Publications by authors named "Shusuke Tomoshige"

24 Publications

  • Page 1 of 1

Functionalization of Human Serum Albumin by Tyrosine Click.

Int J Mol Sci 2021 Aug 12;22(16). Epub 2021 Aug 12.

Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan.

Human serum albumin (HSA) is a promising drug delivery carrier. Although covalent modification of Cys34 is a well-established method, it is desirable to develop a novel covalent modification method that targets residues other than cysteine to introduce multiple functions into a single HSA molecule. We developed a tyrosine-selective modification of HSA. Three tyrosine selective modification methods, hemin-catalyzed, horseradish peroxidase (HRP)-catalyzed, and laccase-catalyzed reactions were performed, and the modification efficiencies and modification sites of the modified HSAs obtained by these methods were evaluated and compared. We found that the laccase-catalyzed method could efficiently modify the tyrosine residue of HSA under mild reaction conditions without inducing oxidative side reactions. An average of 2.2 molecules of functional groups could be introduced to a single molecule of HSA by the laccase method. Binding site analysis using mass spectrometry suggested Y84, Y138, and Y401 as the main modification sites. Furthermore, we evaluated binding to ibuprofen and found that, unlike the conventional lysine residue modification, the inhibition of drug binding was minimal. These results suggest that tyrosine-residue selective chemical modification is a promising method for covalent drug attachment to HSA.
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http://dx.doi.org/10.3390/ijms22168676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8395410PMC
August 2021

In vivo synthetic chemistry of proteolysis targeting chimeras (PROTACs).

Bioorg Med Chem 2021 07 19;41:116221. Epub 2021 May 19.

Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, Miyagi 980-8577, Japan.

Chemical knockdown of therapeutic targets using proteolysis targeting chimeras (PROTACs) is a rapidly developing field in drug discovery, but PROTACs are bifunctional molecules that generally show poor bioavailability due to their relatively high molecular weight. Recent developments aimed at the development of next-generation PROTACs include the in vivo synthesis of PROTAC molecules, and the exploitation of PROTACs as chemical tools for in vivo synthesis of ubiquitinated proteins. This short review covers recent advances in these areas and discusses the prospects for in vivo synthetic PROTAC technology.
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http://dx.doi.org/10.1016/j.bmc.2021.116221DOI Listing
July 2021

Epo-C12 inhibits peroxiredoxin 1 peroxidase activity.

Bioorg Med Chem 2021 07 10;41:116203. Epub 2021 May 10.

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan. Electronic address:

Epo-C12 is a synthetic derivative of epolactaene, isolated from Penicillium sp. BM 1689-P. Epo-C12 induces apoptosis in human acute lymphoblastoid leukemia BALL-1 cells. In our previous studies, seven proteins that bind to Epo-C12 were identified by a combination of pull-down experiments using biotinylated Epo-C12 (Bio-Epo-C12) and mass spectrometry. In the present study, the effect of Epo-C12 on peroxiredoxin 1 (Prx 1), one of the proteins that binds to Epo-C12, was investigated. Epo-C12 inhibited Prx 1 peroxidase activity. However, it did not suppress its chaperone activity. Binding experiments between Bio-Epo-C12 and point-mutated Prx 1s suggest that Epo-C12 binds to Cys and Cys in Prx 1. The present study revealed that Prx 1 is one of the target proteins through which Epo-C12 exerts an apoptotic effect in BALL-1 cells.
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http://dx.doi.org/10.1016/j.bmc.2021.116203DOI Listing
July 2021

Proximity Histidine Labeling by Umpolung Strategy Using Singlet Oxygen.

J Am Chem Soc 2021 May 27;143(20):7726-7731. Epub 2021 Apr 27.

Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Kanagawa 226-8503, Japan.

While electrophilic reagents for histidine labeling have been developed, we report an umpolung strategy for histidine functionalization. A nucleophilic small molecule, 1-methyl-4-arylurazole, selectively labeled histidine under singlet oxygen (O) generation conditions. Rapid histidine labeling can be applied for instant protein labeling. Utilizing the short diffusion distance of O and a technique to localize the O generator, a photocatalyst in close proximity to the ligand-binding site, we demonstrated antibody Fc-selective labeling on magnetic beads functionalized with a ruthenium photocatalyst and Fc ligand, ApA. Three histidine residues located around the ApA binding site were identified as labeling sites by liquid chromatography-mass spectrometry analysis. This result suggests that O-mediated histidine labeling can be applied to a proximity labeling reaction on the nanometer scale.
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http://dx.doi.org/10.1021/jacs.1c01626DOI Listing
May 2021

Synthesis and Cytotoxic Evaluation of -Alkyl-2-halophenazin-1-ones.

