Synthesis and characterization of [¹²⁵I]TZ6544, a promising radioligand for investigating sphingosine-1-phosphate receptor 2

Abstract

Introduction:

Sphingosine-1-phosphate receptor 2 (S1PR2) activation exerts a critical role in biological abnormalities and diseases. A suitable radiotracer will advance our understanding of S1PR2 pathophysiology of diseases. The objective of this study is to evaluate the potential of iodine-125 labeled [¹²⁵I]TZ6544 to be used for screening new compounds binding toward S1PR2, and assessing the changes of S1PR2 expression in the kidney of streptozotocin-induced diabetic rats.

Methods:

[¹²⁵I]TZ6544 was synthesized from borate precursor by copper (II)-catalyzed iodization reaction with [¹²⁵I]NaI. [¹²⁵I]TZ6544 was characterized using human recombinant S1PR2 cell membrane and biodistribution studies of [¹²⁵]TZ6544 were performed on Wistar rats that were euthanized at 5 and 30 minutes post-injection. A rat model of diabetes was induced by IV injection of streptozotocin (55 mg/kg). In vitro autoradiography studies, immunostaining, and enzyme-linked immunosorbent assay (ELISA) analysis were performed in both diabetic and control rats.

Results:

Radiosynthesis of [¹²⁵I]TZ6544 was achieved successfully with good radiochemical yields of ~47% and high radiochemical purity of >99%. [¹²⁵I]TZ6544 is a potent ligand in vitro for S1PR2 with K_d value of 4.31 nM. [¹²⁵I]TZ6544 and [³²P]-labelled endogenous S1P provided comparable IC₅₀ values in radioactive competitive binding assays against known S1PR2 ligands. Compared to control, the kidney of diabetic rats had increased uptake of [¹²⁵I]TZ6544, which could be reduced by a S1PR2 antagonist, JTE-013. Immunostaining and ELISA analysis confirmed that the diabetic rat had increased S1PR2 expression in the kidney.

Conclusions:

[¹²⁵I]TZ6544 was synthesized successfully in high yields, and in vitro evaluation suggested [¹²⁵I]TZ6544 has high potential to be used for screening new S1PR2 compounds and investigating the pathophysiology of S1PR2 functions. The available of [¹²⁵I]TZ6544 may facilitate the development of therapeutics and imaging agents targeting S1PR2.

Advances in Knowledge:

[¹²⁵I]TZ6544 showed increased expression of S1PR2 in diabetic rat kidney and can be used to determine binding potency of S1PR2 compounds.

1. Introduction

Sphingosine-1-phosphate receptor 2 (S1PR2), originally known as endothelial differentiation G-protein coupled receptor 5, was cloned from rat aortic vascular smooth muscle cells and later identified as a high-affinity sphingosine-1-phosphate receptor (S1PR) [1–2]. S1PR2 belongs to a superfamily of S1PR1–5. S1PR2 couples with G_i, G_q, and G_12/13 group of G proteins, which modulate various cellular signaling pathways including cell growth and survival, migration, and adhesion in inflammation, fibrosis, and cancer [3]. Diabetic nephropathy (DN) is characterized by the progressive damage and death of glomerular podocytes, resulting in exudative lesions in glomeruli, renal sclerosis, and renal fibrosis [4–6]. Recent data indicate that activation of sphingosine-kinase-1/S1P/S1PR2 signaling pathway contributes to the development of DN [7–11]. The mRNA and protein levels of S1PR2 receptor was enhanced ~11 and 13-fold in the experimental models of diabetes[12] and the S1PR2 antagonist, JTE-013 (Fig. 1) can attenuate the development of diabetes in animals [12–13].

Fig. 1.

The structures of JTE-013, CYM-5520, and the radiolabeled probes [¹¹C]TZ34125 and [¹²⁵I]TZ6544.

Examination of the pathophysiology of S1PR2 has been hindered due to the lack of highly potent S1PR2 probes. JTE-013 was widely used in preclinical investigations [14–15]; CYM-5520 (Fig 1) was reported as a selective S1PR2 agonist [16]; other S1PR2 compounds are undergoing further characterization [17–20]. A radioactive competitive binding assay that uses a radioligand to determine the binding potency of compounds toward a particular protein (i.e. receptor) can be highly a sensitive and reliable tool for screening new therapeutic candidates. Our group previously reported using a [³²P]S1P competitive binding assay [21] to identify several potent and selective ligands for S1PR1 or S1PR2 [22–28]. However, due to the short half-life of P-32 (t_1/2 = 14 days), [³²P]S1P required to be freshly prepared monthly, which is inefficient for scale-up screening new compounds. In contrast, binding assays using tritiated (H-3 t_1/2 = 12.32 years) or iodinated (I-125 t_1/2 = 60 days) ligands are more efficient for routinely screening new S1PR2 compounds. In addition, ¹²⁵I-labeled radioligands have advantages in characterization of protein expression in tissues because of its half-life, high specific activity, higher counting rates, and cost-efficiency [29]. Herein, we reported our efforts on the radiosynthesis and initial characterization of [¹²⁵I]TZ6544, a highly potent and selective S1PR2 radiotracer [30]. Streptozotocin (STZ) is a chemical that has preferential toxicity toward pancreatic β cells of animals and is widely used to generate rodent models of type 1 diabetes. Therefore, we performed the in vitro autoradiography studies of [¹²⁵I]TZ6544 on the kidney sections of diabetic rats induced by STZ [31–32]. In addition, immunostaining, and enzyme-linked immunosorbent assay (ELISA) analysis of S1PR2 expression in the rat kidney confirmed the increase [¹²⁵I]TZ6544 binding in the kidney of diabetic rats was resulted from the elevated S1PR2 expression. Our data suggest that [¹²⁵I]TZ6544 provides a promising radiotracer for screening S1PR2 compounds. The rodent autoradiography, immunohistochemistry, and ELISA studies support the changes of S1PR2 expression in diabetic rats that can be assessed in vitro using [¹²⁵I]TZ6544.

