Shigemi Terakubo

Enhancement of the T140-based pharmacophores leads to the development of more potent and bio-stable CXCR4 antagonists

A CXCR4 antagonistic peptide, T140, and its bio-stable analogs, such as Ac-TE14011, were previously developed. These peptides inhibit the entry of T cell line-tropic strains of HIV-1 (X4-HIV-1) into T cells. Herein, a series of TE14011 analogs having modifications in the N-terminal region were synthesized to develop effective compounds with increased biostability. Among these analogs, 4F-benzoyl-TE14011 (TF14013) showed the strongest anti-HIV activity derived from CXCR4-antagonism, suggesting that a 4-fluorobenzoyl moiety at the N-terminus of T140 analogs constitutes a novel T140-based pharmacophore for CXCR4 antagonists. Structure-activity relationship (SAR) studies on TE14011 analogs with N(alpha)-acylation by several benzoic acid derivatives have disclosed a significant relationship between the anti-HIV activity and the Hammett constant (sigma) of substituted benzoic acids. TF14013 was found to be stable in mouse serum, but not completely stable in rat liver homogenate due to deletion of the C-terminal Arg14-NH2 from the parent peptide. This biodegradation was completely suppressed by N-alkyl-amidation at the C-terminus. Taken together, the enhancement of the T140-based pharmacophores led to development of a novel CXCR4 antagonist, 4F-benzoyl-TE14011-Me (TF14013-Me), which has very high anti-HIV activity and increased biostability.

Synthesis of potent CXCR4 inhibitors possessing low cytotoxicity and improved biostability based on T140 derivatives

A peptidic CXCR4 antagonist T140 efficiently blocks the entry of T cell line-tropic strains of HIV-1 (X4-HIV-1) into target cells. In this study, a series of T140 derivatives, replacing the basic amino acid residues with Glu (D-Glu) and/or L-citrulline (Cit), were synthesized in order to reduce non-specific binding and cytotoxicity. Among them, TE14011 ([Cit6, D-Glu8]-T140 with the C-terminal amide) exhibited strong anti-HIV activity and low cytotoxicity. TE14011 was found to be stable in mouse serum, but unstable in rat liver homogenate due to the deletion of the N-terminal Arg1-Arg2-L-3-(2-naphthyl)alanine (Nal)3 residues from the parent peptide. N-Terminal acetylation of TE14011 led to the development of a novel lead compound, Ac-TE 14011, which possesses a high selectivity index as well as increased stability in serum and liver homogenate.

Subcellular Location of the Soluble Lytic Transglycosylase Homologue in Staphylococcus aureus

The immunodominant antigen A, IsaA, of Staphylococcus aureus was found to include a putative soluble lytic transglycosylase domain in its C-terminal region. Since the presence of this distinctive domain suggested that the protein might participate in peptidoglycan turnover, as indicated in Gram-negative bacteria, its cellular location was investigated. The protein was found not only in the culture supernatant but also in the cell wall fraction. To estimate its physiological role for the bacterium, its cell surface distribution was studied by immunoelectron microscopy. Protein A-gold particles binding to the immune complex were mainly located on the septal region of the bacterial cell surface. These data suggested that IsaA might be involved in bacterial cell separation through a preferential interaction with peptidoglycan chain.

Topochemical exploration of potent compounds using retro-enantiomer libraries of cyclic pentapeptides

Cyclic pentapeptides have been adopted as conformationally restricted peptide templates to dispose pharmacophores of bioactive peptides. In our recent study, use of two orthogonal cyclic pentapeptide libraries involving conformation-based and sequence-based libraries containing critical residues of a bioactive peptide led to the discovery of potent downsized peptides that possess activity comparable to that of the parent peptide. The present study demonstrates that a third library consisting of retro-enantiomers (retro-inverso peptides) that possess not only all residues with the opposite configuration to those in the corresponding original peptide but also amino acid sequences with reversed arrangement, is important as an alternative library for rationally finding active compounds.

Efficient Utilization of Plant Resources by Alkaline Extraction

As compared to the studies with hot water extracts of plants, those with alkaline extracts were limited. Both alkaline and hot water extracts from green tea leaf, oolong tea leaf and orange flower were compared for their biological activities. Plant materials were successively extracted first with hot-water and then alkaline solution, or extracted directly with alkaline solution. Viable cell number of HIV-infected and UV-irradiated cells was determined by MTT method. Antibacterial activity against Porphyromonas gingivalis 381 was determined by turbidity assay. Cytochrome P-450 (CYP)3A4 activity was measured by β-hydroxylation of testosterone using human recombinant CYP3A4 (Figure 5). Radical intensity of superoxide and hydroxyl radical was determined by ESR spectroscopy. Alkaline extraction recovered twice as much as dried materials as compared with water extraction. Water extracts showed higher anti-bacterial, CYP3A4 inhibitory and superoxide scavenging activities, whereas alkaline extract showed higher anti-HIV and hydroxyl radical scavenging activity. Both water and alkaline extracts showed comparable anti-UV activity. The present study suggests the usefulness of alkaline extraction for the efficient utilization of the natural resources.

