Article Cite This: J. Am. Chem. Soc. 2019, 141, 10409−10416
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Fluorescence Detection of Prostate Cancer by an Activatable Fluorescence Probe for PSMA Carboxypeptidase Activity Minoru Kawatani,†,∇ Kyoko Yamamoto,†,∇ Daisuke Yamada,§,∇ Mako Kamiya,*,†,∥ Jimpei Miyakawa,§ Yu Miyama,⊥ Ryosuke Kojima,†,∥ Teppei Morikawa,⊥ Haruki Kume,§ and Yasuteru Urano*,†,‡,#
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Graduate School of Medicine and ‡Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan § Department of Urology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan ∥ PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan ⊥ Department of Pathology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan # AMED-CREST, Japan Agency for Medical Research and Development, 1-7-1 Otemachi, Chiyoda-ku, Tokyo, 100-0004, Japan S Supporting Information *
ABSTRACT: Prostate cancer (PCa) is a common malignant tumor among adult males, and convenient intraoperative detection of PCa would reduce the risk of leaving positive surgical margins, especially during nerve-sparing procedures. To achieve rapid, fluorescence-based visualization of PCa, we focused on the glutamate carboxypeptidase (CP) activity of prostate-specific membrane antigen (PSMA), a type II transmembrane glycoprotein that is attracting attention as a PCa biomarker. Based on our finding that aryl glutamate conjugates with an azoformyl linker are recognized by PSMA and have a sufficiently low LUMO (lowest unoccupied molecular orbital) energy level to quench the fluorophore through photoinduced electron transfer, we designed and synthesized a first-inclass activatable fluorescence probe for CP activity of PSMA. The developed probe allowed us to visualize the CP activity of PSMA in living cells and in clinical specimens from PCa patients and is expected to be useful for rapid intraoperative detection and diagnosis of PCa.
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INTRODUCTION Prostate cancer (PCa) is one of the most prevalent male cancers worldwide. Radical prostatectomy, in which the entire prostate gland is resected and the bladder and urethra are sutured, is the standard operative strategy for PCa.1,2 However, patients often suffer from postoperative complications such as erectile dysfunction and urinary incontinence as a result of nerve damage or resection around the prostate. Nerve-sparing surgery reduces the risks of such complications3 but involves the risk of recurrence as a result of leaving positive surgical margins,4 since it is quite difficult to distinguish cancerous tissue from normal tissue visually. Therefore, a convenient method to detect cancerous lesions and to determine their precise localization in the tissue during operation would be extremely useful. Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII) or N-acetyl-L-aspartylL-glutamate peptidase I (NAALADase I), is a type II transmembrane glycoprotein that is overexpressed in PCa,5−7 and its expression level is positively correlated with the stage of disease and Gleason score.8,9 Thus, PSMA has attracted considerable attention recently as a target for imaging and therapy of PCa.10 So far, various PSMA ligands, including antibodies, aptamers, and low-molecular-weight ligands, have © 2019 American Chemical Society
been used to target imaging agents such as positron-emitting radionuclides for positron emission tomography (PET),11 a radionuclide for single-photon-emission computed tomography (SPECT), iron oxide for magnetic resonance imaging (MRI),12,13 and fluorophores for fluorescence imaging.14,15 In contrast to these “always-on” probes, there is another category of activatable probes for fluorescence imaging, whose fluorescence can be activated specifically at the tumor site.16−18 But, in spite of the great advantages of activatable probes, such as a high tumor-to-normal (T/N) signal ratio, only a few examples of PSMA-targeting activatable fluorescence probe based on PSMA antibody have been reported so far,19,20 and these do not show an adequate degree of activation and suitable activation kinetics for intraoperative imaging of PCa. Here, we newly focused on the carboxypeptidase (CP) activity of PSMA, which is elevated in PCa,21 and we set out to develop an activatable fluorescence probe with suitable properties for practical application. Although PSMA is known to hydrolyze the C-terminal glutamate of peptides such as N-acetyl-L-aspartyl-L-glutamate (NAAG) or poly-γReceived: April 24, 2019 Published: June 7, 2019 10409
DOI: 10.1021/jacs.9b04412 J. Am. Chem. Soc. 2019, 141, 10409−10416
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Journal of the American Chemical Society glutamated folate,22−24 most of the currently available methods for detecting CP activity are based on HPLC analysis or coupled assay.25−27 Very recently, our group reported for a first time a rational design strategy for activatable fluorescence probes to detect CP activity,28 in which structural conversion from aliphatic carboxamide to aliphatic carboxylate due to CP reaction is transformed into fluorescence change by utilizing the intramolecular spirocyclization of rhodamines. However, unfortunately, a glutamate-conjugated derivative based on this strategy was not recognized as a substrate by PSMA, which presumably has strict substrate specificity. Therefore, we required a different design strategy. Here, we report a newly established design strategy for activatable fluorescence probes to visualize CP activity. We further show that the developed probe can visualize the CP activity of PSMA in living cultured cells and in clinical specimens from PCa patients, and we believe it will be suitable for rapid intraoperative detection and diagnosis of PCa.