2. Material and Methods

2.1. Chemistry

2.1.1. General

Commercially available starting materials, reagents, and solvents were used directly unless otherwise stated. Reactions were monitored by thin-layer chromatography (TLC) carried out on pre-coated glass plates of silica gel 60 F₂₅₄ (Millipore Sigma, Billerica, MA). Visualization was accomplished with ultraviolet light (UV 254 nm). Flash column chromatography was performed using 230–400 mesh silica (SiliCycle Inc., Quebec, Canada). All work-up and purification procedures were carried out with reagent grade solvents. Yields refer to isolate yield; melting points were determined on a MEL-TEMP 3.0 apparatus. ¹H NMR and ¹³C NMR spectra were recorded on a Varian 400 MHz instrument. Chemical shifts are reported in parts per million (ppm) and are calibrated using residual undeuterated solvent as an internal reference. Data are reported as follows: chemical shift, multiplicity, coupling constants (Hz), and integration.

Sodium [¹²⁵I]iodide (specific activity ~629 GBq/mg) in NaOH (10⁻⁵ M) was purchased from Perkin Elmer Life and Analytical Sciences (Boston, MA). Preparative high-performance liquid chromatography (HPLC) was performed using a semi-preparative reverse-phase C18 analytical column (Agilent ZORBAX Eclipse XDB-C18, 250 X 9.4 mm, 5 μm) with UV wavelength at 254 nm and flow rate at 4 mL/min; analytical HPLC was performed on a reverse-phase C18 analytical column (Agilent ZORBAX Eclipse XDB-C18, 250 X 4.6 mm, 5 μm) with UV wavelength at 254 nm and flow rate at 1 mL/min; radioactive detection was carried out using a Bioscan Flowcount radioactive detector (Bioscan Inc, Washington DC); preparative and analytical HPLC were run with a mobile phase of 0.1 M ammonium formate buffer (pH = ~4.5) in acetonitrile.

2.1.2. Synthesis of 5-iodo-2,4-dimethoxyaniline (2)

37% HCl (0.21 mL) diluted in ethanol (3.0 mL) and H₂O (1.5 mL) was added dropwise to a mixture of 1-iodo-2,4-dimethoxy-5-nitrobenzene (1.10 g, 3.6 mmol), iron powder (0.84 g, 15.0 mmol), ethanol (17.5 mL), and H₂O (7.5 mL). The reaction mixture was refluxed for 1 h and monitored by TLC until complete. After cooling to room temperature, the mixture was filtered and the filtrate was concentrated. 5% Na₂CO₃ solution (20 mL) was added to the residue, which was then extracted with ethyl acetate (15 mL x 3). The combined ethyl acetate extracts were washed with saturated brine (20 mL) and dried over anhydrous MgSO₄. After filtration and concentration, the crude product 2 was obtained and used directly for the next step. (0.8 g, 81%) ¹H NMR (400 MHz, CDCl₃) δ 7.10 (s, 1H), 6.44 (s, 1H), 3.85 (s, 3H), 3.82 (s, 3H), 3.54 (br, 2H).

2.1.3. Synthesis of 2-bromo-N-(5-iodo-2,4-dimethoxyphenyl)acetamide (3)

Bromoacetyl bromide (0.60 g, 3.0 mmol) was added dropwise to a solution of 2 (0.80 g, 2.9 mmol) in dichloromethane (20 mL) at 0 °C. After stirring for 5 min, triethylamine (0.60 g, 5.8 mmol) was added and the reaction mixture was warmed to room temperature and stirred overnight. The reaction was monitored by TLC until complete. The mixture was then concentrated and purified by flash chromatography, eluted with hexane/ethyl acetate (3/2, V/V), to afford the intermediate 3 (0.43 g, 37%) as a gray solid. ¹H NMR (400 MHz, CDCl₃) δ 8.67 (s, 1H), 8.53 (s, 1H), 6.46 (s, 1H), 4.01 (s, 2H), 3.93 (s, 3H), 3.88 (s, 3H).

2.1.4. Synthesis of 2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)-N-(4-methoxyphenyl)-acetamide (5)

A mixture of 2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)acetic acid (4) (1.10 g, 5.0 mmol), p-anisidine (0.67 g, 5.5 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (2.85 g, 7.5 mmol), N,N-diisopropylethylamine (DIPEA) (1.61 g, 12.5 mmol), and dichloromethane (20 mL) was stirred at room temperature overnight. The mixture was then concentrated and the residue treated with 1 N HCl (50 mL), the undissolved solid was collected and dried to afford the intermediate 5 (1.0 g, 61%) as a white solid. ¹H NMR (400 MHz, DMSO-d6) δ 11.56 (s, 1H), 10.11 (s, 1H), 7.94 (d, J = 7.9 Hz, 1H), 7.69 (t, J = 7.7 Hz, 1H), 7.47 (d, J = 8.5 Hz, 2H), 7.22 (d, J = 8.2 Hz, 2H), 6.88 (d, J = 8.6 Hz, 2H), 4.67 (s, 2H), 3.71 (s, 3H).

2.1.5. Synthesis of N-(5-iodo-2,4-dimethoxyphenyl)-2-(3-(2-((4-methoxyphenyl)amino)-2-oxoethyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)acetamide (TZ6544)

A mixture of 5 (163 mg, 0.5 mmol), 3 (200 mg, 0.5 mmol), K₂CO₃ (138 mg, 1.0 mmol), and dimethylformamide (3.0 mL) was stirred at room temperature overnight. The mixture was then diluted with H₂O (10 mL), and the precipitate was collected and washed with acetone (10 mL). After air drying, the standard compound TZ6544 was obtained (120 mg, 37%) as a white solid. Mp 260–261 °C. ¹H NMR (400 MHz, DMSO-d6) δ 10.16 (s, 1H), 9.69 (s, 1H), 8.21 (s, 1H), 8.10 (d, J = 7.8 Hz, 1H), 7.80 (t, J = 7.9 Hz, 1H), 7.47 (d, J = 8.5 Hz, 2H), 7.43 – 7.32 (m, 2H), 6.88 (d, J = 8.4 Hz, 2H), 6.77 (s, 1H), 5.07 (s, 2H), 4.75 (s, 2H), 3.91 (s, 3H), 3.85 (s, 3H), 3.72 (s, 3H). ¹³C NMR (101 MHz, DMSO-d6) δ 167.01, 166.11, 162.42, 156.72, 153.01, 151.97, 151.28, 141.70, 137.06, 133.35, 129.41, 124.62, 124.22, 122.02, 121.70, 116.24, 116.00, 115.38, 112.43, 99.45, 57.87, 57.83, 56.61, 47.74, 45.45.

