Teruhiko Ide

Structural basis for binding of human IgG1 to its high-affinity human receptor FcγRI.

Cell-surface Fcγ receptors mediate innate and adaptive immune responses. Human Fcγ receptor I (hFcγRI) binds IgGs with high affinity and is the only Fcγ receptor that can effectively capture monomeric IgGs. However, the molecular basis of hFcγRIs interaction with Fc has not been determined, limiting our understanding of this major immune receptor. Here we report the crystal structure of a complex between hFcγRI and human Fc, at 1.80 Å resolution, revealing an unique hydrophobic pocket at the surface of hFcγRI perfectly suited for residue Leu235 of Fc, which explains the high affinity of this complex. Structural, kinetic and thermodynamic data demonstrate that the binding mechanism is governed by a combination of non-covalent interactions, bridging water molecules and the dynamic features of Fc. In addition, the hinge region of hFcγRI-bound Fc adopts a straight conformation, potentially orienting the Fab moiety. These findings will stimulate the development of novel therapeutic strategies involving hFcγRI.

Antibacterial activity of fluoroalkylated allyl-and diallyl-ammonium chloride oligomers

Abstract A series of fluoroalkylated allyl-and diallyl-ammonium chloride oligomers were prepared using fluoroalkanoyl peroxides as key intermediates, and their antibacterial activities against Staphylococcus aureus and Escherichia coli were studied. Antibacterial activity was found to be sensitive to the structure of these oligomers. Fluoroalkylated allyl-type co-oligomers containing carboxy, sulfo and trimethylsilyl segments were inactive; however, the allyl-or diallyl-type homo-and co-oligomers were in general active. Of these, the perfluoro-l-methyl-2-oxapentylated allylammonium chloride-diallylammonium chloride co-oligomer was found to be the most active against both S. aureus and E. coli . Furthermore, the fluoroalkylated oligomers possessing antibacterial activity were able to reduce the surface tension of water to around 10 mN m 1 . Therefore, these fluoroalkylated oligomers are new attractive functional materials possessing not only unique properties imparted by fluorine but also antibacterial activity.

Anti-HIV-1 activity of thiadiazole derivatives : structure-activity relationship, reverse transcriptase inhibition, and lipophilicity

The structure-activity relationship of the non-nucleoside HIV-1-specific reverse transcriptase (RT) inhibitors 4-phenyl-1,2,5-thiadiazol-3-yl N,N-dialkylcarbamate (TDA) derivatives was investigated with respect to their anti-HIV-1 activity, RT inhibition, and lipophilicity. 4-Phenyl-1,2,5-thiadiazol-3-yl N,N-dimethylcarbamate inhibited HIV-1-induced cytopathic effect (CPE) by 50% at a concentration of 28.8 microM in MT-4 cells. The activity increased more than 100-fold when the hydrogens at the 2-position and the 6-position in phenyl moiety were substituted by chlorines. However, the derivative with a chlorine at the 4-position of phenyl moiety did not show any inhibition of HIV-1 replication at its non-toxic concentrations. All of the 4-(2,6-dichlorophenyl)-1,2,5-thiadiazol-3-yl N-methyl-N-alkylcarbamates proved inhibitory to HIV-1 replication in the nanomolar concentration range. The TDA derivatives that showed anti-HIV-1 activity also inhibited RT activity in an enzymatic assay. However, the TDA derivatives did not show any specific inhibition of a non-nucleoside RT inhibitor (NNRTI)-resistant mutant and its RT activity. When the TDA derivatives were examined for their inhibitory effect on HIV-1 replication in the presence of 50% human serum, the activity significantly decreased depending on-their lipophilicity.

Utilization of wieland furoxan synthesis for preparation of 4-aryl-1,2,5-oxadiazole-3-yl carbamate derivatives having potent anti-HIV activity

Abstract The classical Wieland furoxan synthesis was reinvestigated and this procedure was applied to the preparation of 4-aryl-1,2,5-oxadiazole-3-yl N,N-dialkylcarbamate derivatives, which were found to exhibit potent anti-HIV-1 activity.

