Publications by authors named "Nobutoshi Ito"

55 Publications

Molecular interactions of the CTLA-4 cytoplasmic region with the phosphoinositide 3-kinase SH2 domains.

Mol Immunol 2021 03 30;131:51-59. Epub 2020 Dec 30.

Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan. Electronic address:

During T-cell regulation, T-cell receptors and CD28 lead to signaling activation, while T-lymphocyte antigen 4 (CTLA-4) is known to lead to downregulation, similar to programmed cell death-1 (PD-1). In the cytoplasmic tails of CD28 and CTLA-4, phosphoinositide 3-kinase (PI3K) binds to the consensus sequence including phosphotyrosine via SH2 domains, N- and C-terminal SH2 domains (nSH2 and cSH2), of its regulatory subunit, p85. In this study, we determined the crystal structure of a CTLA-4-derived phosphopeptide in complex with a Cys-substituted mutant of cSH2, C656S/C659V/C670L, at a 1.1 Å resolution. Phosphotyrosine of the bound peptide is tightly accommodated by the residues Arg631, Arg649, Ser651, and Ser652, similar to the cSH2 wild-type recognition mode of CD28, as reported previously. Upon the Cys mutation, the cSH2 thermal stability increased while the CTLA-4 binding affinity slightly changed. The binding experiments also showed that the binding affinity of CTLA-4 by cSH2 was approximately two orders of magnitude lower than that of CD28. Similar to CD28 binding, the CTLA-4 binding affinity of nSH2 was lower than that of cSH2. The complex structure of nSH2 and CTLA-4 was modeled, and compared with the crystal structure of cSH2 mutant and CTLA-4. The difference in the binding affinity between CD28 and CTLA-4, along with the difference between nSH2 and cSH2, could be explained by the 3D structures, which would be closely correlated with the respective T-cell signaling.
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http://dx.doi.org/10.1016/j.molimm.2020.12.002DOI Listing
March 2021

Lithocholic Acid Derivatives as Potent Vitamin D Receptor Agonists.

J Med Chem 2021 01 28;64(1):516-526. Epub 2020 Dec 28.

Department of Chemistry, Faculty of Science, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Bunkyo, Tokyo 112-8610, Japan.

Lithocholic acid () was identified as a second endogenous ligand of vitamin D receptor (VDR), though its activity is very weak. In this study, we designed novel lithocholic acid derivatives based on the crystal structure of VDR-ligand-binding domain (LBD) bound to . Among the synthesized compounds, bearing a 2-hydroxy-2-methylprop-1-yl group instead of the 3-hydroxy group at the 3α-position of showed dramatically increased activity in HL-60 cell differentiation assay, being at least 10 000 times more potent than lithocholic acid () and 3 times more potent than 1α,25-dihydroxyvitamin D (). Although the binding affinities of and its epimer were less than that of , their transactivation activities were greater than that of . X-ray structure analyses of VDR LBD bound to or showed that the binding positions of these compounds in the ligand-binding pocket are similar to that of .
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http://dx.doi.org/10.1021/acs.jmedchem.0c01420DOI Listing
January 2021

Structural basis of mutants of PET-degrading enzyme from Saccharomonospora viridis AHK190 with high activity and thermal stability.

Proteins 2021 May 24;89(5):502-511. Epub 2020 Dec 24.

Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan.

The cutinase-like enzyme from the thermophile Saccharomonospora viridis AHK190, Cut190, is a good candidate to depolymerize polyethylene terephthalate (PET) efficiently. We previously developed a mutant of Cut190 (S226P/R228S), which we designated as Cut190* that has both increased activity and stability and solved its crystal structure. Recently, we showed that mutation of D250C/E296C on one of the Ca -binding sites resulted in a higher thermal stability while retaining its polyesterase activity. In this study, we solved the crystal structures of Cut190* mutants, Q138A/D250C-E296C/Q123H/N202H, designated as Cut190*SS, and its inactive S176A mutant, Cut190*SS_S176A, at high resolution. The overall structures were similar to those of Cut190* and Cut190*S176A reported previously. As expected, Cys250 and Cys296 were closely located to form a disulfide bond, which would assuredly contribute to increase the stability. Isothermal titration calorimetry experiments and 3D Reference Interaction Site Model calculations showed that the metal-binding properties of the Cut190*SS series were different from those of the Cut190* series. However, our results show that binding of Ca to the weak binding site, site 1, would be retained, enabling Cut190*SS to keep its ability to use Ca to accelerate the conformational change from the closed (inactive) to the open (active) form. While increasing the thermal stability, Cut190*SS could still express its enzymatic function. Even after incubation at 70°C, which corresponds to the glass transition temperature of PET, the enzyme retained its activity well, implying a high applicability for industrial PET depolymerization using Cut190*SS.
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http://dx.doi.org/10.1002/prot.26034DOI Listing
May 2021

A Guillain-Barré syndrome-associated SIGLEC10 rare variant impairs its recognition of gangliosides.

J Autoimmun 2021 Jan 19;116:102571. Epub 2020 Nov 19.

Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan. Electronic address:

Guillain-Barré syndrome (GBS), including its variant Miller Fisher syndrome (MFS), is an acute peripheral neuropathy that involves autoimmune mechanisms leading to the production of autoantibodies to gangliosides; sialic acid-containing glycosphingolipids. Although association with various genetic polymorphisms in the major histocompatibility complex (MHC) is shown in other autoimmune diseases, GBS is an exception, showing no such link. No significant association was found by genome wide association studies, suggesting that GBS is not associated with common variants. To address the involvement of rare variants in GBS, we analyzed Siglec-10, a sialic acid-recognizing inhibitory receptor expressed on B cells. Here we demonstrate that two rare variants encoding R47Q and A108V substitutions in the ligand-binding domain are significantly accumulated in patients with GBS. Because of strong linkage disequilibrium, there was no patient carrying only one of them. Recombinant Siglec-10 protein containing R47Q but not A108V shows impaired binding to gangliosides. Homology modeling revealed that the R47Q substitution causes marked alteration in the ligand-binding site. Thus, GBS is associated with a rare variant of the SIGLEC10 gene that impairs ligand binding of Siglec-10. Because Siglec-10 regulates antibody production to sialylated antigens, our finding suggests that Siglec-10 regulates development of GBS by suppressing antibody production to gangliosides, with defects in its function predisposing to disease.
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http://dx.doi.org/10.1016/j.jaut.2020.102571DOI Listing
January 2021

Multiple structural states of Ca2+-regulated PET hydrolase, Cut190, and its correlation with activity and stability.

J Biochem 2021 Mar;169(2):207-213

Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto, Kyoto 606-8522, Japan.

An enzyme, Cut190, from a thermophilic isolate, Saccharomonospora viridis AHK190 could depolymerize polyethylene terephthalate (PET). The catalytic activity and stability of Cut190 and its S226P/R228S mutant, Cut190*, are regulated by Ca2+ binding. We previously determined the crystal structures of the inactive mutant of Cut190*, Cut190*S176A, in complex with metal ions, Ca2+ and Zn2+, and substrates, monoethyl succinate and monoethyl adipate. In this study, we determined the crystal structures of another mutant of Cut190*, Cut190**, in which the three C-terminal residues of Cut190* are deleted, and the inactive mutant, Cut190**S176A, in complex with metal ions. In addition to the previously observed closed, open and engaged forms, we determined the ejecting form, which would allow the product to irreversibly dissociate, followed by proceeding to the next cycle of reaction. These multiple forms would be stable or sub-stable states of Cut190, regulated by Ca2+ binding, and would be closely correlated with the enzyme function. Upon the deletion of the C-terminal residues, we found that the thermal stability increased while retaining the activity. The increased stability could be applied for the protein engineering of Cut190 for PET depolymerization as it requires the reaction above the glass transition temperature of PET.
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http://dx.doi.org/10.1093/jb/mvaa102DOI Listing
March 2021

Mutational effects of Cys113 on structural dynamics of Pin1.

Biophys Physicobiol 2019 29;16:452-465. Epub 2019 Nov 29.

Department of Structural Biology, Medical Research Institute, Tokyo Medical and Dental University (TMDU), Bunkyo-ku, Tokyo 113-8510, Japan.

Pin1 is a peptidyl-prolyl isomerase (PPIase) which catalyzes isomerization of pS/pT-P bond. Its activity is related to various cellular functions including suppression of Alzheimer's disease. A cysteine residue C113 is known to be important for its PPIase activity; a mutation C113A reduced the activity by 130-fold. According to various nuclear magnetic resonance experiments for mutants of C113 and molecular dynamics (MD) simulation of wild-type Pin1, the protonation sate of Sγ of C113 regulates the hydrogen-bonding network of the dual-histidine motif (H59, H157) whose dynamics may affect substrate binding ability. However, it was still unclear why such local dynamic changes altered the PPIase activity of Pin1. In this study, we performed 500 ns of MD simulations of full-length wild-type Pin1 and C113A mutant in order to elucidate why the mutation C113A drastically reduced the PPIase activity of Pin1. The principal component analysis for both MD trajectories clearly elucidated that the mutation C113A suppressed the dynamics of Pin1 because it stabilized a hydrogen-bond between Nɛ of H59 and Oγ of S115. In the dynamics of wild-type protein, the phosphate binding loop (K63-S71) as well as the interdomain hinge showed the closed-open dynamics which correlated with the change of the hydrogen-bonding network of the dual-histidine motif. In contrast, in the dynamics of C113A mutant, the phosphate binding loop took only the closed conformation together with the interdomain hinge. Such closed-open dynamics must be essential for the PPIase activity of Pin1.
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http://dx.doi.org/10.2142/biophysico.16.0_452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6976032PMC
November 2019

Three-dimensional structure of a high affinity anti-(4-hydroxy-3-nitrophenyl)acetyl antibody possessing a glycine residue at position 95 of the heavy chain.

Mol Immunol 2019 10 11;114:545-552. Epub 2019 Sep 11.

Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto, 606-8522, Japan. Electronic address:

Antibodies possessing high affinity and specificity are desired as therapeutic reagents and biosensor materials. Such antibodies are often obtained from immunized animals through the process referred to as affinity maturation where antibody affinity increases with time after immunization. Somatic hypermutation (SHM) was shown to be involved in this process; however, structural basis of affinity maturation has not well been understood yet. We analyzed the crystal structure of a high affinity anti-(4-hydroxy-3-nitrophenyl)acetyl antibody, C6, possessing Gly at position 95 of heavy chain and 17 amino acid replacements by SHM. Here, we discuss how the amino acid residues at position 95, introduced at a junction of V and D gene segments during gene-recombination, as well as those replaced by SHM contribute to increasing the affinity by comparing the C6 structure with that of a germline low affinity antibody, N1G9, possessing Tyr at position 95.
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http://dx.doi.org/10.1016/j.molimm.2019.09.001DOI Listing
October 2019

Development of novel lithocholic acid derivatives as vitamin D receptor agonists.

Bioorg Med Chem 2019 08 3;27(16):3674-3681. Epub 2019 Jul 3.

Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. Electronic address:

Lithocholic acid (2) was identified as the second endogenous ligand of vitamin D receptor (VDR), though its binding affinity to VDR and its vitamin D activity are very weak compared to those of the active metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1). 3-Acylated lithocholic acids were reported to be slightly more potent than lithocholic acid (2) as VDR agonists. Here, aiming to develop more potent lithocholic acid derivatives, we synthesized several derivatives bearing a 3-sulfonate/carbonate or 3-amino/amide substituent, and examined their differentiation-inducing activity toward human promyelocytic leukemia HL-60 cells. Introduction of a nitrogen atom at the 3-position of lithocholic acid (2) decreased the activity, but compound 6 bearing a 3-methylsulfonate group showed more potent activity than lithocholic acid (2) or its acylated derivatives. The binding of 6 to VDR was confirmed by competitive binding assay and X-ray crystallographic analysis of the complex of VDR ligand-binding domain (LBD) with 6.
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http://dx.doi.org/10.1016/j.bmc.2019.07.003DOI Listing
August 2019

Tau aggregation and seeding analyses of two novel MAPT variants found in patients with motor neuron disease and progressive parkinsonism.

Neurobiol Aging 2019 12 1;84:240.e13-240.e22. Epub 2019 Mar 1.

Department of Neurology, Juntendo University, School of Medicine, Tokyo, Japan; Department of Diagnosis, Prevention and Treatment of Dementia, Juntendo University, Graduate School of Medicine, Tokyo, Japan; Research Institute for Diseases of Old Age, Juntendo University, Graduate School of Medicine, Tokyo, Japan. Electronic address:

Variants in the microtubule-associated protein tau (MAPT) gene cause the genetic tauopathies, a subgroup of frontotemporal dementia (FTD) disorders. Through genetic screening of 165 cases possibly associated with tauopathies, including 88 Alzheimer's disease, 26 behavioral variant FTD, eight primary progressive aphasia, nine FTD with motor neuron disease, 21 progressive supranuclear palsy, and 13 corticobasal syndrome, we identified two novel MAPT variants: a heterozygous missense variant, p.P160S, in a patient with FTD with motor neuron disease and a heterozygous insertional variant, p.K298_H299insQ, in three patients with familial progressive supranuclear palsy. The corresponding recombinant tau proteins showed reduced microtubule assembly and increased aggregation by thioflavin S assay. Exon trapping analysis showed that p.K298_H299insQ resulted in the overproduction of 4-repeat tau. In a cell-based model, p.K298_H299insQ had both a higher aggregation ability and seeding activity compared with wild-type tau. These findings indicate that both p.P160S and p.K298_H299insQ may relate to neurodegeneration.
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http://dx.doi.org/10.1016/j.neurobiolaging.2019.02.016DOI Listing
December 2019

Structural and functional properties of Grb2 SH2 dimer in CD28 binding.

Biophys Physicobiol 2019 22;16:80-88. Epub 2019 Feb 22.

Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto 606-8522, Japan.

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein that plays a critical role in cellular signal transduction. It contains a central Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains. Binding of Grb2 SH2 to the cytoplasmic region of CD28, phosphorylated Tyr (pY) containing the peptide motif pY-X-N-X, is required for costimulatory signaling in T cells. In this study, we purified the dimer and monomer forms of Grb2 SH2, respectively, and analyzed their structural and functional properties. Size exclusion chromatography analysis showed that both dimer and monomer exist as stable states. Thermal stability analysis using circular dichroism showed that the dimer mostly dissociates into the monomer around 50°C. CD28 binding experiments showed that the affinity of the dimer to the phosphopeptide was about three fold higher than that of the monomer, possibly due to the avidity effect. The present crystal structure analysis of Grb2 SH2 showed two forms; one is monomer at 1.15 Å resolution, which is currently the highest resolution analysis, and another is dimer at 2.00 Å resolution. In the dimer structure, the C-terminal region, comprising residues 123-152, was extended towards the adjacent molecule, in which Trp121 was the hinge residue. The stable dimer purified using size exclusion chromatography would be due to the C-terminal helix "swapping". In cases where a mutation caused Trp121 to be replaced by Ser in Grb2 SH2, this protein still formed dimers, but lost the ability to bind CD28.
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http://dx.doi.org/10.2142/biophysico.16.0_80DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435016PMC
February 2019

Dual conformation of the ligand induces the partial agonistic activity of retinoid X receptor α (RXRα).

FEBS Lett 2019 01 21;593(2):242-250. Epub 2018 Dec 21.

Department of Chemistry, Rikkyo University, Tokyo, Japan.

1-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)amino]benzotriazole-5-carboxylic acid (CBt-PMN), a partial agonist of retinoid X receptor (RXR), has attracted attention due to its potential to treat type 2 diabetes and central nervous system diseases with reduced adverse effects of existing full agonists. Herein, we report the crystal structure of CBt-PMN-bound ligand-binding domain of human RXRα (hRXRα) and its biochemical characterization. Interestingly, the structure is a tetramer in nature, in which CBt-PMNs are clearly found binding in two different conformations. The dynamics of the hRXRα/CBt-PMN complex examined using molecular dynamics simulations suggest that the flexibility of the AF-2 interface depends on the conformation of the ligand. These facts reveal that the dual conformation of CBt-PMN in the complex is probably the reason behind its partial agonistic activity.
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http://dx.doi.org/10.1002/1873-3468.13301DOI Listing
January 2019

Structural Dynamics of the PET-Degrading Cutinase-like Enzyme from Saccharomonospora viridis AHK190 in Substrate-Bound States Elucidates the Ca-Driven Catalytic Cycle.

Biochemistry 2018 09 27;57(36):5289-5300. Epub 2018 Aug 27.

Graduate School of Life and Environmental Sciences , Kyoto Prefectural University , 1-5 Hangi-cho, Shimogamo , Sakyo-ku, Kyoto , Kyoto 606-8522 , Japan.