2.1.6. Synthesis of 2,4-dimethoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (7)

A mixture of 5-bromo-2,4-dimethoxyaniline (0.46 g, 2.0 mmol), bis(pinacolato)diboron (1.0 g, 4.0 mmol), [1,1’‑bis(diphenylphosphino)ferrocene]palladium(II) dichloride (Pd(dppf)Cl₂) (0.15 g, 0.2 mmol), potassium acetate (1.18 g, 12.0 mmol), and dimethyl sulfoxide (DMSO) (4.0 mL) was stirred at 90 °C overnight. The reaction was monitored by TLC until completed. After cooling to room temperature, the mixture was diluted with ethyl acetate (10 mL) and filtered through Celite®. The filtrate was washed with H₂O (20 mL x 2), saturated brine (20 mL), and dried over anhydrous MgSO₄. After filtration and concentration, the crude product was purified by flash chromatography, eluted with hexane/ethyl acetate (2/1, V/V) to afford the product 7 (0.39 g, 70%). ¹H NMR (400 MHz, CDCl₃) δ 7.07 (s, 1H), 6.43 (s, 1H), 3.87 (s, 3H), 3.79 (s, 3H), 3.48 (s, 2H), 1.33 (s, 12H).

2.1.7. Synthesis of 2-bromo-N-(2,4-dimethoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetamide (8)

Bromoacetyl bromide (280 mg, 1.4 mmol) was added dropwise to a solution of 7 (390 mg, 1.4 mmol) in dichloromethane (15 mL) at 0 °C. After stirring for 5 min, trimethylamine (280 mg, 2.8 mmol) was added; the reaction was then warmed to room temperature and stirred overnight. The reaction was monitored by TLC until completed. The mixture was concentrated and the crude product was purified by flash chromatography, eluted with hexane/ethyl acetate (1/1, V/V), to afford the intermediate 8 (160 mg, 29%). ¹H NMR (400 MHz, CDCl₃) δ 8.47 – 8.35 (m, 2H), 6.43 (s, 1H), 4.00 (s, 2H), 3.90 (s, 3H), 3.82 (s, 3H), 1.31 (s, 12H).

2.1.8. Synthesis of N-(2,4-dimethoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-2-(3-(2-((4-methoxyphenyl)amino)-2-oxoethyl)-2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)acetamide (9)

A mixture of 8 (160 mg, 0.4 mmol), 5 (130 mg, 0.4 mmol), K₂CO₃ (110 mg, 0.8 mmol), and dimethylformamide (3.0 mL) was stirred at room temperature overnight. The mixture was then diluted with ethyl acetate (10 mL), washed with H₂O (20 mL x 2), saturated brine (20 mL), and dried over anhydrous MgSO₄. After filtration and concentration, the crude product was purified by flash chromatography, eluted with hexane/ethyl acetate (1/3, V/V), to afford the precursor product 9 (100 mg, 39%) as white solid. MP: 270–271 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.40 (s, 1H), 8.22 (d, J = 7.7 Hz, 1H), 8.11 (s, 1H), 7.68 (t, J = 7.3 Hz, 1H), 7.59 (s, 1H), 7.38 (t, J = 9.4 Hz, 3H), 7.28 (t, J = 7.6 Hz, 1H), 6.79 (d, J = 9.1 Hz, 2H), 6.35 (s, 1H), 4.91 (s, 4H), 3.83 – 3.70 (m, 9H), 1.28 (s, 12H). ¹³C NMR (101 MHz, DMSO-d6) δ 165.95, 165.05, 161.36, 155.66, 151.96, 150.92, 150.22, 140.64, 136.01, 132.30, 128.35, 123.56, 123.17, 120.96, 120.65, 115.18, 114.94, 114.32, 111.37, 98.39, 56.82, 56.77, 55.55, 46.68, 44.39.

2.2. Radiochemistry

40 μL of precursor (9) in acetonitrile (15 μmol/L) and 10 μL of tetrakis(pyridine)copper(II) triflate/3,4,7,8-tetramethyl-1,10-phenanthroline in methanol (1/1, 12 μmol/L), were added to a 1.5 mL of microcentrifuge tube; this was followed by adding ~40 MBq of [¹²⁵I]NaI (pH 8–11). The reaction mixture was vortexed intermittently and incubated at room temperature for 10 min before loading onto a semi-preparative HPLC reverse phase column (Agilent ZORBAX Eclipse XDB-C18, 250 X 9.4 mm, 5 μm). The HPLC mobile phase was 55% 0.1 M ammonium formate (pH 4.5) in acetonitrile and the retention time for the product was ~21 min. The collected HPLC fraction was diluted with 50 mL of sterile water and the activity was trapped on a Sep-Pak C18 Plus Short Cartridge (Waters, Milford, MA) which was then washed with 20 mL of sterile water. The desired product [¹²⁵I]TZ6544 was eluted from the C-18 cartridge with 0.6 mL of ethanol. The reaction afforded ~18.5 MBq of the desired product which was authenticated by HPLC and co-injecting the cold standard compound. The average radiochemical yield was 47 ± 8% (n = 3). Radiochemical purity was determined by analytical HPLC on a reverse-phase analytical column (Agilent ZORBAX Eclipse XDB-C18, 250 X 4.6 mm, 5 μm), mobile phase 30% 0.1 M ammonium formate (pH 4.5) in acetonitrile; the retention time for the product was ~6.5 min. Because the [¹²⁵I]NaI is carrier-free, the molar activity of [¹²⁵I]TZ6544 can be equal to the molar activity of [¹²⁵I]NaI (~93 GBq/μmol) received from the supplier.

