Design and synthesis of a luminescent iridium complex-peptide hybrid (IPH) that detects cancer cells and induces their apoptosis

Bioorganic & Medicinal Chemistry 26 (2018) 4804–4816

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc

Design and synthesis of a luminescent iridium complex-peptide hybrid (IPH) that detects cancer cells and induces their apoptosis

T

Abdullah-Al Masuma, Kenta Yokoia, Yosuke Hisamatsua, Kana Naitoa, Babita Shashnia, ⁎ Shin Aokia,b, a b

Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan Imaging Frontier Center, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan

A R T I C LE I N FO

A B S T R A C T

Keywords: TRAIL Death receptors Apoptosis Cyclometalated iridium(III) complexes

Tumor necrosis factor related apoptosis inducing ligand (TRAIL) triggers the cell-extrinsic apoptosis pathway by complexation with its signaling receptors such as death receptors (DR4 and DR5). TRAIL is a C3-symmetric type II transmembrane protein, consists of three monomeric units. Cyclometalated iridium(III) complexes such as facIr(tpy)3 (tpy = 2-(4-tolyl)pyridine) also possess a C3-symmetric structure and are known to have excellent luminescence properties. In this study, we report on the design and synthesis of a C3-symmetric and luminescent Ir complex-peptide hybrid (IPH), which contains a cyclic peptide that had been reported to bind to death receptor (DR5). The results of MTT assay of Jurkat, K562 and Molt-4 cells with IPH and co-staining experiments with IPH and an anti-DR5 antibody indicate that IPH binds to DR5 and induces apoptosis in a manner parallel to the DR5 expression level. Mechanistic studies of cell death suggest that apoptosis and necrosis-like cell death are differentiated by the position of the hydrophilic part that connects Ir complex and the peptide units. These findings suggest that IPHs could be a promising tool for controlling apoptosis and necrosis by activation of the extra-and intracellular cell death pathway and to develop new anticancer drugs that detect cancer cells and induce their cell death.

1. Introduction Apoptosis is essential for the maintenance of cell populations in organisms and for maintaining proper homeostasis in metazoan. Perturbation of this normal process alters the balance between cell death and cell proliferation and may lead to the development of various types of disease, in which cancer is most prominent.1–3 Tumor necrosis factor related apoptosis inducing ligand (TRAIL) belongs to TNF superfamily and induces apoptosis in various tumor cells through the cell-extrinsic pathway, independently of p53.4–7 TRAIL is a type II transmembrane protein that has a Zn2+-centered C3-symmetric homotrimeric structure by self-assembly of three monomer units.8–10 It is also known that TRAIL can interact with a complex family of receptors, plasma membrane-expressed TRAIL-R1 (DR4), TRAIL-R2 (DR5), TRAIL-R3 (DcR1), TRAIL-R4 (DcR2) and a soluble receptor, osteoprotegerin (OPG). DR4 and DR5 contain death domain (DD) to convey apoptotic signals, and therefore is denoted as death receptors, whereas DcR1 and DcR2 are unable to induce cell death and regarded as decoy receptors.11–17 Upon binding of TRAIL with DR, TRAIL-DR assemble death inducing signaling complex (DISC) and recruit the adaptor protein (FADD) via ⁎

the death effector domain (DED). These signals activate procaspase-8 to caspase-8 and then caspase-3, which cleaves multiple substrates to execute cell death.3–7 The non-signaling DcR1 does not contains cytoplasmic DD, and DcR2 is very similar to death receptors but contains a truncated form of DD. Therefore, both DcR1 and DcR2 are unable to set up DISC and modulate the activity of DR4 and DR5 and inhibit TRAILmediated apoptosis.13–15 Since TRAIL has low toxicity on normal cells, recombinant TRAIL and agonistic anti-DR4 and anti-DR5 monoclonal antibody (mAb) have been examined for clinical applications.18–26 Development of low molecular weight compounds having TRAIL-like activities could be an alternative and advantageous approach as compared with antibody drugs due to easy modification, easy handling, low cost, and large scale preparation. To date, however, only limited examples of the artificial molecules having TRAIL-like functionalities have been reported.27–35 In addition, details of the extracellular and successive intracellular events induced by TRAIL-DR complexation need further study, regardless of the fact that many studies have been reported on intracellular apoptosis signaling pathways. Therefore, artificial luminescent TRAIL-like agonists would be expected to dually function as

Corresponding author at: Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan. E-mail address: [email protected] (S. Aoki).

https://doi.org/10.1016/j.bmc.2018.08.016 Received 10 May 2018; Received in revised form 19 July 2018; Accepted 11 August 2018 Available online 14 August 2018 0968-0896/ © 2018 Published by Elsevier Ltd.

Bioorganic & Medicinal Chemistry 26 (2018) 4804–4816

A.-A. Masum et al.

Scheme 1. Design of C3-symmetric IPHs as artificial death ligands.

anticancer agents and imaging probes of death signal pathways in cancer cells and related cells. Cyclometalated iridium(III) (Ir(III)) complexes such as fac-Ir(tpy)3 1 (tpy = 2-(4-tolyl)pyridine) (Scheme 1) have been subjects of considerable attention because of their unique photophysical properties36–40 and their analogs have been widely applied to not only organic lightemitting diodes (OLEDs) as phosphorescent emitters,41–46 but also to oxygen sensors,47–48 chemosensors,49–54 and luminescent probes for biological systems.55–73 These are due to their high-luminescence quantum yields, long luminescence lifetimes (τ∼µs) that can eliminate the short lived autofluorescence (τ ∼ns) from biological samples in cellular imaging, and significant Stokes shifts minimize the selfquenching process. The second feature of cyclometalated Ir complexes is their C3-symmetric structure, which could mimic the structures of naturally occurring homotrimeric proteins such as TRAIL, as mentioned above (Scheme 1). We previously reported on the preparation of some Ir(III) complexes that are functionalized as blue ∼ green ∼ red and white color emitters,74–79 pH sensors,74,76-79 and photosensitizers.76–79 We recently reported on the preparation of some Ir complexes having cationic peptides (typically, H2N-KKGG-) such as 2 (Scheme 2) as inducers and detectors of necrosis like cell death of Jurkat cells (a human T-lymphoma cell lines).80–82 These results suggested that Ir complexes are potential agents for the diagnosis and treatment of cancer and related diseases and even for mechanistic studies of cell death processes. Because cyclometalated trishomoleptic Ir(III) complexes such as fac1 possess C3-symmetric structures like TRAIL, we hypothesized that these complexes, when functionalized with DR-binding peptide motifs, would be potent TRAIL mimics for detecting cancer cells and inducing their cell death. Quite recently, we reported on the design and synthesis of Ir complex-peptide hybrids (IPHs) (3–5) (Scheme 2) having cyclic peptides that had been reported to bind DR5 by a research group in Affymax.27–30 Because the solubility of 3 in water is low, a hydrophilic peptide (Ser-Gly-Ser-Gly) was incorporated into the cyclic peptide part to produce more hydrophilic 4 and 5, which were found to induce slow cell death of Jurkat cells as confirmed by MTT assays.83 Staining

Scheme 2. Structure of IPHs.

experiments of Jurkat cells with 4 and 5 indicated that these Ir complexes are internalized with DR5 into the cells, resulting in the slow induction of necrotic type cell death. In this work, on the other hand, the hydrophilic SGSG sequence was incorporated between the Ir complex core and the cyclic peptide part, as shown in 6 (Scheme 2). In 4 and 5, a hydrophilic peptide (Ser-GlySer-Gly) was incorporated at the N-terminus of cyclic peptide part. Next, the same hydrophilic peptide (Ser-Gly-Ser-Gly) was incorporated between the Ir complex core and peptide parts of 6 to change the linker length with minimum effect on its solubility in water. It is reported that 6 induces cell death, possibly apoptosis, of Jurkat cells with a moderate EC50 value and that co-staining of Jurkat cells with 6 and an anti-DR5 antibody confirms that they are co-localized on the cell membrane, suggesting that these two molecules bind to DR5, but at the different sites. Binding of 6 to cancer cells as well as cytotoxicity against cancer cells are dependent on the DR5 expression level of cancer cells. 4805

Bioorganic & Medicinal Chemistry 26 (2018) 4804–4816

A.-A. Masum et al.