A cutinase-type polyesterase from Saccharomonospora viridis AHK190 (Cut190) has been shown to degrade the inner block of polyethylene terephthalate. A unique feature of Cut190 is that its function and stability are regulated by Ca binding. Our previous crystal structure analysis of Cut190S226P showed that one Ca binds to the enzyme, which induces large conformational changes in several loop regions to stabilize an open conformation [Miyakawa, T., et al. (2015) Appl. Microbiol. Biotechnol. 99, 4297]. In this study, to analyze the substrate recognition mechanism of Cut190, we determined the crystal structure of the inactive form of a Cut190 mutant, Cut190*S176A, in complex with calcium ions and/or substrates. We found that three calcium ions bind to Cut190*S176A, which is supported by analysis using native mass spectrometry experiments and 3D Reference Interaction Site Model calculations. The complex structures with the two substrates, monoethyl succinate and monoethyl adipate (engaged and open forms), presumably correspond to the pre- and post-reaction states, as the ester bond is close to the active site and pointing outward from the active site, respectively, for the two complexes. Ca binding induces the pocket to open, enabling the substrate to access the pocket more easily. Molecular dynamics simulations suggest that a post-reaction state in the engaged form presumably exists between the experimentally observed forms, indicating that the substrate would be cleaved in the engaged form and then requires the enzyme to change to the open form to release the product, a process that Ca can greatly accelerate.
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http://dx.doi.org/10.1021/acs.biochem.8b00624DOI Listing
September 2018

The trans isomer of Tau peptide is prone to aggregate, and the WW domain of Pin1 drastically decreases its aggregation.

FEBS Lett 2018 09 13;592(18):3082-3091. Epub 2018 Aug 13.

Medical Research Institute, Tokyo Medical and Dental University, Japan.

In Alzheimer's, the disease-related protein Tau is hyperphosphorylated and aggregates into neurofibrillary tangles (NFT). The cis isomer of the phosphorylated Thr231-Pro232 has been proposed as a precursor of aggregation ('Cistauosis'), but this aggregation scheme is not yet completely accepted. Here, we synthesized peptides comprising a phosphorylated region including Thr231-Pro232 and an aggregation-core region R1 to investigate isomer-specific-aggregation of Tau. The phosphorylated peptide formed amyloid-like aggregation. This aggregation was observed even in the presence of the catalytic domain of the peptidyl-prolyl-isomerase Pin1, which preferentially converts the cis isomer to the trans isomer, but decreased drastically in the presence of the WW domain of Pin1 selectively binding to the trans isomer. These results indicate that the trans isomer is aggregation-prone and that the WW domain of Pin1 effectively inhibits its aggregation.
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http://dx.doi.org/10.1002/1873-3468.13218DOI Listing
September 2018

25 S-Adamantyl-23-yne-26,27-dinor-1α,25-dihydroxyvitamin D: Synthesis, Tissue Selective Biological Activities, and X-ray Crystal Structural Analysis of Its Vitamin D Receptor Complex.

J Med Chem 2018 Aug 23;61(15):6658-6673. Epub 2018 Jul 23.

Department of Biomedical Sciences , Nihon University School of Medicine , Itabashi-ku, Tokyo 173-8610 , Japan.

Both 25 R- and 25 S-25-adamantyl-23-yne-26,27-dinor-1α,25-dihydroxyvitamin D (4a and 4b) were stereoselectively synthesized by a Pd(0)-catalyzed ring closure and Suzuki-Miyaura coupling between enol-triflate 7 and alkenyl-boronic ester 8. The 25 S isomer (4b) showed high vitamin D receptor (VDR) affinity (50% of that of the natural hormone 1α,25-dihydroxyvitamin D, 1) and transactivation potency (kidney HEK293, 90%). In endogenous gene expression, it showed high cell-type selectivity for kidney cells (HEK293, CYP24A1 160% of 1), bone cells (MG63, osteocalcin 64%), and monocytes (U937, CAMP 96%) over intestine (SW480, CYP24A1 8%) and skin (HaCaT, CYP24A1 7%) cells. The X-ray crystal structural analysis of 4b in complex with rat VDR-ligand binding domain (LBD) showed the highest Cα positional shift from the 1/VDR-LBD complex at helix 11. Helix 11 of the 4b and 1 VDR-LBD complexes also showed significant differences in surface properties. These results suggest that 4b should be examined further as another candidate for a mild preventive osteoporosis agent.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00427DOI Listing
August 2018

CD22-Binding Synthetic Sialosides Regulate B Lymphocyte Proliferation Through CD22 Ligand-Dependent and Independent Pathways, and Enhance Antibody Production in Mice.

Front Immunol 2018 19;9:820. Epub 2018 Apr 19.

Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are expressed in various immune cells and most of them carry signaling functions. High-affinity synthetic sialoside ligands have been developed for various Siglecs. Therapeutic potentials of the nanoparticles and compounds that contain multiple numbers of these sialosides and other reagents such as toxins and antigens have been demonstrated. However, whether immune responses can be regulated by monomeric sialoside ligands has not yet been known. CD22 (also known as Siglec-2) is an inhibitory molecule preferentially expressed in B lymphocytes (B cells) and is constitutively bound and functionally regulated by α2,6 sialic acids expressed on the same cell (cis-ligands). Here, we developed synthetic sialosides GSC718 and GSC839 that bind to CD22 with high affinity (IC ~100 nM), and inhibit ligand binding of CD22. When B cells are activated by B cell antigen receptor (BCR) ligation, both GSC718 and GSC839 downregulate proliferation of B cells, and this regulation requires both CD22 and α2,6 sialic acids. This result suggests that these sialosides regulate BCR ligation-induced B cell activation by reversing endogenous ligand-mediated regulation of CD22. By contrast, GSC718 and GSC839 augment B cell proliferation induced by TLR ligands or CD40 ligation, and this augmentation requires CD22 but not α2,6 sialic acids. Thus, these sialosides appear to enhance B cell activation by directly suppressing the inhibitory function of CD22 independently of endogenous ligand-mediated regulation. Moreover, GSC839 augments B cell proliferation that depends on both BCR ligation and CD40 ligation as is the case for B cell responses to antigens, and enhanced antibody production to the extent comparable to CpG oligonuleotides or a small amount of alum. Although these known adjuvants induce production of the inflammatory cytokines or accumulation of inflammatory cells, CD22-binding sialosides do not. Thus, synthetic sialosides that bind to CD22 with high-affinity modulate B cell activation through endogenous ligand-dependent and independent pathways, and carry an adjuvant activity without inducing inflammation.
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http://dx.doi.org/10.3389/fimmu.2018.00820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5917077PMC
June 2019

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

Crystal Structures and Thermodynamic Analysis Reveal Distinct Mechanisms of CD28 Phosphopeptide Binding to the Src Homology 2 (SH2) Domains of Three Adaptor Proteins.

J Biol Chem 2017 01 6;292(3):1052-1060. Epub 2016 Dec 6.

From the Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto 606-8522, Japan,

Full activation of T cells and differentiation into effector T cells are essential for many immune responses and require co-stimulatory signaling via the CD28 receptor. Extracellular ligand binding to CD28 recruits protein-tyrosine kinases to its cytoplasmic tail, which contains a YMNM motif. Following phosphorylation of the tyrosine, the proteins growth factor receptor-bound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (Gads), and p85 subunit of phosphoinositide 3-kinase may bind to pYMNM (where pY is phosphotyrosine) via their Src homology 2 (SH2) domains, leading to downstream signaling to distinct immune pathways. These three adaptor proteins bind to the same site on CD28 with variable affinity, and all are important for CD28-mediated co-stimulatory function. However, the mechanism of how these proteins recognize and compete for CD28 is unclear. To visualize their interactions with CD28, we have determined the crystal structures of Gads SH2 and two p85 SH2 domains in complex with a CD28-derived phosphopeptide. The high resolution structures obtained revealed that, whereas the CD28 phosphopeptide bound to Gads SH2 is in a bent conformation similar to that when bound to Grb2 SH2, it adopts a more extended conformation when bound to the N- and C-terminal SH2 domains of p85. These differences observed in the peptide-protein interactions correlated well with the affinity and other thermodynamic parameters for each interaction determined by isothermal titration calorimetry. The detailed insight into these interactions reported here may inform the development of compounds that specifically inhibit the association of CD28 with these adaptor proteins to suppress excessive T cell responses, such as in allergies and autoimmune diseases.
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http://dx.doi.org/10.1074/jbc.M116.755173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5247639PMC
January 2017

CD72 negatively regulates B lymphocyte responses to the lupus-related endogenous toll-like receptor 7 ligand Sm/RNP.

J Exp Med 2016 11 24;213(12):2691-2706. Epub 2016 Oct 24.

Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo, Tokyo 113-8510, Japan

Toll-like receptor 7 (TLR7) plays an essential role in development of systemic lupus erythematosus by co-stimulating B cells reactive to the endogenous TLR7 ligand Sm/ribonucleoprotein (RNP), a crucial lupus self-antigen. However, how the TLR7-mediated autoimmune response is regulated is not yet known. In this study, we demonstrate that CD72, an inhibitory B cell co-receptor known to prevent development of lupus, recognizes Sm/RNP at the extracellular C-type lectin-like domain (CTLD) and specifically inhibits B cell response to Sm/RNP. Moreover, the CTLD of CD72, a lupus-susceptible allele, binds to Sm/RNP less strongly than that of lupus-resistant CD72 Reduced binding of CD72 is supported by x-ray crystallographic analysis that reveals a considerable alteration in charge at the putative ligand-binding site. Thus, CD72 appears to specifically inhibit B cell response to the endogenous TLR7 ligand Sm/RNP through CTLD-mediated recognition of Sm/RNP, thereby preventing production of anti-Sm/RNP antibody crucial for development of lupus.
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http://dx.doi.org/10.1084/jem.20160560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5110020PMC
November 2016

Identification of hepta-histidine as a candidate drug for Huntington's disease by in silico-in vitro- in vivo-integrated screens of chemical libraries.

Sci Rep 2016 Sep 22;6:33861. Epub 2016 Sep 22.

Department of Neuropathology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.

We identified drug seeds for treating Huntington's disease (HD) by combining in vitro single molecule fluorescence spectroscopy, in silico molecular docking simulations, and in vivo fly and mouse HD models to screen for inhibitors of abnormal interactions between mutant Htt and physiological Ku70, an essential DNA damage repair protein in neurons whose function is known to be impaired by mutant Htt. From 19,468 and 3,010,321 chemicals in actual and virtual libraries, fifty-six chemicals were selected from combined in vitro-in silico screens; six of these were further confirmed to have an in vivo effect on lifespan in a fly HD model, and two chemicals exerted an in vivo effect on the lifespan, body weight and motor function in a mouse HD model. Two oligopeptides, hepta-histidine (7H) and Angiotensin III, rescued the morphological abnormalities of primary neurons differentiated from iPS cells of human HD patients. For these selected drug seeds, we proposed a possible common structure. Unexpectedly, the selected chemicals enhanced rather than inhibited Htt aggregation, as indicated by dynamic light scattering analysis. Taken together, these integrated screens revealed a new pathway for the molecular targeted therapy of HD.
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http://dx.doi.org/10.1038/srep33861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5032119PMC
September 2016

Selective inhibition of the kinase DYRK1A by targeting its folding process.