Thiadiazole Derivatives: Highly Potent and Selective Inhibitors of Human Immunodeficiency Virus Type 1 (HIV‐1) Replications In Vitro

We have recently reported that thiadiazole (TDA) derivatives are highly potent inhibitors of human immunodeficiency virus type 1 (HIV‐1) replication. These compounds belong to the family of nonnucleoside reverse transcriptase inhibitors (NNRTIs). In an attempt to develop more effective and pharmacologically favorable compounds, novel TDA derivatives have been synthesized and examined for their anti‐HIV‐1 activity in vitro. Among them, RD4‐2217 was found to be the most potent inhibitor of HIV‐1 replication. It inhibited replication of the HTLV‐IIIB strain in MT‐4 cells at a concentration of 6 nM. RD4‐2217 was also inhibitory to clinical isolates and zidovudine‐resistant mutants of HIV‐1. The combination of RD4‐2217 with zidovudine or the protease inhibitor A‐75925 synergistically inhibited HIV‐1 replication. Studies on the emergence of drug‐resistant mutants revealed that, although much higher concentrations (1‐10 μM) were required, RD4‐2217 completely suppressed the breakthrough of HIV‐1 in the supernatants during long‐term culturing of infected cells. Furthermore, RD4‐2217 at low concentrations (10 or 100 nM), in combination with zidovudine, also completely inhibited viral breakthrough. In addition, RD4‐2217 had lower lipophilicity and improved protein binding as compared to its congener RD4‐2024 and loviride. These results suggest that RD4‐2217, one of the TDA derivatives, is worth pursuing as a candidate drug for the treatment of HIV‐1 infections.

Characterization of human immunodeficiency virus type 1 strains resistant to the non-nucleoside reverse transcriptase inhibitor RD4-2217.

The non-nucleoside reverse transcriptase (RT) inhibitor RD4–2217 is a thiadiazole derivative that has proved to be a highly potent and selective inhibitor of human immunodeficiency virus type 1 (HIV-1) replication in vitro. In this study we examined genotypic and phenotypic characteristics of RD4–2217-resistant mutants that have been obtained by serial passage of HIV-1 in MT-4 cells in the presence of increasing concentrations (0.05, 0.25, 1 and 10 μM) of the compound. The strains obtained, IIIB/2217RE/0.05 and IIIB/2217RE/0.25, were two-and 15-fold resistant to RD4–2217, respectively, whereas IIIB/2217RE/1 and IIIB/2217RE/10 displayed 161-and >238-fold resistance, respectively. Both IIIB/2217RE/1 and IIIB/2217RE/10 had two amino acid substitutions, V189I and T240I, in the RT. Furthermore, RD4–2217 did not inhibit the replication of an HIV-1 molecular clone, which had the same mutation, at concentrations up to 10 μM, indicating that the V189I plus T240I mutation confers high-level resistance to RD4–2217. Interestingly, the replicability of IIIB/2217RE/1 and IIIB/2217RE/10 appeared to be lower than that of wild-type IIIB in MT-4 cells, suggesting that the V189I plus T240I mutation may impair the enzymatic activity of HIV-1 RT.

RD6-2198, a novel betain-type fluoroalkylated oligomer, inhibits the replications of human immunodeficiency virus type 1 and other enveloped viruses

We have examined a novel betain-type fluoroalkylated oligomer, RD6-2198, for its inhibitory effects on the replication of human immunodeficiency virus type 1 (HIV-1) and other enveloped viruses, including herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively) and respiratory syncytial virus (RSV) in cell cultures. We have found that the compound is a potent and selective inhibitor of these viruses. RD6-2198 inhibited the replication of HIV-1IIIB at a concentration of 0.85 microg/ml with a selectivity index greater than 59 in MT-4 cells. Furthermore, its 50% effective concentration (EC50) values for HSV-1, HSV-2 and RSV, were 0.51, 0.94 and 3.0 microg/ml, respectively. We found that the RD6-2198 suppressed the gp120-CD4 interaction (as monitored by an enzyme-linked immunosorbent assay (ELISA) method). RD6-2198 also inhibited the binding of anti-gp120 monoclonal antibody to gp120 expressed on MOLT-4/IIIB cells (MOLT-4 cells chronically infected with HIV-1IIIB). However, the compound did not inhibit the interaction of anti-CD4 antibody with CD4. These results suggest that RD6-2198 interacts with the viral envelope glycoprotein and thereby inhibits the viral adsorption process. In addition, RD6-2198 was also found to suppress the proliferation of MOLT-4/IIIB cells. When applied topically, RD6-2198 at a concentration of 10 mg/ml completely protected mice from an intravaginal HSV-2 infection.

