An HDAC-Targeted Imaging Probe LBH589âCy5.5 for Tumor

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An HDAC-Targeted Near-Infrared Probe LBH589Cy5.5 for Tumor Detection and Therapy Evaluation Qingqing Meng, Zhiyi Liu, Feng Li, Jianjun Ma, He Wang, Yi Huan, and Zheng Li Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00167 • Publication Date (Web): 01 Jun 2015 Downloaded from http://pubs.acs.org on June 15, 2015

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Molecular Pharmaceutics

An HDAC-Targeted Near-Infrared Probe LBH589-Cy5.5 for Tumor Detection and Therapy Evaluation

Qingqing Meng†, Zhiyi Liu†, Feng Li†, Jianjun Ma‡, He Wang‡, Yi Huan†‡, Zheng Li†,* †

Department of Translational Imaging, Houston Methodist Research Institute, Houston TX

77030, USA; ‡Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi 710038, China; †‡Department of Radiology, Xijing Hospital, Fouth Millitary Medical University Xi’an, Shaanxi 710038, China KEYWORDS: Histone deacetylases, NIR fluorescent imaging, LBH589, therapy evaluation, triple-negative breast cancer

ABSTRACT: Histone deacetylases (HDACs) are overexpressed in various cancers. In vivo HDAC imaging to measure the expression and functions of HDACs in tumor plays an important role for the tumor diagnosis and HDAC-targeted therapy evaluation. The development of stable and highly sensitive HDAC targeting probe is highly desirable. Near-infrared (NIR) fluorescence optical imaging is a powerful technology for visualizing disease at the molecular level in vivo with high sensitivity and no ionizing radiation. We report here the design, synthesis and bioactivity evaluation of LBH589-Cy5.5 for in vivo NIR fluorescence imaging of HDACs. The IC50 value of resulting NIR probe to HDACs was determined to be 9.6 nM. In vitro fluorescence microscopic studies using a triple-negative breast cancer cell line, MDA-MB-231 established the binding specificity of LBH589-Cy5.5 to HDACs. An in vivo imaging study performed in MDAMB-231 tumor xenografts demonstrated accumulation of the probe in tumor with good contrast

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from 2h to 48h post-injection. Furthermore, the fluorescent signal of LBH589-Cy5.5 in tumor was successfully blocked by co-injection of non-fluorescent LBH589 with the probe. In a following therapy evaluation study, the administration of SAHA, a clinically used HDAC inhibitor, decreased LBH589-Cy5.5 accumulation in tumor, demonstrating the potential application of LBH589-Cy5.5 for evaluating therapeutic response of HDAC inhibitors in cancer treatment.

INTRODUCTION Histone deacetylases (HDACs) are crucial enzymes found in both the nucleus and the cytoplasm. It has a wide spectrum of cellular functions which include regulating gene transcription by acetylation/deacetylation of the histones to modulate chromatin structure (i.e. epigenetic regulation) and manipulating protein stability, translocation of transcriptional factors, and apoptosis by altering the acetylation status of various proteins (non-histone effects).1 The known 18 isoforms of HDACs are classified into four groups, based on their catalytic dependence and sequence homology: Class I, II and IV are zinc-dependent, and Class III is NAD+-dependent.2 At present, abnormally high expression of HDACs has been observed in various cancers including breast cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell cancer, and hepatocellular carcinoma that preclude the transcription of tumor suppressor genes.3-8 Inhibition of HDACs has now emerged as a powerful strategy for cancer therapy. Meanwhile, non-invasive HDAC-targeted imaging to measure expression and functions of these enzymes is highly desirable. Visualizing the dynamics of HDACs during tumorigenesis can greatly enhance our understanding on tumor epigenetic status, and may play a critical role for personalized HDAC-targeted cancer therapy.