Nat Commun 2016 Apr 22;7:11391. Epub 2016 Apr 22.

Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Kyoto 606-8501, Japan.

Autophosphorylation of amino-acid residues is part of the folding process of various protein kinases. Conventional chemical screening of mature kinases has missed inhibitors that selectively interfere with the folding process. Here we report a cell-based assay that evaluates inhibition of a kinase at a transitional state during the folding process and identify a folding intermediate-selective inhibitor of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), which we refer to as FINDY. FINDY suppresses intramolecular autophosphorylation of Ser97 in DYRK1A in cultured cells, leading to its degradation, but does not inhibit substrate phosphorylation catalysed by the mature kinase. FINDY also suppresses Ser97 autophosphorylation of recombinant DYRK1A, suggesting direct inhibition, and shows high selectivity for DYRK1A over other DYRK family members. In addition, FINDY rescues DYRK1A-induced developmental malformations in Xenopus laevis embryos. Our study demonstrates that transitional folding intermediates of protein kinases can be targeted by small molecules, and paves the way for developing novel types of kinase inhibitors.
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http://dx.doi.org/10.1038/ncomms11391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4844702PMC
April 2016

Crystal Structure of the Vitamin D Receptor Ligand-Binding Domain with Lithocholic Acids.

Vitam Horm 2016 8;100:117-36. Epub 2015 Dec 8.

Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan. Electronic address:

The secondary bile acid lithocholic acid (LCA) and its derivatives act as selective modulators of the vitamin D receptor (VDR), although their structures fundamentally differ from that of the natural hormone 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). The complexes of the ligand-binding domain of rat VDR (VDR-LBD) with LCA and its derivatives revealed that the ligands bound to the same ligand-binding pocket (LBP) of VDR-LBD that 1,25(OH)2D3 binds to, but in the opposite orientation; their A-ring was positioned at the top of the LBP, whereas their acyclic tail was located at the bottom of the LBP. However, most of the hydrophobic and hydrophilic interactions observed in the complex with 1,25(OH)2D3 were reproduced in the complexes with LCA and its derivatives. Additional interactions between VDR-LBD and the C-3 substituents of the A-ring were also observed in the complexes, probably related to the observed difference in the potency among the LCA-type ligands. Recently, zebrafish VDR has been reported to have the second LBP on the outside of the canonical LBP, although its physiological function is unclear.
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http://dx.doi.org/10.1016/bs.vh.2015.10.004DOI Listing
January 2017

Modeling and experimental assessment of a buried Leu-Ile mutation in dengue envelope domain III.

Biochem Biophys Res Commun 2016 Feb 28;471(1):163-8. Epub 2016 Jan 28.

Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16, Nakamachi, Koganei-shi, Tokyo, 184-8588, Japan. Electronic address:

Envelope protein domain III (ED3) of the dengue virus is important for both antibody binding and host cell interaction. Here, we focused on how a L387I mutation in the protein core could take place in DEN4 ED3, but cannot be accommodated in DEN3 ED3 without destabilizing its structure. To this end, we modeled a DEN4_L387I structure using the Penultimate Rotamer Library and taking the DEN4 ED3 main-chain as a fixed template. We found that three out of seven Ile(387) conformers fit in DEN4 ED3 without introducing the severe atomic clashes that are observed when DEN3 serotype's ED3 is used as a template. A more extensive search using 273 side-chain rotamers of the residues surrounding Ile(387) confirmed this prediction. In order to assess the prediction, we determined the crystal structure of DEN4_L387I at 2 Å resolution. Ile(387) indeed adopted one of the three predicted rotamers. Altogether, this study demonstrates that the effects of single mutations are to a large extent successfully predicted by systematically modeling the side-chain structures of the mutated as well as those of its surrounding residues using fixed main-chain structures and assessing inter-atomic steric clashes. More accurate and reliable predictions require considering sub-angstrom main-chain deformation, which remains a challenging task.
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http://dx.doi.org/10.1016/j.bbrc.2016.01.159DOI Listing
February 2016

Synthesis, Biological Activities, and X-ray Crystal Structural Analysis of 25-Hydroxy-25(or 26)-adamantyl-17-[20(22),23-diynyl]-21-norvitamin D Compounds.

J Med Chem 2015 Dec 15;58(24):9510-21. Epub 2015 Dec 15.

Department of Biomedical Sciences, Nihon University School of Medicine , 30-1 Ohyaguchikami-machi, Itabashi-ku, Tokyo 173-8610, Japan.

Novel 19-norvitamin D analogues (ADYW1-4, 5a-d) in which an adamantyl diyne side chain is attached directly to the 17-position of the D ring are designed and stereoselectively synthesized. The adamantane ring of these analogues was expected to interfere with helix 12 (H12, activation function 2) of the vitamin D receptor (VDR) to modulate its activities. The analogue 5b binds to the VDR (7% of the natural hormone) and shows significant partial agonistic activity in transactivation assay. Compound 5b showed considerable selectivity in VDR target genes expressions in vitro, it was taken up by target cells 2-3 times more readily, and its lifetime was three times longer than the natural hormone. The X-ray crystal structure of 5b in complex with VDR reveals that the ligand binds similarly to the natural hormone, but the diyne moiety is slightly bent (angles around the diyne 5° to 8°) with respect to the original diyne vitamin D compound 6 in VDR (<1°) due to steric hindrance with helix 12.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00792DOI Listing
December 2015

Fine tuning of agonistic/antagonistic activity for vitamin D receptor by 22-alkyl chain length of ligands: 22S-Hexyl compound unexpectedly restored agonistic activity.

