Shinya Ariyasu

Design and synthesis of 8-hydroxyquinoline-based radioprotective agents.

In radiation therapy, adverse side effects are often induced due to the excessive cell death that occurs in radiosensitive normal cells. The radiation-induced cell death of normal cells is caused, at least in part, by apoptosis, which undergoes via activation of p53 and increase in the p53 protein, a zinc-containing transcriptional factor, in response to cellular damage. Therefore, radioprotective drugs that can protect normal cells from radiation and thus suppress adverse side effects would be highly desirable. We report herein on the radioprotective activity of 8-hydroxyquinoline (8HQ) derivatives that were initially designed so as to interact with the Zn(2+) in p53. Indeed, the 5,7-bis(methylaminosulfonyl)-8HQ and 8-methoxyquinoline derivatives considerably protected MOLT-4 cells against γ-ray radiation (10 Gy), accompanied by a low cytotoxicity. However, mechanistic studies revealed that the interaction of these drugs with p53 is weak and the mechanism for inhibiting apoptosis appears to be different from that of previously reported radioprotectors such as bispicen, which inhibits apoptosis via the denaturation of p53 as well as by blocking both transcription-dependent and -independent apoptotic pathways.

Enrichment of circulating tumor cells in tumor-bearing mouse blood by a deterministic lateral displacement microfluidic device.

Concentration of real tumor cells leaking into blood from cancer was attempted by a deterministic lateral displacement (DLD) microfluidic device. Spiked cultured cell line tumor cells are often used to verify performance of the circulating tumor cells (CTCs) separation methods. Cultured tumor cells are obviously larger than most of hematocytes and considered not to be appropriate as CTC mimics, while there is uncertainty in identifying real CTCs from clinical samples and there is no practical way to examine CTCs leakage into benign cells during the sorting. In this work, blood samples were prepared from tumor-bearing mice whose tumors were induced by implanting cells with GFP expression to living mice. Therefore, CTCs were identified by their fluorescence emission. We succeeded in the enrichment of tumor cells to 0.05% from the blood, in which CTCs were negligibly detected among three million blood cells, and little loss of CTCs was observed.

Selective Capture and Collection of Live Target Cells Using a Photoreactive Silicon Wafer Device Modified with Antibodies via a Photocleavable Linker

A device for the capture and recollection of live target cells is described. The platform was a silicon (Si) wafer modified with an anti-HEL antibody (anti-HEL-IgG, HEL = hen egg lysozyme) through a photocleavable 3-amino-3-(2-nitrophenyl)propionic acid (ANP) linker. The modification processes of the Si wafer surface were monitored by Fourier transform infrared spectroscopy-attenuated total reflection (FTIR-ATR) and fast-scanning atomic force microscopy (FS-AFM). The attachment of IgG and its release reaction on the Si surface via the photochemical cleavage of the ANP linker were observed directly by FS-AFM. The results of an enzyme-linked immunosorbent assay (ELISA) indicated that the photorelease of the complex of anti-HEL-IgG with the secondary antibody-alkaline phosphatase hybrid (secondary IgG-AP) from the Si surface occurs with minimum damage. Furthermore, it was possible to collect SP2/O cells selectively that express HEL on their cell membranes (SP2/O-HEL) on the Si wafer device. Photochemical cleavage of the ANP linker facilitated the effective release of living SP2/O cells whose viability was verified by staining experiments using tripan blue. Moreover, it was possible to reculture the recovered cells. This methodology represents an effective strategy for isolating intact target cells in the biological and medicinal sciences and related fields.

AS-2, a novel inhibitor of p53-dependent apoptosis, prevents apoptotic mitochondrial dysfunction in a transcription-independent manner and protects mice from a lethal dose of ionizing radiation

