysis. Although it is necessary to take into account the presence of competitive unlabeled substrates, substrate labeling efficiency is quite high by the position-specific labeling method used in this study. Short peptide substrates for protease assays often show limited solubility and cause undesirable aggregations. Introduction of large fluorophores and/or a quencher may also boost these solubility problems. The highly soluble, C-terminal protein (EF-Ts) on our substrates enhanced their water solubility. Furthermore, ideal protease activity could be evaluated by referring to the schema in Fig. 1, where the original protein substrate was used instead of a peptide containing the cleavage site. In conclusion, the combination of the protein substrate reported here and FCS is quite suitable for detecting activities of various proteases and should be a powerful tool for high-throughput screening to identify protease inhibitors. Fig. 5. Inhibition of MMP-9 using GM6001 (A), actinonin (B), and 1-azakenpaullone (C), as analyzed by FCS. Each value represents the mean ± SD of five measurements.
strongly inhibited cleavage activity (diffusion time = 547 ls). Cleavage was also inhibited by actinonin (diffusion time = 475 ls), which is a known aminopeptidase M inhibitor [20] but is not known to be an MMP-9 inhibitor. Several compounds shortened the diffusion time of the substrate without MMP-9 (see Supplementary Table 2). This is why the substrate with these compounds exhibited considerably short diffusion times (<240 ls) (Fig. 4B). Inhibition of MMP-9 activity by GM6001 and actinonin MMP-9 activities were measured with three compounds: GM6001, actinonin, and 1-azakenpaullone (negative control) (Fig. 5). The diffusion time for the MMP-9 substrate increased with increasing amounts of GM6001. GM6001 strongly inhibited MMP9, with an estimated IC50 of 9.1 nM. High concentrations of actinonin weakly inhibited MMP-9 activity, whereas 1-azakenpaullone did not inhibit MMP-9 activity at all. Discussion We developed novel substrates labeled with TAMRA for an FCS-based protease assay (Fig. 1). For sensitive detection of a cleavage reaction by FCS, changes in the substrate molecular size
Acknowledgments For SCADS inhibitor kits, we thank the Screening Committee of Anticancer Drugs supported by a Grant-in-Aid for Scientific Research on Priority Area ‘‘Cancer” (MEXT, Japan). We thank Ryoji Abe (Protein Express, Japan), Yoshifumi Yamada (Olympus, Japan), and Mitsushiro Yamaguchi (Olympus) for their help. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ab.2009.03.049. References [1] S. Netzel-Arnett, S.K. Mallya, H. Nagase, H. Birkedal-Hansen, H.E. Van Wart, Continuously recording fluorescent assays optimized for five human matrix metalloproteinases, Anal. Biochem. 195 (1991) 86–92. [2] E.D. Matayoshi, G.T. Wang, G.A. Krafft, J. Erickson, Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer, Science 247 (1990) 954–958. [3] T. Hohsaka, D. Kajihara, Y. Ashizuka, H. Murakami, M. Sisido, Efficient incorporation of nonnatural amino acids with large aromatic groups into streptavidin in in vitro protein synthesizing systems, J. Am. Chem. Soc. 121 (1999) 34–40. [4] T. Ohtsuki, T. Manabe, M. Sisido, Multiple incorporation of non-natural amino acids into a single protein using tRNAs with non-standard structures, FEBS Lett. 579 (2005) 6769–6774.