Scheme 3. Synthesis of IPH 6.

11. Condensation of 11 with β-alanine ethyl ester hydrochloride and the subsequent ester hydrolysis yielded 12, which was converted to the corresponding N-hydroxysuccinimide (NHS) ester 13. Condensation of 13 with 10 through the N-terminus of the H2N-SGSG- part of 10 afforded 6, which was purified by reversed-phase HPLC column with a continuous gradient of H2O (0.1%TFA)/CH3CN (0.1%TFA) and was lyophilized to give the corresponding TFA salt in the form of a yellow powder.

Mechanistic studies suggest that 6 induces apoptosis, while 4 and 5 induce necrotic type cell death. 2. Results and discussion 2.1. Design and synthesis of Ir complex-peptide hybrids (IPHs). Synthesis of 3–5 was reported in our previous paper83 and 6 was prepared in a similar manner, as shown in Scheme 3. Boc-NH-Ser-GlySer-Gly-OH 7 was synthesized by solid phase peptide synthesis and the C-terminus of this peptide was converted to the activated ester 8, which was coupled with cyclic peptide 927–30 to give 10. The Vilsmeier reaction of 1 (fac-Ir(tpy)3) and the following Pinnick oxidation80–82 gave

2.2. UV/Vis and luminescence spectra of Ir complex-peptide hybrids (IPHs) UV/Vis and luminescence spectra of the Ir complex 6 (10 µM) in DMSO at 25 °C are shown in Fig. 1 and its photophysical data are 4806

Bioorganic & Medicinal Chemistry 26 (2018) 4804–4816

A.-A. Masum et al.

Figure 2. Time course of frequency change (Δ F (Hz)) of IPHs-DR5 complexation on 27 MHz QCM. Conditions: temperature: 25 °C, solvent: phosphate buffered saline (PBS). An aliquot of solutions of TRAIL (plain curve) (200 µg/mL), 6 (bold curve), 14 (bold dashed curve) and 2 (plain dashed curve) (10 mM solution in DMSO) was added to DR5 fixed on the sensor chip. Addition time of analytes to DR5 are indicated by the plain arrows.

2.3. Complexation of 6 with DR5 evaluated by 27 MHz QCM To confirm the binding of 6 to DR5, 27 MHz quartz-crystal microbalance (QCM) measurements were carried out. DR5 was immobilized on a sensor chip, to which 6 was added. From the Δ F decay curves of 6 shown in Fig. 2, the complexation constant (Kapp) of 6 with DR5 (and dissociation constants, Kd) was determined to be (4.6 ± 0.2) x 105 M−1 (Kd = 2.2 ± 0.1 µM), which is almost identical to those of 4 and 5 (Table 2).83 In the X-ray crystal structure of (TRAIL)3-(DR)3 complex,8–10 there are many intermolecular peptide-peptide interactions (PPI) between them, which possibly cause this strong complexation. On the other hand, the DR binding sites of 6 are possibly limited to its cyclic peptide units that may recognize their counterpart moieties in DR5, which have not been identified yet. It is assumed that these are one of the reasons of lower affinity of 6 with DR than that of TRAIL with DR. Very weak interactions were observed for the Ir complex 14 (Scheme 4), which lacks the DR-binding peptide, and for 2 that contains KKGG peptide (Scheme 2).81–82 Negligible interactions of 6 with calmodulin (it is reported that 2 binds to calmodulin)81–82 and albumin (data not shown) was observed, suggesting the selective binding of 6 to DR5.

Figure 1. (a) UV/Vis spectra of 3 (bold curve), 4 (plain curve), 5 (bold dashed curve) and 6 (dashed curve). (b) Emission spectra of 3 (bold curve), 4 (plain curve), 5 (bold dashed curve) and 6 (dashed curve) in degassed DMSO at 25 °C ([Ir complex] = 10 μM, excitation at 366 nm). A.u. is arbitrary unit. (Inset of Fig. 1b) Photograph showing emission of 4, 5 and 6 (excitation at 365 nm).

Table 1 Photophysical properties of Ir complexes, 2–6, in degassed DMSO at 25 °C ([Ir complex] = 10 µM, excitation at 366 nm).

b

Compound

λmax (absorption)

λmax (emission)

Φa

τb

2 3 4 5 6

280, 285, 285, 286, 286,

509 nm 505 nm 506 nm 506 nm 506 nm

0.55 0.39 0.33 0.36 0.35

1.7 µs 1.1 µs 1.3 µs 1.2 µs 1.0 µs

362 nm 363 nm 361 nm 360 nm 360 nm

a Quinine sulfate in 0.1 M H2SO4 (Φ = 0.55) was used as a reference. Lifetime of luminescence emission.

2.4. Cancer cell death induced by IPHs was evaluated by MTT Assay. First, the cytotoxicity of 6 against Jurkat cells was compared to those of 3–5 by MTT assay (MTT = 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide). Jurkat cells were incubated with 3–6 at the given concentrations in 10% FCS (fetal calf serum)-

summarized in Table 1 in comparison with those of 2–5. A strong green emission with a maxima at ca. 506 nm was observed for 6, which is almost same as those of 3–5 (Fig. 1b). The strong absorption bands at 270–300 nm were assigned to the 1π-π* transition of the tpy ligands and weak shoulder bands at 320–450 nm were assigned as spin-allowed singlet-to-singlet metal-to-ligand charge transfer (1MLCT) transitions, spin-forbidden singlet-to-triplet (3MLCT) transitions, and 3π-π* transitions. The luminescence quantum yields (Φ) of 3–6 were determined to be 0.33–0.39, and their luminescence lifetimes are 1.0 ∼ 1.3 µs, as summarized in Table 1. The concentrations of the Ir complexes in stock solutions (DMSO) were determined by the molar extinction coefficient at 380 nm (ɛ380nm = 1.43 ± 0.03 × 104 M−1 cm1 ) of 3–6, which are almost identical to those of typical Ir(tpy)3 derivatives such as 2, as we previously reported.80–81

Table 2 Complexation Constants of IPHs (assuming a 1:1 complexation). Analyte TRAIL 4 5 6 2 14

4807

Kapp (M−1) (2.3 ± (3.8 ± (4.0 ± (4.6 ± < 104 < 104

Kd 8

0.05) x 10 0.1) x 105 0.2) x 105 0.2) x 105

4.3 ± 0.1 nM 2.7 ± 0.1 µM 2.5 ± 0.1 µM 2.2 ± 0.1 µM > 100 µM > 100 µM

Bioorganic & Medicinal Chemistry 26 (2018) 4804–4816

A.-A. Masum et al.

Scheme 4. Structure of 14.