ACS Omega 2020 Oct 16;5(42):27667-27674. Epub 2020 Oct 16.

Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yaamazaki, Noda, Chiba 278-8510, Japan.

In this study, the synthesis of -alkyl-2-halophenazin-1-ones has been established. Six -alkyl-2-halophenazin-1-ones, including WS-9659 B and marinocyanins A and B, were synthesized by the direct oxidative condensation of 4-halo-1,2,3-benzenetriol with the corresponding -alkylbenzene-1,2-diamines. One of the most significant features of the present method is that it can be successfully applied to the synthesis of -alkyl-2-chlorophenazin-1-ones. The traditional chlorination of -alkyl-phenazin-1-ones with -chlorosuccinimide selectively occurs at the 4-position to afford the undesired -alkyl-4-chlorophenazin-1-ones. Our synthetic route successfully circumvents this problem, culminating in the first chemical synthesis of WS-9659 B. The cytotoxicity of six -alkyl-2-halophenazin-1-ones and three -alkylphenazin-1-ones against human promyelocytic leukemia HL-60, human lung cancer A549, and normal MRC-5 cells was evaluated. Among the compounds tested in this study, 2-chloropyocyanin possesses significant selectivity toward A549 cells. The cytotoxic evaluation provides structural insights into the potency and selectivity of these compounds for cancer cells.
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http://dx.doi.org/10.1021/acsomega.0c04253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594318PMC
October 2020

Investigation on the Epoxidation of Piperitenone, and Structure-activity Relationships of Piperitenone Oxide for Differentiation-inducing Activity.

J Oleo Sci 2020 Aug 9;69(8):951-958. Epub 2020 Jul 9.

Department of Applied Biological Science, Tokyo University of Science.

Piperitenone oxide, a major chemical constituent of the essential oil of spearmint, Mentha spicata, induces differentiation in human colon cancer RCM-1 cells. In this study, piperitenone oxide and trans-piperitenone dioxide were prepared as racemic forms by epoxidation of piperitenone. The relative configuration between two epoxides in piperitenone dioxide was determined to be trans by H NMR analysis and nuclear Overhauser effect spectroscopy (NOESY) in conjunction with density functional theory (DFT) calculations. Optical resolution of (±)-piperitenone oxide by high-performance liquid chromatography (HPLC) using a chiral stationary phase (CSP) afforded both enantiomers with over 98% enantiomeric excess (ee). Evaluation of the differentiation-inducing activity of the synthetic compounds revealed that the epoxide at C-1 and C-6 in piperitenone oxide is important for the activity, and (+)-piperitenone oxide has stronger activity than (-)-piperitenone oxide. The results obtained in this study provide new information on the application of piperitenone oxide and spearmint for differentiation-inducing therapy. Furthermore, natural piperitenone oxide was isolated from M. spicata. The enantiomeric excess of the isolated natural piperitenone oxide was 66% ee. Epoxidation of piperitenone with hydrogen peroxide proceeded in a phosphate buffer under weak basic conditions to give (±)-piperitenone oxide. These results suggest that the nonenzymatic epoxidation of piperitenone, which causes a decrease in the enantiomeric excess of natural piperitenone oxide, is accompanied by an enzymatic epoxidation in the biosynthesis of piperitenone oxide.
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http://dx.doi.org/10.5650/jos.ess19278DOI Listing
August 2020

PROTACs and Other Chemical Protein Degradation Technologies for the Treatment of Neurodegenerative Disorders.

Angew Chem Int Ed Engl 2021 02 18;60(7):3346-3354. Epub 2020 Aug 18.

Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, 980-8577, Japan.

Neurodegenerative disorders (NDs) are a group of diseases that cause neural cell damage, leading to motility and/or cognitive dysfunctions. One of the causative agents is misfolded protein aggregates, which are considered as undruggable in terms of conventional tools, such as inhibitors and agonists/antagonists. Indeed, there is currently no FDA-approved drug for the causal treatment of NDs. However, emerging technologies for chemical protein degradation are opening up the possibility of selective elimination of target proteins through physiological protein degradation machineries, which do not depend on the functions of the target proteins. Here, we review recent efforts towards the treatment of NDs using chemical protein degradation technologies, and we briefly discuss the challenges and prospects.
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http://dx.doi.org/10.1002/anie.202004746DOI Listing
February 2021

Selective Degradation of Target Proteins by Chimeric Small-Molecular Drugs, PROTACs and SNIPERs.