Protease inhibitor discovery method / H. Nakata et al. / Anal. Biochem. 390 (2009) 121–125 [5] D. Kajihara, R. Abe, I. Iijima, C. Komiyama, M. Sisido, T. Hohsaka, FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids, Nat. Methods 3 (2006) 923– 929. [6] Y. Doi, T. Ohtsuki, Y. Shimizu, T. Ueda, M. Sisido, Elongation factor Tu mutants expand amino acid tolerance of protein biosynthesis system, J. Am. Chem. Soc. 129 (2007) 14458–14462. [7] E. Haustein, P. Schwille, Fluorescence correlation spectroscopy: novel variations of an established technique, Annu. Rev. Biophys. Biomol. Struct. 36 (2007) 151–169. [8] W. Al-Soufi, B. Reija, S. Felekyan, C.A. Seidel, M. Novo, Dynamics of supramolecular association monitored by fluorescence correlation spectroscopy, ChemPhysChem 9 (2008) 1819–1827. [9] T. Hesterkamp, J. Barker, A. Davenport, M. Whittaker, Fragment based drug discovery using fluorescence correlation spectroscopy techniques: challenges and solutions, Curr. Top. Med. Chem. 7 (2007) 1582–1591. [10] H. Nagase, C.G. Fields, G.B. Fields, Design and characterization of a fluorogenic substrate selectively hydrolyzed by stromelysin 1 (matrix metalloproteinase3), J. Biol. Chem. 269 (1994) 20952–20957. [11] G. Murphy, H. Nagase, Progress in matrix metalloproteinase research, Mol. Aspects Med. 29 (2008) 290–308. [12] E.I. Deryugina, J.P. Quigley, Matrix metalloproteinases and tumor metastasis, Cancer Metastasis Rev. 25 (2006) 9–34. [13] N. Johansson, M. Ahonen, V.M. Kahari, Matrix metalloproteinases in tumor invasion, Cell. Mol. Life Sci. 57 (2000) 5–15.
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[14] M. Garcia-Calvo, E.P. Peterson, B. Leiting, R. Ruel, D.W. Nicholson, N.A. Thornberry, Inhibition of human caspases by peptide-based and macromolecular inhibitors, J. Biol. Chem. 273 (1998) 32608–32613. [15] K. Saito, I. Wada, M. Tamura, M. Kinjo, Direct detection of caspase-3 activation in single live cells by cross-correlation analysis, Biochem. Biophys. Res. Commun. 324 (2004) 849–854. [16] G. Murphy, Q. Nguyen, M.I. Cockett, S.J. Atkinson, J.A. Allan, C.G. Knight, F. Willenbrock, A.J. Docherty, Assessment of the role of the fibronectin-like domain of gelatinase A by analysis of a deletion mutant, J. Biol. Chem. 269 (1994) 6632–6636. [17] M. Kawada, H. Inoue, T. Masuda, D. Ikeda, Insulin-like growth factor I secreted from prostate stromal cells mediates tumor–stromal cell interactions of prostate cancer, Cancer Res. 66 (2006) 4419–4425. [18] A. Tsuchiya, E. Tashiro, M. Yoshida, M. Imoto, Involvement of protein phosphatase 2A nuclear accumulation and subsequent inactivation of activator protein-1 in leptomycin B-inhibited cyclin D1 expression, Oncogene 26 (2007) 1522–1532. [19] R.E. Galardy, M.E. Cassabonne, C. Giese, J.H. Gilbert, F. Lapierre, H. Lopez, M.E. Schaefer, R. Stack, M. Sullivan, B. Summers, et al., Low molecular weight inhibitors in corneal ulceration, Ann. NY Acad. Sci. 732 (1994) 315–323. [20] H. Umezawa, T. Aoyagi, T. Tanaka, H. Suda, A. Okuyama, H. Naganawa, M. Hamada, T. Takeuchi, Production of actinonin, an inhibitor of aminopeptidase M, by actinomycetes, J. Antibiot. 38 (1985) 1629–1630. [21] T. Kohl, E. Haustein, P. Schwille, Determining protease activity in vivo by fluorescence cross-correlation analysis, Biophys. J. 89 (2005) 2770–2782.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
A protease inhibitor discovery method using fluorescence correlation spectroscopy with position-specific labeled protein substrates Hidetaka Nakata a,b, Takashi Ohtsuki a,*, Masahiko Sisido a a b
Department of Bioscience and Biotechnology, Okayama University, Okayama 700-8530, Japan Olympus Corporation, Tokyo 192-8512, Japan
a r t i c l e
i n f o
Article history: Received 23 January 2009 Available online 24 April 2009 Keywords: Fluorescence correlation spectroscopy Protease assay Protease substrate Four-base codon Position-specific labeling
a b s t r a c t We developed novel substrates for protease activity evaluation by fluorescence correlation spectroscopy (FCS). Substrates were labeled in a position-specific manner with a fluorophore near the N terminus and included a C-terminal, 30 kDa, highly soluble protein (elongation factor Ts [EF-Ts]). The C-terminal protein enhanced the substrate peptide solubility and increased the molecular weight, enabling sensitive detection by FCS. Using the labeled substrates, caspase-3 and matrix metalloproteinase-9 (MMP-9) activities were confirmed by FCS. To demonstrate the suitability of this FCS-based assay for high-throughput screening, we screened various chemical compounds for MMP-9 inhibitors. The screening results confirmed the inhibitory activity of one compound and also revealed another potential MMP-9 inhibitor. Thus, this combination of position-specific labeled protein substrates and FCS may serve as a useful tool for evaluating activities of various proteases and for protease inhibitor screening. Ó 2009 Elsevier Inc. All rights reserved.