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Currently, despite the critical role of in vivo imaging of HDACs, limited probes were reported for cancer and neurological disease diagnosis. [18F]fluoroacetamide-1-hexanoicanilide (FAHA) and [11C]MS-275 were reported for brain cerebral HDAC imaging.9-11

18

F-suberoylanilide

hydroxamic acid (18F-SAHA) was reported to visualize the HDAC level in a murine ovarian cancer model by positron emission tomography (PET).12 Wang et al had incorporated carbon-11 to the structure of HDAC inhibitors (HDACi), Belinostat (PXD101), Panobinostat (LBH-589) and PCI 34051 to explore the brain permeability of hydroxamic acid-based HDACi using PET.13 However, low brain uptake of the probes and the short half-life of carbon-11(20 min) limited their clinical applications. Recently, Hooker’s group reported a 11C radiotracer, [11C]Martinostat for quantitative imaging of HDAC density in the brain and in peripheral organs in nonhuman primate.14 Panobinostat (LBH589) is a potent pan-deacetylase inhibitor that can block multiple cancer-related pathways and reverse epigenetic events implicated in cancer progression.15 It has potent inhibitory activity at low nanomolar concentrations against all zinc-dependent HDAC enzymes, suggesting the bona fide pan-HDAC activity.15-18 It has shown favorable clinical response in various clinical trials with minimal toxicity and just received FDA approval for multiple myeloma treatment.19-23 In this study, given its high HDAC-binding affinity and potent therapeutic effect in breast cancer treatment15, 16, 18, LBH589 was chosen as the targeting ligand for the development of HDACs imaging probe for triple-native breast cancer detection and HDACi therapy evaluation. Small molecules like LBH589 have advantages in developing the imaging moiety owing to their high affinity to target, low immunogenicity, and flexibility in chemical modification.24



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Near-infrared fluorescence optical imaging is a powerful technology for visualizing disease at the molecular level in vivo. It is a relatively inexpensive imaging modality with high sensitivity and no ionizing radiation, in which the lower limit of detection can reach picomolar concentrations of contrast agents.25, 26 Recent development in molecular imaging has established NIR imaging as a noninvasive imaging modality for monitoring the biological activity of a wide variety of molecular targets.27, 28 NIR optical imaging plays a significant role in tumor detection and staging, therapy response evaluation, and drug development in preclinical models. In this study, based on the binding mode of LBH589 to HDAC protein29, an optical probe LBH589Cy5.5 was successful developed for in vivo tumor HDAC imaging using a triple-negative breast cancer orthotropic xenograft model that does not express estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2).30

EXPERIMENTAL PROCEDURES Chemistry. General methods. All chemicals and solvents are purchased from either SigmaAldrich (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). LBH589 (Panobinostat) was purchased from Selleck chemicals (Houston, TX). The NHS ester of Cy5.5 was purchased from GE Healthcare (Piscataway, NJ). Mass spectrometry was recorded on a Thermo Finnigan LCQ Fleet using electrospray as the ionized method. High performance liquid chromatography (HPLC) purification and analysis wer

e performed on a semi-preparative reversed-phase Agilent 1260 HPLC system equipped with a diode array UV-vis absorbance detector.


