PLK1-Targeted Fluorescent Tumor Imaging with High Signal-to

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PLK1-Targeted Fluorescent Tumor Imaging with High Signal-Background-Ratio Ji-Ting Hou, Kyung-Phil Ko, Hu Shi, Wen Xiu Ren, Peter Verwilst, Seyoung Koo, Jin Yong Lee, Sung-Gil Chi, and Jong Seung Kim ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00544 • Publication Date (Web): 18 Sep 2017 Downloaded from http://pubs.acs.org on September 19, 2017

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PLK1-Targeted Fluorescent Tumor Imaging with High SignalBackground-Ratio Ji-Ting Hou,a,d,† Kyung-Phil Ko,b,† Hu Shi,c,† Wen Xiu Ren,a Peter Verwilst,a Seyoung Koo,a Jin Yong Lee,c,* Sung-Gil Chi,b,* Jong Seung Kima,* a

Department of Chemistry, Korea University, Seoul 02841, Korea. Department of Life Sciences, Korea University, Seoul 02841, Korea. c Department of Chemistry, Sungkyunkwan University, Suwon 16419, Korea d Hubei Collaborative Innovation Center for Biomass Conversion and Utilization, Hubei Engineering University, Xiaogan 432000, China b

Supporting Information Placeholder ABSTRACT: As significantly expressed during cell division, polo-like kinase 1 (PLK1) plays crucial roles in numerous mitotic events and has attracted interest as a potential therapeutic marker in oncological drug discovery. We prepared two small molecular fluorescent probes, 1 and 2, conjugated to SBE13 (a type II PLK1 inhibitor) to investigate the PLK1-targeted imaging of cancer cells and tumors. Enzymatic docking studies, molecular dynamics simulations, in vitro and in vivo imaging experiments, all supported the selective targeting and visualization of PLK1 expressing cells by probes 1 and 2, and probe 2 was successfully demonstrated to image PLK1-upregualted tumors with a remarkable signal to background ratios. These findings represent the first example of small-molecule based fluorescent imaging of tumors using PLK1 as a target, which could provide new avenues for tumor diagnosis and precision therapeutics. KEYWORDS: PLK1 • SBE13 • targeted imaging • high SBR ratio • fluorescence

Precision therapeutics, selectively and efficiently killing cancer cells, holds great promise in the treatment of cancer, but requires equally powerful imaging tools to differentiate cancerous tissues from normal cells.1 In this regard, the use of tumor imaging agents, enabling the visualization of tumor tissues, greatly augments the chances of exhaustive tumor ablation. Ideal tumor imaging agents should satisfy two criteria: cancer recognition and normal structure preservation, both requiring a high signalbackground ratio (SBR).2 Among the available imaging modalities, biological fluorescence imaging with smart imaging agents, combining high sensitivity, spatial and temporal resolution and versatility with selective tumor tissue recognition,3,4 has been attracting increasing attention5-10 Recently, a strategy to use proteins or membrane–bound receptors that are up-regulated in cancer cells as targets to design tumor-targeted fluorescent probes has been widely employed, namely affinity-based probes. These probes have been directed at the folate receptor,11 the integrin receptor,12 the prostate-specific membrane antigen,13 and the cyclooxygenase-2 enzyme,14 However, in particular inhomogeneous expression of the target receptors, due to tumor heterogeneity and increased background signals, originating from the unbound probes are major limitations to this methodology. Whereas background signals as a result of the

unbound probes’ fluorescence are hard to eliminate, the use of novel targets with a wider, ideally universal expression in various tumors would undoubtedly increase the applicability of affinitybased probes. Hence, the identification of novel targets for the selective imaging of tumors is an urgent and important matter. Polo-like kinases (PLKs), a subfamily of serine/threonine kinases consist of five members: named PLK1‒5. PLKs regulate a number of events associated with the progression of the cell cycle, differentiation, as well as cell death or survival.15,16 PLK1, in particular, is involved in a number of crucial events related to mitosis and is notably expressed during cell division,17and is also believed to be involved in the malignant transformation of cells as a key factor in cell devision.18 Furthermore, a large proportion of cancers exhibit elevated levels of PLK1, and thus this kinase has been regarded as a common proto-oncogene with potential applications as a prognostic marker.19 Thus, fluorescent imaging of PLK1 would enable the discrimination of suspected malignant cells via the visualization of this marker. Examples of PLK1targeted tumor imaging, however are rare,20 and no reports employ small fluorescent labeling of PLK1 by synthetic dyes. In view of their synthetic ease, as well as non-immunogenic nature and fast target recognition, small molecule-based probes targeting PLK1 would represent a significant advantage to currently employed methods. Thus we herein describe a small molecular PLK1 affinitive probe able to discern tumors from normal tissues. SBE13, a type II PLK1 inhibitor developed by Keppner et al., stabilizes the inactive DFG-out conformation of PLK1 with excellent selectivity over other PLKs, with an inhibitory concentration of 0.2 nM.21,22 Thus, we designed the fluorescent imaging agent 1 with a coumarin derivative as the fluorophore and SBE13 as the binding ligand, to selectively and potently bind PLK1 (Scheme 1). To minimize the influence of the fluorophore on the affinity of SBE13 for PLK1, an alkyl chain with six carbon atoms was inserted between the fluorophore and the ligand, to avoid the steric obstruction of the cargo.23 Coumarin was chosen for its high photostability, large Stokes shift, and facile functionalization.24-26

