Publications by authors named "Ikuo Fujii"

62 Publications

A "ligand-targeting" peptide-drug conjugate: Targeted intracellular drug delivery by VEGF-binding helix-loop-helix peptides via receptor-mediated endocytosis.

PLoS One 2021 25;16(2):e0247045. Epub 2021 Feb 25.

Department of Biological Science, Osaka Prefecture University, Sakai, Osaka, Japan.

As a new alternative to antibody-drug conjugates, we generated "ligand-targeting" peptide-drug conjugates (PDCs), which utilize receptor-mediated endocytosis for targeted intracellular drug delivery. The PDC makes a complex with an extracellular ligand and then binds to the receptor on the cell surface to stimulate intracellular uptake via the endocytic pathway. A helix-loop-helix (HLH) peptide was designed as the drug carrier and randomized to give a conformationally constrained peptide library. The phage-displayed library was screened against vascular endothelial growth factor (VEGF) to yield the binding peptide M49, which exhibited strong binding affinity (KD = 0.87 nM). The confocal fluorescence microscopy revealed that peptide M49 formed a ternary complex with VEGF and its receptor, which was then internalized into human umbilical vein endothelial cells (HUVECs) via VEGF receptor-mediated endocytosis. The backbone-cyclized peptide M49K was conjugated with a drug, monomethyl auristatin E, to afford a PDC, which inhibited VEGF-induced HUVEC proliferation. HLH peptides and their PDCs have great potential as a new modality for targeted molecular therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0247045PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906330PMC
February 2021

Environmental pH stress influences cellular secretion and uptake of extracellular vesicles.

FEBS Open Bio 2021 03 18;11(3):753-767. Epub 2021 Feb 18.

Keio University School of Medicine, Tsukuba, Japan.

Exosomes (extracellular vesicles/EVs) participate in cell-cell communication and contain bioactive molecules, such as microRNAs. However, the detailed characteristics of secreted EVs produced by cells grown under low pH conditions are still unknown. Here, we report that low pH in the cell culture medium significantly affected the secretion of EVs with increased protein content and zeta potential. The intracellular expression level and location of stably expressed GFP-fused CD63 (an EV tetraspanin) in HeLa cells were also significantly affected by environmental pH. In addition, increased cellular uptake of EVs was observed. Moreover, the uptake rate was influenced by the presence of serum in the cell culture medium. Our findings contribute to our understanding of the effect of environmental conditions on EV-based cell-cell communication.
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http://dx.doi.org/10.1002/2211-5463.13107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7931216PMC
March 2021

An Immune-Stimulatory Helix-Loop-Helix Peptide: Selective Inhibition of CTLA-4-B7 Interaction.

ACS Chem Biol 2020 02 8;15(2):360-368. Epub 2020 Jan 8.

Department of Biological Science, Graduate School of Science , Osaka Prefecture University , 1-1 Gakuen-cho, Naka-ku , Sakai , Osaka 599-8531 , Japan.

Molecular-targeting peptides and mini-proteins are promising alternatives to antibodies in a wide range of applications in bioscience and medicine. We have developed a helix-loop-helix (HLH) peptide as an alternative to antibodies to inhibit specific protein interactions. Cytotoxic T lymphocyte antigen-4 (CTLA-4) downregulates immune responses of cytotoxic T-cells by interaction with B7-1, a co-stimulatory molecule expressed on antigen presenting cells (APCs). To induce immune stimulatory activity, we used directed evolution methods to generate a HLH peptide that binds to CTLA-4, inhibiting the CTLA-4-B7-1 interaction and inducing immune stimulatory activity. Yeast-displayed libraries of HLH peptides were constructed and screened against CTLA-4 and identified the binding peptide Y-2, which exhibits a moderate affinity. The affinity of Y-2 was improved by affinity maturation to afford a stronger binder, ERY2-4. Peptide ERY2-4 specifically bound to CTLA-4 with a of 196.8 ± 2.3 nM, comparable to the affinity of the CTLA-4-B7-1 interaction. Furthermore, ERY2-4 inhibited the CTLA-4-B7-1 interaction with an IC of 1.1 ± 0.03 μM and blocked the interaction between CTLA-4 and dendritic cells (DCs) presenting B7 on their surface. Importantly, ERY2-4 showed no cross-reactivity against CD28, suggesting it does not suppress T-cell activation. Finally, in a mixed lymphocyte reaction assay with DCs and T cells, ERY2-4 enhanced an allogeneic lymphocyte response. Since CTLA-4 is a critical immune checkpoint for restricting the cancer immune response, this inhibitory HLH peptide represents a new class of drug candidates for immunotherapy.
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http://dx.doi.org/10.1021/acschembio.9b00743DOI Listing
February 2020

Author Correction: BiFC-based visualisation system reveals cell fusion morphology and heterokaryon incompatibility in the filamentous fungus Aspergillus oryzae.

Sci Rep 2019 Oct 31;9(1):16078. Epub 2019 Oct 31.

Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-019-52663-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821747PMC
October 2019

Intracellular target delivery of cell-penetrating peptide-conjugated dodecaborate for boron neutron capture therapy (BNCT).