Pharmaceuticals (Basel) 2020 Apr 21;13(4). Epub 2020 Apr 21.

Division of Molecular Target and Gene Therapy Products, National Institute of Health Sciences, 3-25-26, Tonomachi, Kawasaki, Kanagawa 210-9501, Japan.

New therapeutic modalities are needed to address the problem of pathological but undruggable proteins. One possible approach is the induction of protein degradation by chimeric drugs composed of a ubiquitin ligase (E3) ligand coupled to a ligand for the target protein. This article reviews chimeric drugs that decrease the level of specific proteins such as proteolysis targeting chimeric molecules (PROTACs) and specific and nongenetic inhibitor of apoptosis protein (IAP)-dependent protein erasers (SNIPERs), which target proteins for proteasome-mediated degradation. We cover strategies for increasing the degradation activity induced by small molecules, and their scope for application to undruggable proteins.
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http://dx.doi.org/10.3390/ph13040074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7243126PMC
April 2020

Isolation, synthesis, and biological activities of a bibenzyl from var. .

Biosci Biotechnol Biochem 2020 Jan 6;84(1):31-36. Epub 2019 Sep 6.

Department of Applied Biological Science, Tokyo University of Science, Chiba, Japan.

4-(2-Hydroxyphenethyl)-2,6-dimethoxyphenol, a bibenzyl, was isolated from the leaves of var. , collected from Mount Tateyama. Japanese rock ptarmigans frequently eat the leaves and fruits of this plant. The structure of the bibenzyl was confirmed by NMR spectroscopic analysis and fully characterized. A synthesis of this compound was accomplished by coupling 2-hydroxyphenylacetic acid with syringaldehyde, decarboxylation of the resultant isoaurones, and hydrogenation of the double bond in the corresponding stilbene. This compound displayed cytotoxic activity against human cancer cells (HCT116 and Hela cells) and leukemia cells (HL-60 cells). The present study suggests that this plant serves as a source of biologically active natural products. Also, our findings provide information on the secondary metabolites in the diet of Japanese rock ptarmigans.
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http://dx.doi.org/10.1080/09168451.2019.1662279DOI Listing
January 2020

Application of protein knockdown strategy targeting β-sheet structure to multiple disease-associated polyglutamine proteins.

Bioorg Med Chem 2020 01 31;28(1):115175. Epub 2019 Oct 31.

Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-032, Japan; Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan. Electronic address:

Polyglutamine diseases are a class of neurodegenerative diseases associated with the accumulation of aggregated mutant proteins. We previously developed a class of degradation-inducing agents targeting the β-sheet-rich structure typical of such aggregates, and we showed that these agents dose-, time-, and proteasome-dependently decrease the intracellular level of mutant huntingtin with an extended polyglutamine tract, which correlates well with the severity of Huntington's disease. Here, we demonstrate that the same agents also deplete other polyglutamine disease-related proteins: mutant ataxin-3 and ataxin-7 in cells from spino-cerebellar ataxia patients, and mutant atrophin-1 in cells from dentatorubral-pallidoluysian atrophy patients. Targeting cross-β-sheet structure could be an effective design strategy to develop therapeutic agents for multiple neurodegenerative diseases.
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http://dx.doi.org/10.1016/j.bmc.2019.115175DOI Listing
January 2020

Synthetic and Biological Studies of Juglorubin and Related Naphthoquinones.

J Org Chem 2019 11 23;84(21):13957-13966. Epub 2019 Oct 23.

Department of Applied Biological Science, Faculty of Science and Technology , Tokyo University of Science , 2641 Yamazaki , Noda, Chiba 278-8510 , Japan.

