A Novel Substrate Radiotracer for Molecular Imaging of SIRT2 Expression and Activity with Positron Emission Tomography

Purpose: The purpose of this study was to develop a SIRT2-specific substrate-type radiotracer for non-invasive PET imaging of epigenetic regulatory processes mediated by SIRT2 in normal and disease tissues. Procedures: A library of compounds containing tert-butyloxycarbonyl-lysineaminomethylcoumarin backbone was derivatized with fluoroalkyl chains 3–16 carbons in length. SIRT2 most efficiently cleaved the myristoyl, followed by 12-fluorododecanoic and 10fluorodecanoic groups (Kcat/Km 716.5 ± 72.8, 615.4 ± 50.5, 269.5 ± 52.1/s mol, respectively). Radiosynthesis of 12- [18F]fluorododecanoic aminohexanoicanilide (12-[18F]DDAHA) was achieved by nucleophilic radiofluorination of 12-iododecanoic-AHA precursor. Results: A significantly higher accumulation of 12-[18F]DDAHA was observed in MCF-7 and MDA-MB-435 cells in vitro as compared to U87, MiaPaCa, and MCF10A, which was consistent with levels of SIRT2 expression. Initial in vivo studies using 12-[18F]DDAHA conducted in a 9L glioma-bearing rats were discouraging, due to rapid defluorination of this radiotracer upon intravenous administration, as evidenced by significant accumulation of F-18 radioactivity in the skull and other bones, which confounded the interpretation of images of radiotracer accumulation within the tumor and other regions of the brain. Conclusions: The next generation of SIRT2-specific radiotracers resistant to systemic defluorination should be developed using alternative sites of radiofluorination on the aliphatic chain of DDAHA. A SIRT2-selective radiotracer may provide information about SIRT2 expression and activity in tumors and normal organs and tissues, which may help to better understand the roles of SIRT2 in different diseases.

serum (FCS), penicillin and streptomycin, at 37°C in a humidified atmosphere with 5% CO2. In vitro radiotracer uptake studies were performed as previously described 8 . Briefly, prior to in vitro radiotracer uptake experiment, the cells were inspected for confluency and viability. Each of the four cell plates for a given cell line had the media removed by pipette aspiration, and fresh media (15mL) containing the radiotracer (1-2uCi/mL) was added to cells in the culture plates. The tumor cell monolayers were incubated with the radioactivity-containing medium for 30 minutes. The cells were then harvested by gentle unidirectional scraping and the suspensions of cells in the media were transferred into 15mL conical centrifuge tubes and pelleted by centrifugation (3000 rpm for 2 min). 50uL samples of supernatant and cell pellets flash-frozen on dry ice were placed in pre-weighed scintillation vials, weighed, and the radioactivity was measured using Pakard 5500 gamma counter (Perkin Elmer, CA). The background-corrected and decay-corrected radioactivity concentrations in individual cell pellets (cpm/g) was divided by corresponding cell culture media samples (cpm/g) to determine the fold increase of radiotracer accumulation in cells versus the cell culture media. All experiments were conducted in triplicate.

Intracerebral glioma model in rats.
All studies in rats were performed under a protocol approved by the Institutional Animal Care and Use Committee of Wayne State University. The 9L cells were obtained from ATCC and propagated in MEM supplemented with non-essential amino acids and 10% FCS in at 37 o C in humidified atmosphere with 5% CO2. For preparation of cellular suspension for intracerebral injection, the 9L cells were dislodged from cell culture flasks using Hank's Balanced Salt solution (HBSS, 5 mL) for 1-2 minutes, centrifuged to obtain the cellular pellet, and re-suspended in cell culture medium without FCS at 1x10 4