Susceptibilities of Fluoroquinolone-Resistant Haemophilus parainfluenzae Isolates from Japanese Patients with Respiratory Infections to Five Fluoroquinolones and Other Antimicrobial Agents

Respiratory tract infections remain a common cause of morbidity and mortality worldwide, and Haemophilus spp. have been the important pathogens causing respiratory tract infections (2, 3). Haemophilus parainfluenzae is one of the pathogens that are frequently isolated from respiratory specimens, but the clinical isolates are usually neglected as indigenous bacteria of the human respiratory tract. However, recent case reports have indicated that H. parainfluenzae might also be the causative organism of certain respiratory diseases, such as acute pneumonia (4, 7). We have focused on this bacterium by studying its prevalence in patients, as well as the antimicrobial susceptibilities of the clinical isolates. Furthermore, we identified some fluoroquinolone-resistant strains of H. parainfluenzae (FRHP) in our collection. Respiratory specimens (e.g., sputum, pharyngeal swabs, and tonsillar swabs) were collected from patients visiting the outpatient departments of 382 facilities all over Japan in 1998 to 2000. H. influenzae (1,213 strains), H. parainfluenzae (920 strains), H. haemolyticus (232 strains), and H. parahaemolyticus (111 strains) were isolated from the samples collected in the 3 years. The MICs of some antimicrobial agents, including levofloxacin, were determined for all of the strains by the NCCLS standard broth microdilution method (5). Ampicillin was purchased from Wako Chemicals (Osaka, Japan), and all other antimicrobial agents were kindly provided by the corresponding manufacturers. We determined the fluoroquinolone-resistant strains of Haemophilus spp. with the NCCLS breakpoint of levofloxacin, >2 μg/ml (6). The numbers of FRHP isolates were 2 (2.4%), 16 (3.6%), and 13 (3.0%) in 1998, 1999, and 2000, respectively, while only one or two fluoroquinolone-resistant strains of the other Haemophilus spp. were isolated. The distributions of drug MICs for H. parainfluenzae, 20 fluoroquinolone-susceptible strains of H. parainfluenzae (FSHP), and 30 FRHP isolates are summarized in Table ​Table1.1. All five fluoroquinolones exhibited excellent antimicrobial activity against FSHP (MICs for 90% of the strains tested [MIC90s], ≤0.015 to 0.06 μg/ml), whereas each MIC90 of fluoroquinolones for FRHP was more than 100-fold higher than that that for FSHP. Sitafloxacin exhibited a clearly lower MIC90 (1 μg/ml) than did the other fluoroquinolones. The susceptibilities of FRHP to other, nonfluoroquinolone antimicrobial agents were similar to those of FSHP, while two FRHP isolates exhibited lower susceptibilities to ampicillin (MIC, >32 μg/ml). TABLE 1. Distribution of the MICs of five fluoroquinolones and four nonfluoroquinolones for FSHP and FRHPa To our knowledge, only a few studies previously examined the drug susceptibility of H. parainfluenzae. The recently reported surveillance studies in Europe (2) and the United States (3) indicated that most H. parainfluenzae isolates were susceptible to levofloxacin and other fluoroquinolones. This is the first report regarding clinical isolates of FRHP. It is conceivable that the fluoroquinolone resistance of FRHP described in this report is caused by genetic mutations in the gyrA and/or the parC gene which likely occur in some other bacteria, such as Streptococcus pneumoniae and H. influenzae (1, 8). This bacterial resistance might be related to the selective pressure derived from the prevalent use of fluoroquinolones in Japan (8). Careful monitoring of changes in the drug susceptibility pattern of H. parainfluenzae, as well as those of many other pathogens, is important. (This study was previously presented in part [42nd Intersci. Conf. Antimicrob. Agents Chemother., abstr. C2-1994, p. 124].)

Therapeutic potential of solubilized nanolignin against oral diseases

Abstract Lignocellulosic biomass is a complex biopolymer that is primarily composed of cellulose, hemicellulose, and lignin. Lignin, the main natural aromatic polymer, has a very complex and complicated structure that depends on the separation method and plant species. Their molecular weights fluctuate largely from monolignols to nanofibers, depending on pH, extraction conditions (pH, temperature, Fe 2+ / 3+ ) and shearing process. Limited digestion of LCC demonstrated the importance of lignin moiety, but not carbohydrate moiety for antihuman-immunodeficiency virus (HIV) activity induction. Dehydrogenation polymers of p -coumaric acid, felluric acid, or caffeic acid showed much higher anti-HIV activity than their respective monomers, suggesting the importance of highly polymerized structures. [ 125 I]-labeled LCC rapidly bound to the virus and reduced its infectivity. Bioavailability of orally administered [ 125 I]-LCC was very low, validating its application through oral mucosa. Alkaline extraction was superior to hot water extraction in recovering anti-HIV activity from green tea, black tea, and oolong tea-leaves and licorice root. Oral intake of alkaline extract of Sasa senanensis Rehder leaf (SE) (OTC drug) and LCC/vitamin C tablet significantly improved the symptoms of lichenoid dysplasia and herpes simplex virus (HSV)-patients, respectively. Use of SE-enriched tooth-paste significantly reduced halitosis. These data suggest the applicability of solubilized nanolignin against various oral diseases.

Biological activity of an Indian medical plant, Indigofera cordifolia

The ethanol extract of Indigofera cordifolia was studied for in vivo gastroprotective activity, cytotoxic activity against oral tumor and normal cells, multidrug resistance (MDR) reversal activity, anti-human immunodeficiency virus (HIV) activity and radical scavenging activity. The extract of I. cordifolia showed potent gastric mucosal protective activity against stomach injury induced by HCl/EtOH solution. However, the gastroprotective activity could not be related to the radical mechanism, because the extract weakly scavenged both OH radical and . The extract also showed promising levels of MDR-reversing activity. This study demonstrates the tumor-specific cytotoxic action of the plant extract. However, the extract had no anti-HIV activity. From above results, the study suggests the medicinal importance of I. cordiforia extract.