The binding of soluble recombinant human Fcγ receptor I for human immunoglobulin G is conferred by its first and second extracellular domains.

Human FcγRI is a high affinity receptor for the Fc portion of human immunoglobulin G (IgG), and has extracellular, transmembrane and cytoplasmic regions. The extracellular region of human FcγRI, which is the part that interacts with human IgG, is comprised of three immunoglobulin-like domains. Unlike low affinity Fcγ receptors (FcγRII and FcγRIII), FcγRI has a unique third extracellular domain (D3). This study investigated the contribution of D3 to the binding between recombinant human FcγRI (rhFcγRI) and human IgG. The three extracellular domains and the first and second extracellular domains of human FcγRI were expressed by Escherichia coli as rhFcγRI and rhFcγRI-D1D2, respectively. The binding specificity of rhFcγRI-D1D2 to human IgG subclasses was the same as that of rhFcγRI. From surface plasmon resonance analysis, the binding affinity of rhFcγRI-D1D2 for human IgG1/κ was high (the equilibrium dissociation constant: KD=8.04 × 10(-10)M), but slightly lower than that of rhFcγRI (KD=2.59 × 10(-10)M). While the association of rhFcγRI-D1D2 with human IgG1/κ was same as that of rhFcγRI, the dissociation of rhFcγRI-D1D2 was faster than that of rhFcγRI. From these results, D3 of rhFcγRI would not contribute directly to the binding specificity and association of rhFcγRI, but to the holding bound human IgG.

Cellulomonas soli sp. nov. and Cellulomonas oligotrophica sp. nov., isolated from soil.

Two novel bacterial strains, designated Kc1(T) and Kc5(T), were isolated from soil in Japan. Cells of the novel strains were Gram-reaction-positive, aerobic or facultatively anaerobic, motile rods. Phylogenetic analyses based on 16S rRNA gene sequences indicated that both strains belonged to the genus Cellulomonas. The 16S rRNA gene sequences of strains Kc1(T) and Kc5(T) showed closest similarity to that of Cellulomonas terrae DB5(T) (98.1 % and 98.4 % similarity, respectively), and the 16S rRNA gene similarity between the two novel strains was 97.8 %. In both strains, the major menaquinone was MK-9(H(4)), the predominant polar lipids were diphosphatidylglycerol and phosphatidylinositol mannosides, and the peptidoglycan contained ornithine and glutamic acid. Cell-wall sugars were identified as rhamnose, galactose and mannose in strain Kc1(T) and rhamnose and glucose in strain Kc5(T). The DNA G+C contents of strains Kc1(T) and Kc5(T) were 73.6 mol% and 75.8 mol%, respectively. Based on the chemotaxonomic and physiological data and the results of DNA-DNA hybridizations, the two strains represent two novel species within the genus Cellulomonas, for which the names Cellulomonas soli sp. nov. (type strain Kc1(T) =DSM 24484(T) =JCM 17535(T)) and Cellulomonas oligotrophica sp. nov. (type strain Kc5(T) =DSM 24482(T) =JCM 17534(T)) are proposed.

Engineering of erythropoietin receptor for use as an affinity ligand

Recombinant human erythropoietin receptor (rhEPOR) has applicability as an affinity ligand for purification of recombinant human erythropoietin (rHuEPO) because of its specific binding to rHuEPO. For application of rhEPOR as a ligand for purification of rHuEPO, soluble rhEPOR was expressed in the periplasm of Escherichia coli and engineered by directed evolution through random mutagenesis and integration of mutations. From the screening of random mutagenesis, we identified an amino acid mutation (H114Y) contributing to rHuEPO binding and four amino acid mutations (R76S, A132D, A162D, and C181Y) contributing to expression of soluble rhEPOR. However, the rHuEPO that binds to engineered rhEPOR having H114Y mutation is difficult to dissociate from the engineered rhEPOR. Therefore, H114Y mutation was not suitable for the construction of the rhEPOR ligand. As a rhEPOR ligand, engineered rhEPOR containing four amino acid mutations (EPORm4L) was constructed by integration of mutations except for H114Y. The expression of EPORm4L (127mgl(-1) of culture medium) was markedly increased in comparison with wild-type rhEPOR (2mgl(-1) of culture medium). Small-scale affinity chromatography demonstrated that EPORm4L worked as an affinity ligand for purification of rHuEPO.