Chem Commun (Camb) 2019 Nov;55(93):13955-13958

Graduate School of Science, Osaka Prefecture University, 1-1, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.

In this study, we designed and synthesized organelle-targeted cell-penetrating peptide (CPP)-conjugated boron compounds to increase their cellular uptake and to control the intracellular locations for the induction of sophisticated anticancer biological activity in boron neutron capture therapy (BNCT), leading to anticancer effects with ATP reduction and apoptosis when irradiated with neutrons in an in vitro BNCT assay.
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http://dx.doi.org/10.1039/c9cc03924dDOI Listing
November 2019

Effects of gefitinib treatment on cellular uptake of extracellular vesicles in EGFR-mutant non-small cell lung cancer cells.

Int J Pharm 2019 Dec 11;572:118762. Epub 2019 Oct 11.

NanoSquare Research Institute, Research Center for the 21st Century, Organization for Research Promotion, Osaka Prefecture University, Sakai, Osaka 599-8570, Japan; Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8570, Japan. Electronic address:

Extracellular vesicles (exosomes, EVs) are cell membrane particles (30-200 nm) secreted by virtually all cells. During intercellular communication in the body, secreted EVs play crucial roles by carrying functional biomolecules (e.g., microRNAs and enzymes) into other cells to affect cellular function, including disease progression. We previously reported that the macropinocytosis pathway contributes greatly to the efficient cellular uptake of EVs. The activation of growth factor receptors, such as epidermal growth factor receptor (EGFR), induces macropinocytosis. In this study, we demonstrated the effects of gefitinib, a tyrosine kinase inhibitor of EGFR, on the cellular uptake of EVs. In EGFR-mutant HCC827 non-small cell lung cancer (NSCLC) cells, which are sensitive to gefitinib, macropinocytosis was suppressed by gefitinib treatment. However, the cellular uptake of EVs was increased by gefitinib treatment, whereas that of liposomes was reduced. In accordance with the results of the cellular uptake studies, the anti-cancer activity of doxorubicin (DOX)-loaded EVs in HCC827 cells was significantly increased in the presence of gefitinib, whereas the activity of DOX-loaded liposomes was reduced. The digestion of EV proteins by trypsin did not affect uptake, suggesting that the cellular uptake of EVs might not be mediated by EV proteins. These results suggest that gefitinib can enhance cell-to-cell communication via EVs within the tumor microenvironment. In addition, EVs show potential as drug delivery vehicles in combination with gefitinib for the treatment of patients harboring EGFR-mutant NSCLC tumors.
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http://dx.doi.org/10.1016/j.ijpharm.2019.118762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899172PMC
December 2019

Generation of Novel Anti-MUC1 Monoclonal Antibodies with Designed Carbohydrate Specificities Using MUC1 Glycopeptide Library.

ACS Omega 2017 Nov 1;2(11):7493-7505. Epub 2017 Nov 1.

Shionogi Pharmaceutical Research Center, Shionogi & Co., Ltd., 3-1-1 Futaba-cho, Toyonaka, Osaka 561-0825, Japan.

Numerous anti-mucin 1 (anti-MUC1) antibodies that recognize -glycan core structures have already been developed. However, most of them show low specificities toward -glycan structures and/or low affinity toward a monovalent epitope. In this study, using an MUC1 glycopeptide library, we established two novel anti-MUC1 monoclonal antibodies (1B2 and 12D10) with designed carbohydrate specificities. Compared with previously reported anti-MUC1 antibodies, 1B2 and 12D10 showed quite different features regarding their specificities, affinities, and reactivity profiles to various cell lines. Both antibodies recognized specific -glycan structures at the PDT*R motif (the asterisk represents an -glycosylation site). 1B2 recognized -glycans with an unsubstituted -6 position of the GalNAc residue (Tn, T, and 23ST), whereas 12D10 recognized Neu5Ac at the same position (STn, 26ST, and dST). Neither of them bound to glycopeptides with core 2 -glycans that have GlcNAc at the -6 position of the GalNAc residue. Furthermore, 1B2 and 12D10 showed a strong binding to not only native MUC1 but also 20-mer glycopeptide with a monovalent epitope. These anti-MUC1 antibodies should thus become powerful tools for biological studies on MUC1 -glycan structures. Furthermore, the strategy of using glycopeptide libraries should enable the development of novel antibodies with predesigned -glycan specificities.
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http://dx.doi.org/10.1021/acsomega.7b00708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6044872PMC
November 2017

Highly Sensitive and Practical Fluorescent Sandwich ELISA for Ciguatoxins.

Anal Chem 2018 06 29;90(12):7318-7324. Epub 2018 May 29.

Department of Biological Science, Graduate School of Science , Osaka Prefecture University , Osaka 599-8570 , Japan.