Scheme 1. Structures of SBE13 and probe 1.

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First, we explored the binding of PLK1 to probe 1 to confirm the feasibility of our design. To identify the active sites and investigate the interactions between probe 1 and PLK1 at an atomic level, docking and molecular dynamics (MD) simulations were performed (Figure 1). The free energy of PLK1 binding to probe 1 was calculated to be -71.8 kcal/mol from the MD simulation. Four residues (L59, C67, M98, and F183) were found to play an important role in the active site (Figure S6). It is worth to mention that C67 and F183 are important for maintaining the selectivity of inhibitors towards PLK and other kinases. Our simulations support that the binding of the probes are unlike to be sterically hindered by the linker and fluorescent group. As probe 1 was predicted to significantly bind PLK1, probe 1 was synthesized as illustrated in the Supporting Information (Scheme S1). A

B M98

L59 C67

F183

Figure 1. Structure (A) and PLK1 binding sites (B) of probe 1. The absorption and emission spectra of probe 1 were examined in phosphate buffered saline (PBS) (10 mM, pH = 7.4, containing 0.2% DMSO). Probe 1 showed an absorption maximum at 425 nm, characteristic of the coumarin moiety, and fluoresced strongly at 480 nm, with a Stokes shift of 55 nm (Figure S1). The quantum yield was determined to be 0.13, using fluorescein as the standard.27 The fluorescence intensity of probe 1 changed negligibly in the presence of biologically relevant potential interferences such as Na+, K+, Mg2+, GSH, Cys and H2O2 (Figure S2). The timedependent irradiation with visible light also showed little influence on the emission intensity of probe 1 (Figure S3), indicating a desired level of stability and robustness. Next, the primary intracellular imaging ability of probe 1 was tested in HeLa cells. After 30 min incubation, strong fluorescence was observed from the cells with a probe concentration of 5 µM, confirming an excellent cell permeability of the probe (Figure S4). Subsequently, we investigated the correlation between the emission intensity of probe 1 and PLK1 expression levels in five human cells, namely MCF10A (breast epithelial cell), HaCaT (immortalized keratinocyte), HCT116 (colon cancer), MDAMB231 (breast cancer), and MCF7 (breast cancer) cells. An immunoblot assay revealed that among the five cells we tested, MCF10A cells express the lowest level of PLK1 protein and both HaCaT and HCT116 cells express the highest levels (Figure 2A). As predicted, probe 1 emitted the lowest fluorescence in MCF10A and much brighter fluorescence in other four cell types, which is in accordance with the PLK1 expression levels in these cells (Figure 2B and C). This supports that the intracellular fluorescence intensity of probe 1 is tightly associated with PLK1 expression levels. Moreover, the co-localization of probe 1 with PLK1 in cells was explored upon co-incubation with a fluorescently labeled anti-PLK1 antibody. Interestingly, confocal microscopy revealed that the fluorescence of probe 1 was completely contained in that of anti-PLK1. Since PLK1 has been reported to accumulate at the central spindle microtubules during anaphase and cytokinesis,28 we proposed that probe 1 bound predominantly with microtubule-enriched PLK1 in dividing cells, certifying the excellent PLK1 targetability of probe 1 (Figure 2D).