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RESULTS AND DISCUSSION Evaluation of Aryl Glutamate Conjugates as PSMA Substrates. In order to develop an activatable fluorescence probe for the CP activity of PSMA, we started by searching the literature for CP substrates in which substrate amino acids or peptides are conjugated to aryl groups via π-conjugation. We found that amino acids conjugated to aryl groups directly (amide bond29) or via self-immolative linkers such as urea,30 carbamate,29,30 or azoformyl31−33 can be recognized by various types of CPs, though it is not known whether human PSMA also recognizes these conjugates. Therefore, we newly prepared five aryl-glutamate conjugates 1-5 with several linkers (urea, 1 and 2; carbamate, 3; azoformyl, 4; amide, 5) and examined their reactivity with PSMA by means of fluorescent derivatization of the released glutamate with o-phthaldialdehyde and β-mercaptoethanol (Figure 1a,b and Scheme S1)34 using NAAG peptide as a positive control. We found that, among the compounds tested, only conjugate 4 with an azoformyl linker (Ph-AF-Glu) is recognized by PSMA and hydrolyzed to afford glutamate. Further, this reaction was inhibited in the presence of a PSMA inhibitor, 2(phosphonomethyl)pentanedioic acid (2-PMPA)35,36 (Figure 1b, Figure S1). Phenyl- or anisyl-azoformyl compounds have been developed as CPA (carboxypeptidase A) and CPB (carboxypeptidase B) substrates, enabling the enzyme reactions to be monitored by measuring the decrease of absorption at 350 nm due to loss of the azo chromophore.37 However, these azoformyl substrates are not suitable for livecell imaging of CP activity due to the limited sensitivity of absorption spectrophotometry. Considering that enzymecatalyzed hydrolysis of the amino acid of a phenylazoformyl conjugate is followed by self-decomposition to afford molecular carbon dioxide, nitrogen, and a phenyl group, together with the strong electron-withdrawing ability of the azoformyl group, we thought that CP-mediated hydrolysis would induce a significant change in the electron density of the aryl group, which could be translated into significant fluorescence activation via a photoinduced electron transfer (PeT) process. Specifically, we aimed to develop an activatable CP probe whose fluorescence would be quenched through a donor-excited PeT (d-PeT) process,38 i.e., electron transfer from the fluorophore to the phenyl group, but which would be converted to a highly fluorescent molecule after hydrolysis by the enzyme owing to a drastic change in the lowest unoccupied
Figure 1. Evaluation of aryl glutamate conjugates as PSMA substrates. (a) Chemical structures of aryl glutamate conjugates 1−5 and NAAG (endogenous substrate of PSMA). (b) In vitro reaction of conjugates 1−5 with recombinant human PSMA, followed by the fluorescent derivatization of released glutamate with o-phthalaldehyde with βmercaptoethanol. A 50 μM solution of conjugates 1−5 or NAAG was incubated with recombinant human PSMA (1.45 μg/mL) for 1 h at 37 °C in the absence or presence of 2-PMPA (1 μM), a specific inhibitor of PSMA. *P < 0.0001, **P < 0.001 (Student’s t test, n = 3). (c) Molecular design of activatable fluorescence probes for CP activity of PSMA based on the d-PeT process. The probe fluorescence is quenched by d-PeT before reaction with PSMA, but PSMA catalyzed hydrolysis of the glutamate moiety, followed by the release of molecular carbon dioxide and nitrogen, affords a highly fluorescent molecule.