Juglorubin, juglorescein, and juglocombins A/B are naturally occurring naphthoquinone dimers isolated from sp. These dimers are proposed to be biogenetically derived from juglomycin C, a monomeric naphthoquinone isolated from the same sp. In this study, the dimerization of a juglomycin C derivative, a key step in the total syntheses of these natural products, was investigated. Juglorubin was synthesized from the minor product of the dimerization via the formation of the juglocombin A/B stereoisomers. A mechanism for the dimerization reaction as well as a plausible biosynthetic pathway to obtain juglorubin from juglomycin C are proposed. Furthermore, the antibacterial and cytotoxic activities of five synthetic compounds were evaluated. Among the compounds tested in this study, 1'--methyljuglocombin B dimethyl ester and juglomycin C exhibited antibacterial activity against . 1'--Methyljuglocombin B dimethyl ester and juglomycin C showed cytotoxicity against human colon carcinoma HCT116 cells and human leukemia HL-60 cells. 1'--Methyljuglocombin B dimethyl ester exhibited cytotoxicity against human normal MRC-5 cells as strong as that against human cancer cells. In contrast, juglomycin C was less toxic against normal MRC-5 cells, indicating a significant selectivity toward cancer cells.
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http://dx.doi.org/10.1021/acs.joc.9b02119DOI Listing
November 2019

Synthesis of nucleotide analogues, EFdA, EdA and EdAP, and the effect of EdAP on hepatitis B virus replication.

Biosci Biotechnol Biochem 2020 Feb 7;84(2):217-227. Epub 2019 Oct 7.

Department of Applied Biological Science, Tokyo University of Science, Chiba, Japan.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) and 4'-ethynyl-2'-deoxyadenosine (EdA) are nucleoside analogues which inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. EdAP, a cyclosaligenyl (cycloSal) phosphate derivative of EdA, inhibits the replication of the influenza A virus. The common structural feature of these compounds is the ethynyl group at the 4'-position. In this study, these nucleoside analogues were prepared by a common synthetic strategy starting from the known 1,2-di--acetyl-D-ribofuranose. Biological evaluation of EdAP revealed that this compound reduced hepatitis B virus (HBV) replication dose-dependently without cytotoxicity against host cells tested in this study.
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http://dx.doi.org/10.1080/09168451.2019.1673696DOI Listing
February 2020

Total Syntheses of Pyocyanin, Lavanducyanin, and Marinocyanins A and B.

Org Lett 2019 09 28;21(18):7311-7314. Epub 2019 Aug 28.

Department of Applied Biological Science, Faculty of Science and Technology , Tokyo University of Science , 2641 Yamazaki , Noda , Chiba 278-8510 , Japan.

Total syntheses of pyocyanin, lavanducyanin, and marinocyanins A and B have been accomplished. The -substituted phenazin-1-one skeleton, a common framework of these natural products, was constructed through the oxidative condensation of pyrogallol with -substituted benzene-1,2-diamine under an oxygen atmosphere in a single step. Regioselective bromination with -bromosuccinimide at the C-2 position of -alkylated phenazin-1-ones afforded brominated natural products.
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http://dx.doi.org/10.1021/acs.orglett.9b02601DOI Listing
September 2019

Unified Approach toward Syntheses of Juglomycins and Their Derivatives.

ACS Omega 2019 Jul 5;4(7):11737-11748. Epub 2019 Jul 5.

Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.

A unified and common intermediate strategy for syntheses of juglomycins and their derivatives is reported. The use of a 1,4-dimethoxynaphthalene derivative as a key intermediate enabled easy access to various juglomycin derivatives. In this study, juglomycins A-D, juglomycin C amide, khatmiamycin and its 4-epimer, and the structure proposed for juglomycin Z were synthesized from this intermediate. The absolute configuration of natural khatmiamycin has been established to be 3,4 through our synthesis. Unfortunately, the spectroscopic data for synthetic juglomycin Z were not consistent with the data reported for the natural one, strongly suggesting a structural misassignment.
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http://dx.doi.org/10.1021/acsomega.9b01376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6682012PMC
July 2019

Synthesis, antibacterial and cytotoxic evaluation of flavipucine and its derivatives.

Bioorg Med Chem Lett 2019 06 26;29(11):1390-1394. Epub 2019 Mar 26.

Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan. Electronic address:

The antibacterial and cytotoxic activity of seven racemic lactams and both enantiomers of flavipucine were evaluated. Of the compounds tested in this study, flavipucine and phenylflavipucine displayed bactericidal activity against Bacillus subtilis. These results indicate that the pyridione epoxide moiety is a pharmacophore for antibacterial activity against B. subtilis. Flavipucine showed cytotoxic activity against several cancer cells. The cytotoxic activity of flavipucine against human leukemia HL-60 cells is as strong as that of SN-38, the active metabolite of irinotecan. In contrast, the cytotoxic activity of flavipucine against nonneoplastic HEK293 cells and human normal MRC-5 cells is weaker than that of SN-38. No significant differences in the biological activity of the racemates or enantiomers of flavipucine were observed.
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http://dx.doi.org/10.1016/j.bmcl.2019.03.034DOI Listing
June 2019

Slt, MltD, and MltG of Pseudomonas aeruginosa as Targets of Bulgecin A in Potentiation of β-Lactam Antibiotics.

ACS Chem Biol 2019 02 18;14(2):296-303. Epub 2019 Jan 18.

Department of Chemistry and Biochemistry , University of Notre Dame , 352 McCourtney Hall , Notre Dame , Indiana 46556 , United States.

The interplay between the activities of lytic transglycosylases (LTs) and penicillin-binding proteins (PBPs) is critical for the health of the bacterial cell wall. Bulgecin A (a natural-product inhibitor of LTs) potentiates the activity of β-lactam antibiotics (inhibitors of PBPs), underscoring this intimate mechanistic interdependence. Bulgecin A in the presence of an appropriate β-lactam causes bulge deformation due to the formation of aberrant peptidoglycan at the division site of the bacterium. As Pseudomonas aeruginosa, a nefarious human pathogen, has 11 LT paralogs, the answer as to which LT activity correlates with β-lactam potentiation is important and is currently unknown. Growth of P. aeruginosa PAO1 strains harboring individual transposon-insertion mutants at each of the 11 genes for LTs, in the presence of the β-lactam antibiotic ceftazidime or meropenem, implicated the gene products of slt, mltD, and mltG (of the 11), in bulge formation and potentiation. Hence, the respective enzymes would be the targets of inhibition by bulgecin A, which was indeed documented. We further demonstrated by imaging in real time and by SEM that cell lysis occurs by the structural failure of this bulge. Upon removal of the β-lactam antibiotic prior to lysis, P. aeruginosa experiences delayed recovery from the elongation and bulge phenotype in the presence of bulgecin A. These observations argue for a collaborative role for the target LTs in the repair of the aberrant cell wall, the absence of activities of which in the presence of bulgecin A results in potentiation of the β-lactam antibiotic.
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http://dx.doi.org/10.1021/acschembio.8b01025DOI Listing
February 2019

Total Syntheses of Bulgecins A, B, and C and Their Bactericidal Potentiation of the β-Lactam Antibiotics.

ACS Infect Dis 2018 06 7;4(6):860-867. Epub 2018 May 7.

Department of Chemistry & Biochemistry , University of Notre Dame , 352 McCourtney Hall , Notre Dame , Indiana 46556 , United States.

The bulgecins are iminosaccharide secondary metabolites of the Gram-negative bacterium Paraburkholderia acidophila and inhibitors of lytic transglycosylases of bacterial cell-wall biosynthesis and remodeling. The activities of the bulgecins are intimately intertwined with the mechanism of a cobiosynthesized β-lactam antibiotic. β-Lactams inhibit the penicillin-binding proteins, enzymes also critical to cell-wall biosynthesis. The simultaneous loss of the lytic transglycosylase (by bulgecin) and penicillin-binding protein (by β-lactams) activities results in deformation of the septal cell wall, observed microscopically as a bulge preceding bacterial cell lysis. We describe a practical synthesis of the three naturally occurring bulgecin iminosaccharides and their mechanistic evaluation in a series of microbiological studies. These studies identify potentiation by the bulgecin at subminimum inhibitory concentrations of the β-lactam against three pathogenic Gram-negative bacteria and establish for the first time that this potentiation results in a significant increase in the bactericidal efficacy of a clinical β-lactam.
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http://dx.doi.org/10.1021/acsinfecdis.8b00105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5996343PMC
June 2018

Exolytic and endolytic turnover of peptidoglycan by lytic transglycosylase Slt of .

Proc Natl Acad Sci U S A 2018 04 9;115(17):4393-4398. Epub 2018 Apr 9.