In a previous study, we reported that some tetradentate zinc(II) chelators inhibit p53 through the denaturation of its zinc-requiring structure but a chelator, Bispicen, a potent inhibitor of in vitro apoptosis, failed to show any efficient radioprotective effect against irradiated mice because the toxicity of the chelator to mice. The unsuitability of using tetradentate chelators as radioprotectors prompted us to undertake a more extensive search for p53-inhibiting agents that are weaker zinc(II) chelators and therefore less toxic. Here, we show that an 8-hydroxyquinoline (8HQ) derivative, AS-2, suppresses p53-dependent apoptosis through a transcription-independent mechanism. A mechanistic study using cells with different p53 characteristics revealed that the suppressive effect of AS-2 on apoptosis is specifically mediated through p53. In addition, AS-2 was less effective in preventing p53-mediated transcription-dependent events than pifithrin-μ (PFTμ), an inhibitor of transcription-independent apoptosis by p53. Fluorescence visualization of the extranuclear distribution of AS-2 also supports that it is ineffective on the transcription-dependent pathway. Further investigations revealed that AS-2 suppressed mitochondrial apoptotic events, such as the mitochondrial release of intermembrane proteins and the loss of mitochondrial membrane potential, although AS-2 resulted in an increase in the mitochondrial translocation of p53 as opposed to the decrease of cytosolic p53, and did not affect the apoptotic interaction of p53 with Bcl-2. AS-2 also protected mice that had been exposed to a lethal dose of ionizing radiation. Our findings indicate that some types of bidentate 8HQ chelators could serve as radioprotectors with no substantial toxicity in vivo.

Investigation of Thermally Induced Cellular Ablation and Heat Response Triggered by Planar MoS2-Based Nanocomposite

In comparison to conventional tumor treatment methods, photothermal therapy (PTT) is one of the innovative therapeutic strategies that employs light to produce localized heat for targeted ablation of cancer cells. Among the various kinds of heat generation nanomaterials, transition metal dichalcogenide nanosheets, especially molybdenum disulfide (MoS2), have recently been investigated as one of the promising PTT candidates because of their strong absorbance in the near-infrared (NIR) tissue transparency window and excellent photothermal conversion capability. In line with the great potential of MoS2-based nanomaterials in biomedical applications, their intrinsic therapeutic performance and corresponding cellular response are required to be continually investigated. In order to further improve MoS2-based PTT efficacy and dissect the molecular mechanism during heat stimuli, in this study, we successfully designed a novel and effective PTT platform by integration of MoS2 nanosheets with peptide-based inhibition molecules to block the function of heat shock proteins (Hsp90), one type of chaperone proteins that play protective roles in living systems against cellular photothermal response. Such a combined nanosystem could effectively induce cell ablation and viability assays indicated approximately 5-fold higher PTT treatment efficacy (8.8% viability) than that of MoS2 itself (48% viability) upon 808 nm light irradiation. Moreover, different from the case based on MoS2 alone that could cause tumor ablation through the process of necrosis, the detailed mechanism analysis revealed that the inhibition of Hsp90 could significantly increase the photothermally mediated apoptosis, hence resulting in remarkable enhancement of photothermal treatment. Such promising studies provide the great opportunity to better understand the cellular basis of light-triggered thermal response. Moreover, they can also facilitate the rational design of new generations of PTT platforms toward future theranostics.

Development of a novel sulfonate ester-based prodrug strategy.

A self-immolative γ-aminopropylsulfonate linker was investigated for use in the development of prodrugs that are reactive to various chemical or biological stimuli. To demonstrate their utility, a leucine-conjugated prodrug of 5-chloroquinolin-8-ol (5-Cl-8-HQ), which is a potent inhibitor against aminopeptidase from Aeromonas proteolytica (AAP), was synthesized. The sulfonate prodrug was considerably stable under physiological conditions, with only enzyme-mediated hydrolysis of leucine triggering the subsequent intramolecular cyclization to simultaneously release 5-Cl-8-HQ and form γ-sultam. It was also confirmed that this γ-aminopropylsulfonate linker was applicable for prodrugs of not only 8-HQ derivatives but also other drugs bearing a phenolic hydroxy group.

Site-Specific Dual Functionalization of Cysteine Residue in Peptides and Proteins with 2-Azidoacrylates

Herein, we report use of 2-azidoacrylates to perform site-specific dual functionalization of the cysteine residue of peptides and bovine serum albumin (BSA), a native protein containing one free cysteine residue. The sulfhydryl group of the cysteine residue could be conjugated with 2-azidoacrylates bearing various functionalities, such as fluorescent dyes under physiological aqueous buffer conditions, to afford peptide and protein conjugates anchoring an azide moiety. Successive azide-alkyne cycloaddition enables installation of the second functionality, thus affording dual-functionalized peptide- and protein-based materials.