cells/µL concentration. Sprague-
Dawley rats (200-250 g, N=3) were anesthetized by inhalation of isoflurane in oxygen (3-4% isoflurane for induction; 1.5-2.5% isoflurane for maintenance). The body temperature was maintained using electronically-controlled heating pad (M2M Imaging, Cleveland, OH) set at 37 o C. The rats were placed into a stereotaxic apparatus (Kopf Tujunga, CA). A midline scalp incision was made to reveal bregma and a burr hole 2 mm in diameter was drilled in the skull till dura mater and the bone hemostasis was achieved by application of bone wax. Then, a shortbeveled 30-ga needle connected to a 100 μL syringe (Hamilton, CA) loaded with the cell suspension was inserted in to the brain using the following stereotactic coordinates relative to bregma; AP: -1.5, LAT: -4, DV: -6. A 5 µL tumor cell suspension was then injected slowly over 10 minutes, then the needle was slowly withdrawn 2 mm and the remaining 5 µL was injected slowly over 10 minutes for a total of 1x10 5 cells in 10 µL.
MR Imaging. The rats were anesthetized and body temperature was maintained at 37 o C, as described above. The animals' head was fixed in position using a bite bar and ear bars with a receive-only surface coil 2-element phased array placed dorsally on top of the head. MR images were acquired using a 7T ClinScan system (Bruker, UK) controlled by the Syngo software (Siemens, Knoxville, TN). A localizing T1-weighted scan was performed and adjustments to head position were made as needed. T2 images were obtained with TR 3530 ms, TE 38 ms, and FOV 3.2 cm x 3.2 cm x 2.4 cm, resulting in spatial resolution of 125 µm x 125 µm x 1 mm. Images were processed using ImageJ software (NIH, Bethesda, MD).
PET/CT Imaging. The rats were anesthetized and maintained under anesthesia throughout the PET/CT imaging studies, as described above for MRI and surgical procedures. Anesthetized rats were placed in stereotactic head holder made of polycarbonate plastic (Kopf-Tujunga, Germany) and attached to the bed the microPET R4 scanner (Siemens, Knoxville, TN) in the supine position with the long axis of the animal parallel to the long axis of the scanner and the brain positioned in the center of the field of view (FOV). [ 18 F]-12FDDAHA (300-500 µCi/animal) was administered in saline via the tail-vein in a total volume ≤1ml, as a slow bolus injection over the period of 1 min. Dynamic PET images were obtained over 60 minutes. After PET imaging, the positioning bed with the affixed anesthetized animal was transferred to the Inveon SPECT/CT scanner (Siemens, Knoxville, TN) and CT images and 4 overlapping frames (2 min each) were acquired covering the whole body using X-ray tube settings of 80 kV and 500 uA with exposure time of 300-350 milliseconds of each of the 360 rotational steps. Following completion of the reaction, the solvent was evaporated and the compound was purified by column chromatography using a gradient of 0.5-2% methanol/dichloromethane for elution. The product was obtained in 85% yield as an off-white powder. 1
The resulting mixture was evaporated and purified by column chromatography using 10% ethyl acetate in hexane as the eluent. The ethyl protecting group was hydrolyzed by 100ul of 15wt% sodium methoxide in methanol. The following mixture was evaporated and 2 mL of thionyl chloride (SOCl2) was added to the residue. The resulting mixture was stirred at 60°C, under reflux, for 3 hours. Following the reaction, all SOCl2 was evaporated and the product was washed with toluene before being evaporated once more. Boc-Lys-AMC (Bachem (92 mg, 0.24 mmol) was dissolved in 10 mL of dichloromethane with 1 equivalent of TEA. The mixture was added dropwise to the flask containing the acetyl chloride residue, and the reaction was stirred continuously at 22°C, under argon, for 12 hours. Following completion of the reaction, the solvent was evaporated and the compound was purified by column chromatography using a gradient of 3-5% methanol/dichloromethane for elution. The product was obtained in 20% yield as an off white powder. 1

2-yl)carbamate (3)
10-Fluorodecanoic acid (45 mg, 0.24 mmol) was dissolved in excess SOCl2 (1 mL, 1.3 mmol) and stirred at 60°C, under reflux, for 3 hours. Following the reaction, all SOCl2 was evaporated and the product was washed with toluene before being evaporated once more. Boc-Lys-AMC (96 mg, 0.24 mmol) was dissolved in 7 mL of dichloromethane with 1 equivalent of TEA. The mixture was added dropwise to the flask containing the acetyl chloride residue, and the reaction was stirred continuously at 22°C, under argon, for 12 hours. Following completion of the reaction, the solvent was evaporated and the compound purified by column chromatography using a gradient of 3-5% methanol/dichloromethane for elution. The product was obtained in 55% yield as an off white powder. 1   and stirred at 60°C, under reflux, for 3 hours. Following the reaction, all SOCl2 was evaporated and the product was washed with toluene before being evaporated once more. Boc-Lys-AMC (36 mg, 0.09mmol) was dissolved in 7 mL of dichloromethane with 1 equivalent of triethylamine (TEA). The mixture was added dropwise to the flask containing the acetyl chloride residue, and the reaction was stirred continuously at 22°C, under argon, for 12 hours. Following completion of the reaction, the solvent was evaporated and the compound was purified by column chromatography using a gradient of 3-8% methanol/dichloromethane for elution. The product was obtained in 20% yield as an off white powder. 1