Protease inhibitor discovery is an important step in drug discovery. Short peptides are generally used as model substrates for original substrate proteins. Peptides with a tryptophan or other fluorophore–quencher pair can be used to detect substrate cleavage, where cleavage is monitored by the fluorescence intensity increase following quencher release [1,2]. Although peptide substrates are inexpensive and easy to handle, they often show limited solubility and cause undesirable aggregations that may lead to incorrect conclusions. The introduction of a large-sized fluorophore–quencher pair may boost these solubility problems and possibly impede protease access. Such solubility problems can be overcome by the use of soluble protein substrates instead of short peptide substrates. In this study, we developed novel protein substrates with a fluorophore at a specific position (Fig. 1) by a cell-free translation system with an expanded genetic code [3–6]. Cleavage of the fluorescently labeled substrate by proteases generates a small, fluorophore-bearing peptide fragment. We performed protease assays to detect the small peptide fragment using fluorescence correlation spectroscopy (FCS),1 a powerful tool for studying interactions
among biomolecules at the single-molecule level in solution [7–9]. In FCS experiments, changes in the diffusion times of the labeled molecules are considered. The observed data can be interpreted by changes in the molecular weights or conformations of the biomolecules. FCS is advantageous for high-throughput screening because samples on a multiwell plate can be automatically measured by the FCS instrument. In addition, FCS requires only a small volume at a low concentration (1010–108 M) of the fluorophore-bearing substrate compared with measurements using a peptide substrate bearing a fluorophore–quencher pair (106–105 M) [2,10]. We attempted to detect the activity of caspase-3 and matrix metalloproteinase (MMP)-9 using fluorescently labeled substrates and FCS. To demonstrate the suitability of our FCS-based assay for screening, various chemical compounds were screened for MMP-9 inhibitors. Because MMPs are related to many diseases [11], particularly tumor metastasis and invasion [12,13], MMPs such as MMP-9 have attracted attention as targets for drug development. Materials and methods
* Corresponding author. Fax: +81 86 251 8219. E-mail address: [email protected] (T. Ohtsuki). 1 Abbreviations used: FCS, fluorescence correlation spectroscopy; MMP, matrix metalloproteinase; EF-Ts, elongation factor Ts; PCR, polymerase chain reaction; TAMRA-X-aminophenylalanine, 4-(6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoyl)aminophenylalanine; TAMRA–tRNA, TAMRA-X-aminophenylalanyl– tRNACCCG; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; EDTA, ethylenediaminetetraacetic acid; EGTA, ethyleneglycoltetraacetic acid; DTT, dithiothreitol; DMSO, dimethyl sulfoxide; FRET, fluorescence resonance energy transfer; FCCS, fluorescence cross-correlation spectroscopy. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.03.049
Constructing a vector expressing a caspase-3 substrate The coding sequence of the Escherichia coli elongation factor Ts (EF-Ts) gene was polymerase chain reaction (PCR)-amplified from EasyXpress–Positive Control DNA (Qiagen, Germany) using two primers: 50 -aaa ccg ggt cta atg aga cca tgg ctg aaa tta ccg cat ccc30 and 50 -aac ccc ccc cgg gag act gct tgg aca tcg cag-30 . The
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Protease inhibitor discovery method / H. Nakata et al. / Anal. Biochem. 390 (2009) 121–125
FCS measurements
Fig. 1. Design of a fluorescently labeled substrate for a protease (upper panel). Position-specific labeling of proteins with TAMRA was performed by introducing TAMRA-X-aminophenylalanine into proteins in response to a CGGG four-base codon.