molecular orbital (LUMO) energy level of the phenyl group (Figure 1c). First-in-Class Activatable Fluorescence Probe for PSMA. In order to examine whether the phenylazoformyl (Ph-AF) group works as an electron acceptor from the fluorophore, we calculated the LUMO energy levels of some Ph-AF derivatives and compared the values with those of compounds previously reported to quench the fluorescence of fluorescein through d-PeT. As the electron donor, we chose a xanthene moiety, the fluorophore of fluorescein derivatives. We found that the LUMO energy levels of the Ph-AF derivatives were lower than that of trimellitic acid trimethyl ester (Figure S2 and Table S1). Since the fluorescein derivative bearing trimellitic acid trimethyl ester as the benzene moiety has a low fluorescence quantum yield (QY) of 0.001,38 we 10410
DOI: 10.1021/jacs.9b04412 J. Am. Chem. Soc. 2019, 141, 10409−10416
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Journal of the American Chemical Society expected that a fluorescein derivative bearing Ph-AF as the benzene moiety would show suppressed fluorescence. Encouraged by this result, we prepared two fluorescein derivatives, 4GluAF-Fl and 5GluAF-Fl, in which glutamate was conjugated to a benzene moiety through an AF linker at the 4 and 5 position, respectively (Figure 2a and Scheme S2).
Table 1. Photophysical Properties of the Developed Fluorescence Probes for CP Activity of PSMA and the Putative Hydrolysis Products compd 4GluAF-Fla 5GluAF-Fla 5GluAF-FMa 5GluAF2MeTGa Fluoresceinb FMc 2MeTGb
absorbance maximum (nm)
emission maximum (nm)
fluorescence quantum yield
494 494 496 495
521 526 521 516
0.003 0.002 0.004 0.002
492 494 491
511 518 510
0.85 0.76 0.85
a c
Measured in 0.2 M sodium phosphate buffer, pH 7.4. bReference 41. Reference 38.
fluorescent fluorescein, together with small quantity of byproducts, while 4GluAF-Fl was not hydrolyzed (Figure S3). It has been reported that compounds with structural similarities to PSMA substrates are not always recognized by PSMA;25,39 thus, the difference in reactivity between 4GluAFFl and 5GluAf-Fl presumably reflects strict substrate specificity of PSMA. The PSMA-catalyzed hydrolysis of 5GluAF-Fl was inhibited in the presence of 2-PMPA (Figure 2b and Figure S4a). Next, in order to examine whether 5GluAF-Fl shows reactivity toward cancer cells expressing PSMA, we incubated 5GluAF-Fl with lysates of two PCa cell lines, LNCaP cells (PSMA-positive) and PC3 cells (PSMA-negative). 5GluAF-Fl showed a large fluorescence increase when incubated with LNCaP cell lysate, but not when incubated with PC3 cell lysate or in the presence of 2-PMPA (Figure 2c). Further, when 5GluAF-Fl was applied to living LNCaP and PC3 cells seeded in 96-well plates and the fluorescence change from each well was recorded on a plate reader, fluorescence activation was observed only in the presence of LNCaP cells, suggesting that the CP activity of PSMA in living cells can be successfully monitored with 5GluAF-Fl (Figure S5). However, when we performed live-cell imaging using confocal fluorescence microscopy, we did not observe a marked fluorescence increase inside LNCaP cells (Figure 2d, Figure S6). Considering that the active site of PSMA is located in the extracellular domain40 and the hydrolysis product, fluorescein, is highly hydrophilic, it seems likely that 5GluAF-Fl reacted with PSMA on the cell surface to produce cell-impermeable fluorescein, resulting in a fluorescence increase exclusively in the extracellular space. Thus, we need to improve the cell permeability of the reaction product to visualize PSMApositive cells. Live-Cell Imaging of the CP Activity of PSMA. For improving the cell permeability