2.3. In vitro binding studies

2.3.1. The binding potency of TZ6544 toward S1PRs determined using [³²P]S1P

The binding potency of TZ6544 for S1PR2 was first determined by a [³²P]S1P competitive binding assay using our previously reported procedure [21]. Human recombinant S1PR1, 2, 3, and 5 lysophospholipid receptor membranes were purchased from Millipore (Millipore, Billerica, MA) and human recombinant S1PR4 lysophospholipid receptor membrane was purchased from Muiltispan (Multispan, Hayward, CA). Briefly, increasing concentrations of TZ6544 (0.01, 0.1, 1.0, 10, 100, and 1000 nM) were incubated with S1PR2 lysophospholipid receptor membrane (~1 μg/well) and [³²P]S1P (0.1 nM) in a 96-well poly-L-lysine microplate for 60 min. The reaction was terminated by collecting the membranes onto 96-well glass fiber filtration plates (Millipore, Billerica, MA). After washing with assay buffer (50 mM HEPES Na, pH 7.5, 5 mM MgCl₂, 1 mM CaCl₂, and 0.5% fatty acid-free bovine serum albumin), filter-bound radioactivity was measured by a Beckman LS 3801 scintillation counter using Cherenkov counting. The binding affinity of TZ6544 toward the other four sphingosine-1-phosphate receptors was similarly determined using the above procedure. IC₅₀ values were calculated using GraphPad Prism (GraphPadSoftware, Inc., San Diego, CA).

2.3.2. Characterization of the radioligand [¹²⁵I]TZ6544

2.3.2.1. Saturation binding assay using [¹²⁵I]TZ6544

We determined the dissociation constant, K_d (nM) of [¹²⁵I]TZ6544 through a saturation radioligand binding study using escalating concentrations of [¹²⁵I]TZ6544 to directly measure its binding toward S1PR2. The S1PR2 receptor membrane (Millipore, Billerica, MA) was diluted with assay buffer (50 mM HEPES Na, pH 7.5, 5 mM MgCl₂, 1 mM CaCl₂, and 0.5% fatty acid-free bovine serum albumin) and placed in a 96-well poly-L-lysine microplate (50 μL, 0.5 μg/well). Then, a 50 μL aliquot of increasing concentrations of [¹²⁵I]TZ6544 (0.2, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 16.0, and 20.0 nM) was added to each well; each concentration of [¹²⁵I]TZ6544 was calculated from the radioactivity and molar activity. For the determination of total binding and nonspecific binding, an additional 50 μL of assay buffer and 30 μM of JTE-013, a well-known S1PR2 antagonist was added to each well, respectively. The mixture was incubated at room temperature for 1 h on a shaker and then transferred onto 96-well glass fiber filtration plates. The unbound activity was filtered and rapidly washed away using 200 μL/well of ice-cold assay buffer for 5 times. After drying under the vacuum, the filter bound radionuclide was measured using a Beckman 8000 automated gamma counter. Data analysis was accomplished using GraphPad Prism (GraphPadSoftware Inc., San Diego, CA). The specific binding was calculated by subtracting the non-specific binding from the total binding. The binding assay data were curve-fit to a one-site binding equation to generate the dissociation constant (K_d) of the [¹²⁵I]TZ6544.

2.3.2.2. Determining the IC₅₀ values of known S1PR2 compounds using [¹²⁵I]TZ6544

Radiotracer [¹²⁵I]TZ6544 was used to determine the IC₅₀ values of several known S1PR2 compounds to confirm whether [¹²⁵I]TZ6544 is a suitable radioligand for assessing the other compounds binding potency toward S1PR2. The IC₅₀ values of several reported S1PR2 ligands including JTE-013, S1P, and TZ34125 were determined using [¹²⁵I]TZ6544 according to the following procedure. Human recombinant S1PR2 cell membrane was diluted with assay buffer (50 mM HEPES Na, pH 7.5, 5 mM MgCl₂, 1 mM CaCl₂, and 0.5% fatty acid-free bovine serum albumin) and placed in a 96-well poly-L-lysine microplate (50 μL, ~1.0 μg/well). Then, 50 μL of different concentrations (0.03, 0.15, 0.3, 1.5, 3.0, 15, 30, 150, 300, 1500, and 3000 nM) of each ligand and 50 μL of [¹²⁵I]TZ6544 (6.0 nM) was added to each well. The mixture was incubated for 1 h on a shaker at room temperature and then transferred onto a 96-well glass fiber filtration plates. The unbound activity was filtered and rapidly washed using ice-cold assay buffer (5 × 200 μL/well). After drying under reduced pressure, the filter bound radionuclide was measured by a Beckman 8000 automated gamma counter. The binding potency (IC₅₀) was calculated using GraphPad Prism.

2.4. In vitro studies with the STZ-induced rat model of diabetes

All rodent studies were performed in compliance with the United States Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals as described in the Guide for the Care and Use of Laboratory Animals, under protocols approved by the Washington University in St Louis School of Medicine Institutional Animal Care and Use Committee. Safety procedures for working with STZ in research animals were additionally reviewed and approved by the Institutional Biosafety Committee. STZ is an N-nitro derivative of glucosamine that is toxic to the insulin-producing pancreatic β-cells, and is widely used to generate rodent models of type 1 diabetes [31–32]. Adult male Sprague-Dawely (SD) rats were purchased from Charles River Laboratories (Wilmington, MA) and allowed to acclimate in the institutional animal facility for at least a week before being used. For the 12-week, model of STZ-induced type 1 diabetes, SD rats (~8–10 weeks old) were fasted overnight and IV injected with STZ (55 mg/kg) in sodium citrate buffer (1 mL/kg); sham control rats were similarly injected with citrate buffer. Following treatment, rats were given drinking water supplemented with sucrose (15 g/L) for 48 h to limit early mortality as stores of insulin are released from damaged pancreatic islet cells. Control and treated rats were weighed and blood glucose levels checked weekly to monitor the progression of diabetes. Diabetic rats were euthanized after 12-weeks and tissues harvested; kidneys from each rat were harvested tissues were fixed in 10% formalin and paraffin-embedded for subsequent staining and immunohistochemistry or snap-frozen and stored at −80 °C until used in the in vitro studies described below.