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Molecular Modeling: The X-ray structure of HDAC1 (PDB code: 4BKX) reported by Millard et al29 was employed in the docking calculation. The protein and the ligand were respectively prepared using the protein preparation wizard and ligprep modules of the Schrodinger Suite 2009. Glide SP was used for the docking calculation. The docked poses were evaluated by GlideScore. Synthesis of LBH589-Cy5.5 conjugate. Cy5.5-NHS (0.7 mg, 0.4 µmol) was dissolved in dry DMSO (0.5mL) in a 2-mL vial, LBH589 (1.0 mg, 2.9 µmol) and NEt3 (20 µL) were then added subsequently. The reaction was stirred in dark at room temperature overnight and was quenched by adding trifluoroacetic acid (40 µL). The crude product was purified by a semi-preparative reversed-phase HPLC employing a Phenomenex Luna C-18 column (250 mm × 10 mm) with the mobile phase starting from 95% solvent A (0.05 M ammonia acetate in water) and 5% solvent B (acetonitrile) to 5% solvent A and 95% solvent B at 20 min, and the flow rate is 4 mL/min. Fractions containing LBH589-Cy5.5 were collected, lyophilized, redissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 µmol/mL, and stored in the dark at -20 °C until use. The purified LBH589-Cy5.5 was provided as deep blue solid over 70% yield after lyophilization and over 98% purity checked by HPLC under 254 nm and 675 nm channels (see Supporting information). The purified LBH589-Cy5.5 conjugate was characterized by mass spectroscopy (MS). MS (electrospray): m/z 1245.81([M-H]-, calculated 1247.34); 622.89 ([M-2H]2-, 100%). Biology. HDAC inhibition assay. HDAC Fluorimetric Assay/Drug Discovery Kit (Enzo Life Sciences, NY) was used to determine the HDAC inhibition ability of LBH589-Cy5.5. Assays were carried out according to the manufacturer’s protocol. Trichostatin A was used as positive control. HeLa cell nuclear extracts were used as a source of mixed HDAC enzymes. Briefly, assay buffer (25 µL in blank, 10 µL in control) or inhibitor (ranged from 0.5 nM to 5 mΜ in 10



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µL assay buffer) was added to 96-well microtiter plate. HeLa cell nuclear extracts (0.5 µL in 15 µL assay buffer) was added to all wells except in the no-enzyme control. Then fluor de lysTM substrate was added to all wells to get 50 µM final concentration. Allow HDAC reaction to proceed at 25 °C for 45 min and stop them by addition of fluor de lysTM developer (50 µL containing 2 µM TSA). Incubate plate at 25 °C for 10-15 min and the fluorescence was read using a FLUOstar OPTIMA Micro plate reader (Durham, NC) with excitation at 360 nm and emission at 460 nm. The IC50 value was calculated by nonlinear regression analysis using GraphPad Prism 5.0 (Graph-Pad Software, Inc., San Diego, CA, USA). Each data point is the mean of triplicate measurements. Cell line and tumor model. Triple-negative human breast cancer MDA-MB-231 cells were obtained from American Type Culture Collection and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C in humidified atmosphere with 5% CO2. Animal procedures were performed according to a protocol approved by Houston Methodist Research Institute Animal Care and Use Committee. MDA-MB-231 cells (5 × 106) were suspended in 100 µL PBS and injected orthotopically into the lower right mammary fat pad of 5-6 weeks female BALB/c nu/nu mice (Charles River). Mice were subjected to the in vivo imaging studies when tumors reached 4-6 mm in diameter. In vitro fluorescence microscopic studies. MDA-MB-231 cells were seeded on 8-well Nunc Lab-Tek II Chamber Slide System (Thermo Scientific, MA). After 24h, cells were washed with PBS and incubated in different concentrations of LBH589-Cy5.5 (1 µM, 0.5 µM, 0.2µM) with or without 100-fold excess LBH589 (100µM, 50µM, 20 µM) as the blocking agent at 37 °C for 60 min, respectively. The cells were washed three times with ice-cold PBS and mounted with