of the reaction product, we modified 5GluAF-Fl by changing the 2′-substituent from carboxylic acid to methoxycarbonyl or methyl, affording 5GluAF-FM or 5GluAF-2MeTG, which would produce a more hydrophobic hydrolysis product: fluorescein methyl ester (FM) or 2′Me TokyoGreen (2MeTG),41 respectively (Figure 3a and Schemes S3 and S4). The photophysical properties indicated that the fluorescence of these derivatives was well quenched, and the fluorescence QYs were as low as that of 5GluAF-Fl (Table 1). We confirmed that both probes can be hydrolyzed by recombinant PSMA and PSMA in cell lysate to produce 2MeTG and FM; the hydrolysis kinetics were in the order 5GluAF-2MeTG > 5GluAF-FM > 5GluAF-Fl (Figure
Figure 2. First-in-class activatable fluorescence probe for PSMA. (a) Chemical structures of 4GluAF-Fl and 5GluAF-Fl and the enzymatic reaction with PSMA. (b) Fluorescence spectra of 4GluAF-Fl (left) and 5GluAF-Fl (right) before and after reaction with recombinant human PSMA. A 10 μM probe solution in tris-buffered saline, pH 7.4, was incubated with recombinant human PSMA (0.44 μg) for 10 h at 37 °C in the absence or presence of 2-PMPA (10 μM) and then diluted in tris-buffered saline, pH 7.4, to give a final probe concentration of 0.67 μM for fluorescence measurement. (c) Time course of fluorescence activation of 5GluAF-Fl upon addition of lysate from LNCaP cells (PSMA-positive) or PC3 cells (PSMA-negative). A 10 μM probe solution was incubated with cell lysate (0.36 mg/mL) for 10 h at 37 °C in the absence or presence of 2-PMPA (10 μM). Error bars represent standard deviation (SD) from a single experiment in triplicate. (d) Live-cell imaging of LNCaP cells or PC3 cells treated with 5GluAF-Fl by confocal microscopy. Cells were incubated with 10 μM 5GluAF-Fl for 5 h at 37 °C, and bright-field (BF) and fluorescence images were captured. Ex/em: 490/500−600 nm. Scale bars, 50 μm.
As expected, both 4GluAF-Fl and 5GluAF-Fl are well quenched, and the fluorescence QY values were calculated to be 0.003 and 0.002, respectively (Table 1). When incubated with recombinant human PSMA, 5GluAF-Fl showed a large fluorescence increase (>200 fold), while 4GluAF-Fl did not show fluorescence activation (Figure 2b). LC−MS analysis confirmed that 5GluAF-Fl was mainly converted to highly 10411
DOI: 10.1021/jacs.9b04412 J. Am. Chem. Soc. 2019, 141, 10409−10416
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Figure 3. Live-cell imaging of CP activity of PSMA. (a) Chemical structures of 5GluAF-FM and 5GluAF-2MeTG, and the enzymatic reaction with PSMA. (b) Fluorescence spectra of 5GluAF-FM (left) and 5GluAF-2MeTG (right) before and after reaction with recombinant human PSMA. A 10 μM probe solution in tris-buffered saline, pH 7.4, was incubated with recombinant human PSMA (0.44 μg) for 10 h at 37 °C in the absence or presence of 2-PMPA (10 μM) and then diluted in tris-buffered saline, pH 7.4, to make a final probe concentration of 0.67 μM for fluorescence measurement. (c) Time courses of fluorescence activation of 5GluAF-FM (left) and 5GluAF-2MeTG (right) upon addition of lysate from LNCaP cells (PSMA-positive) or PC3 cells (PSMA-negative). A 10 μM probe solution was incubated with cell lysate (0.36 mg/mL) for 10 h at 37 °C in the absence or presence of 2-PMPA (10 μM). Error bars represent standard deviation (SD) from a single experiment conducted in triplicate. (d) Live cell imaging of LNCaP cells and PC3 cells treated with 5GluAF-FM (left) and 5GluAF-2MeTG (right) by confocal microscopy. Cells were incubated with 10 μM 5GluAF-FM or 5GluAF-2MeTG for 5 h at 37 °C, and bright-field (BF) and fluorescence images were captured. Ex/em: 490/500−600 nm. Scale bars, 50 μm.