PCR-amplified sequence was inserted between the NcoI and SmaI sites of a pROX–FL vector, which was included in an In vitro Pinpoint Fluorescence Labeling Kit 543 (Olympus, Japan), according to the insertion protocol of the QuickChange Site-Directed Mutagenesis Kit (Stratagene, USA). The sequence 50 -gac gaa gtt gac-30 , encoding the caspase-3 cleavage site (amino acid sequence DEVD [14]), was inserted between a CGGG four-base codon and the coding sequence of the E. coli EF-Ts gene. The CGGG codon was used to encode TAMRA-X-aminophenylalanine, (4-(6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoyl)aminophenylalanine), as described below. The vector included a C-terminal His tag and an N-terminal tag (MSKQIEVNXSNET, where X = TAMRA-labeled amino acid) containing a CGGG four-base codon. Constructing a vector expressing an MMP-9 substrate The coding sequence of the E. coli EF-Ts gene was PCR-amplified from EasyXpress–Positive Control DNA by using two primers, 50 gcg agc tca tgg ctg aaa tta ccg cat ccc-30 and 50 -aac ccc ccc cgg gag act gct tgg aca tcg cag-30 , and cloned into the SacI and SmaI sites of a pROX–FL vector. The sequence 50 -ccg ctg ggt atg tgg tct cgt-30 , encoding the MMP-9 cleavage site (amino acid sequence PLGMWSR [1]), was cloned into the NdeI and XhoI sites.
FCS measurements were performed at 25 °C with an MF20 instrument (Olympus) using a UAPO 40/NA 1.15 water immersion objective (Olympus). Sample solutions were distributed into the wells of a 384-well glass-bottomed microplate with a flat thickness of 0.175 ± 0.02 mm, and measurements were carried out in each well containing 30 ll of sample solution. To detect TAMRA-labeled molecules, an He–Ne laser (543 nm) and an HQ590/60 filter were used. Repeated FCS measurements showed that the fluorescence of TAMRA remained constant (data not shown). To ensure the stable output (100 lW) of the laser beam for excitation, the laser was warmed up for more than 2 h before performing the measurements. All experiments were performed under identical conditions, with a data acquisition time of 10 s for each measurement, and the measurements were repeated five times at each well. To detect cleavage of the caspase-3 substrate, 1 nM labeled substrate was incubated with 1 U/ll of caspase-3 (Calbiochem, Germany) at 37 °C for 1 h in 20 mM Hepes-K (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethyleneglycoltetraacetic acid (EGTA), and 10 mM dithiothreitol (DTT) [15] in the presence or absence of 1 mM Ac-DEVDCHO (Biomol, USA), a caspase-3 inhibitor. The reaction mixtures were used for FCS measurements. To detect cleavage of the MMP-9 substrate, 1 nM labeled substrate was incubated with 0.3 lg/ml of MMP-9 (Calbiochem) at 25 °C for 1 h in 50 mM Tris–HCl (pH 7.5), 175 mM NaCl, 10 mM CaCl2, 0.05% Brij-35, 4.75% methanol, and 0.5% dimethyl sulfoxide (DMSO) [16]. Solutions (10 lM) of each compound from SCADS inhibitor kits I, II, and III [17,18] were used as candidates for MMP-9 inhibitors. The kits were kindly provided by the Screening Committee of Anticancer Drugs (Japan) and contained a total of 282 chemical compounds. After heat inactivation of MMP-9 at 95 °C for 5 min, FCS measurements were performed. SDS–PAGE analysis of caspase-3 substrate cleavage The reaction mixture (10 ll) contained 0.1 pmol of caspase-3 substrate and 100 U of caspase-3 in 20 mM Hepes-K (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 10 mM DTT. The reaction mixture was incubated at 37 °C for 1 h in the presence or absence of 1 mM Ac-DEVD-CHO and was then analyzed by 20% SDS–PAGE. The gel was visualized with an FMBIOIII fluorescence imager (Hitachi Software Engineering, Japan) with excitation at 532 nm and emission at 580 nm.