Ciguatera fish poisoning (CFP) caused by the consumption of fish that have accumulated ciguatoxins (CTXs) affects more than 50000 people annually. The spread of CFP causes enormous damage to public health, fishery resources, and the economies of tropical and subtropical endemic regions. The difficulty in avoiding CFP arises from the lack of sensitive and reliable analytical methods for the detection and quantification of CTXs in contaminated fish, along with the normal appearance, smell, and taste of fish contaminated with the causative toxins. Thus, an accurate, sensitive, routine, and portable detection method for CTXs is urgently required. We have successfully developed a highly sensitive fluorescent sandwich ELISA, which can detect, differentiate, and quantify four major CTX congeners (CTX1B, CTX3C, 51-hydroxyCTX3C, and 54-deoxyCTX1B) with a detection limit of less than 1 pg/mL. The ELISA protocol, using one microtiter plate coated with two mAbs (10C9 and 3G8), and ALP-linked 8H4, can detect any of the four CTX congeners in a single operation. CTX1B spiked into fish at the FDA guidance level of 0.01 ppb CTX1B equivalent toxicity in fish from Pacific regions was also proven to be reliably detected by this ELISA. Furthermore, the efficiency of extraction/purification procedures and the matrix effect of contaminants in fish were evaluated in detail, since pretreatment and matrix effects are critical for ELISA analysis.
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http://dx.doi.org/10.1021/acs.analchem.8b00519DOI Listing
June 2018

Structural basis of the broad substrate tolerance of the antibody 7B9-catalyzed hydrolysis of p-nitrobenzyl esters.

Bioorg Med Chem 2018 05 19;26(8):1412-1417. Epub 2017 Aug 19.

Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan. Electronic address:

Catalytic antibody 7B9, which was elicited against p-nitrobenzyl phosphonate transition-state analogue (TSA) 1, hydrolyzes a wide range of p-nitrobenzyl monoesters and thus shows broad substrate tolerance. To reveal the molecular basis of this substrate tolerance, the 7B9 Fab fragment complexed with p-nitrobenzyl ethylphosphonate 2 was crystallized and the three-dimensional structure was determined. The crystal structure showed that the strongly antigenic p-nitrobenzyl moiety occupied a relatively shallow antigen-combining site and therefore the alkyl moiety was located outside the pocket. These results support the observed broad substrate tolerance of 7B9 and help rationalize how 7B9 can catalyze various p-nitrobenzyl ester derivatives. The crystal structure also showed that three amino acid residues (Asn, Ser, and Arg) were placed in key positions to form hydrogen bonds with the phosphonate oxygens of the transitions-state analogue. In addition, the role of these amino acid residues was examined by site-directed mutagenesis to alanine: all mutants (AsnAla, SerAla, and ArgAla) showed no detectable catalytic activity. Coupling the findings from our structural studies with these mutagenesis results clarified the structural basis of the observed broad substrate tolerance of antibody 7B9-catalyzed hydrolyses. Our findings provide new strategies for the generation of catalytic antibodies that accept a broad range of substrates, aiding their practical application in synthetic organic chemistry.
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http://dx.doi.org/10.1016/j.bmc.2017.07.050DOI Listing
May 2018

BiFC-based visualisation system reveals cell fusion morphology and heterokaryon incompatibility in the filamentous fungus Aspergillus oryzae.

Sci Rep 2018 02 13;8(1):2922. Epub 2018 Feb 13.

Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.

Aspergillus oryzae is an industrially important filamentous fungus used for Japanese traditional food fermentation and heterologous protein production. Although cell fusion is important for heterokaryon formation and sexual/parasexual reproduction required for cross breeding, knowledge on cell fusion and heterokaryon incompatibility in A. oryzae is limited because of low cell fusion frequency. Therefore, we aimed to develop a BiFC system to specifically visualise fused cells and facilitate the analysis of cell fusion in A. oryzae. The cell fusion ability and morphology of 15 A. oryzae strains were investigated using heterodimerising proteins LZA and LZB fused with split green fluorescence protein. Morphological investigation of fused cells revealed that cell fusion occurred mainly as conidial anastomosis during the early growth stage. Self-fusion abilities were detected in most industrial A. oryzae strains, but only a few strain pairs showed non-self fusion. Protoplast fusion assay demonstrated that almost all the pairs capable of non-self fusion were capable of heterokaryon formation and vice versa, thus providing the first evidence of heterokaryon incompatibility in A. oryzae. The BiFC system developed in this study provides an effective method in studying morphology of fused cells and heterokaryon incompatibility in the filamentous fungal species with low cell fusion efficiency.
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http://dx.doi.org/10.1038/s41598-018-21323-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5811552PMC
February 2018

Gefitinib Enhances Mitochondrial Biological Functions in NSCLCs with Mutations at a High Cell Density.

Anticancer Res 2017 09;37(9):4779-4788

NanoSquare Research Institution, Research Center for the 21st Century, Organization for Research Promotion, Osaka Prefecture University, Sakai, Japan

Background/aim: Gefitinib is a tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR) and has been approved for the treatment of non-small cell lung cancers (NSCLCs) with EGFR mutations. Here we demonstrated that gefitinib induced a significantly enhanced biological activity of succinate-tetrazolium reductase (STR) in mitochondria and mitochondrial membrane potential in HCC827 cells (EGFR mutation NSCLCs, sensitive to gefitinib) at a high cell density.

Materials And Methods: We assessed the biological activity (STR, mitochondrial membrane potential, expression level of Bcl-2 family proteins) of gefitinib on NSCLCs at different cell densities.