Affinity Clusters: An Adenine-Coated Gold Cluster Binds to Thymine Loops in DNA

Functionalization of the surface ligand is of primary importance for the specific binding of nanoparticles (NPs) to biological and nonbiological molecular architectures. The tailored arrangement of NPs in twoor three-dimensional space exploits new optical, electronic, and catalytic resources for the development of revolutionary devices and sensing systems. In constructing such devices, it is essential to control the spatial positioning of NPs. DNA is generally recognized as a programmable substrate in attempts to control NP arrangement. The majority of such attempts have been based on complementary hybridization between two single-stranded (ss) DNAs: one as the template and the other tagged with gold (Au) NPs. Recently, template DNAs have developed from one-dimensional double-stranded (ds) DNAs to two-dimensional DNAs made of doublecrossover (dx) or triple-crossover (tx) DNAs. In order to utilize these new DNA architectures without being based on complementary hybridization, we previously reported a method in which we synthesized a fusion protein composed of zinc-finger (ZF) and metallothionein motifs—which recognize DNA and AuNPs, respectively—and was formed in a parallelogram arrangement of four [Au11 ACHTUNGTRENNUNG(PPh3)4 ACHTUNGTRENNUNG(S-Cys)6] clusters on a dxDNA comprising four corresponding ZFsrecognizing sequences. Herein, we report a third method to tailor the arrangement of gold clusters on DNA, based on the finding that adenine (A)-coated gold substrates trap thymine (T) molecules. With the aim of developing affinity clusters capable of selectively binding to thymine loops (Tn) knitted into the DNA architectures via A–T complementary recognition (Figure 1), we synthesized two gold clusters, [Au11 ACHTUNGTRENNUNG(PPh3)6(MA)2]+ 1 and [Au25ACHTUNGTRENNUNG(PPh3)8(MA)6Cl2]2+ 2, where MA= 8-mercaptoadenine. The Au MA clusters were synthesized by treating [AuPPh3Cl] and MA with NaBH4 in N,N-dimethylformamide, following the method reported by Zheng et al. N,Ndimethylformamide (DMF) was selected for two reasons: 1) it is a good solvent for MA, and 2) its CO N group provides an environment similar to polypyrrolidone, which is known to metastabilize gold clusters. The product ratio 2/1 increased with the reaction temperature. The identification of clusters 1 and 2 was carried out by UV/Vis, TEM, MALDI-TOF mass spectrometry, XPS, and H NMR spectroscopy. The UV/Vis spectra of 1 showed a peak at 420 nm that is characteristic of Au11 clusters; [10] for example, cluster [Au11 ACHTUNGTRENNUNG(PPh3)8Cl3] (Au11) also shows a peak at 420 nm. On the other hand, cluster 2 exhibited a broadband spectrum with no peaks that were assignable to discrete energy levels originating from molecular orbitals or surface plasmon resonances (see the Supporting Information). This observation indicates that the core number of 2 was larger than 11 but smaller than those of NPs with radii of approximately 5 nm. Considering the continuous distribution of the core numbers in the reaction mixture, Au25 was the first candidate composition of 2 (Au39, Au55, and other comparatively large clusters would also be candidates); however, it was impossible to reach an immediate conclusion because the 420 nm peak of Au11 diminishes or disappears in the presence of thiol ligands, whereas Au25 clusters with thiolate ligands show an absorption maximum at approximately 670 nm. This observation led us to obtain TEM images of 1 and 2 (Figure 2 a, b). The average diameters of 1 and 2 sampled from these images were 0.8 ACHTUNGTRENNUNG( 0.2) nm and 1.1 ACHTUNGTRENNUNG( 0.2) nm, respectively (Figure 2 c,d). These values strongly indicate that 1 and 2 are Au11 and Au25 clusters, respectively, considering the core numbers of familiar gold clusters. The EDX spectra [a] Prof. T. Yamamura Department of Chemistry, Faculty of Science Tokyo University of Science (TUS) Kagurazaka, Shinjuku-ku, Tokyo 162-0825 (Japan) Fax: (+81) 03-5228-8251 E-mail : tyamamur@rs.kagu.tus.ac.jp [b] Dr. S. Ariyasu Center for Technologies against Cancer Tokyo University of Science (TUS) Yamazaki 2641, Noda, Chiba 278-8510 (Japan) [c] Prof. R. Sakamoto Department of Chemistry,Graduate School of Science The University of Tokyo Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033 (Japan) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201000894.