Figure 4. The results of MTT assay: cell viability of Jurkat cells after incubation with TRAIL (150 ng/mL), 6 (75 µM) for 16 h, and 4–5 (75 µM) for 24 h, in the absence of Z-VAD-FMK (closed bars) and in the presence of Z-VAD-FMK (open bars).

high SS, which was disappeared in the cells pretreated with Z-VAD-FMK (Fig. 5f). As for 4, there was no difference between Z-VAD-FMK pretreated or untreated cells (Fig. 5c–d). Flow cytomeric analyses of Jurkat cells that had been treated with 6 and then stained with propidium iodide (PI) were also conducted. Jurkat cells were pretreated with Z-VAD-FMK (15 µM) at 37 °C for 1 h, incubated with 6 (25/35 µM)/TRAIL (150 ng/mL) at 37 °C for 6 or 12 h, and then incubated with PI (30 µM) to stain the dead cells for flow cytometric analysis. Cells that were not pretreated with Z-VAD-FMK were stained with PI (red bars in Fig. 6), but the cells that were pretreated with Z-VAD-FMK were weakly stained with PI. These results suggest that 6 induces apoptosis similar to TRAIL.

Figure 3. The results of MTT assay: cell viability of Jurkat cells (% of control at [Ir complex] = 0 µM) in the presence of TRAIL (150 ng/ml), 3, 4, 5, 6, 9, and 14 (75 µM) after incubation at 37 °C for 16 h.

containing RPMI 1640 (MTG free) medium at 37 °C for 16 h and treated with MTT reagent. For comparison, the cytotoxicity of TRAIL was also evaluated by MTT assay. TRAIL induced up to 70% cell death at 150 ng/mL and 4, 5 and 6 (75 µM) induced cell death up to 33%, 42% and 55%, respectively (Fig. 3) (EC50 values of 4, 5 and 6 are 114 µM, 89 µM and 68 µM, respectively). 3 negligibly induces cell death, possibly due to its low solubility, as described in our previous paper.83 Monomer peptide 9 and Ir complex 14, which lacks the receptor binding peptide, negligibly induce cell death. It is known that the Ir complexes function as photosensitizers to activate triplet oxygen (3O2) to singlet oxygen (1O2), which induces necrosis-like cell death of adhesive cell lines such as HeLa cells.75–77 We also examined the effect of the photoirradiation on the cell viability of Jurkat cells and it was found that Jurkat cells undergo cell death after photoirradiation (465 nm) for 3–5 min in the absence of Ir complexes, suggesting that Jurkat cells are very sensitive to photoirradiation.82 Therefore, the effect of Ir complexes on the cell viability was examined on the luminescence microscopy as quickly as possible.

2.6. Luminescence staining and cell death induction of cancer cells by IPH Luminescent staining of DR5-expressed Jurkat cells was performed using the Ir complex 6. Jurkat cells were incubated with 6 for 1 h at 4 °C and 37 °C, respectively, and observed by luminescence microscopy, as displayed in Fig. 7. After incubation at 4 °C, negligible emission (Fig. 7a–c) was observed. In contrast, significant green emission spots from 6 were observed in the cytosol of the cells after incubation at 37 °C in a parallel manner to the increasing concentration of 6. Co-staining of Jurkat cells with 6 and an anti-DR5 antibody (conjugated with a red fluorochrome) was carried out. Jurkat cells were first treated with the anti-DR5 antibody and then with 6 and observed on fluorescence microscopy. Fig. 8b–c shows the green emission from 6 and Fig. 8d–e shows red emission from the anti-DR5 antibody. Overlays of these images (Fig. 8f,g) exhibit yellow spots implying that the binding of 6 and anti-DR5 antibody to DR5 without significant competition. In the co-staining assay, anti-DR5 antibody binds to DR5 and then 6 to form a complex comprising three molecules, DR5, anti-DR5 antibody and 6. The movement of this ternary complex (DR5, anti-DR5 antibody and 6) is possibly slower than that of DR5-6 complex. We assume that this is one of the reasons why 6 seems to be localized in the cytosol in Fig. 7(d–f) and 6 seems to be localized mainly on the cell membrane in Fig. 8. Competitive assay of 6 with the DR5 binding peptide 9 (Scheme 3)27–29 was conducted. Jurkat cells were treated with 9 and then with 6. Binding of 6 to Jurkat cells significantly was decreased after the pretreatment of cells with 9, which was observed as a reduction in the emission from 6 (Figs. S1 and S2 in Supporting Information). These facts support the binding of 6 to DR5.

2.5. Mechanistic study of cell death induced by IPHs. It has been established that apoptotic signaling activates several caspases that cleave multiple substrates to execute cell death.3–7 Broad caspase inhibitors such as Z-VAD-FMK84 have been reported to inhibit apoptosis. Jurkat cells were thus treated with Z-VAD-FMK (15 µM) for 1 h and then incubated with IPHs or TRAIL (as a positive control). As shown in Fig. 4, the cell death of Jurkat cells by TRAIL and 6 was effectively inhibited by Z-VAD-FMK, while that by 4 and 5 was negligibly inhibited. Cell death induced by IPHs was also checked by flow cytometry measurements. Jurkat cells incubated with TRAIL (150 ng/mL) and 6 (75 µM) produced a large population of cells with high side scatter (SS) in the absence of Z-VAD-FMK as indicated by a red dashed circle (in Fig. 5a and 5e), while these is smaller number of plots in the corresponding position of Fig. 5b. Similarly, Jurkat cells incubated with 6 in the absence of Z-VAD-FMK produced a large population of cells with a 4808

Bioorganic & Medicinal Chemistry 26 (2018) 4804–4816

A.-A. Masum et al.

Figure 5. Flow cytometric distribution of Jurkat cells after treatment (a) with TRAIL (150 ng/ml) at 37 °C for 6 h, (b) with Z-VAD-FMK (15 µM) at 37 °C for 1 h and then with TRAIL (150 ng/ml) at 37 °C for 6 h, (c) with 4 (75 µM) at 37 °C for 24 h, (d) with Z-VAD-FMK (15 µM) at 37 °C for 1 h and then with 4 (75 µM) at 37 °C for 24 h. (e) with 6 (75 µM) at 37 °C for 6 h, (f) with Z-VAD-FMK (15 µM) at 37 °C for 1 h and then with 6 (75 µM) at 37 °C for 6 h. Dashed circle indicate the population of cells with side scatter (SS).

to which PI (30 µM) was added to stain the dead cells for analysis by flow cytometer. As summarized in Fig. 9, it was indicated that cell death induction of these three cell lines by 6 was parallel to the DR5 expression level. For comparison, cellular uptake and cytotoxicity of IPHs in negative control cells was also studied. Human embryonic kidney (HEK293) cells, which were derived by transformation of the epithelial cells,30,33,85 were incubated with 4 and 6 (10 µM) at 37 °C for 1 h and observed by luminescence microscopy. Very weak emission was observed from the HEK293 cells after incubation with IPHs (Fig. 10). Cytotoxicity of IPHs on HEK293 cells was studied by MTT assay. HEK293 cells were incubated with IPHs (4 and 6) (75 µM) and TRAIL

We next performed cell staining of different types of cancer cells such as Jurkat, K562 and Molt-4 cells. First, the DR5 expression in Jurkat, K562 and Molt-4 cells was evaluated by staining with an antiDR5 antibody and analyzed by flow cytometer and it was found that Jurkat cells express considerably higher levels of DR5 than those of K562 and Molt-4 cells (Fig. S3 in Supporting Information). These three cell lines were then stained with 6 at 37 °C for 1 h and then analyzed by flow cytometer. Jurkat cells were highly stained by 6, while K562 and Molt-4 cells were weakly stained (Fig. S3 in Supporting Information). The relationship between the cell death inducing activity of 6 and DR5 expression of cancer cells was studied. Jurkat, K562 and Molt-4 cells were individually incubated with 6 (25/75 µM) at 37 °C for 16 h, 4809

Bioorganic & Medicinal Chemistry 26 (2018) 4804–4816

A.-A. Masum et al.