could visualize PCa in clinical tissue specimens. Resected specimens from prostate cancer patients who had not received any hormone therapy, chemotherapy, or radiotherapy were collected during radical prostatectomy. The specimens were incubated with a 50 μM solution of 5GluAF-2MeTG on a heating plate (40 °C) for 30 min, which would be an acceptable duration for incorporating this imaging into actual surgical procedures, and the fluorescence images were captured with a Maestro fluorescence imaging system. Although there was a substantial fluorescence signal at 0 min due to the combination of tissue autofluorescence and background fluorescence of the probe, we observed heterogeneous fluorescence activation in the specimens during 30 min (Figure 4a and Figure S11). In order to examine the correlation between the fluorescence activation and the results of pathological/immunohistochemical analysis, several representative ROIs (regions of interest) were selected, cut out, and pathologically examined for the existence of PCa and the histological grade (Gleason score).42 In addition, the PSMA expression level was determined by immunohistochemical analysis. We found that most of the strongly fluorescent
3b,c, Figures S3b,c and S7 and Table S2). Further, when we performed live-cell imaging of LNCaP cells, 5GluAF-2MeTG showed a significant fluorescence increase in the cells, while 5GluAF-FM did not (Figure 3d and Figure S8). LC−MS analysis of the extracellular solution revealed that 2MeTG was mainly produced during incubation of 5GluAF-2MeTG, while in the case of 5GluAF-FM (m/z = 548), we observed a mass peak whose molecular weight is higher by 2 units (m/z = 550), suggesting that 5GluAF-FM tends to be reduced in the cellular environment (Figure S9). The difference in susceptibility to reduction might be accounted for by the difference in the LUMO energy level of the azoformyl benzene moiety (Figure S2). The observed fluorescence activation of 5GluAF-2MeTG in LNCaP cells was inhibited in the presence of 2-PMPA (Figure S10), and no activation was observed when the probe was applied to PC3 cells (Figure 3d and Figure S8). These results demonstrated that 5GluAF-2MeTG is a first-in-class activatable fluorescence probe for the CP activity of PSMA and is capable of visualizing PSMA-expressing living cells. Fluorescence Detection of PCa in Resected Clinical Specimens. We next examined whether 5GluAF-2MeTG 10412
DOI: 10.1021/jacs.9b04412 J. Am. Chem. Soc. 2019, 141, 10409−10416
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Figure 4. Ex vivo fluorescence imaging of PCa in surgically resected clinical specimen from prostate cancer patients. (a) White-light and fluorescence images of resected tissue after incubation with 5GluAF-2MeTG for 0 and 30 min. The specimen was incubated with a 50 μM solution of 5GluAF-2MeTG in tris-buffered saline, pH 7.4, for 30 min on a heating plate (40 °C). Fluorescence images were captured with a Maestro InVivo imaging system (PerkinElmer) using the blue-filter setting (excitation, 465/30 nm; emission, 515 nm long-pass). The fluorescence image at 540 nm was extracted and presented. The specimen corresponds to case 4 in (e). Scale bars, 1 cm. (b) Time course of fluorescence activation during incubation for 30 min with 5GluAF-2MeTG at the ROIs indicated in (a). (c) Table of fluorescence increase, cancer existence, Gleason score, and PSMA expression level at each ROI. (d) Representative images of hematoxylin and eosin (HE) staining and immunohistochemistry (IHC) of PSMA. Images at ROI #7 and ROI #1 are presented as a cancer region with high PSMA expression and a normal region with low PSMA expression, respectively. Asterisks (*) indicate stromal cells, in which PSMA expression was not observed. Scale bars, 100 μm. (e) Fluorescence increase at ROIs in 4 clinical specimens incubated with 5GluAF-2MeTG were analyzed and ordered by activity. Each case was from a different prostate cancer patient (cases 1−4), and # represents the ROI number in each specimen. Red bars show cancer-containing regions, and blue bars show noncancerous regions.