Results: The 3D cell culture experiments showed the enhanced mitochondrial biological activity in clustered cell culture treated with gefitinib. Interestingly, the expression levels of Bcl-x and Bax, were affected by the cellular number and gefitinib treatment. We also found that gefitinib prevented additive anticancer activity in the combinational treatment with doxorubicin, which induces mitochondria-dependent apoptotic cell death.

Conclusion: Our results indicate that gefitinib may work as a mitochondrial protector against combinational treatment with mitochondria-dependent anticancer agents in high-cell-density.
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http://dx.doi.org/10.21873/anticanres.11884DOI Listing
September 2017

Arginine-rich cell-penetrating peptide-modified extracellular vesicles for active macropinocytosis induction and efficient intracellular delivery.

Sci Rep 2017 05 16;7(1):1991. Epub 2017 May 16.

Institute for Chemical Research, Kyoto University, Uji, Kyoto, 611-0011, Japan.

Extracellular vesicles (EVs) including exosomes have been shown to play crucial roles in cell-to-cell communication because of their ability to carry biofunctional molecules (e.g., microRNAs and enzymes). EVs also have pharmaceutical advantages and are highly anticipated to be a next-generation intracellular delivery tool. Here, we demonstrate an experimental technique that uses arginine-rich cell-penetrating peptide (CPP)-modified EVs to induce active macropinocytosis for effective cellular EV uptake. Modification of arginine-rich CPPs on the EV membrane resulted in the activation of the macropinocytosis pathway, and the number of arginine residues in the peptide sequences affected the cellular EV uptake efficiency. Consequently, the ribosome-inactivating protein saporin-encapsulated EVs modified with hexadeca-arginine (R16) peptide effectively attained anti-cancer activity.
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http://dx.doi.org/10.1038/s41598-017-02014-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5434003PMC
May 2017

Effects of substrate conformational strain on binding kinetics of catalytic antibodies.

Biophys Physicobiol 2016 14;13:135-138. Epub 2016 Jul 14.

Graduate School of Science, Osaka Prefecture University, Sakai, Osaka 599-8570, Japan.

We analyzed the correlation between the conformational strain and the binding kinetics in antigen-antibody interactions. The catalytic antibodies 6D9, 9C10, and 7C8 catalyze the hydrolysis of a nonbioactive chloramphenicol monoester derivative to generate a bioactive chloramphenicol. The crystal structure of 6D9 complexed with a transition-state analog (TSA) suggests that 6D9 binds the substrate to change the conformation of the ester moiety to a thermodynamically unstable twisted conformation, enabling the substrate to reach the transition state during catalysis. The present binding kinetic analysis showed that the association rate for 6D9 binding to the substrate was much lower than that to TSA, whereas those for 9C10 and 7C8 binding were similar to those to TSA. Considering that 7C8 binds to the substrate with little conformational change in the substrate, the slow association rate observed in 6D9 could be attributed to the conformational strain in the substrate.
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http://dx.doi.org/10.2142/biophysico.13.0_135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042168PMC
July 2016

Site-Directed Chemical Mutations on Abzymes: Large Rate Accelerations in the Catalysis by Exchanging the Functionalized Small Nonprotein Components.

ACS Chem Biol 2016 10 29;11(10):2803-2811. Epub 2016 Aug 29.

Department of Biological Science, Graduate School of Science, Osaka Prefecture University , 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.

Taking advantage of antibody molecules to generate tailor-made binding sites, we propose a new class of protein modifications, termed as "site-directed chemical mutation." In this modification, chemically synthesized catalytic components with a variety of steric and electronic properties can be noncovalently and nongenetically incorporated into specific sites in antibody molecules to induce enzymatic activity. Two catalytic antibodies, 25E2 and 27C1, possess antigen-combining sites which bind catalytic components and act as apoproteins in catalytic reactions. By simply exchanging these components, antibodies 25E2 and 27C1 can catalyze a wide range of chemical transformations including acyl-transfer, β-elimination, aldol, and decarboxylation reactions. Although both antibodies were generated with the same hapten, phosphonate diester 1, they showed different catalytic activity. When phenylacetic acid 4 was used as the catalytic component, 25E2 efficiently catalyzed the elimination reaction of β-haloketone 2, whereas 27C1 showed no catalytic activity. In this work, we focused on the β-elimination reaction and examined the site-directed chemical mutation of 27C1 to induce activity and elucidate the catalytic mechanism. Molecular models showed that the cationic guanidyl group of Arg in 27C1 makes a hydrogen bond with the P═O oxygen in the hapten. This suggested that during β-elimination, Arg of 27C1 would form a salt bridge with the carboxylate of 4, thus destroying reactivity. Therefore, we utilized site-directed chemical mutation to change the charge properties of the catalytic components. When amine components 7-10 were used, 27C1 efficiently catalyzed the β-elimination reaction. It is noteworthy that chemical mutation with secondary amine 8 provided extremely high activity, with a rate acceleration [(k/K 2)/k] of 1 000 000. This catalytic activity likely arises from the proximity effect, plus general-base catalysis associated the electrostatic interactions. In 27C1, the cationic guanidyl group of Arg is spatially close to the nitrogen of the amine components. In this microenvironment, the intrinsic pK of the amine is perturbed and shifts to a lower pK, which efficiently abstracts the α-proton during the reaction. This mechanism is consistent with the observed kinetic isotope effect (E2 or E1cB mechanism). Thus, site-directed chemical mutation provides a better understanding of enzyme functions and opens new avenues in biocatalyst research.
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http://dx.doi.org/10.1021/acschembio.6b00574DOI Listing
October 2016

Vectorization of biomacromolecules into cells using extracellular vesicles with enhanced internalization induced by macropinocytosis.