Minimal motif peptide structure of metzincin clan zinc peptidases in micelles

It is well known that the functions of metalloproteins generally originate from their metal‐binding motifs. However, the intrinsic nature of individual motifs remains unknown, particularly the details about metal‐binding effects on the folding of motifs; the converse is also unknown, although there is no doubt that the motif is the core of the reactivity for each metalloprotein. In this study, we focused our attention on the zinc‐binding motif of the metzincin clan family, HEXXHXXGXXH; this family contains the general zinc‐binding sequence His–Glu–Xaa–Xaa–His (HEXXH) and the extended GXXH region. We adopted the motif sequence of stromelysin‐1 and investigated the folding properties of the Trp‐labeled peptides WAHEIAHSLGLFHA (STR‐W1), AWHEIAHSLGLFHA (STR‐W2), AHEIAHSLGWFHA (STR‐W11), and AHEIAHSLGLFHWA (STR‐W14) in the presence and absence of zinc ions in hydrophobic micellar environments by circular dichroism (CD) measurements. We accessed successful incorporation of these zinc peptides into micelles using quenching of Trp fluorescence. Results of CD studies indicated that two of the Trp‐incorporated peptides, STR‐W1 and STR‐W14, exhibited helical folding in the hydrophobic region of cetyltrimethylammonium chloride micelle. The NMR structural analysis of the apo STR‐W14 revealed that the conformation in the C‐terminus GXXH region significantly differred between the apo state in the micelle and the reported Zn‐bound state of stromelysin‐1 in crystal structures. The structural analyses of the qualitative Zn‐binding properties of this motif peptide provide an interesting Zn‐binding mechanism: the minimum consensus motif in the metzincin clan, a basic zinc‐binding motif with an extended GXXH region, has the potential to serve as a preorganized Zn binding scaffold in a hydrophobic environment. Copyright

A chemical modulator of p53 transactivation that acts as a radioprotective agonist

Inhibiting p53-dependent apoptosis by inhibitors of p53 is an effective strategy for preventing radiation-induced damage in hematopoietic lineages, while p53 and p21 also play radioprotective roles in the gastrointestinal epithelium. We previously identified some zinc(II) chelators, including 8-quinolinol derivatives, that suppress apoptosis in attempts to discover compounds that target the zinc-binding site in p53. We found that 5-chloro-8-quinolinol (5CHQ) has a unique p53-modulating activity that shifts its transactivation from proapoptotic to protective responses, including enhancing p21 induction and suppressing PUMA induction. This p53-modulating activity also influenced p53 and p53-target gene expression in unirradiated cells without inducing DNA damage. The specificity of 5CHQ for p53 and p21 was demonstrated by silencing the expression of each protein. These effects seem to be attributable to the sequence-specific alteration of p53 DNA-binding, as evaluated by chromatin immunoprecipitation and electrophoretic mobility shift assays. In addition, 5-chloro-8-methoxyquinoline itself had no antiapoptotic activity, indicating that the hydroxyl group at the 8-position is required for its antiapoptotic activity. We applied this remarkable agonistic activity to protecting the hematopoietic and gastrointestinal system in mouse irradiation models. The dose reduction factors of 5CHQ in total-body and abdominally irradiated mice were about 1.2 and 1.3, respectively. 5CHQ effectively protected mouse epithelial stem cells from a lethal dose of abdominal irradiation. Furthermore, the specificity of 5CHQ for p53 in reducing the lethality induced by abdominal irradiation was revealed in Trp53-KO mice. These results indicate that the pharmacologic upregulation of radioprotective p53 target genes is an effective strategy for addressing the gastrointestinal syndrome. Mol Cancer Ther; 17(2); 432–42. ©2017 AACR. See all articles in this MCT Focus section, “Developmental Therapeutics in Radiation Oncology.”