Figure 2. PLK1-related fluorescence imaging of probe 1 in human cells. (A) PLK1 levels in five human cell lines. Immunoblot assay was performed using anti-PLK1 antibody. Tubulin was used as the loading control. (B) Fluorescence microscopic images of probe 1 or 11-treated cells. Cells were incubated with 20 µM of probe 1 or 11 for 20 h. (Scale bar: 20 µm. Magnification: 200X.) (C) Relative fluorescence intensity of 1-treated cells. Fluorescence emission was determined at 480 nm. (D) Co-localization of probe 1 and PLK1 in HCT116. Cells were stained with anti-PLK1 antibody (red), probe 1 (green), and DAPI (blue). Bars represent the average and SD of triplicate measurements. DIC: differential interface contrast. (Scale bar: 20 µm. Magnification: 400X.) A WST-1 assay was conducted and probe 1 was found to evoke no detectable cytotoxic effect up to 72 h post-treatment (20 µM) in the tested cells (Figure S5A and B). The results indicated that probe 1 is a non-cytotoxic agent that allows a sensitive detection and discrimination of PLK1-expressing human cells from multiple tissue origins. Further explorations were performed to examine the ability of probe 1 to monitor the PLK1 level variations in cells. Nocodazole is an antineoplastic agent which triggers the G2 phase-specific cell cycle arrest and consequently induces PLK1 accumulation.29 As shown in Figure 3, a dose-associated elevation of PLK1 was observed when HCT116 cells were pre-treated with nocodazole. Consistently, the fluorescence of probe 1 was markedly enhanced in HCT116 cells treated by nocodazole. However, the fluorescence of compound 11, an analogue of 1 without the PLK1targeted ligand (Scheme S1), showed negligible emission in HCT116 cells and manifested no changes after the nocodazole treatment (Figure 3C), suggesting that the PLK1 targetability of SBE13 prompted the accumulation of 1 inside cells and the emission enhancement of probe 1 in the presence of nocodazole is induced by the elevation in PLK1 levels. In addition, the fluorescence response of 1 under PLK1 depletion, using small-interfering (si) RNA-mediated knockdown of PLK1, showed a clear reduction in the intracellular fluorescence as depicted in Figure 3D and E. The PLK1 dependence on the fluorescence was clearly visible at concentrations of as little as 0.5 µM of 1, clearly showing the high sensitivity of the PLK1 affinitive probe (Figure 3F). By contrast, the fluorescence of 11 was virtually unresponsive to PLK1 depletion.

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ACS Sensors kept steady in the presence of biologically relevant potential interferences and under visible light irradiation (Figure S7 and 8).

Figure 4. (A) The structure of probe 2; (B) The normalized absorption and emission spectra of probe 2 in PBS.

Figure 3. PLK1-dependent fluorescence response of probe 1. (A) Immunoblot assay for PLK1 expression upon nocodazole treatment. (B) HCT116 cells were pre-exposed to nocodazole (50 ng/mL, 15 h) and incubated with probe 1 or 11 (1 µM, 3 h). PC: phase contrast. (Scale bar: 20 µm. Magnification: 200X.) (C) A nocodazole dose-associated fluorescence enhancement of probe 1 in cells. Compound 11 was used as a negative control. (D) Immunoblot assay of si-PLK1-mediated PLK1 knockdown in HCT116 cells. (E) HCT116 cells transfected with si-PLK1 (4 µM) or control si-RNA were incubated with probe 1 (1 µM, 3 h). (Scale bar: 20 µm. Magnification: 200X.) (F) si-PLK1 doseassociated decrease in the fluorescence of probe 1. Bars represent the average and SD of triplicate measurements. The overexpression of PLK1 in various tumors, should result in the accumulating of 1 in tumors, allowing for the visualization of the pathogenic tissues. However, the short absorption and emission wavelengths of this probe would be prohibitive for in vivo applications, therefore probe 2, was prepared with 3-cyano-4phenyl-2(5H)-furanon as a strong electron-withdrawing group, as a long wavelength analogue of 1. The free energy of PLK1 with probe 2 was calculated to be -81.9 kcal/mol (Figure S6). Although both the probes are sterically bulky, they dock well in the active site of PLK1, and the L59, C67, M98, and F183 residues, located in the active cleft, were significant contributors to the binding interaction of both probes, indicating their importance in the molecular recognition of the SBE13 bearing probes with PLK1. As illustrated in Figure 4, probe 2 displayed a maximum absorption peak at around 555 nm and an emission maximum at 660 nm in PBS, representing a dramatic bathochromic shift, relative to probe 1, as a result of the strong intramolecular charge transfer (ICT) nature of the new fluorophore. With the red-shifted emission and absorption, the probe fulfills the wavelength requirements for in vivo imaging. With the large Stokes shift of 105 nm an increased contrast and thus a higher SBR can be anticipated as well. Like probe 1, the fluorescence intensity of probe 2 almost