2.4.1. In vitro autoradiography studies

In vitro autoradiography studies of [¹²⁵I]TZ6544 were performed on snap-frozen kidney sections from diabetic rats and sham control rats to determine if binding of [¹²⁵I]TZ6544 reflects the S1PR2 expression. Kidneys were snap-frozen and stored at −80 °C then sectioned crosswise at 14 microns with a Leica cryostat, sections were mounted on glass slides. The autoradiography experiment was performed by incubating frozen rat kidney tissue mounted on glass slides and 1.85 KBq/slide of [¹²⁵I]TZ6544 in 500 μL of binding buffer (50 mM HEPES-Na pH 7.5, 5 mM MgCl₂, and 1 mM CaCl₂, 0.5% fatty acid-free bovine serum albumin). The blocking study was performed by incubating slides in the above solution with 10 μM of JTE-013 [14]. After incubation for 60 min at room temperature, the slides were washed for 2 min each using the following buffers sequentially: 1X TBST, 15% ethanol in 1X TBST, 30% ethanol in 1X TBST, 1X TBST (1X TBST buffer: 20 mM of Tris, 150 mM of NaCl, 0.1% of Tween 20). Slides were then placed into a cassette and exposed to a phosphor sensor sheet for 24 h. Autoradiography signal was visualized using a Typhoon FLA 9500 phosphor imaging system (GE Healthcare Life Sciences, Uppsala, Sweden). Autoradiography signal on the renal tissues (cortex and medulla, not including the renal papilla) was measured and quantified with the MultiGauge software program (Fujifilm, Tokyo, Japan). The data were background-corrected and expressed as photo-stimulated luminescence signals per square millimeter (PSL/mm²). For statistical comparison, the student t-test (unpaired) was applied using MS Excel, p < 0.05 was considered statistically significant.

2.4.2. Immunostaining study and ELISA analysis

Immunostaining study was carried out using a rabbit anti-rat S1PR2 antibody (ThermoFisher, Waltham, MA) to confirm if the binding of [¹²⁵I]TZ6544 to diabetic rat kidney correlated with increased S1PR2 expression in the rats with STZ-induced type 1 diabetes. As briefly described above, kidney tissue was fixed with 10% formalin and embedded in paraffin. The tissue block was then cut into 5 μm sections which were mounted on glass slides. Sections were deparaffinized, and endogenous peroxidase activity was quenched before the slides were incubated in a blocking buffer to block nonspecific binding. Upon the completion of the blocking, the slides were incubated overnight with a 1:100 dilution of a rabbit anti-rat S1PR2 antibody at 4 °C. The primary antibody binding was detected using an anti-rabbit HRP-DAB staining kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. A Nikon E600 microscope coupled with a charge-coupled device camera was used to obtain all photomicrographs.

In addition to the immunostaining, an ELISA was performed to evaluate the expression level of S1PR2 in the rat kidney tissues. All samples were assayed in triplicate. In brief, approximately 200 mg of frozen kidney tissue was placed in RIPA Lysis and Extraction Buffer (ThermoFisher, Waltham, MA ) containing 200 mg/mL 1X protease inhibitor cocktail (ThermoFisher, Waltham, MA) and homogenized on ice using a TissueRuptor (Qiagen, Germantown, MD). The supernatant was collected after centrifugation of the homogenate at 21,000 x g for 1 h at 4 °C. Protein concentration was assessed with the MicroBCA Protein Assay Kit (ThermoFisher, Waltham, MA). Twenty micrograms of total protein diluted in 100 μL of coating buffer (50 mM carbonate-bicarbonate buffer, pH 9.6) was coated to 96-well Nunc MaxiSorp flat-bottom plates (ThermoFisher, Waltham, MA), incubated overnight at 4 °C and then blocked with 4% bovine serum albumin in PBS for 1 h at room temperature. The sample was then incubated with anti-rat S1PR2 or anti-rat β-actin antibody (ThermoFisher, Waltham, MA) at 4 °C overnight, washed, and incubated with HRP conjugated anti-rabbit or anti-mouse antibody (Cell Signaling, Danvers, MA) for 2 h at room temperature. After the final wash, the assay was developed by adding 100 μL of 1-Step Ultra TMB-ELISA Substrate Solution (ThermoFisher, Waltham, MA) and the absorbance was read on a Synergy 2 plate reader (BioTek) at 650 nm. The relative expression level of S1PR2 of each well was normalized by dividing the expression level of β-actin; an unpaired student t-test was performed to evaluate the difference between diabetic rats and the control group.

2.5. Biodistribution study of [¹²⁵I]TZ6544

Adult male Wistar rats from Charles River were used for initial biodistribution studies of [¹²⁵I]TZ6544 in normal rats at 5 and 30 min post-injection. The concentrated solution of [¹²⁵I]TZ6544 was diluted to ~0.037 MBq/100 μL which was formulated for injection in 10% ethanol in 5% Macrogol (15)-hydroxystearate (Kolliphor® HS 15, Millipore Sigma, Billerica, MA) and normal saline solution, in order to ensure that the radiotracer was solubilized well. Rats were injected via the lateral tail vein under 2–3% isoflurane/oxygen anesthesia, then allowed to recover until euthanized under isoflurane/oxygen anesthesia at the appropriate time point post-injection. Tissues including blood, lung, liver, kidney, muscle, fat, pancreas, spleen, kidney, liver, thyroid, thymus, brain, and small intestines were collected, weighed, and counted in a Beckman Gamma 8000 counter, counts were corrected for background and decay corrected. Tracer uptake in tissues and organs was calculated as percent injected dose per gram of tissue (%ID/g).