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ProLong Gold anti-fade reagent containing DAPI (Invitrogen, CA). The fluorescent signal was recorded using FluoView 1000 laser scanning confocal microscope (Olympus). In vivo fluorescence imaging and ex vivo biodistribution study. In vivo and ex vivo fluorescence imaging were performed with IVIS200 imaging system, and quantified by Living Imaging software (Xenogen, CA). Excitation and emission filters were set at 675 nm and 720 nm respectively as suggested by the system for image acquisition. For in vivo studies (n = 5), stock solutions (∼1 mM) of LBH589-Cy5.5 conjugate in DMSO was diluted with PBS for injection. Each tumor-bearing mouse was injected intravenously with 10 nmol of LBH589-Cy5.5 conjugate. Images were acquired at 2 h, 6 h, 24 h and 48 h post-injection (p.i.). For blocking studies (n = 5), mice were co-injected with 10 nmol of LBH589-Cy5.5 and unlabeled HDACi LBH589 (dosage = 20 mg/kg) following the same procedures described above. Regions of interest (ROI) were drawn over the tumor and adjacent normal tissue to calculate the in vivo tumor/normal tissue ratio. In the ROI analysis, mean fluorescence intensities of the tumor area (tumor) defined as radiance (photons/s/cm2/sr) and of the area at the adjacent normal tissue (muscle) were calculated. After the 48 h imaging, mice were sacriﬁced, and tumors, muscles and major organs (i.e., liver, spleen, kidney, heart, lung and intestines) were excised for ex vivo imaging acquisition. ROI analysis and quantification of the ﬂuorescence signals (n = 5) were performed with Living Imaging (Xenogen, CA). Immunohistochemistry. Tumors were fixed in 10% (w/v) neutral buffered formalin. The fixed tissues were embedded in paraffin from which continuous 4-µm sections were prepared. Hematoxylin and eosin (H&E) staining was performed according to standard protocol. For immunohistochemistry, heat-induced antigen retrieval was performed in sodium citrate buffer



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(pH 6.0). A rabbit polyclonal anti-HDAC1 antibody (Cat. ab7028, Abcam, USA) was used at a 1:100 dilution. Therapeutic evaluation study. MDA-MB-231 tumor-bearing mice (n = 4) were treated with SAHA by intraperitoneal injection for consecutive 3 days at the dose of 50 mg/kg/mouse/day. Following SAHA treatment, fluorescence imaging was performed by intravenous injection of 10 nmol of LBH589-Cy5.5, and images were acquired at 24h p.i. For control group (n = 4), mice were treated with PBS, and the fluorescence images were acquired at the same time point (24h p.i.). All images were scaled to the same maximum intensity for direct comparisons. Statistical analysis. Statistical analysis was carried out with Microsoft Office Excel 2007. Studies involving two groups were subjected to unpaired Student's t-test, with P < 0.05 considered statistically significant. RESULTS Design and Synthesis of the Probe. LBH589 is a potent hydroxamic acid pan-HDAC inhibitor with nanomolar range IC50 values for all HDACs. It has a hydroxamic acid zinc binding motif, a linker, and a capping motif. 31 To design the optical imaging probe from the structure of LBH589 with retained HDAC affinity, we first examined the binding mode of LBH589 to the HDAC1 protein. The reported X-ray structure of HDAC1 (PDB code: 4BKX)29 was used in our molecular modeling. The preferred coordination mode of LBH589 was presented in Figure 1. The hydroxamic acid group extends toward the zinc ion at the bottom of the entrance channel with a Zn-O distance of ∼1.99 Å. The hydrocarbon linker fills the hydrophobic channel with the N atom located at the entrance of the active pocket. These results indicated the modification of N atom in the middle of the hydrocarbon chain may not interfere the binding of LBH589 to the HDAC1. LBH589-Cy5.5 was successfully synthesized by conjugating NHS-activated ester of


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Cy5.5 to the secondary amine group of LBH589 adjacent to the capping motif. The crude product was purified by semi-preparative HPLC to give LBH589-Cy5.5 conjugate in 70% yield which was then confirmed by mass spectrometry. To determine whether Cy5.5 conjugation may have any effect on HDACs binding characteristics of HDACi LBH589, the HDAC inhibitory activity of LBH589-Cy5.5 conjugate was measured using Fluorimetric Assay/Drug Discovery Kit, and the IC50 value of LBH589-Cy5.5 to the HDAC-rich HeLa nuclear extracts was measured as 9.6 nM as shown in Figure 2, which is comparable to the reported IC50 value of LBH589.15



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Figure 1. Synthetic scheme of LBH589 and LBH-Cy5.5 (A); Coordination mode of LBH589 to the HDAC1 (PDB code: 4BKX) active site. LBH589 are shown in blue sticks and HDAC1 are shown in cartoon. Zn was shown in red spheres. The blue arrow indicated the N atom which could be modified (B).