(150 ng/mL) in 10% FCS (fetal calf serum)-containing RPMI 1640 (MTG free) medium at 37 °C for 16 h and treated with MTT reagent. IPHs and TRAIL exhibit negligible cytotoxicity on HEK293 cells as summarized in Fig. S4 in Supporting Information, implying the selectivity of detection and apoptosis induction of 6 to Jurkat cells over HEK293 cells.

2.7. Annexin V and PI staining of cell death induced by IPHs. It is known that phosphatidylserine (PS), which is located in the inner surface of the plasma membrane, is exposed to the outer surface in early stage of apoptosis, which can be detected by Annexin V and is not stained by propidium iodide (PI).86 At the late stage of apoptosis or necrosis, cells lose their membrane integrity, and can then detected with PI.87 We conducted Annexin V-Cy3 and PI staining experiments, in which Jurkat cells were treated with 4 and 6 at at 37 °C for 2 h and then incubated with Annexin V-Cy3 or PI. The cells treated with 6 were stained with Annexin V-Cy3 but not PI (Fig. 11ba–be and da–de), while negligible emission was observed from the cells treated with 4 (Fig. 11aa-ae and 11ca-ce). After incubation of Jurkat cells with 4 and 6 for 24 h, both cells were strongly stained with PI (Fig. 11ea–fe). These results suggest that 6 induces apoptotic cell death, whereas 4 induces not apoptosis, possibly necrotic cell death, as we previously reported.83 We also studied the fragmentation of DNA to examine apoptosis. Negligible DNA ladders were observed after treatment of Jurkat cells with IPHs (Fig. S5 in Supporting Information). However, DNA ladder might not be considered be an exclusive indicator to for apoptosis, because it has been reported that apoptosis can occur without oligonucleosomal DNA fragmentation.88

Figure 6. The results of flow cytometry assay of PI staining of Jurkat cells treated with 6/TRAIL. Red bars correspond to the cells incubated with 6 (25/ 35 µM) or TRAIL (150 ng/mL) at 37 °C for 6 or 12 h, and then treated with PI (30 µM) for 10 min at room temperature. Green bars correspond to the cells treated with Z-VAD-FMK (15 µM) at 37 °C for 1 h and then 6 (25/35 µM) or TRAIL (150 ng/mL) at 37 °C for 6 or 12 h, and then treated with PI (30 µM) for 10 min at room temperature. The relative intensity was calculated against the geometric mean values of luminescence intensity to blank (relative intensity = 1).

Figure 7. Luminescence microscopy images (Biorevo, BZ-9000, Keyence) of Jurkat cells (x 40) stained with 6. (a–c) Jurkat cells after incubation with 6 (10 µM) at 4 °C for 1 h. (d–f) Jurkat cells after incubation with 6 (5 µM) at 37 °C for 1 h. (g–i) Jurkat cells after incubation with 6 (10 µM) at 37 °C for 1 h. (a) bright image, (b) emission image (emission from 6), (c) overlay image of (a) & (b), (d) bright image, (e) emission image (emission from 6), (f) overlay image of (d) & (e), (g) bright image, (h) emission image (emission from 6), (i) overlay image of (g) & (h). Scale bar (white) = 10 µm.

4810

Bioorganic & Medicinal Chemistry 26 (2018) 4804–4816

A.-A. Masum et al.

Figure 8. Luminescence microscopy images (Biorevo, BZ-9000, Keyence) of Jurkat cells (x40) after treatment with the anti-DR5 antibody (15 µg/ml) at 4 °C for 15 min and then 6 (5 µM) at 37 °C for 1 h. (a) bright image, (b) emission image (emission from 6), (c) overlay image of (a) & (b), (d) emission image (emission from anti-DR5 antibody), (e) overlay image of (a) & (d), (f) overlay image of (b) & (d), and (g) overlay image of (a) & (f). Scale bar (white) in Fig. 8a = 10 µm.

complexes containing DR5 binding cyclic peptide. Studies demonstrate that 6 binds to DR5 on the cell membrane of cancer cells such as Jurkat cells and induces TRAIL-like apoptosis of cancer cells in a parallel manner to the DR5 expression level, whereas our previous IPH such as 4 that has a hydrophilic SGSG at the N-terminus of the cyclic peptide part induces necrotic type cell death. To the best of our knowledge, 6 is the first example of an artificial luminescent TRAIL mimic that induces apoptosis-like cell death, although its EC50 is not so small. It should be mentioned that moderate cytotoxicity (not so small EC50 values) and/or slow cell death induction are appropriate for the detection and cell death induction of cancer cells. Our study suggests that IPHs represent good candidates to study TRAIL mediated apoptosis pathway, understanding of the mechanism of cell death mediated by death receptor as well as cancer cell imaging, which could be a new strategy for the treatment of cancer. Figure 9. Results of cell death assay (PI assay) of 6 against Molt-4 cells (blue bars), K562 cells (green bars) and Jurkat cells (red bars). The relative intensity was calculated against the geometric mean values of luminescence intensity to blank (relative intensity = 1).

4. Materials and methods 4.1. General Information All reagents and solvents were of the highest commercial quality and were used without further purification. TRAIL/Apo2L (human recombinant) was purchased from WAKO Pure Chemical Industries. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) was purchased from Dojindo. Z-VAD-FMK (Z-Val-Ala-Asp(OMe)

3. Conclusions Herein, we report on the design and synthesis of an Ir complexpeptide hybrid (IPH) 6 consisted of C3-symmetric triscyclometalated Ir 4811

Bioorganic & Medicinal Chemistry 26 (2018) 4804–4816

A.-A. Masum et al.

Figure 10. Luminescence microscopy images (Biorevo, BZ-9000, Keyence) of HEK293 cells (x40) stained with 4 and 6. (a–c) HEK293 cells after incubation with 4 (10 µM) at 37˚C for 1 h. (d–f) HEK293 cells after incubation with 6 (10 µM) at 37˚C for 1 h. (a) bright image, (b) emission image (c) overlay image of (a) & (b), (d) bright image, (e) emission image (f) overlay image of (d) & (e). Scale bar (white) = 10 µm.

fluoromethylketone) was purchased from Peptide Institute. The AntiDR5 antibody [DR5-01-1] (phycoerythrin) (ab55863) was purchased from Abcam. Annexin V-Cy3 was purchased from Biovision Inc. Propidium iodide was purchased from Nacalai tesque. All aqueous solutions were prepared using deionized and distilled water. IR spectra were recorded on a Perkin-Elmer FT-IR spectrophotometer (Spectrum100). 1H NMR (300 MHz) spectra were recorded on a JEOL Always 300 spectrometer. Tetramethylsilane (TMS) was used as an internal reference for 1H measurements in CDCl3 and CD3OD and 3(Trimethylsilyl)-propionic-2,2,3,3-d4 acid (TSP) sodium salt was used as an external reference for 1H NMR measurement in D2O. Mass spectral measurements were performed on JEOL JMS-SX102A and Varian TQFT spectrometers. UV spectra were recorded on a JASCO V-550 spectrophotometer, equipped with a temperature controller unit at 25 ± 0.1 °C. Emission spectra were recorded on a JASCO FP-6200 and FP-6500 spectrometers. Luminescence imaging studies were performed using fluorescent microscope (Biorevo, BZ-9000, Keyence). Thin-layer chromatographies (TLC) and silica gel column chromatographies were performed using Merck Art. 5554 (silica gel) TLC plate and Fuji Silysia Chemical FL-100D, respectively. HPLC experiments were carried out using a system consisting of two PU-980 intelligent HPLC pumps (JASCO, Japan), a UV-970 intelligent UV-visible detector (JASCO), a Rheodine injector (Model No. 7125) and a Chromatopak C-R6A (Shimadzu, Japan). For analytical HPLC, Senshu Pak Pegasil ODS column (Senshu Scientific Co., Ltd.) (4.6φx 250 mm, No. 07051001) was used. For preparative HPLC, Senshu Pak Pegasil ODS SP100 column (Senshu Scientific Co., Ltd.) (20φx 250 mm, No. 1302014G) was used. Lyophilization was performed with freeze dryer FD-5 N (EYELA).