Sci Rep 2016 10 17;6:34937. Epub 2016 Oct 17.

Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.

Extracellular vesicles (EVs, exosomes) are approximately 30- to 200-nm-long vesicles that have received increased attention due to their role in cell-to-cell communication. Although EVs are highly anticipated to be a next-generation intracellular delivery tool because of their pharmaceutical advantages, including non-immunogenicity, their cellular uptake efficacy is low because of the repulsion of EVs and negatively charged cell membranes and size limitations in endocytosis. Here, we demonstrate a methodology for achieving enhanced cellular EV uptake using arginine-rich cell-penetrating peptides (CPPs) to induce active macropinocytosis. The induction of macropinocytosis via a simple modification to the exosomal membrane using stearylated octaarginine, which is a representative CPP, significantly enhanced the cellular EV uptake efficacy. Consequently, effective EV-based intracellular delivery of an artificially encapsulated ribosome-inactivating protein, saporin, in EVs was attained.
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http://dx.doi.org/10.1038/srep34937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5066177PMC
October 2016

A Cyclized Helix-Loop-Helix Peptide as a Molecular Scaffold for the Design of Inhibitors of Intracellular Protein-Protein Interactions by Epitope and Arginine Grafting.

Angew Chem Int Ed Engl 2016 08 28;55(36):10612-5. Epub 2016 Jul 28.

Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1, Gakuen-cho, Naka-ku, Osaka, 599-8531, Japan.

The design of inhibitors of intracellular protein-protein interactions (PPIs) remains a challenge in chemical biology and drug discovery. We propose a cyclized helix-loop-helix (cHLH) peptide as a scaffold for generating cell-permeable PPI inhibitors through bifunctional grafting: epitope grafting to provide binding activity, and arginine grafting to endow cell-permeability. To inhibit p53-HDM2 interactions, the p53 epitope was grafted onto the C-terminal helix and six Arg residues were grafted onto another helix. The designed peptide cHLHp53-R showed high inhibitory activity for this interaction, and computational analysis suggested a binding mode for HDM2. Confocal microscopy of cells treated with fluorescently labeled cHLHp53-R revealed cell membrane penetration and cytosolic localization. The peptide inhibited the growth of HCT116 and LnCap cancer cells. This strategy of bifunctional grafting onto a well-structured peptide scaffold could facilitate the generation of inhibitors for intracellular PPIs.
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http://dx.doi.org/10.1002/anie.201603230DOI Listing
August 2016

Macrocyclization and labeling of helix-loop-helix peptide with intramolecular bis-thioether linkage.

Biopolymers 2016 Nov;106(4):415-21

Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka, 599-8531, Japan.

Conformationally constrained peptides have been developed as an inhibitor for protein-protein interactions (PPIs), and we have de novo designed cyclized helix-loop-helix (cHLH) peptide with a disulfide bond consisting of 40 amino acids to generate molecular-targeting peptides. However, synthesis of long peptides has sometimes resulted in low yield according to the respective amino acid sequences. Here we developed a method for efficient synthesis and labeling for cHLH peptides. First, we synthesized two peptide fragments and connected them by the copper-mediated alkyne and azide cycloaddition (CuAAC) reaction. Cyclization was performed by bis-thioether linkage using 1,3-dibromomethyl-5-propargyloxybenzene, and subsequently, the cHLH peptide was labeled with an azide-labeled probe. Finally, we designed and synthesized a peptide inhibitor for the p53-HDM2 interaction using a structure-guided design and successfully labeled it with a fluorescent probe or a functional peptide, respectively, by click chemistry. This macrocyclization and labeling method for cHLH peptide would facilitate the discovery of de novo bioactive ligands and therapeutic leads. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 415-421, 2016.
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http://dx.doi.org/10.1002/bip.22826DOI Listing
November 2016

Development of a novel immunoassay specific for mouse intact proinsulin.

Anal Biochem 2015 Sep 28;484:91-8. Epub 2015 May 28.