2-yl)amino)-1-oxohexan-2-yl)carbamate (7)
Boc-Lys-AMC (232 mg, 0.45mmol) was dissolved in trifluoroacetic acid (TFA) to remove the boc protecting group. The mixture was stirred for 30 minutes at 22°C and the TFA (2.5 mL) was evaporated. The resulting residue was dissolved in dichloromethane (DCM) and 1eq of TEA was added to neutralize the product. The mixture was evaporated and the product was washed with water and dried to remove salts.   in the SIRT2 active site 11 . Therefore, we hypothesize that the distance between the amide bond carbonyl carbon and the nicotinamide cleavage site of NAD + plays an important role in substrate catalytic efficiency of SIRT enzymes. The mechanism of SIRT2 mediated cleavage of acylated lysine moieties involves simultaneous cleavage of nicotinamide and activation and subsequent cleavage of the acyl carbonyl carbon amide bond. Therefore, the selection of site for with substitution fluorine atom (strongly electronegative and electron withdrawing) is crucial for preservation of catalytic activity of SIRT2. For example, if a fluorine atom (or another electron withdrawing group) were to be placed in close proximity to the α-carbon, catalytic efficiency would decrease, as reported by us previously for fluro-and trifluoro-acetamidohexanoicanilide 12 .

Regarding the role of SIRT2 in oncogenesis and progression
The role of SIRT2 in oncogenesis and progression of brain gliomas and other primary brain tumors is not yet well understood. Although some studies demonstrated downregulation of SIRT2 expression in about 70% of gliomas 13 , it appears that the expression levels alone don't necessarily determine the enzymatic activity and mechanistic roles of SIRT2 in gliomagenesis, progression, and development of resistance to chemo-and radio-therapy. Furthermore, there is a controversy in the literature regarding the glioma promoting or suppressing roles of SIRT2. For example, one study 14 demonstrated that both genomic shRNA-mediated knockdown or pharmacologic inhibition (by AGK2) of SIRT2 expression-activity caused necrosis and caspase-3-dependent apoptosis of C6 rat glioma cells. In contrast, another study 15 demonstrated that in T98G, U87MG, and U251 cells the shRNA-mediated SIRT2 knockdown promoted colony formation, while adenoviralmediated overexpression of SIRT2 repressed colony formation in vitro. The latter study has revealed the mechanism of SIRT2-mediated suppression of glioma cell growth occurred via negative regulation of miR-21 expression, which is known to be upregulated in gliomas and to promote glioma growth 16 and resistance to chemo-radiotherapy 17 . Therefore, additional studies are required to determine whether the genetic knock-down or pharmacologic inhibition of SIRT2 expression in glioma cells in vivo will be therapeutically effective of will result in the accelerated growth and invasion. Such studies will be greatly facilitated by multi-modal molecular imaging with PET/CT/MRI and a SIRT2-specific radiotracer.

Regarding SIRT2 expression in various cancers
The development of a second-generation SIRT2-selective substrate-type radiotracer is very important because several recent studies demonstrated that the labeling index of nuclear-localized SIRT2 is significantly higher in glioblastomas (grade IV), as compared to astrocytomas (grade II) and normal brain tissue and strongly correlated with malignant progression and the overall survival of patients with glioblastomas 18 . The immunohistochemical staining of many tissues in the Human Protein Atlas displays intense staining of SIRT2 protein in all glioma tissues with greater magnitude than in breast or pancreatic cancer patient derived tissue samples (http://www.proteinatlas.org/search/sirt2). Higher levels of expression and higher labeling index for SIRT2 is also associated with progression and poor prognosis in patients with non-small cell lung cancer (NSCLC) 19 and cervical carcinoma 20 . Breast carcinomas with increased levels of SIRT2 expression have poorer prognosis as compared to those with lower expression levels 18 .
Some other tumor types (i.e., melanomas), may harbor mutations in SIRT2 gene resulting in reduction of enzymatic activity by 80-90% compared to the wild-type protein, however consequences to tumor progression and overall prognosis are yet unknown 21 . Therefore, molecular imaging with PET using SIRT2-specific radiotracers should provide information about the location and magnitude of SIRT2 expression and activity in tumors non-invasively and in real time. This may help to determine the mechanistic, therapeutic, and prognostic roles of SIRT2 in different cancers as well as monitoring novel SIRT2 targeted therapies.