4.3. Synthesis of Ir Complex-Peptide hybrid (IPH) Ir complexes 1, 11, 12, 13 and cyclic peptide 9 were prepared according to our previously reported method.74,83 Linker Peptide 7: (Boc-Ser(tBu)-Gly-Ser(tBu)-Gly-OH) The Fmoc group of Fmoc-Gly-2-Cl-Trt-Resin (500 mg (0.53 mmol)) was deprotected by treatment with 20% piperidine/DMF. Each Fmocprotected amino acid (Fmoc-Xaa-OH) (4 eq. against Gly on the resin) was coupled with peptides attached to the resin at 45 °C for 30 min in the presence of DIC (4 eq.), HOBt (8 eq.), and DMF (2.5 mL). After repetition of the deprotection and coupling steps, the N-terminus was protected with Boc using Boc2O (4 eq.) and DIEA (4 eq.) in DMF. The peptide was then cleaved from the resin in a mixture of 1,1,1,3,3,3hexafluoro-2-propanol/CH2Cl2 (60/40). After stirring for 4 h, the resin was filtered and washed several times with CH2Cl2. Evaporation of the filtrate gave the C-terminal free peptide, which was purified by silica gel column chromatography (CHCl3/MeOH = 50/1 ∼ 30/1) to give a colorless amorphous. IR (ATR): ν = 3300, 2975, 2933, 2876, 2104, 1720, 1653, 1512, 1364, 1235, 1163, 1090, 1022, 956, 879, 749, 636 cm–1. 1H NMR (CDCL3, 300 MHz), δ 7.45 (s, 1H), 5.48 (s, 1H), 4.59 (s, 1H), 4.25 (s, 1H), 4.05 (m, 2H), 3.95 (m, 1H), 3.79 (m, 2H), 3.49 (t, 2H), 1.45 (s, 9H), 1.18 (s, 18H) ppm. ESI-MS (m/z) Calcd for C23H43N4O9 [M + H]1+: 519.30300 Found: 519.30185. 8 (NHS ester of Boc-Ser(tBu)-Gly-Ser(tBu)-Gly-OH): Linker peptide 7 (120 mg, 0.23 mmol) was dissolved in DMF (12 mL), to which EDC (1335 mg, 6.9 mmol), and NHS (798 mg, 6.9 mmol) were added. After stirring for 24 h, the reaction mixture was concentrated under reduced pressure and then extracted with CHCl3/NH4Cl. The organic layer was dried over Na2SO4 and the remaining residue was precipitated by adding hexane and the precipitation was filtrated to obtain 8 as a white solid (93 mg, 67%). IR (ATR): ν = 3321, 2974, 2929, 2850, 2282, 1822, 1785, 1738, 1625, 1573, 1435, 1364, 1310, 1242, 1196, 1086, 1046, 892, 754, 641 cm–1. 1H NMR (CDCL3, 300 MHz), δ 8.01 (s, 1H), 7.60 (s, 1H), 5.48 (s, 1H), 4.41 (m, 1H), 4.39 (m, 1H), 3.75 (m, 2H), 3.73 (m, 1H), 3.45 (m, 2H), 3.42 (m, 2H), 2.88 (s, 4H), 1.45 (m, 9) 1.19 (s, 18H) ppm. ESI-MS (m/z) Calcd for C27H45N5O11Na [M + Na]1 +: 638.30133 Found: 638.30026. Cyclic Peptide 10: Cyclic peptide 927-29,83 (130 mg, 0.64 mmol) was dissolved in distilled DMF (4 mL) and DIEA (337 µL, 1.93 mmol) was added. Then NHS ester of Boc-Ser(tBu)-Gly-Ser(tBu)-Gly-OH 8

4.2. Abbreviations used for chemical names: DIC: N,N'-Diisopropylcarbodiimide. DIEA: N,N-Diisopropylethylamine. DMF: N,N-Dimethylformamide. EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide. HOBt: 1-Hydroxybenzotriazole. NHS: NHydroxysuccinimide. PyBOP: Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate. TFA: Trifluoroacetic acid. THF: Tetrahydrofuran. TIPS: Triisopropylsilane. TMSCl: Trimethylsilyl chloride. 4812

Bioorganic & Medicinal Chemistry 26 (2018) 4804–4816

A.-A. Masum et al.

Figure 11. Luminescence microscopic images of (Biorevo, BZ-9000, Keyence) of Jurkat cells treated with 4 and 6 and stained with Annexin V-Cy3 and PI (30 µM). (aa–ae) Jurkat cells incubated with 4 (35 µM) at 37 °C for 2 h and then with Annexin V-Cy3 for 5 min at room temperature. (ba–be) Jurkat cells incubated with 6 (35 µM) at 37 °C for 2 h and then with Annexin V-Cy3 for 5 min at room temperature. (ca–ce) Jurkat cells incubated with 4 (35 µM) at 37˚C for 2 h, then with PI (30 µM) for 10 min at room temperature. (da-de) Jurkat cells incubated with 6 (35 µM) at 37˚C for 2 h, then with PI (30 µM) for 10 min at room temperature. (ea–ee) Jurkat cells incubated with 4 (35 µM) at 37 °C for 24 h, then with PI (30 µM) for 10 min at room temperature. (fa–fe) Jurkat cells incubated with 6 (35 µM) at 37 °C for 24 h, then with PI (30 µM) for 10 min at room temperature. Scale bar (white) = 10 µm.

The deprotection of 10 was carried out in a mixture of TFA/H2O/ TIPS/Thioanisole (85/2.5/1/2.5). After 4 h of stirring, the reaction mixture was evaporated and then precipitated by adding cold Et2O and centrifuged (2000 rpm for 5 min at 0 °C). The crude product was purified by preparative HPLC (Senshu Pak PEGASIL ODS 20 ϕ X 250 mm) (H2O (0.1% TFA)/CH3CN (0.1% TFA) = 80/20 → 50/50 (30 min), flow rate: 10 mL/min), lyophilized to give 10 (30 mg, 27% over two steps) as white solid. IR (ATR): ν = 3279, 3074, 2962, 2162, 2050, 1980, 1635, 1522, 1469, 1412, 1199, 1180, 1132, 835, 800, 743, 720, 544, 517 cm–1. 1H NMR (D2O, 300 MHz) δ = 7.52 (m, 1H), 7.41(m, 1H), 7.20 (m, 2H), 7.08 (m, 1H), 4.41 (m, 13H), 4.05 (m, 13H), 3.18 (m, 14H), 2.82 (m, 15H), 2.07 (m, 14H), 1.65 (m, 36H), 0.92 (m, 46H)