3. Results

3.1. Chemistry

The syntheses of reference standard TZ6544 and precursor 9 are depicted in Scheme 1. First, the commercially available 1-iodo-2,4-dimethoxy-5-nitrobenzene (1) was treated with iron powder and hydrochloride (HCl) to yield aniline 2, which was reacted with bromoacetyl bromide to give the bromide 3. Meanwhile, commercially available 2-(2,4-dioxo-1,4-dihydroquinazolin-3(2H)-yl)acetic acid (4) was coupled with p-anisidine to afford intermediate 5 using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) as the condensation agent. The nucleophilic substitution of bromide 3 and intermediate 5 gave the reference standard TZ6544.

Scheme 1.

Reagents and conditions: (a) iron powder, 37% HCl, ethanol, H₂O, reflux, 1 h; (b) bromoacetyl bromide, trimethylamine, dichloromethane, 0 °C-RT, 10 h; (c) HATU, DIPEA, p-anisidine, dichloromethane, RT, overnight; (d) K₂CO₃, dimethylformamide, RT, 12 h; (e) bis(pinacolato)diboron, [1,1’-bis(diphenylphosphino)ferrocene]dichloropalladium(II), potassium acetate, DMSO; (f) 5, K₂CO₃, dimethylformamide, RT, 12 h.

The radiolabeling precursor 9 was prepared using a similar procedure described for TZ6544. 5-Bromo-2,4-dimethoxyaniline (6) was converted to borate 7 by a palladium-catalyzed Miyaura borylation reaction with bis(pinacolato)diboron. After treating with bromoacetyl bromide and coupling with intermediate 5, the borate precursor 9 was obtained. The structures of all the compounds were confirmed by nuclear magnetic resonance (NMR) spectroscopy (see Supplementary Material).

3.2. Radiochemistry

The radiosynthesis of [¹²⁵I]TZ6544 was accomplished as shown in Scheme 2. The borate precursor 9 was readily ¹²⁵I-labeled with [¹²⁵I]NaI using the copper-catalyzed reaction at room temperature. [¹²⁵I]TZ6544 was obtained in >99% radiochemical purity and with a radiochemical yield of ~47% (n = 3). The identity of [¹²⁵I]TZ6544 was confirmed by HPLC using a coinjection of an aliquot of the radioactive product with cold reference standard TZ6544. The molar activity of [¹²⁵I]TZ6544 was calculated as ~93 GBq/μmol, based on the molar activity of [¹²⁵I]NaI provided by the supplier.

Scheme 2.

Radiosynthesis of [¹²⁵I]TZ6544.

3.3. In vitro binding studies

3.3.1. The IC₅₀ values of TZ6544 binding to S1PR1, 2,3,4 and 5 determined using [³²P]S1P

A competitive binding assay using [³²P]S1P was performed to measure the binding potency of compound TZ6544 toward S1PR2. As shown in Fig. 2, TZ6544 is a potent S1PR2 antagonist with an average IC₅₀ value of 2.8 ± 0.5 nM. The [³²P]S1P assay was also used to measure the binding activity of TZ6544 toward the other four sphingosine-1-phosphate receptors (1, 3, 4, and 5), and the IC₅₀ values of TZ6544 toward S1PR1, 3, 4, and 5 were > 1000 nM (Fig. 2), indicating TZ6544 is a potent and selective ligand for S1PR2 and may merit further evaluation as a therapeutic agent. These data encouraged our efforts to radiosynthesize [¹²⁵I]TZ6544 for further characterization its binding properties.

Fig. 2.

Representative binding curve of TZ6544 binding toward S1PR2 and IC₅₀ values of TZ6544 toward S1PR2–5 determined using [³²P]S1P. Data were generated from at least three independent experiments, each run was performed in duplicate.

3.3.2. Characterization of the radioligand [¹²⁵I]TZ6544

3.3.2.1. Saturation binding [¹²⁵I]TZ6544

The saturation binding study results are shown in Fig. 3. The binding of [¹²⁵I]TZ6544 to S1PR2 membrane was saturable (Total binding, Fig. 3A) with escalating concentrations of [¹²⁵I]TZ6544. Nonspecific binding was determined using 10 μM of JTE-013 as a blocking agent (Fig. 3A). The specific binding curve was calculated by subtracting nonspecific binding from total binding. In addition, the result of Scatchard plot analysis (Fig. 3B) indicated that [¹²⁵I]TZ6544 binding to human S1PR2 cell membranes fit well into a one-site binding model with a K_d value of 4.31 ± 0.39 nM.

Fig. 3.

Scatchard analysis of [¹²⁵I]TZ6544 binding to the human recombinant S1PR2 membrane: (A) Representative saturation binding curves for [¹²⁵I]TZ6544 which show the total binding, non-specific binding, and specific binding. (B) Representative Scatchard plot used to determine K_d value.

3.3.2.2. The IC₅₀ values of serval known compounds determined using [¹²⁵I]TZ6544

When using [¹²⁵I]TZ6544 as the radioligand to determine the IC₅₀ of known S1PR2 ligands, we found that the IC₅₀ values of JTE-013, S1P, TZ34125, and TZ6544 binding toward S1PR2 were 29.4, 5.81, 8.63, and 5.06 nM, respectively, as shown in Fig. 4. We reported the IC₅₀ values of these compounds were 58.4, 3.6, 9.5, and 2.8 nM, respectively determined using [³²P]S1P (Table 1). The IC₅₀ values of these known S1PR2 compounds determined using [¹²⁵I]TZ6544 are comparable to that using [³²P]S1P. It was observed that JTE-013 and TZ6544 show ~2-fold difference. However, in our studies, we previously found different batches of [³²P]S1P could lead a 2-fold IC₅₀ value difference. That is the reason we would like to develop a relatively long half-life I-125 radiotracer as a screening ligand.