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556;

β-Lactam antibiotics inhibit cell-wall transpeptidases, preventing the peptidoglycan, the major constituent of the bacterial cell wall, from cross-linking. This causes accumulation of long non-cross-linked strands of peptidoglycan, which leads to bacterial death. , a nefarious bacterial pathogen, attempts to repair this aberrantly formed peptidoglycan by the function of the lytic transglycosylase Slt. We document in this report that Slt turns over the peptidoglycan by both exolytic and endolytic reactions, which cause glycosidic bond scission from a terminus or in the middle of the peptidoglycan, respectively. These reactions were characterized with complex synthetic peptidoglycan fragments that ranged in size from tetrasaccharides to octasaccharides. The X-ray structure of the wild-type apo Slt revealed it to be a doughnut-shaped protein. In a series of six additional X-ray crystal structures, we provide insights with authentic substrates into how Slt is enabled for catalysis for both the endolytic and exolytic reactions. The substrate for the exolytic reaction binds Slt in a canonical arrangement and reveals how both the glycan chain and the peptide stems are recognized by the Slt. We document that the apo enzyme does not have a fully formed active site for the endolytic reaction. However, binding of the peptidoglycan at the existing subsites within the catalytic domain causes a conformational change in the protein that assembles the surface for binding of a more expansive peptidoglycan between the catalytic domain and an adjacent domain. The complexes of Slt with synthetic peptidoglycan substrates provide an unprecedented snapshot of the endolytic reaction.
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http://dx.doi.org/10.1073/pnas.1801298115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5924928PMC
April 2018

Degradation of huntingtin mediated by a hybrid molecule composed of IAP antagonist linked to phenyldiazenyl benzothiazole derivative.

Bioorg Med Chem Lett 2018 02 12;28(4):707-710. Epub 2018 Jan 12.

Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Electronic address:

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by aggregation of mutant huntingtin (mHtt), and removal of mHtt is expected as a potential therapeutic option. We previously reported protein knockdown of Htt by using hybrid small molecules (Htt degraders) consisting of BE04, a ligand of ubiquitin ligase (E3), linked to probes for protein aggregates. Here, in order to examine the effect of changing the ligand, we synthesized a similar Htt degrader utilizing MV1, an antagonist of the inhibitor of apoptosis protein (IAP) family (a subgroup of ubiquitin E3 ligases), which is expected to have a higher affinity and specificity for IAP, as compared with BE04. The MV1-based hybrid successfully induced interaction between Htt aggregates and IAP, and reduced mHtt levels in living cells. Its mode of action was confirmed to be the same as that of the BE04-based hybrid. However, although the affinity of MV1 for IAP is greater than that of BE04, the efficacy of Htt degradation by the MV1-based molecule was lower, suggesting that linker length between the ligand and probe might be an important determinant of efficacy.
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http://dx.doi.org/10.1016/j.bmcl.2018.01.012DOI Listing
February 2018

Discovery of Small Molecules that Induce the Degradation of Huntingtin.

Angew Chem Int Ed Engl 2017 09 9;56(38):11530-11533. Epub 2017 Aug 9.

Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the aggregation of mutant huntingtin (mHtt), and removal of toxic mHtt is expected to be an effective therapeutic approach. We designed two small hybrid molecules (1 and 2) by linking a ligand for ubiquitin ligase (cellular inhibitor of apoptosis protein 1; cIAP1) with probes for mHtt aggregates, anticipating that these compounds would recruit cIAP1 to mHtt and induce selective degradation by the ubiquitin-proteasome system. The synthesized compounds reduced mHtt levels in HD patient fibroblasts and appear to be promising candidates for the development of a treatment for HD.
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http://dx.doi.org/10.1002/anie.201706529DOI Listing
September 2017

Improvement in Aqueous Solubility of Retinoic Acid Receptor (RAR) Agonists by Bending the Molecular Structure.

Chem Asian J 2016 Aug 20;11(15):2210-7. Epub 2016 Jul 20.

Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.

Aqueous solubility is a key requirement for many functional molecules, e. g., drug candidates. Decrease of the partition coefficient (log P) by chemical modification, i.e., introduction of hydrophilic group(s) into molecules, is a classical strategy for improving aqueous solubility. We have been investigating alternative strategies for improving the aqueous solubility of pharmaceutical compounds by disrupting intermolecular interactions. Here, we show that introducing a bend into the molecular structure of retinoic acid receptor (RAR) agonists by changing the substitution pattern from para to meta or ortho dramatically enhances aqueous solubility by up to 890-fold. We found that meta analogs exhibit similar hydrophobicity to the parent para compound, and have lower melting points, supporting the idea that the increase of aqueous solubility was due to decreased intermolecular interactions in the solid state as a result of the structural changes.
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http://dx.doi.org/10.1002/asia.201600744DOI Listing
August 2016

Efficient protein knockdown of HaloTag-fused proteins using hybrid molecules consisting of IAP antagonist and HaloTag ligand.