(39.71 mg, 0.64 mmol) was added and stirred for 6 h. The crude compound was diluted with 0.1%TFA H2O and then purified by preparative HPLC (Senshu Pak PEGASIL ODS 20 ϕ X 250 mm) (H2O (0.1% TFA)/ CH3CN (0.1% TFA) = 80/20 → 50/50 (30 min), flow rate: 10 mL/min), lyophilized to give the protected precursor of 10. IR (ATR): ν = 3283, 3063, 2977, 2878, 2773, 2115, 1779, 1634, 1532, 1469, 1394, 1366, 1339, 1290, 1175, 1130, 1023, 929, 873, 833, 799, 719, 594 cm–1. 1H NMR (D2O, 300 MHz) δ = 7.51 (m, 1H), 7.37 (m, 1H), 7.21 (m, 2H), 7.08 (m, 1H), 4.41 (m, 15H), 4.04 (m, 19H), 3.87 (m, 32H), 2.81 (m, 2H), 2.07 (m, 11H), 1.85 (m, 33H), 1.62 (m, 17H), 0.92 (m, 46H) ppm. ESI-MS (m/z) Calcd for C108H183N34O31S2 [M + 3H]3+: 839.10914 Found: 839.10728. 4813

Bioorganic & Medicinal Chemistry 26 (2018) 4804–4816

A.-A. Masum et al.

ppm. ESI-MS (m/z) Calcd for C95H159N34O29S2 [M + 3H] 3 +: 768.38033 Found: 768.38192. Ir Complex 6: Ir complex 1374,83 (6 mg, 0.0044 mmol) was added to a solution of 10 (30 mg, 0.013 mmol) and DIEA (23 µL, 0.134 mmol) in DMF (600 μL), and the reaction mixture was then stirred for 24 h at room temperature in the dark. The reaction mixture was diluted with H2O (0.1%TFA) and purified by preparative HPLC (Senshu Pak PEGASIL ODS 20 ϕ X 250 mm) (H2O (0.1% TFA)/CH3CN (0.1% TFA) = 80/20 → 50/50, flow rate: 10 mL/min). Lyophilized to give 6 as a yellow powder (10.7 mg, 23%). IR (ATR): ν = 3284, 2961, 1638, 1535, 1471, 1425, 1200, 1136, 1071, 914, 837, 800, 782, 721, 633, 595 cm–1. 1 H NMR. (D2O, 400 MHz) δ = 7.73 (m, 3H), 7.46 (m, 3H), 7.21 (m, 6H), 6.82 (m, 3H), 6.68 (m, 3H), 4.41 (m, 33H), 3.77 (m, 13H), 3.75 (m, 19H), 3.73 (m, 20H), 3.23 (m, 27H), 3.21 (m, 16H), 2.09 (m, 7H), 1.87 (m, 17H), 1.51 (m, 31H), 1.36 (m, 44H), 1.33 (m, 186H), 0.91 (m, 74H) ppm. ESI-MS (m/z) Calcd for C333H515IrN108O93S6 [M + 8H] 8 +: 987.96917 Found: 987.96163.

response was subtracted to obtain apparent complexation constants Kapp and dissociation constants Kd (= 1/Kapp).

4.6. Cell culture Jurkat cells, Molt-4 cells, K562 cells and HEK293 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), L-glutamine, HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid, pKa = 7.5), 2-mercaptoethanol and penicillin/streptomycin and monothioglycerol (MTG) in a humidified 5% CO2 incubator at 37 °C.

4.7. MTT assay Jurkat cells (1.0 x 105 cells/ml) were incubated in 1% DMSO 10% FCS RPMI1640 medium (MTG free) containing solution of IPHs 3–6 (0–75 µM) under 5% CO2 at 37 ˚C for 16 h in 96 well plates (BD Falcon), and 0.5% MTT reagent in PBS buffer (10 µL) was then added to the cells. After incubation under 5% CO2 at 37 °C for 4 h, formazan lysis solution (10% SDS in 0.01 N HCl) (100 µL) was added and incubated overnight under same condition, followed by measurement of absorbance at 570 nm by a microplate reader (BIO-RAD). For MTT assay of HEK293 cells, 1.0 x 105 cells/ml of HEK293 cells were seeded in 96 well plate in 10% FCS RPMI1640 medium. On the next day the medium was removed, the cells were softly washed with PBS buffer, to which a given IPH (in 1% DMSO 10% FCS RPMI1640 medium (MTG free)) was added and incubated under 5% CO2 at 37 °C for 16 h. 0.5% MTT reagent in PBS buffer (10 µL) was then added to the cells. After incubation under 5% CO2 at 37 °C for 4 h, formazan lysis solution (10% SDS in 0.01 N HCl) (100 µL) was added and incubated overnight under same conditions, followed by measurement of absorbance at 570 nm by a microplate reader (BIO-RAD).

4.4. Measurements of UV/Vis absorption and luminescence spectra UV/Vis spectra were recorded on a JASCO V-550 UV/Vis spectrophotometer equipped with a temperature controller and emission spectra were recorded on a JASCO FP-6200 spectrofluorometer at 25 °C. Before the luminescence measurements, ample solutions were degassed by Ar bubbling for 10 min in quartz cuvettes equipped with Teflon septum screw caps. Concentrations of all the Ir complexes in stock solutions (DMSO) were determined based on a molar extinction coefficient of 380 nm (ε380 nm = 1.08 ± 0.07 × 104 M−1 cm-1). Quantum yields for luminescence (Φ ) were determined by comparing with the integrated corrected emission spectrum of a quinine sulfate standard, whose emission quantum yield in 0.1 M H2SO4 was assumed to be 0.55 (excitation at 366 nm). Equation (1) was used to calculate the emission quantum yields, in which Φ s and Φ r denote the quantum yields of the sample and reference compounds, ηs and η r are the refractive indexes of the solvents used for the measurements of the sample and reference, and As and Ar are the absorbance of the sample and the reference, and Is and Ir stand for the integrated areas under the emission spectra of the sample and reference, respectively (all of the Ir compounds were excited at 366 nm for luminescence measurements in this study).

Φs = Φr (ηs2 Ar Is)/(ηr2 As Ir )

4.8. MTT assay in the presence of caspase inhibitor (Z-VAD-FMK) Jurkat cells (1.0 × 105 cells/mL) were incubated in 10% FCS RPMI 1640 medium (MTG free) containing a solution Z-VAD-FMK (15 µM) (under 5% CO2 at 37 °C for 1 h in 96 well plates (BD Falcon). Then IPHs (75 µM) was added and incubated under 5% CO2 at 37 °C for 16/24 h, then 0.5% MTT reagent in PBS buffer (10 µL) was added to the cells and incubated under 5% CO2 at 37 °C for 4 h. A formazan lysis solution (10% SDS in 0.01 N HCl) (100 µL) was added and the resulting solution incubated overnight under the same conditions, followed by measurement of absorbance at 570 nm with a microplate reader (BIO-RAD).

(1)

The luminescence lifetimes of sample solutions were measured on a TSP1000-M-PL (Unisoku, Osaka, Japan) instrument by using THG (355 nm) of Nd: YAG laser, Minilite I (Continuum, CA, USA), at 25 °C in degassed aqueous solutions. An R2949 photomultiplier were used to monitor the signals. Data were analyzed using the nonlinear leastsquares procedure.

4.9. Fluorescent microscopy studies of Jurkat cells with IPHs Jurkat cells (1.0 × 106 cells/mL) were incubated in the absence or presence of IPHs in 10% FCS-containing RPMI 1640 medium (MTG free) for specified time under 5% CO2 at 37 °C. The cells were then washed twice with ice-cold PBS with 0.1% NaN3 and 0.5% FCS and placed on Greiner CELLviewTM petri dish (35 × 10 mm) and mounted on fluorescent microscope for observation (Biorevo, BZ-9000, Keyence) (excitation 377 ± 25 nm, emission 520 ± 35 nm, FF01 filter). For fluorescence microscopy studies of HEK293 cells, 1.0 x 105 cells/ml of HEK293 cells were seeded in 96 well plate in 10% FCS RPMI1640 medium. On the next day the medium was removed, the cells were softly washed with PBS buffer, to which a given IPH (in 1% DMSO 10% FCS RPMI1640 medium (MTG free)) was added and incubated under 5% CO2 at 37 °C for 1 h. The cells were then washed thrice with ice-cold PBS with 0.1% NaN3 and 0.5% FCS and mounted on fluorescent microscope for observation (Biorevo, BZ-9000, Keyence) (excitation 377 ± 25 nm, emission 520 ± 35 nm, FF01 filter).