Fig. 4.

Representative S1PR2 binding curves of several knows compounds using [¹²⁵I]TZ6544. Data were generated by at least three independent experiments.

Table 1.

The S1PR2 IC₅₀ (nM ± SD) values determined with [³²P]S1P or [¹²⁵I]TZ6544

Compounds	[32P]S1P competition assay IC50 (nM)	[125I]TZ6544 competition assay IC50 (nM)a
JTE-013	58.4 ± 7.4b	29.4 ± 2.2
S1P	3.6 ± 0.5b	5.8 ± 0.4
TZ34125	9.5 ± 0.7c	8.6 ± 0.3
TZ6544	2.8 ± 0.5	4.4 ± 0.2

^aIC₅₀ values were determined at least three independent experiments, each run was performed in duplicate;

^breference[23];

^creference[22],

3.4. In vitro studies with the STZ-induced diabetic rat model

3.4.1. In vitro autography study of [¹²⁵I]TZ6544

The diabetic rats were treated with STZ at 55 mg/kg 12 weeks prior to euthanasia and tissue harvesting. Bodyweight and blood glucose levels were measured weekly in control and treated animals elevated blood glucose is indicative of diabetes (Fig. 5A). Representative in vitro autoradiography of frozen kidney sections is shown in Fig. 5B. The kidney radioactivity of [¹²⁵I]TZ6544 was 103.5 PSL/mm² (n = 5) for the diabetic rats, and 72.0 PSL/mm², (n = 4) for the control rats. The binding of [¹²⁵I]TZ6544 to the kidney of diabetic rats was increased by 44% compared to that observed in the control rats. In addition, the binding of [¹²⁵I]TZ6544 to kidney tissue in both groups could be blocked using 10 μM of JTE-013, indicating that the binding is S1PR2 specific (Fig. 5C).

Fig. 5.

In vitro autoradiography of [¹²⁵I]TZ6544 on kidney sections from control and diabetic rats (STZ). A: Blood glucose (mg/dL) from control and diabetic rats; B: Representative autoradiography images of control and diabetic kidney sections with and without blocking agent JTE-013 (10 μM). C: Quantification of average autoradiography signal (PSL/mm²) from each group of kidney sections incubated with [¹²⁵I]TZ6544 (STZ n = 5, control n = 4, p = 0.009).

3.4.2. Immunostaining and ELISA of S1PR2 in the kidney of diabetic and control rats

Immunostaining of S1PR2 was performed to confirm the increased expression of S1PR2 in the kidney of STZ-induced diabetic rats. As shown in Fig. 6, positive staining for S1PR2 (brown color) was observed in the kidney of both control and diabetic rats. The diabetic group (STZ) showed higher staining intensity, indicating the expression of S1PR2 in the kidney of diabetic rats is upregulated compared to controls. The amount of S1PR2 in the kidney was also evaluated by ELISA analysis. ELISA is a commonly used analytical biochemistry assay to detect the expression of protein in cells and tissues. As shown in Fig. 7, the level of S1PR2 was statistically higher in the diabetic rats than the control rats (p = 0.0195, unpaired t-test).

Fig. 6.

Representative immunostaining of S1PR2 in kidney sections of control and diabetic (STZ) rats (200X).

Fig. 7.

ELISA analysis of S1PR2 in the kidney tissue homogenates of control and diabetic (STZ) rats. Unpaired t-test indicated that the expression level of S1PR2 in diabetic rats was 20% higher than control rats (p = 0.0195).

3.5. Ex vivo biodistribution study of [¹²⁵I]TZ6544

The pilot biodistribution study of [¹²⁵I]TZ6544 in normal rats is displayed in Table 2. At 5 min, the initial uptake (%ID/g) of [¹²⁵I]TZ6544 in blood, heart, lung, kidney, and liver was 0.19, 0.39, 0.64, 0.58, and 2.20, respectively. Brain uptake is very low (0.01 %ID/g), suggesting that [¹²⁵I]TZ6544 does not cross the blood-brain barrier. At 30 minute post-injection, rat biodistribution data shows clearance from most organs, including heart, kidney, and pancreas, but much higher levels of radioactivity in the small intestine, indicating hepatobiliary clearance may happen in vivo. Activity in the thyroid is low, suggesting that the compound is metabolically stable to in vivo deiodination.

Table 2.

Biodistribution study of [¹²⁵I]TZ6544 in Wistar rats (% ID/g values, mean ± SD, n = 4).

Organs	Uptake (ID %/gram)
	5 min control	30 min control
Blood	0.19 ± 0.05	0.08 ± 0.02
Heart	0.39 ± 0.08	0.14 ± 0.02
Lung	0.64 ± 0.01	0.22 ± 0.04
Thyroid	0.32 ± 0.11	0.53 ± 0.09
Muscle	0.12 ± 0.01	0.08 ± 0.01
Fat	0.07 ± 0.02	0.09 ± 0.01
Pancreas	0.40 ± 0.04	0.14 ± 0.01
Spleen	0.29 ± 0.06	0.06 ± 0.01
Kidney	0.58 ± 0.02	0.15 ± 0.02
Liver	2.20 ± 0.32	0.36 ± 0.07
Thymus	0.10 ± 0.01	0.10 ± 0.02
Brain	0.01 ± 0.00	0.01 ± 0.00
Small intestines	1.09 ± 0.23	5.32 ± 0.27

4. Discussion

The goal of our study was to evaluate [¹²⁵I]TZ6544 as a promising S1PR2 radioligand that could be used to screen other compounds, as well as being used to investigate the functions of S1PR2 in animal models of diseases such as STZ-induced diabetic rats.