Bioorg Med Chem 2015 Nov 26;23(22):7274-81. Epub 2015 Oct 26.

Laboratory of Drug Design and Medicinal Chemistry, Showa Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen, Machida, Tokyo 194-8543, Japan. Electronic address:

1α,25-Dihydroxyvitamin D3 exerts its actions by binding to vitamin D receptor (VDR). We are continuing the study related to the alteration of pocket structure of VDR by 22-alkyl substituent of ligands and the relationships between the alteration and agonistic/antagonistic activity. Previously we reported that compounds 2 (22-H), 3 (22S-Et), and 4 (22S-Bu) are VDR agonist, partial agonist and antagonist, respectively. Here, we describe the synthesis and biological evaluation of 22S-hexyl analog 5 (22S-Hex), which was designed to be a stronger VDR antagonist than 4. Unexpectedly, 5 showed partial agonistic but not antagonistic activity when bound to VDR, indicating that it is not necessarily true that the bulkier the side chain is, the stronger the antagonistic activity will be. X-ray crystallographic analysis of the VDR-ligand-binding domain (VDR-LBD) accommodating compound 5 indicated that the partial agonist activity of 5 is dependent on the mixed population of the agonistic and antagonistic conformations. Binding of compound 5 may not bring the complex into the only antagonistic conformation due to the large conformational change of the VDR-LBD. From this study it was found that fine tuning of agonistic/antagonistic activity for VDR is possible by 22-alkyl chain length of ligands.
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http://dx.doi.org/10.1016/j.bmc.2015.10.026DOI Listing
November 2015

Structural and biophysical analysis of sero-specific immune responses using epitope grafted Dengue ED3 mutants.

Biochim Biophys Acta 2015 Oct 6;1854(10 Pt A):1438-43. Epub 2015 Jul 6.

Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan. Electronic address:

Dengue fever is a re-emerging tropical disease and its severe form is caused by cross-reactivity between its four serotypes (DEN1, DEN2, DEN3 and DEN4). The third domain of the viral envelope protein (ED3) contains the two major putative epitopes and is a highly suitable model protein for examining the molecular determinants of a virus' sero-specificity. Here we examine d the sero-specificity and cross-reactivity of the immune response against DEN3 and DEN4 ED3 using six epitope grafted ED3 variants where the surface-exposed epitope residues from DEN3 ED3 were switched to those of DEN4 ED3 and vice versa. We prepared anti-DEN3 and anti-DEN4 ED3 serum by immunizing Swiss albino mice and measured their reactivities against all six grafted mutants. As expected, both sera exhibited strong reactivity against its own serotype's ED3, and little cross-reactivity against their counterpart serotype's ED3s. E2 played a major role in the sero-specificity of anti-DEN3 serum, whereas E1 was important for DEN4 ED3's sero-specificity. Next, the reactivity patterns corroborated our working hypothesis that sero-specificity could be transferred by grafting the surface exposed epitope residues from one serotype to the other. To analyze the above results from a structural viewpoint, we determined the crystal structure of a DEN4 ED3 variant, where E2 was grafted from DEN3 ED3, at 2.78Å resolution and modeled the structures of the five remaining grafted variants by assuming that the overall backbone remained unchanged. The examination of the electrostatic and molecular surfaces of the variants suggested some further rationale for the sero-specificity of the immune responses.
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http://dx.doi.org/10.1016/j.bbapap.2015.07.004DOI Listing
October 2015

Design and synthesis of a potent inhibitor of class 1 DYRK kinases as a suppressor of adipogenesis.

Bioorg Med Chem 2015 Aug 14;23(15):4434-4441. Epub 2015 Jun 14.

Department of Anatomy and Developmental Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan. Electronic address:

Dysregulation of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) has been demonstrated in several pathological conditions, including Alzheimer's disease and cancer progression. It has been recently reported that a gain of function-mutation in the human DYRK1B gene exacerbates metabolic syndrome by enhancing obesity. In the previous study, we developed an inhibitor of DYRK family kinases (INDY) and demonstrated that INDY suppresses the pathological phenotypes induced by overexpression of DYRK1A or DYRK1B in cellular and animal models. In this study, we designed and synthesized a novel inhibitor of DYRK family kinases based on the crystal structure of the DYRK1A/INDY complex by replacing the phenol group of INDY with dibenzofuran to produce a derivative, named BINDY. This compound exhibited potent and selective inhibitory activity toward DYRK family kinases in an in vitro assay. Furthermore, treatment of 3T3-L1 pre-adipocytes with BINDY hampered adipogenesis by suppressing gene expression of the critical transcription factors PPARγ and C/EBPα. This study indicates the possibility of BINDY as a potential drug for metabolic syndrome.
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http://dx.doi.org/10.1016/j.bmc.2015.06.018DOI Listing
August 2015

Identification of a Dual Inhibitor of SRPK1 and CK2 That Attenuates Pathological Angiogenesis of Macular Degeneration in Mice.

Mol Pharmacol 2015 Aug 20;88(2):316-25. Epub 2015 May 20.

Department of Ophthalmology and Visual Sciences (S.M., N.Y.), Department of Anatomy and Developmental Biology (S.M., I.K., Ke.N., Ma.H.), and Medical Research Support Center (Y.O., M.D.), Graduate School of Medicine, Kyoto University, Kyoto, Japan; Laboratory of Structural Biology, Medical Research Institute (Mi.H., No.I., T.I.), and Laboratory of Chemical Bioscience, Institute of Biomaterials and Bioengineering (S.Y., T.H.), Tokyo Medical and Dental University, Tokyo, Japan; Open Innovation Center for Drug Discovery, The University of Tokyo, Tokyo, Japan (T.O., H.K., T.N.); PharmaDesign, Inc., Tokyo, Japan (Na.I., T.F.); and Department of Ophthalmology, Graduate School of Medicine, Hokkaido University, Sapporo, Japan (J.F., Ko.N., S.I.)