4.5. 27 MHz quartz crystal microbalance (27 MHz QCM) analysis QCM analysis was performed on an Affinix-Q4 apparatus (Initium Inc., Japan). After cleaning the Au (4.9 mm2) electrode, equipped on quartz crystal, with 1% SDS and piranha solution, was incubated at room temperature for 60 min with an aqueous solution of 3,3′-dithiodipropionic acid (3 mM, 4 μL). The surface was then washed with distilled water and was then activated by treatment with a mixture of EDC·HCl (0.52 M) and N-hydroxysuccinimide (0.87 M) for 30 min, washed with distilled water, then treated with DR5 (100 μg/mL, 4 μL) at room temperature for 60 min. After washing with distilled water, an aqueous solution of 1 M ethanolamine (5 μL) was added as a blocking reagent. After washing with distilled water, the cell was filled with phosphate buffered saline (PBS) (500 μL). The apparent binding constants (Kapp) for IPHs with DR5 in PBS were calculated from the decrease in frequency. From the frequency decrease curve the nonspecific 4814

Bioorganic & Medicinal Chemistry 26 (2018) 4804–4816

A.-A. Masum et al.

References

4.10. Annexin V-Cy3 staining The cells were incubated with IPHs for 2 h, and then washed with PBS buffer. The cells were then suspended in 1X binding buffer and Annexin V-Cy3 was added and incubate at room temperature for 5 min. After that the cells were observed on a fluorescent microscope (Biorevo, BZ-9000, Keyence) (excitation 540 ± 25 nm, emission 605 ± 55 nm, TRICT filter).

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

4.11. Propidium iodide (PI) staining The cells were incubated with IPHs for a given time, washed with PBS buffer and then incubated with PI (30 µM) in PBS buffer at room temperature for 10–15 min. The cells were then again washed with PBS buffer and observed on a fluorescent microscope (Biorevo, BZ-9000, Keyence) (excitation 540 ± 25 nm, emission 605 ± 55 nm, TRICT filter) or analyzed by flow cytometer (Beckman Coulter Gallios Flow Cytometer, detector: FL2, excitation 488 nm, emission 575 ± 20 nm).

16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

4.12. Anti-DR5 antibody staining Cells were incubated with anti-DR5 antibody at 4 °C for 15 min on ice. The cells were then washed twice with ice-cold PBS containing 0.5% FCS and 0.1% NaN3 and were and mounted on a fluorescent microscope (Biorevo, BZ-9000, Keyence) (excitation 540 ± 25 nm, emission 605 ± 55 nm, TRICT filter) or analyzed by flow cytometer (Beckman Coulter Gallios Flow Cytometer, detector: FL2, excitation 488 nm, emission 575 ± 20 nm).

28.

29. 30. 31. 32. 33. 34.

4.13. Flow cytometry analysis of staining and cell death induction assay Jurkat cells, K562 cells or Molt-4 cells (3.0 × 105 cells) were incubated in the absence or the presence of IPHs in 10% FCS RPMI 1640 medium (MTG free) for specified time under 5% CO2 at 37 °C. After that the cells were washed twice with ice cold FACS buffer and then suspended in 450 µL FACS buffer. The cells were analyzed by flow cytometer ((Beckman Coulter Gallios Flow Cytometer, detector: FL2, excitation 488 nm, emission 575 ± 20 nm to detect PI staining or antiDR5 antibody staining, detector: FL10, excitation 405 nm, emission 550 ± 40 nm to detect Ir complexes staining).

35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46.

Acknowledgments This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (No. 24890256, 26860016 and 16K18851 for Y.H., and No. 24650011, 24640156, 15K00408, and 17K08225 for S.A.), the Uehara Memorial Foundation for Y.H. the Tokyo Biochemical Research Foundation, Tokyo, Japan for S.A. and High-Tech Research Center Project for Private Universities (matching fund subsidy from MEXT). S.A also thanks to the TUS (Tokyo University of Science) fund for strategic research areas. Special thanks to Prof. Dr. Takeshi Inukai (Department of Pediatrics, School of Medicine, University of Yamanashi), for valuable suggestion and Prof. Yoichiro Iwakura, Research Institute for Biomedical Sciences, Tokyo University of Science, for providing us HEK293 cell lines. We sincerely acknowledge Ms. Fukiko Hasegawa and Ms. Noriko Sawabe (Faculty of Pharmaceutical Sciences, Tokyo University of Science) for mass spectra and NMR measurements.

47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64.

Appendix A. Supplementary data

65. 66. 67.