Figure 2. Inhibition curve of HDAC enzyme activities by LBH589-Cy5.5. Each point represents mean ± s.d. of triplicate measurements.

In Vitro Fluorescence Microscopic Studies. To validate LBH589-Cy5.5 as an HDACspecific ligand in live cells, a triple-negative breast cancer cell line MDA-MB-231 known to overexpress HDACs32 were incubated with LBH589-Cy5.5 at increased concentrations of 0.2 µM, 0.5 µM and 1 µM, and then subjected to confocal laser microscopy. As shown in Figure 3, fluorescent signal in the cells strengthened when incubated with increased concentrations of LBH589-Cy5.5, demonstrating the receptor-mediated binding. The cell uptake of LBH589Cy5.5 was effectively blocked by co-incubation of non-fluorescent LBH589 at all conditions. This result provided strong supporting evidence that LBH589-Cy5.5 bound specifically to the HDACs overexpressed in the MDA-MB-231 tumor cells.


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Figure 3. Targeting specificity of LBH589-Cy5.5 in vitro. Cells were stained with DAPI (nuclear staining) (blue) and LBH589-Cy5.5 probe (red). All images were acquired by confocal laser microscopy at 600× magnification. The scale bar represents 20 µm. (A) MDA-MB-231 cells incubated with LBH589-Cy5.5 at 0.2 µM, 0.5 µM, and 1 µM. (B) MDA-MB-231 cells



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incubated with LBH589-Cy5.5 at 0.2 µM, 0.5 µM, and 1 µM in the presence of 100-fold excess LBH589.

In Vivo Fluorescence Imaging and Ex Vivo Biodistribution Study. We validated LBH589Cy5.5 for in vivo detection of tumor HDACs using a triple-negative MDA-MB-231 breast cancer xenograft model. Figure 4A showed the NIR fluorescence images of nude mice bearing orthotropic MDA-MB-231 breast tumors after intravenous injection of 10 nmol of LBH589Cy5.5. The whole body images showed relatively strong fluorescent signals at an early time point (2h) compared to the following time points (6h, 24h and 48h) due to the rapid blood clearance of the probe. The in vivo imaging results indicated that LBH589-Cy5.5 probe could rapidly accumulate in the tumor as early as 2h p.i., and the tumor fluorescence signal was still clearly visible at 48 hr p.i. As shown in Figure 4B, the tumor/normal tissue ratios in the imaging group were 2.03 ± 0.24 at 2h, 2.09 ± 0.13 at 6h, and reached 3.11 ± 0.31 at 24h and 3.07 ± 0.35 at 48h p.i. (n = 5). To validate the specificity of LBH589-Cy5.5 targeting HDACs, an in vivo blocking study was performed by co-injection of 10 nmol of LBH589-Cy5.5 with HDACi LBH589 (20 mg/kg, 1 µmol). As shown in Figure 4C, the tumor uptake of LBH589-Cy5.5 for the blocking group decreased dramatically in comparison with the imaging group at 6h p.i. Figure 4D showed the fluorescent intensities of tumors defined as photons per second per centimeter squared per steradian (p/s/cm2/sr) at 2h, 6h and 24h p.i. for both imaging and blocking groups. The data showed that the tumor uptake reached maximum at 2h p.i. and slowly washed out over time. The tumor fluorescent intensities in the blocking group (1.64 ± 0.32 at 2h, 1.92 ± 0.38 at 6h, 2.32 ± 0.35 at 24h) are significantly lower than those in the imaging group


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(6.36 ± 1.85 at 2h, 4.45 ± 1.15 at 6h, 2.98 ± 0.80 at 24h, P

An HDAC-Targeted Imaging Probe LBH589âCy5.5 for Tumor

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An HDAC-Targeted Imaging Probe LBH589âCy5.5 for Tumor