Preparation of fluorescently labeled protease substrates
Results
The TAMRA-labeled substrates were synthesized using an RTS 100 E. coli HY Kit (Roche Diagnostics, Switzerland) and the TAMRA–tRNA (TAMRA-X-aminophenylalanyl–tRNACCCG) included in the In vitro Pin-point Fluorescence Labeling Kit 543. The reaction mixture for in vitro translation (150 ll) contained 15 ll of the plasmid DNA (100 ng/ll), 15 ll of TAMRA–tRNA, 36 ll of amino acids, 3 ll of methionine, 30 ll of reaction mix, and 36 ll of E. coli lysate. Amino acids, methionine, reaction mix, and E. coli lysate were included in the RTS 100 E. coli HY Kit. The reaction mixture was incubated at 30 °C for 2 h. Expressed TAMRA-labeled substrates were purified using His Spin Trap Columns (GE Healthcare, UK) according to the supplier’s instructions. Substrates were concentrated with UltraFree-0.5 centrifugal devices (Millipore, USA), and the substrate sizes and concentrations were confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and FCS.
FCS analysis of the protease reaction and its inhibition The diffusion time of the fluorescently labeled caspase-3 substrate was measured by FCS after substrate cleavage with caspase-3 in either the presence or absence of the caspase-3 inhibitor Ac-DEVD-CHO (Fig. 2). Two-component fit analysis of the separation of the substrate and the free TAMRA amino acids showed that the substrate was almost completely free of TAMRA amino acids. The remaining free TAMRA amino acids represented 3% of the total TAMRA dye in the substrate. The diffusion time of the substrate was 551 ls, a reasonable value considering the substrate size (34 kDa). When the substrate was cleaved with caspase3, the diffusion time was significantly decreased (264 ls). The diffusion time of the small fragment (2 kDa) generated by cleavage with caspase-3 was estimated to be 244 ls by performing threecomponent fit analysis of the separation of uncleaved substrate,
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Fig. 2. FCS analysis of the cleavage of a caspase-3 substrate. FCS analysis was performed after incubation of 1 nM labeled substrate at 37 °C for 1 h in the presence or absence of 1 U/ll of caspase-3 and 1 mM Ac-DEVD-CHO (inhibitor). Values represent means ± SD of five measurements.
cleaved substrate, and free TAMRA amino acid. Cleavage of caspase-3 substrate was confirmed by SDS–PAGE (72% of the substrate was cleaved [Fig. 3]). However, the diffusion time was not different from that of the intact substrate when the substrate cleavage reaction was successfully inhibited (as confirmed by SDS–PAGE [Fig. 3]) by Ac-DEVD-CHO (Fig. 2). These results indicate that the FCS-based assay is useful for detecting protease activity and applicable for compound screening for protease inhibitors. Screening compounds by FCS to identify MMP-9 inhibitors Substrate cleavage with MMP-9 was also detected by FCS (Fig. 4A). The diffusion time of the substrate (35 kDa) was 535 ls, and the diffusion time significantly decreased (286 ls) when the substrate was cleaved with MMP-9. The diffusion time of the small fragment (3 kDa) generated by cleavage with MMP-9 was estimated to be 276 ls by three-component fit analysis. To demonstrate that the FCS-based assay was suitable for screening, we screened 278 chemical compounds (see Supplementary Table 1
Fig. 3. SDS–PAGE analysis of caspase-3 substrate cleavage. The excitation and emission wavelengths for visualization were 532 and 580 nm, respectively.