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.bmc.2018.08.016. 4815

Jacobson MD, Weil M, Raff MC. Cell. 1997;88:347–354. Nagata S. Cell. 1997;88:355–365. Fesik SW. Cell. 2000;103:273–282. Ashkenazi A, Dixit VM. Science. 1998;281:1305–1308. Bodmer J-L, Schneider P, Tschopp J. Trends Biochem Sci. 2002;27:19–26. Merino D, Lalaoui N, Morizot A, Solary E, Micheau O. Expert Opin Ther Targets. 2007;11:1299–1314. Takeda K, Stagg J, Yagita H, Okamura K, Smith MJ. Oncogene. 2007;26:3745–3757. Hymowitz SG, Christinger HW, Fuh G, et al. Mol Cell. 1999;4:563–571. Hymowitz SG, O’Connell M, Ultsch MH, et al. Biochemistry. 2000;39:633–640. Cha S-S, Sung B-J, Kim Y-A, et al. J Biol Chem. 2000;275:31171–31177. Pan G, O’Rourke K, Chinnaiyan AM, et al. Science. 1997;276:111–113. Walczak H, Delgi-Esposti MA, Johnson RS, et al. EMBO J. 1997;16:5386–5397. Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM. Science. 1997;277:815–818. Delgi-Esposti MA, Smolak PJ, Walczak H, et al. J Exp Med. 1997;186:1165–1170. Delgi-Esposti MA, Dougall WC, Smolak PJ, Waugh JY, Smith CA, Goodwin RG. Immunity. 1997;7:813–820. Simonet WS, Lacey DL, Dunstan CR, et al. Cell. 1997;89:309–319. MacFarlane M, Kohlhass SL, Sutcliffe MJ, Dyer MJS, Cohen GM. Cancer Res. 2005;65:11265–11270. Ashkenazi A, Pai RC, Fong S, et al. J Clin Invest. 1999;104:155–162. Walczak H, Miller RE, Ariall K, et al. Nat Med. 1999;5:157–163. Kelly SK, Ashkenazi A. Curr Opin Pharmacol. 2004;4:333–339. Ashkenazi A, Holland P, Eckhardt SG. J Clin Oncol. 2008;26:3621–3630. Ichikawa K, Liu W, Zhao L, et al. Nat Med. 2001;7:954–960. Jin H, Yang R, Fong S, et al. Cancer Res. 2004;64:4900–4905. Camidge DR. Expert Opin Biol Ther. 2008;8:1167–1176. Li J, Knee DA, Wang Y, et al. Drug Dev Res. 2008;69:69–82. Wiezorek J, Holland P, Graves J. Clin Cancer Res. 2011;16:1701–1708. Angell YM, Bhandari A, Chakrabarti A, et al. In: American Peptide Symposia vol. 9: Understanding Biology Using Peptides (Ed.: S. E. Blondelle), Springer, New York, 2006, pp. 405–406. Angell YM, Bhandari A, De Francisco MN, et al. In: Advances in Experimental Medicine and Biology, vol. 611: Peptides for Youth, (Eds.: S. DelValle, E. Escher, W. D. Lubell), Springer, New York, 2009, pp. 101–103. Angell YM, Bhandari M, De Francisco MN, et al. Adv Exp Med Biol. 2009;611:101–103. Pavet V, Beyrath J, Pardin C, et al. Cancer Res. 2010;70:1101–1110. Lamanna G, Smulski CR, Chekkat N, et al. Chem Eur J. 2013;19:1762–1768. Pulka-Ziach K, Pavet V, Chekkat N, et al. ChemBioChem. 2015;16:293–301. Valldorf B, Fittler H, Deweid L, et al. Angew Chem Int Ed. 2016;55:1–6. Kaga C, Okochi M, Nakanishi M, Hayashi H, Kato R, Honda H. Biochem. Biophys Res Commun. 2007;362:1063–1068. Wang G, Wang X, Yu H, et al. Nat Chem Biol. 2013;9:84–89. Tamayo AB, Alleyne BD, Djurovich PI, et al. J Am Chem Soc. 2003;125:7377–7387. Dedeian K, Djurovich PI, Garces FO, Carlson G, Watts R. J Inorg Chem. 1991;30:1685–1687. Lowry MS, Bernhard S. Chem Eur J. 2006;12:7970–7977. Flamigni L, Barbieri A, Sabatini C, Ventura B, Barigelletti F. Curr. Chem. 2007;281:143–203. Evans RC, Douglas P, Winscom CJ. Coord Chem Rev. 2006;250:2093–2126. Ulbricht C, Beyer B, Friebe C, Winter A, Schubert US. Adv Mater. 2009;21:4418–4441. Farinola GM, Ragni R. Chem Soc Rev. 2011;40:3467–3482. Chi Y, Chou P-T. Chem Soc Rev. 2010;39:638–655. Lamansky S, Djurovich P, Murphy D, et al. J Am Chem Soc. 2001;123:4304–4312. Baldo MA, O’Brien DF, You Y, et al. Nature. 1998;395:151–154. Yersin H. Highly Efficient OLEDs with Phosphorescent Materials. Weinheim, Germany: Wiley-VCH; 2008. Marco GD, Lanza M, Mamo A, et al. Anal Chem. 1998;70:5019–5023. DeRosa MC, Mosher PJ, Yap GPA, Focsaneanu K-S, Crutchley RJ, Evanc CEB. Inorg Chem. 2003;42:4864–4872. Licini M, Williams JAG. Commun. 1999:1943–1944. Arm KJ, Leslie W, Williams JAG. Inorg Chim Acta. 2006;359:1222–1232. Ho M-L, Hwang F-M, Chen P-N, et al. Org Biomol Chem. 2006;4:98–103. Konishi K, Yamaguchi H, Harada A. Chem Lett. 2006;35:720–721. Schmittel M, Lin H. Inorg Chem. 2007;46:9139–9145. Zhao Q, Cao T, Li F, et al. Organomet. 2007;26:2077–2081. Lo KK-W, Louie M-W, Zhang KY. Coord Chem Rev. 2010;254:2603–2622. Lo KK-W, Li SP-Y, Zhang KY. New J Chem. 2011;35:265–287. Leung S-K, Kwok KY, Zhang KY, Lo KK-W. Inorg Chem. 2010;49:4984–4995. Lo KK-W, Li SP-Y. RSC Adv. 2014;24:10560–10585. Zhang KY, Li SP-Y, Zhu N, et al. Inorg Chem. 2010;49:2530–2540. Lee P-K, Law WH- T, Liu H-W, Lo KK-W. Inorg Chem. 2011;50:8570–8579. Zhang KY, Liu H-W, Fong TT-H, Chen X-G, Lo KK-W. Inorg Chem. 2010;49:5432–5443. Zhao Q, Huang C, Li F. Chem Soc Rev. 2011;40:2508–2524. Zhao Q, Yu M, Shi L, et al. Organometallics. 2010;29:1085–1091. Chunyan L, Mengxiao Y, Yun Sun, Yongquan W, Chunhui H, Fuyou L. J Am Chem Soc. 2011;133:11231–11239. You Y, Lee S, Kim T, et al. J Am Chem Soc. 2011;133:18328–18342. Wu Y, Jing H, Dong Z, Zhao Q, Wu H, Li F. Inorg Chem. 2011;50:7412–7420. Murphy L, Congreve A, Palsson L-O, Williams JAG. Chem Commun.

Bioorganic & Medicinal Chemistry 26 (2018) 4804–4816

A.-A. Masum et al.

2016;55:3829–3843. 80. Hisamatsu Y, Shibuya A, Suzuki N, Abe R, Aoki S. Bioconjugate Chem. 2015;26:857–879. 81. Hisamatsu Y, Suzuki N, Masum A, et al. Bioconjugate Chem. 2017;28:507–523. 82. Yokoi K, Hisamatsu Y, Naito K, Aoki S. Eur J Inorg Chem. 2017:5295–5309. 83. Masum A, Hisamatsu Y, Yokoi K, Aoki S, Bioinorg Chem Appl. Article ID: 7578965 (DOI:org/10.1155/2018/ 2018 7578965). 84. Slee EA, Zhu H, Chow SC, MacFarlane M, Nicholson DW, Cohen GM. Biochem J. 1996;315:21–24. 85. Shaw G, Morse S, Ararat M, et al. FASEB J. 2002;16:866–871. 86. Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH. Blood. 1994;84:1415–1420. 87. Vermes I, Haanen C, Steffens-Nakken H, Reutellingsperger C. J Immunol Methods. 1995;184:39–51. 88. Kroemer G, Galluzzi L, Vandenabeele P, et al. Cell Death Differ. 2009;16:3–11.

2010;46:8743–8745. Baggaley E, Weinstein JA, Williams JAG. Coord Chem Rev. 2012;256:1762–1785. You Y. Curr Opin Chem Biol. 2013;17:699–707. Cao R, Jia J, Ma X, Zhou M, Fei H. J Med Chem. 2013;56:3636–3644. Zhou Y, Jia J, Li W, Fei H, Zhou M. Chem Commun. 2013;49:3230–3232. Zhang S, Hosaka M, Yoshihara T, et al. Cancer Res. 2010;70:4490–4498. Tobita S, Yoshihara T. Curr Opin Chem Biol. 2016;33:39–45. Aoki S, Matsuo Y, Ogura S, et al. Inorg Chem. 2011;50:806–818. Hisamatsu Y, Aoki S. Eur J Inorg Chem. 2011:5360–5369. Moromizato S, Hisamatsu Y, Suzuki T, Matsuo Y, Abe R, Aoki S. Inorg Chem. 2012;51:12697–12706. 77. Nakagawa A, Hisamatsu Y, Moromizato S, Kohno M, Aoki S. Inorg Chem. 2014;53:409–422. 78. Kando A, Hisamatsu Y, Ohwada H, Moromizato S, Kohno M, Aoki S. Inorg Chem. 2015:5342–5357. 79. Kumar S, Hisamatsu Y, Tamaki Y, Ishitani O, Aoki S. Inorg Chem. 68. 69. 70. 71. 72. 73. 74. 75. 76.

4816