Bioorg Med Chem 2016 07 20;24(14):3144-8. Epub 2016 May 20.

Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Electronic address:

We previously reported a protein knockdown system for HaloTag-fused proteins using hybrid small molecules consisting of alkyl chloride, which binds covalently to HaloTag, linked to BE04 (2), a bestatin (3) derivative with an affinity for cellular inhibitor of apoptosis protein 1 (cIAP1, a kind of ubiquitin ligase). This system addressed several limitations of prior protein knockdown technology, and was applied to degrade two HaloTag-fused proteins. However, the degradation activity of these hybrid small molecules was not potent. Therefore, we set out to improve this system. We report here the design, synthesis and biological evaluation of novel hybrid compounds 4a and 4b consisting of alkyl chloride linked to IAP antagonist MV1 (5). Compounds 4a and 4b were confirmed to reduce the levels of HaloTag-fused tumor necrosis factor α (HaloTag-TNFα), HaloTag-fused cell division control protein 42 (HaloTag-Cdc42), and unfused HaloTag protein in living cells more potently than did BE04-linked compound 1b. Analysis of the mode of action revealed that the reduction of HaloTag-TNFα is proteasome-dependent, and is also dependent on the linker structure between MV1 (5) and alkyl chloride. These compounds appear to induce ubiquitination at the HaloTag moiety of HaloTag-fused proteins. Our results indicate that these newly synthesized MV1-type hybrid compounds, 4a and 4b, are efficient tools for protein knockdown for HaloTag-fused proteins.
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http://dx.doi.org/10.1016/j.bmc.2016.05.035DOI Listing
July 2016

Degradation of HaloTag-fused nuclear proteins using bestatin-HaloTag ligand hybrid molecules.

Org Biomol Chem 2015 Oct;13(38):9746-50

Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

We have developed a protein knockdown technology using hybrid small molecules designed as conjugates of a ligand for the target protein and a ligand for ubiquitin ligase cellular inhibitor of apoptosis protein 1 (cIAP1). However, this technology has several limitations. Here, we report the development of a novel protein knockdown system to address these limitations. In this system, target proteins are fused with HaloTag to provide a common binding site for a degradation inducer. We designed and synthesized small molecules consisting of alkyl chloride as the HaloTag-binding degradation inducer, which binds to HaloTag, linked to BE04 (2), which binds to cIAP1. Using this system, we successfully knocked down HaloTag-fused cAMP responsive element binding protein 1 (HaloTag-CREB1) and HaloTag-fused c-jun (HaloTag-c-jun), which are ligand-unknown nuclear proteins, in living cells. HaloTag-binding degradation inducers can be synthesized easily, and are expected to be useful as biological tools for pan-degradation of HaloTag-fused proteins.
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http://dx.doi.org/10.1039/c5ob01395jDOI Listing
October 2015

Structure-activity relationships of bisphenol A analogs at estrogen receptors (ERs): discovery of an ERα-selective antagonist.

Bioorg Med Chem Lett 2013 Jul 30;23(14):4031-6. Epub 2013 May 30.

Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Tokyo 113-0032, Japan.

Our multi-template approach for drug discovery, focusing on protein targets with similar fold structures, has yielded lead compounds for various targets. We have also shown that a diphenylmethane skeleton can serve as a surrogate for a steroid skeleton. Here, on the basis of those ideas, we hypothesized that the diphenylmethane derivative bisphenol A (BPA) would bind to the ligand-binding domain of estrogen receptors (ERs) in a similar manner to estradiol and act as a steroid surrogate. To test this idea, we synthesized a series of BPA analogs and evaluated their structure-activity relationships, focusing on agonistic/antagonistic activities at ERs and ERα/ERβ subtype selectivity. Among the compounds examined, 18 was found to be a potent ERα-antagonist with high selectivity over ERβ and androgen receptor under our assay conditions. A computational docking study suggested that 18 would bind to the antagonistic conformation of ERα. ERα-selective antagonists, such as 18, are candidate agents for treatment of breast cancer.
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http://dx.doi.org/10.1016/j.bmcl.2013.05.067DOI Listing
July 2013
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