regions contained cancer strongly expressing PSMA, while weakly fluorescent regions were mostly normal prostate tissue or cancer-containing regions with weak PSMA expression (Figure 4b−d and Figure S11). We also confirmed the absence of PSMA expression in stromal cells (Figure 4d). Figure 4e summarizes the results of applying 5GluAF-2MeTG to four clinical specimens, arranged in descending order of fluorescence increase. Although there are some false-negatives and false-positives, probably due to limited PSMA expression in cancer tissue and substantial PSMA expression in normal prostate tissue, the fluorescence activation of 5GluAF-2MeTG after incubation for 30 min was mostly well correlated with the presence of cancer, reflecting the expression level of PSMA in tissues. It is particularly noteworthy that tiny millimeter-sized cancer regions such as ROI #7 in Figure 4 or ROI #6 in Figure S11a can be clearly visualized as fluorescent spots; such small
cancer regions would be difficult to detect by means of conventional preoperative diagnostic methods such as CT and MRI. These results demonstrate that 5GluAF-2MeTG can detect the CP activity of PSMA even in small cancer regions of clinical specimens, and therefore should be useful as an intraoperative diagnostic tool for PCa.
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CONCLUSION In conclusion, we have designed and synthesized 5GluAF2MeTG as a first-in-class activatable fluorescence probe for CP activity of PSMA and succeeded in applying it for fluorescence visualization of PCa in cultured cells and in clinical specimens. We first established that Ph-AF-Glu is a substrate of PSMA and then developed 5GluAF-Fl, whose fluorescence was controlled by d-PeT. But, although 5GluAF-Fl showed significant fluorescence activation upon reaction with PSMA, it failed to 10413
DOI: 10.1021/jacs.9b04412 J. Am. Chem. Soc. 2019, 141, 10409−10416
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Journal of the American Chemical Society
fluorescence microscope (TCS SP 8 STED, Leica). After imaging, the extracellular solution was collected and centrifuged at 15000 rpm at 4 °C, and the supernatant was stored at −80 °C for LC−MS analysis. Prostate Cancer Patients. This study was conducted with the approval of the Research Ethics Committee of the University of Tokyo (registration number: 11623). All experiments were performed in accordance with guidelines and regulations approved by the Research Ethics Committee of the University of Tokyo. Informed consent was obtained from all patients. Prostate cancer patients examined or treated at the University of Tokyo Hospital in Tokyo, Japan, were prospectively included in this study. Ex Vivo Fluorescence Imaging of Freshly Resected Prostate Specimens. Surgical specimens were rinsed with tris-buffered saline, pH 7.4, and a 50 μM solution of 5GluAF-2MeTG in tris-buffered saline, pH 7.4, was added to cover the entire specimen, which was then incubated on a heating plate (40 °C). Fluorescence images were captured with a Maestro In-Vivo imaging system (PerkinElmer) for 30 min, using the blue-filter setting (excitation, 465/30 nm; emission, 515 nm long-pass). Histological Analysis and Immunohistochemistry. Resected specimens were evaluated pathologically. Hematoxylin and eosin (HE) staining was used to confirm the presence of cancer and the histological grade (Gleason score),42 together with immunohistochemistry (IHC), to determine the PSMA expression level. IHC staining was performed by an automated IHC slide-staining instrument (Ventana BenchMark XT, Roche), using primary antibody against PSMA (mouse monoclonal antibody, clone 3E6, Dako M3620) at 1:200 dilution and an I-VIEW DAB universal kit (760− 041, Roche). 3,3′-Diaminobenzidine (DAB) was used as a chromogen, and hematoxylin was applied for counterstaining. PSMA expression level in epithelial cells (either normal or cancerous) was scored from 1 (weak) to 4 (strong) by a pathologist who was blinded to the results of fluorescence imaging.