Shionogi Pharmaceutical Research Center, Shionogi & Company, Toyonaka, Osaka 561-0825, Japan. Electronic address:

The blood concentration of intact proinsulin, but not total proinsulin, has been suggested to be a diagnostic marker for type 2 diabetes mellitus (T2DM), but a sensitive assay specific for rodent intact proinsulin is lacking. Here, a novel enzyme-linked immunosorbent assay (ELISA) for mouse intact proinsulin was developed. The developed ELISA detected mouse intact proinsulin with the working range of 8.3 to 2700pg/ml. Cross-reactivity with mouse split-32,33 proinsulin was approximately 100times lower than the reactivity with mouse intact proinsulin, and no cross-reactivity with mouse insulin was detected. The developed ELISA was sufficiently sensitive to detect low levels of intact proinsulin in normal mouse plasma. The measurement by the developed ELISA revealed that intact proinsulin was elevated in the plasma of type 2 diabetic db/db mice as mice aged, and the ratio of intact proinsulin/insulin in plasma was correlated with levels of glycated hemoglobin A1c as seen in T2DM patients. These results suggest that the plasma level of intact proinsulin, but not total proinsulin, is a sensitive marker for pancreatic dysfunction and the ensuring diabetic disease progression of db/db mice. This ELISA could aid nonclinical evaluation of therapeutic interventions in T2DM.
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http://dx.doi.org/10.1016/j.ab.2015.05.012DOI Listing
September 2015

'Turn On/Off' fluorescence probe for the screening of unactivated Bruton's tyrosine kinase.

Bioorg Med Chem Lett 2015 30;25(10):2141-5. Epub 2015 Mar 30.

Drug Discovery Research, Carna Biosciences, Inc., 3rd Floor, BMA, 1-5-5 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.

BTK has emerged as a promising target for treating B-cell malignancies and autoimmune diseases, and there has been a growing demand to identify selective BTK inhibitors efficiently. In this Letter, we have designed and synthesized a new fluorescent probe to screen compounds that preferentially bind to an unactivated state of BTK (BTK [U]). The fluorescence of the probe was turned on in the presence of BTK [U], and quenched by the addition of compounds which preferentially bind to BTK [U]. This unique fluorescent probe was successfully applied to the screening of a kinase focused compound library. The results suggest that this new method is a simple and easy-to-perform assay to screen inhibitors of BTK [U].
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http://dx.doi.org/10.1016/j.bmcl.2015.03.063DOI Listing
January 2016

Development of an ultrasensitive immunoassay using affinity maturated antibodies for the measurement of rodent insulin.

Anal Biochem 2015 Mar 15;473:72-9. Epub 2014 Dec 15.

Shionogi Pharmaceutical Research Center, Shionogi & Company, Toyonaka, Osaka 561-0825, Japan. Electronic address:

The measurement of plasma insulin is important for clinical diagnosis of diabetes and for preclinical research of metabolic diseases, especially in rodent models used in drug discovery research for type 2 diabetes. Fasting immunoreactive insulin (F-IRI) concentrations are used to calculate the homeostasis model assessment ratio (HOMA-R), an index of insulin sensitivity. However, even the most sensitive commercially available enzyme-linked immunosorbent assay (ELISA) kits cannot measure the very low F-IRI concentrations in normal rats and mice. Therefore, we sought to develop a new rodent insulin ELISA with greater sensitivity for low F-IRI concentrations. Despite repeated efforts, high-affinity antibodies could not be generated by immunizing mice with mouse insulin (self-antigen). Therefore, we generated two weak monoclonal antibodies (13G4 and 26B2) that were affinity maturated and used to develop a highly sensitive ELISA. The measurement range of the sandwich ELISA with the affinity maturated antibodies (13G4m1 and 26B2m1) was 1.5 to 30,000 pg/ml, and its detection limit was at least 10 times lower than those of commercially available kits. In conclusion, we describe the development of a new ultrasensitive ELISA suitable for measuring very low plasma insulin concentrations in rodents. This ELISA might be very useful in drug discovery research in diabetes.
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http://dx.doi.org/10.1016/j.ab.2014.12.003DOI Listing
March 2015

Preparation of anti-ciguatoxin monoclonal antibodies using synthetic haptens: sandwich ELISA detection of ciguatoxins.

J AOAC Int 2014 Mar-Apr;97(2):373-9

Ciguatera fish poisoning (CFP) is a form of food poisoning caused by the consumption of fish that have accumulated a type of sodium channel activator toxin called ciguatoxins (CTXs), which are produced by dinoflagellates of the genus Gambierdiscus through the food chain. CFP affects more than 50000 people each year. The extremely low level of CTXs in tainted fish has hampered the development of antibodies for the detection of these toxins. Monoclonal antibodies (mAbs) specific against major congeners of CTX3C, 51-hydroxyCTX3C, CTX1B, and 54-deoxyCTX1B were prepared by immunization of mice with protein conjugates of rationally designed synthetic haptens in place of the natural toxins. We found that haptenic groups possessing a surface area larger than 400 angstroms2 were required to produce mAbs that can bind strongly to CTXs. Direct sandwich ELISA utilizing two different monoclonal antibodies that bind specifically to one of the two wings of a CTX were established to detect CTXs. No cross-reactivity was observed against the other marine toxins tested, including brevetoxin A, brevetoxin B, okadaic acid, and maitotoxin.
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http://dx.doi.org/10.5740/jaoacint.sgetsumurayaDOI Listing
June 2014

High-throughput de novo screening of receptor agonists with an automated single-cell analysis and isolation system.

Sci Rep 2014 Feb 28;4:4242. Epub 2014 Feb 28.

Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan.