We first synthesized the TZ6544 and determined its S1PR2 binding potency using [³²P]S1P binding assay. The results showed TZ6544 was a potent and highly selectively S1PR2 ligand with an IC₅₀ value of 2.8 nM; this is much more potent than the other known S1PR2 compounds such as S1P, JTE-013, and TZ34125. Inspired by the high potency of TZ6544, we first designed the tin precursor for the radiolabeling of [¹²⁵I]TZ6544. Although radioiodination is frequently accomplished through the reaction of a tin precursor with [¹²⁵I]NaI in the presence of an oxidant, either hydroperoxide or peroxyacetic acid [33], synthesis of the tin precursor needed for our planned iodination reaction was very challenging. Coincidentally, a rapid Cu-catalyzed [¹²⁵I]iodination of borate at room temperature was recently reported in the literature as a practical approach to radioiodination of suitable ligands [34]. We, therefore, synthesized the corresponding borate precursor; using the Cu-catalyzed [¹²⁵I]iodination of borate precursor, [¹²⁵I]TZ6544 was successfully achieved with a high radiochemical yield (~47%) and high radiochemical purity (> 99%).

As mentioned above, the high potency of TZ6544 in the [³²P]S1P assay inspired the synthesis of the iodinated ligand. Our initial direct binding study showed [¹²⁵I]TZ6544 has a high binding affinity with a K_d value of 4.31 nM for human recombinant S1PR2 membrane. The binding data fit into a one-site binding model very well and the Scatchard plot was linear, demonstrating that [¹²⁵I]TZ6544 only has one binding site with S1PR2. We then used [¹²⁵I]TZ6544 to measure the IC₅₀ values of several reported S1PR2 ligands; the results were comparable to that determined using [³²P]S1P [22–23]. Although values for JTE-013 and TZ6544 were not completely consistent with the expected results, we have observed up to a 2-fold difference in S1PR2 IC₅₀ values for the same compound under the same conditions with different batches of [³²P]S1P in our binding assay. Based on our experience, we feel the IC₅₀ values presented here for both JTE-013 and TZ6544 generated using [¹²⁵I]TZ6544 and [³²P]S1P represent acceptable variability. Given the advantages afforded by the longer half-life of I-125 (t_1/2 = 60 days) compared with P-32 (t_1/2 = 14 days), and its high selectivity for S1PR2 in the [³²P]S1P binding assay, [¹²⁵I]TZ6544 is a suitable competitive radioligand for screening S1PR2 compounds. One batch of [¹²⁵I]TZ6544 can be used for much longer than [³²P]S1P, which will not only reduce the inherent between-batch variability but also improve the cost-efficiency.

It has been previously reported that mRNA and expression of S1PR2 in diabetic rat kidneys is increased at 12 weeks after STZ treatment [12]. In our STZ-induced rat model of diabetes, 12 weeks after treatment, diabetic rats at had elevated blood glucose levels ranging from 566–800 mg/dL, blood glucose levels in the control rats were between 150–200 mg/dL, blood glucose was measured weekly after treatment and remained elevated in all diabetic rats. Our S1PR2 immunohistochemistry staining result (Fig. 6) indicates the expression of S1PR2 is essentially in all rat kidney tissues (cortex and medulla, not renal pelvis) with strong signal in the vascular endothelial cells, which matches our in vitro autoradiography study of [¹²⁵I]TZ6544. The autoradiography study showed ~44% increased binding of [¹²⁵I]TZ6544 in the diabetic rat kidney compared to the control tissue (Fig. 5C). Additionally, a blocking study carried out in the presence of JTE-013 (10 μM) reduced the binding of [¹²⁵I]TZ6544 in the diabetic rat kidney; binding of [¹²⁵I]TZ6544 in the control rat was also reduced by JTE-013. This is understandable because the kidney expresses S1PR2 during normal homeostatic conditions. Our immunohistochemistry staining and ELISA studies confirmed increased kidney S1PR2 expression in the diabetic rats compared to control tissue (Fig. 6 and 7).

The preliminary ex vivo rat biodistribution study showed that [¹²⁵I]TZ6544 has relatively high initial uptake in the lung, kidney, and liver, consistent with the expression of S1PR2 in these organs and with previous reports [13, 35–37]. The uptake of [¹²⁵I]TZ6544 in most of the organs was significantly decreased from 5 min to 30 min, indicating a quick clearance rate of [¹²⁵I]TZ6544 in vivo; tracer levels in the small intestines suggest that [¹²⁵I]TZ6544 may be excreted through the bile [38]. Although minor accumulation was observed in the thyroid, this I-125 radiotracer didn’t show significant deiodination in vivo, which is a concern for most of iodine-125/124 or 131 labeled radioligands for in vivo studies [33, 39]. Further studies are needed to assess the in vivo pharmacokinetics of [¹²⁵I]TZ6544 and to investigate the S1PR2 functions in animal model of STZ-induced diabetes and other diseases.

5. Conclusion

In summary, we successfully developed a potent S1PR2 ligand TZ6544 and radiosynthesized [¹²⁵I]TZ6544 with good radiochemical yield (∼47%) and high radiochemical purity (>99%). The in vitro binding assay using [³²P]S1P showed that TZ6544 is a potent and selectively S1PR2 ligand with an IC₅₀ value of 2.8 nM. The in vitro study showed that [¹²⁵I]TZ6544 has K_d value of 4.31 nM for S1PR2. The IC₅₀ values of several known S1PR2 ligands determined using [¹²⁵I]TZ6544 were comparable to the IC₅₀ values determined using [³²P]S1P. The in vitro autoradiography, immunohistochemistry staining, and ELISA data revealed that [¹²⁵I]TZ6544 can detect increase of S1PR2 expression in the kidney of diabetic rats. Our initial biodistribution study of [¹²⁵I]TZ6544 suggested that [¹²⁵I]TZ6544 didn’t happen significant deiodination in vivo and this radiotracer may have bile excretion. Together, [¹²⁵I]TZ6544 has the potential to be a radioligand for screening the other S1PR2 compounds and investigating the S1PR2 expression changes in disease. Further structural optimization of [¹²⁵I]TZ6544 could develop a PET radiotracer as a non-invasive imaging tool for investigating the pathophysiology of S1PR2 functions in diabetes and other diseases.