Excessive angiogenesis contributes to numerous diseases, including cancer and blinding retinopathy. Antibodies against vascular endothelial growth factor (VEGF) have been approved and are widely used in clinical treatment. Our previous studies using SRPIN340, a small molecule inhibitor of SRPK1 (serine-arginine protein kinase 1), demonstrated that SRPK1 is a potential target for the development of antiangiogenic drugs. In this study, we solved the structure of SRPK1 bound to SRPIN340 by X-ray crystallography. Using pharmacophore docking models followed by in vitro kinase assays, we screened a large-scale chemical library, and thus identified a new inhibitor of SRPK1. This inhibitor, SRPIN803, prevented VEGF production more effectively than SRPIN340 owing to the dual inhibition of SRPK1 and CK2 (casein kinase 2). In a mouse model of age-related macular degeneration, topical administration of eye ointment containing SRPIN803 significantly inhibited choroidal neovascularization, suggesting a clinical potential of SRPIN803 as a topical ointment for ocular neovascularization. Thus SRPIN803 merits further investigation as a novel inhibitor of VEGF.
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http://dx.doi.org/10.1124/mol.114.097345DOI Listing
August 2015

CDK9 inhibitor FIT-039 prevents replication of multiple DNA viruses.

J Clin Invest 2014 Aug 8;124(8):3479-88. Epub 2014 Jul 8.

A wide range of antiviral drugs is currently available; however, drug-resistant viruses have begun to emerge and represent a potential public health risk. Here, we explored the use of compounds that inhibit or interfere with the action of essential host factors to prevent virus replication. In particular, we focused on the cyclin-dependent kinase 9 (CDK9) inhibitor, FIT-039, which suppressed replication of a broad spectrum of DNA viruses through inhibition of mRNA transcription. Specifically, FIT-039 inhibited replication of herpes simplex virus 1 (HSV-1), HSV-2, human adenovirus, and human cytomegalovirus in cultured cells, and topical application of FIT-039 ointment suppressed skin legion formation in a murine HSV-1 infection model. FIT-039 did not affect cell cycle progression or cellular proliferation in host cells. Compared with the general CDK inhibitor flavopiridol, transcriptome analyses of FIT-039-treated cells revealed that FIT-039 specifically inhibited CDK9. Given at concentrations above the inhibitory concentration, FIT-039 did not have a cytotoxic effect on mammalian cells. Importantly, administration of FIT-039 ameliorated the severity of skin lesion formation in mice infected with an acyclovir-resistant HSV-1, without noticeable adverse effects. Together, these data indicate that FIT-039 has potential as an antiviral agent for clinical therapeutics.
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http://dx.doi.org/10.1172/JCI73805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4109542PMC
August 2014

Combination of triple bond and adamantane ring on the vitamin D side chain produced partial agonists for vitamin D receptor.

J Med Chem 2014 May 7;57(10):4073-87. Epub 2014 May 7.

Department of Chemistry, ‡Research Center for Smart Molecules, Faculty of Science, Rikkyo University , Toshima-ku, Tokyo 171-8501, Japan.

Vitamin D receptor (VDR) ligands are therapeutic agents that are used for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism and have immense potential as therapeutic agents for autoimmune diseases, cancers, and cardiovascular diseases. However, the major side effect of VDR ligands, the development of hypercalcemia, limits their expanded use. To develop tissue-selective VDR modulators, we have designed vitamin D analogues with an adamantane ring at the side chain terminal, which would interfere with helix 12, the activation function 2, and modulate the VDR potency. Here we report 25- or 26-adamantyl-23,23,24,24-tetradehydro-19-norvitamin D derivatives (ADTK1-4, 4b,a and 5a,b). These compounds showed high VDR affinities (90% at maximum), partial agonistic activities (EC50 10(-9)-10(-8) M with 40-80% efficacy) in transactivation, and tissue-selective activity in target gene expressions. We investigate the structure-activity relationships of these compounds on the basis of their X-ray crystal structures.
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http://dx.doi.org/10.1021/jm401989cDOI Listing
May 2014

High resolution crystal structure of the Grb2 SH2 domain with a phosphopeptide derived from CD28.

PLoS One 2013 30;8(9):e74482. Epub 2013 Sep 30.

Research Institute for Biomedical Sciences, Tokyo University of Science, Noda-shi, Chiba, Japan.

Src homology 2 (SH2) domains play a critical role in cellular signal transduction. They bind to peptides containing phosphotyrosine (pY) with various specificities that depend on the flanking amino-acid residues. The SH2 domain of growth-factor receptor-bound protein 2 (Grb2) specifically recognizes pY-X-N-X, whereas the SH2 domains in phosphatidylinositol 3-kinase (PI3K) recognize pY-X-X-M. Binding of the pY site in CD28 (pY-M-N-M) by PI3K and Grb2 through their SH2 domains is a key step that triggers the CD28 signal transduction for T cell activation and differentiation. In this study, we determined the crystal structure of the Grb2 SH2 domain in complex with a pY-containing peptide derived from CD28 at 1.35 Å resolution. The peptide was found to adopt a twisted U-type conformation, similar to, but distinct from type-I β-turn. In all previously reported crystal structures, the peptide bound to the Grb2 SH2 domains adopts a type-I β-turn conformation, except those with a proline residue at the pY+3 position. Molecular modeling also suggests that the same peptide bound to PI3K might adopt a very different conformation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0074482PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3787023PMC
May 2014