Reconstitution of signaling pathways involving single mammalian transmembrane receptors has not been accomplished in yeast cells. In this study, intact EGF receptor (EGFR) and a cell wall-anchored form of EGF were co-expressed on the yeast cell surface, which led to autophosphorylation of the EGFR in an EGF-dependent autocrine manner. After changing from EGF to a conformationally constrained peptide library, cells were fluorescently labeled with an anti-phospho-EGFR antibody. Each cell was subjected to an automated single-cell analysis and isolation system that analyzed the fluorescent intensity of each cell and automatically retrieved each cell with the highest fluorescence. In ~3.2 × 10(6) peptide library, we isolated six novel peptides with agonistic activity of the EGFR in human squamous carcinoma A431 cells. The combination of yeast cells expressing mammalian receptors, a cell wall-anchored peptide library, and an automated single-cell analysis and isolation system might facilitate a rational approach for de novo drug screening.
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http://dx.doi.org/10.1038/srep04242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937795PMC
February 2014

Peptide-based immunoadsorbents: molecular grafting of IgG-Fc-binding epitopes of Protein A onto a de novo-designed helix-loop-helix peptide.

Bioorg Med Chem 2014 Mar 6;22(6):1845-9. Epub 2014 Feb 6.

Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan. Electronic address:

The development of immunoadsorbents that have high specificity for immunoglobulin and no immunogenicity is essential for immunoadsorption treatment of autoimmune diseases. In this study, we designed peptide immunoadsorbents by molecular grafting of the IgG-Fc binding epitopes of Protein A onto a de novo-designed helix-loop-helix peptide. Linear (linG7A5) and cyclic (cyG7A5) grafted peptides were synthesized to test their binding affinity and specificity. Peptide cyG7A5 demonstrated high specificity for human IgG-Fc, with a K(D) of 19 μM, and demonstrated no affinity to other plasma proteins, human serum albumin, or fibrinogen. To evaluate their immunoadsorbance efficiency, the grafted peptides and Protein A were conjugated to polyvinyl acetate resin and tested in a batch-wise process for adsorption removal of IgG from human plasma. The IgG capture capacities of the peptides correlated well with their binding affinities. Interestingly, cyG7A5 showed a higher binding specificity for IgG than did Protein A.
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http://dx.doi.org/10.1016/j.bmc.2014.01.053DOI Listing
March 2014

Phage selection of peptide "microantibodies".

Curr Protoc Chem Biol 2013 ;5(3):171-94

Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Osaka, Japan.

A bioactive peptide capable of inhibiting protein-protein interactions has the potential to be a molecular tool for biological studies and a therapeutic by disrupting aberrant interactions involved in diseases. We have developed combinatorial libraries of peptides with helix-loop-helix structure, from which the isolated peptides have the constrained structure to reduce entropy costs in binding, resulting in high binding affinities for target molecules. Previously, we designed a de novo peptide of helix-loop-helix structure that we termed a "microantibody." Using the microantibody as a library scaffold, we have constructed a phage-display library to successfully isolate molecular-targeting peptides against a cytokine receptor (granulocyte colony-stimulating factor receptor), a protein kinase (Aurora-A), and a ganglioside (GM1). Protocols in this article describe a general procedure for the library construction and the library screening.
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http://dx.doi.org/10.1002/9780470559277.ch130039DOI Listing
March 2015

Antibody-catalyzed decarboxylation and aldol reactions using a primary amine molecule as a functionalized small nonprotein component.

Bioorg Med Chem 2013 Nov 19;21(22):7011-7. Epub 2013 Sep 19.

Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan; Department of System Chemotherapy and Molecular Science, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto 606-8501, Japan.

Catalytic antibody 27C1 bears binding sites for both a substrate- and a functionalized small nonprotein component in the active site. We investigated the possibility of exploiting imine and enamine intermediates using a primary amine molecule into the active site of antibody 27C1. The antibody catalyzed β-keto acid decarboxylation with a rate enhancement (kcat/Km/kuncat) of 140,000, as well as highly regioselective cross-aldol reactions of ketones and p-nitrobenzaldehyde. These studies provide new strategies for the generation of catalytic antibodies possessing binding sites for functionalized components.
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http://dx.doi.org/10.1016/j.bmc.2013.09.015DOI Listing
November 2013

The crystal structure of multidrug-resistance regulator RamR with multiple drugs.

Nat Commun 2013 ;4:2078

Laboratory of Microbiology and Infectious Diseases, Division of Special Projects, Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan.

RamR is a transcriptional repressor of the gene-encoding RamA protein, which controls the expression of the multidrug efflux system genes acrAB-tolC. RamR is an important multidrug-resistance factor, however, its structure and the identity of the molecules to which it responds have been unknown. Here, we report the crystal structure of RamR in complex with multiple drugs, including berberine, crystal violet, dequalinium, ethidium bromide and rhodamine 6G. All compounds are found to interact with Phe155 of RamR, and each compound is surrounded by different amino acid residues. Binding of these compounds to RamR reduces its DNA-binding affinity, which results in the increased expression of ramA. Our results reveal significant flexibility in the substrate-recognition region of RamR, which regulates the bacterial efflux participating in multidrug resistance.
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http://dx.doi.org/10.1038/ncomms3078DOI Listing
December 2013

An automated system for high-throughput single cell-based breeding.