visualize PSMA-positive cells, apparently due to poor cellular permeability of the hydrolysis product. Therefore, we modified the chemical structure to afford 5GluAF-2MeTG, which produces cell-permeable 2MeTG as its PSMA-catalyzed hydrolysis product, making it possible to visualize PSMApositive cells. To our knowledge, this is the first example of an activatable fluorescence probe detecting the CP activity of PSMA in living cells. When 5GluAF-2MeTG was applied to resected specimens from PCa patients, we observed significant fluorescence activation, which corresponded well with the pathological localization of PCa. These results demonstrate the potential utility of our probe to visualize PCa in clinical situations. Nerve-sparing surgery is an important treatment option to preserve nerve function and urinary function postoperatively in cases where invasion of cancer into the neurovascular bundles can be ruled out based on preoperative diagnostics such as ultrasound, CT, MRI, and needle biopsy. However, it is difficult to detect all cancerous tissue preoperatively, especially tiny regions, and so there has been concern about the risk of increased rates of recurrence if positive surgical margins are left during nerve-sparing radical prostatectomy. In this context, our newly developed probe might be useful to improve the accuracy of intraoperative cancer detection, enabling efficient nerve-sparing by clearly distinguishing cancerous regions near the neurovascular bundle. Thus, we believe that our probe might enable more confident minimally invasive prostate resection, without increased risk of complications such as erectile dysfunction and urinary incontinence. Furthermore, it was recently reported that PSMA is highly expressed in the neovasculature of other cancers, such as breast cancer,43 lung cancer,44 gastric cancer,45 colorectal cancer,45 and bladder cancers,46 and it is also expressed in a variety of neurological diseases.47 Thus, we think that 5GluAF-2MeTG could also be useful as a tool to study the roles of PSMA in the pathophysiology of these diseases, as well as in the development of therapeutics. Our newly established design strategy should also be applicable to develop activatable fluorescence probes for other carboxypeptidase activities by replacing the substrate moiety as well as red-shifted analogues by changing the fluorophore.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.9b04412. Synthesis, experimental details, and photophysical properties of the compounds (PDF)
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AUTHOR INFORMATION
Corresponding Authors
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Synthesis. For synthetic protocols, see the Supporting Information. Cell Lines and Culture. LNCaP and PC3 cells were obtained from RIKEN Cell Bank. Both cell lines were cultured in RPMI 1640 medium (Wako) supplemented with 10% (v/v) FBS and 1% (v/v) penicillin streptomycin (Invitrogen) in an incubator at 37 °C under 5% CO2 in air. Live-Cell Confocal Imaging. An eight-well chamber (μ-slide, eight-well; Ibidi) was coated with 30 μg/mL fibronectin solution (063-05591, Wako) in DPBS (−) at 37 °C for 2 h. Then LNCaP cells or PC3 cells were seeded at 2.5 × 104 cells per well and cultured overnight at 37 °C under 5% CO2 in air. The medium of each well was replaced with 150 μL of HBSS (+), then 50 μL of a 40 μM probe solution in HBSS (+) was added to each well, and incubation was continued under the same conditions. Fluorescence images were captured for 5 h with a confocal fluorescence microscope (TCS SP 8 STED, Leica) equipped with a CO2 incubator. Excitation and emission wavelengths were 490 nm and 500−600 nm, respectively. HC PL APO CS2 40x/1.30 Oil was used as the objective lens. For the inhibitor assay, the cells were incubated with 10 μM probe solution in HBSS (+) in the presence of 10 μM 2-PMPA at 37 °C under 5% CO2 in air for 15 h. Fluorescence images were captured with a confocal
Mako Kamiya: 0000-0002-5592-1849 Yasuteru Urano: 0000-0002-1220-6327 Author Contributions ∇
M.K., K.Y., and D.Y. contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported in part by AMED/P-CREATE under Grant No. JP18 cm0106409 (to Y.U.), by JST/PRESTO Grant No. JPMJPR14F8 (to M. Kamiya), by MEXT/JSPS KAKENHI Grant Nos. JP16H02606, JP26111012 (to Y.U.), and JP15H05951 “Resonance Bio” and JP19H02826 (to M. Kamiya), by JSPS Core-to-Core Program, A. Advanced Research Networks, by Astellas Foundation for Research on Metabolic Disorders (to M. Kamiya), and Japan Foundation for Applied Enzymology (to M. Kamiya), as well as a stipend from Graduate Program for Leaders in Life Innovation (GPLLI) (to M. Kawatani) and a JSPS stipend (to K.Y.). 10414
DOI: 10.1021/jacs.9b04412 J. Am. Chem. Soc. 2019, 141, 10409−10416
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Journal of the American Chemical Society
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The authors thank Dr. Yusuke Saito, Dr. Taketo Kawai, Dr. Masaki Nakamura, Dr. Motofumi Suzuki, and Dr. Akihiko Matsumoto for their support in collecting clinical specimens.
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