in supplementary material) from the SCADS inhibitor kits for MMP-9 inhibitors. Three compounds from the kits could not be used for FCS measurement due to their high autofluorescence, and one compound could not be used because of its low concentration. After the cleavage reaction was performed in microtubes, the reaction solution was transferred onto a multiwell plate and the plate was set in the FCS instrument for high-throughput measurement. Fig. 4B shows the diffusion times of the fluorescently labeled substrate after incubation with MMP-9 in the presence of the chemical compounds. The largest SD value among those measured using the MMP-9 substrate was 38 ls (see Supplementary Tables 1 and 2). In general, a value larger than standard value + 3 SD value is considered to be significantly larger than the standard value. Thus, compounds that exhibited diffusion times longer than 400 ls (286 + 3 38) can be considered to have inhibitory activity. We confirmed that GM6001, a known MMP-9 inhibitor [19],
Fig. 4. Screening of 278 chemical compounds by FCS to identify MMP-9 inhibitors. (A) Diffusion times of the substrate only and of the substrate with MMP-9 without the inhibitor candidate. (B) Diffusion times of the substrate with MMP-9 and with inhibitor candidates. Chemical compounds as candidates for MMP-9 inhibitors are numbered as shown in Supplementary Table 1 (see Supplementary material). The number under each bar corresponds to that of each compound. Each value represents the mean of five measurements.
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are very important. In this study, considerable changes in the molecular size of the substrate through its cleavage were achieved by fusion of a 30-kDa protein (EF-Ts) to the substrate. As examples, the substrates for caspase-3 and MMP-9 were synthesized and cleaved with these proteases. The diffusion times of the substrate decreased significantly after the cleavage reaction (Figs. 2 and 4A). The EF-Ts protein successfully enhanced the molecular weight. In the screening of compounds by FCS to identify MMP-9 inhibitors, we found some possible inhibitors (Fig. 4B), indicating that the FCS-based protease assay is suitable for inhibitor screening. The developed fluorescently labeled substrates had a single fluorophore near the N terminus that was proteolytically cleaved from the C-terminal region, including the EF-Ts protein. In fluorescence analysis using a fluorophore–quencher pair or fluorescence resonance energy transfer (FRET) analysis using two fluorophores, incomplete substrate labeling can lead to a narrow dynamic range. Protease activity analyses can also be performed using fluorescence cross-correlation spectroscopy (FCCS), an extension of FCS [15,21]. FCCS does not depend on molecular size changes but does require two fluorescent proteins or fluorophores. FCS analysis using a single fluorophore does not have the problem of incomplete labeling because the unlabeled substrate is ignored by the FCS analysis. Although it is necessary to take into account the presence of competitive unlabeled substrates, substrate labeling efficiency is quite high by the position-specific labeling method used in this study. Short peptide substrates for protease assays often show limited solubility and cause undesirable aggregations. Introduction of large fluorophores and/or a quencher may also boost these solubility problems. The highly soluble, C-terminal protein (EF-Ts) on our substrates enhanced their water solubility. Furthermore, ideal protease activity could be evaluated by referring to the schema in Fig. 1, where the original protein substrate was used instead of a peptide containing the cleavage site. In conclusion, the combination of the protein substrate reported here and FCS is quite suitable for detecting activities of various proteases and should be a powerful tool for high-throughput screening to identify protease inhibitors. Fig. 5. Inhibition of MMP-9 using GM6001 (A), actinonin (B), and 1-azakenpaullone (C), as analyzed by FCS. Each value represents the mean ± SD of five measurements.
strongly inhibited cleavage activity (diffusion time = 547 ls). Cleavage was also inhibited by actinonin (diffusion time = 475 ls), which is a known aminopeptidase M inhibitor [20] but is not known to be an MMP-9 inhibitor. Several compounds shortened the diffusion time of the substrate without MMP-9 (see Supplementary Table 2). This is why the substrate with these compounds exhibited considerably short diffusion times (<240 ls) (Fig. 4B). Inhibition of MMP-9 activity by GM6001 and actinonin MMP-9 activities were measured with three compounds: GM6001, actinonin, and 1-azakenpaullone (negative control) (Fig. 5). The diffusion time for the MMP-9 substrate increased with increasing amounts of GM6001. GM6001 strongly inhibited MMP9, with an estimated IC50 of 9.1 nM. High concentrations of actinonin weakly inhibited MMP-9 activity, whereas 1-azakenpaullone did not inhibit MMP-9 activity at all. Discussion We developed novel substrates labeled with TAMRA for an FCS-based protease assay (Fig. 1). For sensitive detection of a cleavage reaction by FCS, changes in the substrate molecular size
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