Sci Rep 2013 1;3:1191. Epub 2013 Feb 1.

Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan.

When establishing the most appropriate cells from the huge numbers of a cell library for practical use of cells in regenerative medicine and production of various biopharmaceuticals, cell heterogeneity often found in an isogenic cell population limits the refinement of clonal cell culture. Here, we demonstrated high-throughput screening of the most suitable cells in a cell library by an automated undisruptive single-cell analysis and isolation system, followed by expansion of isolated single cells. This system enabled establishment of the most suitable cells, such as embryonic stem cells with the highest expression of the pluripotency marker Rex1 and hybridomas with the highest antibody secretion, which could not be achieved by conventional high-throughput cell screening systems (e.g., a fluorescence-activated cell sorter). This single cell-based breeding system may be a powerful tool to analyze stochastic fluctuations and delineate their molecular mechanisms.
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http://dx.doi.org/10.1038/srep01191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561619PMC
August 2013

Development of a neutralizing antibody specific for the active form of matrix metalloproteinase-13.

Biochemistry 2012 Nov 23;51(44):8877-84. Epub 2012 Oct 23.

Shionogi Pharmaceutical Research Center, Shionogi & Company, Ltd., 3-1-1 Futaba-cho, Toyonaka, Osaka 561-0825, Japan.

Matrix metalloproteinase-13 (MMP-13) is important in the pathology of osteoarthritis (OA). Although MMP-13 is considered a therapeutic target for OA, it is unclear how MMP-13 activity is regulated by the system that comprises various proteinases and their inhibitors. MMP-13 neutralizing antibodies could be a useful tool for investigating the involvement of MMP-13 in cartilage degeneration in OA-affected joints because antibodies possess high affinity and specificity compared with low-molecular weight chemical compounds. On the basis of three-dimensional structure and amino acid sequence information on MMP-13, we selected an appropriate antigen peptide and generated a neutralizing antibody by immunizing mice with the antigen. The most significant property of monoclonal antibody 14D10 was the specific binding to the active form of MMP-13, but not to the latent form, or other MMPs. With this property, active MMP-13 was measured selectively by an enzyme-linked immunosorbet assay. Furthermore, 14D10 suppressed the cleavage of type II collagen in human articular chondrocyte cultures, and 14D10 is thought to inhibit MMP-13 activity effectively. Thus, the highly selective MMP-13 neutralizing antibody (14D10) might be a useful tool for investigating the mechanism of type II collagen degradation in arthritic pathology.
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http://dx.doi.org/10.1021/bi301228dDOI Listing
November 2012

Development of a monoclonal antibody against the left wing of ciguatoxin CTX1B: thiol strategy and detection using a sandwich ELISA.

Toxicon 2012 Sep 7;60(3):348-57. Epub 2012 May 7.

Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Osaka 599-8531, Japan.

Ciguatera fish poisoning (CFP) is a form of food poisoning caused by the ingestion of a variety of reef fish that have accumulated trace amounts of ciguatoxins produced by dinoflagellates of the genus Gambierdiscus through the food chain. CFP affects more than 50,000 people each year. The extremely low level of the causative neurotoxins, ciguatoxins, in fish has hampered the preparation of antibodies for detecting the toxins. In this paper, we describe a thiol strategy for synthesizing a keyhole limpet hemocyanin (KLH)-conjugate (20) of the ABCDE-ring fragment of the Pacific ciguatoxins, CTX1B (1) and 54-deoxyCTX1B (4). We succeeded in producing a monoclonal antibody (3G8) against the left wings of these ciguatoxins by immunizing mice with the hapten-KLH conjugate (20) as the synthetic antigen. The most promising mAb, 3G8, does not cross-react with other related marine toxins. Sandwich enzyme-linked immunosorbent assay (ELISA) utilizing 3G8 and the previously prepared monoclonal antibody (8H4) enabled us to detect 1 specifically at less than 0.28 ng/mL.
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http://dx.doi.org/10.1016/j.toxicon.2012.04.347DOI Listing
September 2012

Design and combinatorial synthesis of a novel kinase-focused library using click chemistry-based fragment assembly.

Bioorg Med Chem Lett 2012 Jan 30;22(1):591-6. Epub 2011 Oct 30.

Drug Discovery Research, Carna Biosciences, Inc., 3rd Floor, BMA, 1-5-5 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.

Fragment-based lead discovery is a new approach for lead generation that has emerged in the past decade. Because the initial fragments identified in the fragment screening typically show weak binding affinity, an intensive medicinal chemistry effort would be required to grow initial fragments into a potential lead compound. Here we demonstrate a kinase focused evolved fragment (KFEF) library, constructed by click chemistry-based fragment assembly, that is a valuable source of kinase inhibitors. This combinatorial assembly of two fragments, kinase-privileged alkyne fragments and diversified azide fragments, by two cycloaddition reactions shows a unique potential for the one-step synthesis of structurally diverse evolved fragments. The screening of this triazole-based KFEF library allowed the rapid identification of potent lead candidates for FLT3 and GSK3β kinase.
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http://dx.doi.org/10.1016/j.bmcl.2011.10.076DOI Listing
January 2012
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