Analogous to the experiments described above for 1, probe 2 showed no significant toxicity in the tested cell lines (Figure S9) and the fluorescence in cells loaded with 2 was increased or decreased as a result of nocodazole treatment or si-RNA dependent PLK1 knockdown (Figure S10). The basic fluorophore skeleton (16), not bearing the SBE13 subunit was used as a negative control (Figure S10) and, like 11, the fluorescence from cells subjected to 16 did not show any dependence on the PLK1 content. Together, these results strongly support the notion of probes 1 and 2 as highly PLK1-dependent cellular imaging agents, and importantly the targetability of SBE13 was not compromised by the accompanying fluorophore conjugation. Finally, the ability of probe 2 to detect tumors in vivo was evaluated using a mouse tumor xenograft assay. 5 × 106 HCT116 cells were injected subcutaneously into four-week-old immunedeficient mice (nu/nu) to induce tumor formation. After 2 weeks of visible tumor growth, DMSO or nocodazole (5 mg/kg) was injected intraperitoneally, and probe 2 was administered intravenously (5 mg/kg) to the mice. As seen in Figure 5A, site-specific fluorescence was detected predominantly in tumor tissues in probe 2-injected mice, and the fluorescence intensity significantly increased when nocodazole was pre-administrated. Moreover, a comparative analysis of excised organ tissues demonstrated that strong fluorescence only emanated from the tumor site, while the heart, kidney or spleen showed only weak fluorescence, suggesting that PLK1 level in tumor tissues is much higher than that in normal organs. Importantly, even in the liver, a vital organ for xenobiotic metabolism, the fluorescence intensity was very weak (Figure 5B). The fluorescence intensity ratio of Ftumor/Fliver could reach up to 17.6 in the nocodazole-pretreated mouse, while this value was still found to be 3.3 in only 2-injected mice (Figure 5C). The excellent SBR observed in tissues of mice subjected to affinity-based probe 2 is quite rare, with most affinity-based always-on probes suffering from severe background fluorescence as a result of the accumulation of these probes in other normal organs, like the liver and kidneys, distinctly limiting their applicability.14,30,31 A tumor-weight associated fluorescence change was observed in the 2-injected mice (Figure 5D), which obviously resulted from the higher levels of 2 in tumors of larger size. In conclusion, we presented two PLK1-targeted, coumarinbased fluorescent imaging agents 1 and 2, comprising the PLK1 inhibitor SBE13 as the protein binding ligand. The ability of the probes to bind to PLK1 was demonstrated by docking studies. These compounds were non-cytotoxic and their fluorescence intensities in various human cells exhibited sensitive responses to PLK1 levels. Furthermore, probe 2, exhibiting near-infrared emission, was successfully applied in selective tumor imaging in the mouse tumor xenograft model with outstanding SBR. Our results suggest, that affinity-based fluorescent imaging agents can achieve selective tumor recognition with high SBR upon targeting

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appropriate cellular factors. Thus, our work might provide new avenues for tumor diagnosis and precision therapeutics.

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This work was supported by the Ministry of Science, ICT & Future Planning (MSIP) of Korea (No. 2009-0081566 for JSK) National Research Foundation of Korea (NRF20158A2A1A01005389 for SGC).

REFERENCES

Figure 5. Mouse tumor xenograft assay for in vivo fluorescence imaging of probe 2. (A) White light (top) and fluorescence (bottom) images of HCT116-xenograft tumor bearing mice injected with probe 2, pretreated with DMSO (left) and nocodazole (right). (B) Dissected organs and tumors and the corresponding fluorescent images form probe 2-injected tumor-bearing mice. (C) Quantification of the fluorescence intensity of the excised organ tissues and tumors of probe 2-injected tumor-bearing mice (D) Tumor weight-associated fluorescence of 2. Bars represent the average and SD of triplicate measurements. λex = 560 nm, λem = 660 nm.

ASSOCIATED CONTENT Supporting Information Experimental details, characterization data, and other materials are available online free of charge as SUPPORTING INFORMATION. Additional experimental methods, NMR spectra, and other figures (PDF)

AUTHOR INFORMATION Corresponding Author [email protected] (J. S. Kim); [email protected] (S.-G. Chi) [email protected] (J. Y. Lee)

Author Contributions †

These authors contributed equally.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT

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