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Biomedical subjects

A Itai

Publications and source records attributed to A Itai.

At least 19 recordsLinked to original sources
PubMed2003

Rapid identification of 1-aminocyclopropane-1-carboxylate (ACC) synthase genotypes in cultivars of Japanese pear (Pyrus pyrifolia Nakai) using CAPS markers.

In Japanese pear (Pyrus pyrifolia Nakai), fruit storage potential is closely related to the amount of ethylene produced. We have developed a rapid and accurate method for analyzing genes involved in high ethylene production during fruit ripening in Japanese pear. This involves cleaved-amplified polymorphic sequences (CAPS) of two 1-aminocyclopropane-1-carboxylate (ACC) synthase genes (PPACS1 and PPACS2). Two CAPS markers (A for PPACS1 and B for PPACS2), associated with the amount of ethylene produced, were identified. Marker A was associated with high ethylene producers and marker B with moderate ethylene producers. The absence of these two markers enabled the identification of low ethylene producers. Using these markers, we have identified ethylene genotypes for 40 Japanese pear cultivars and two Chinese pear (P. bretschneideri) cultivars that are commercially important and used in breeding programs. Furthermore, we performed linkage analysis of these two genes in the F(2) population, which revealed that the recombination frequency between the two markers was 20.8 +/- 3.6%. This information is critical to the selection of parents and in breeding strategies to improve storage ability of Japanese pears.

Alleles↗
PubMed2001

Discovery of novel aldose reductase inhibitors using a protein structure-based approach: 3D-database search followed by design and synthesis.

Aldose reductase (AR) has been implicated in the etiology of diabetic complications. Due to the limited number of currently available drugs for the treatment of diabetic complications, we have carried out structure-based drug design and synthesis in an attempt to find new types of AR inhibitors. With the ADAM&EVE program, a three-dimensional database (ACD3D) was searched using the ligand binding site of the AR crystal structure. Out of 179 compounds selected through this search followed by visual inspection, 36 compounds were purchased and subjected to a biological assay. Ten compounds showed more than 40% inhibition of AR at a 15 microg/mL concentration. In a subsequent lead optimization, a series of analogues of the most active compound were synthesized based on the docking mode derived by ADAM&EVE. Many of these congeners exhibited higher activities compared to the mother compound. Indeed, the most potent, synthesized compound showed an approximately 20-fold increase in inhibitory activity (IC(50) = 0.21 vs 4.3 microM). Furthermore, a hydrophobic subsite was newly inferred, which would be useful for the design of inhibitors with improved affinity for AR.

Aldehyde Reductase↗
PubMed2001

Structure-Activity study of retinoid agonists bearing substituted dicarba-closo-dodecaborane. Relation between retinoidal activity and conformation of two aromatic nuclei.

We have investigated the structure activity relationships of the potent retinoid agonist, 4-[4-(2-propyl-1,2-dicarba-closo-dodecaboran-l-yl)phenylamino]benzoic acid (BR403), which we have previously reported. Substitution of a methyl group on the aromatic nucleus or a methyl group on the nitrogen atom, or replacement of the amino group with ether, methylene, carboxyl or 1,1-ethylene greatly decreased the activity. The relatively planar conformation at the phenyl-N-phenyl moiety seems to play a critical role in the appearance of the biological activity.

Animals↗
PubMed2001

Potent estrogen agonists based on carborane as a hydrophobic skeletal structure. A new medicinal application of boron clusters.

BACKGROUND: Carboranes (dicarba-closo-dodecaboranes) are a class of carbon-containing polyhedral boron-cluster compounds having remarkable thermal stability and exceptional hydrophobicity. Applications of the unique structural and chemical properties offered by icosahedral carboranes in boron neutron capture therapy have received increasing attention over the past 30 years. However, these features of carboranes may allow another application as a hydrophobic pharmacophore in biologically active molecules that interact hydrophobically with receptors. RESULTS: We have designed candidate estrogen-receptor-binding compounds having carborane as a hydrophobic skeletal structure and synthesized them. The most potent compound bearing a carborane cage exhibited activity at least 10-fold greater than that of 17beta-estradiol in the luciferase reporter gene assay. Estrogen receptor-alpha-binding data for the compound were consistent with the results of the luciferase reporter gene assay. The compound also showed potent in vivo effects on the recovery of uterine weight and bone loss in ovariectomized mice. CONCLUSION: Further development of the potent carborane-containing estrogenic agonists described here, having a new skeletal structure and unique characteristics, should yield novel therapeutic agents, especially selective estrogen receptor modulators. Furthermore, the suitability of the spherical carborane cage for binding to the cavity of the estrogen receptor-alpha ligand-binding domain should provide a basis for a similar approach to developing novel ligands for other steroid receptors.

Animals↗
PubMed2001

Inhibition of RXR and PPARgamma ameliorates diet-induced obesity and type 2 diabetes.

PPARgamma is a ligand-activated transcription factor and functions as a heterodimer with a retinoid X receptor (RXR). Supraphysiological activation of PPARgamma by thiazolidinediones can reduce insulin resistance and hyperglycemia in type 2 diabetes, but these drugs can also cause weight gain. Quite unexpectedly, a moderate reduction of PPARgamma activity observed in heterozygous PPARgamma-deficient mice or the Pro12Ala polymorphism in human PPARgamma, has been shown to prevent insulin resistance and obesity induced by a high-fat diet. In this study, we investigated whether functional antagonism toward PPARgamma/RXR could be used to treat obesity and type 2 diabetes. We show herein that an RXR antagonist and a PPARgamma antagonist decrease triglyceride (TG) content in white adipose tissue, skeletal muscle, and liver. These inhibitors potentiated leptin's effects and increased fatty acid combustion and energy dissipation, thereby ameliorating HF diet-induced obesity and insulin resistance. Paradoxically, treatment of heterozygous PPARgamma-deficient mice with an RXR antagonist or a PPARgamma antagonist depletes white adipose tissue and markedly decreases leptin levels and energy dissipation, which increases TG content in skeletal muscle and the liver, thereby leading to the re-emergence of insulin resistance. Our data suggested that appropriate functional antagonism of PPARgamma/RXR may be a logical approach to protection against obesity and related diseases such as type 2 diabetes.

3T3 Cells↗
PubMed2001

Protein structure-based de novo design and synthesis of aldose reductase inhibitors.

Aldose reductase has been implicated in the etiology of diabetic complications. Due to the limited number of currently available drugs for the treatment of diabetic complications, we have carried out a structure-based de novo design and synthesis in an attempt to identify new aldose reductase inhibitors. With the LEGEND program, we have designed 200 chemical structures that fit into the ligand binding site of the crystal structure of the enzyme. After their visual inspection and assessment of synthetic feasibility, four compounds were chosen to be synthesized. These compounds were all found to have reasonable inhibitory activities (IC50 = 17-91 microM), indicating the first successful generation of nonpeptide drug leads that have been obtained using a rational de novo design approach.

Aldehyde Reductase↗
PubMed2000

Novel design of nonpeptide AVP V(2) receptor agonists: structural requirements for an agonist having 1-(4-aminobenzoyl)-2,3,4, 5-tetrahydro-1H-1-benzazepine as a template.

The discovery of a series of nonpeptide arginine vasopressin V(2) receptor agonists is described. After identifying the aniline derivative 8 as our lead compound from the metabolites of compound 7 that showed antidiuretic activity by po administration to Brattleboro rats, improvements in the in vitro potency involving evaluations of the structural requirements for agonist action and optimizing the structure of the benzoyl moiety have been intensively undertaken. These studies led to compounds 16g, 19a, and 23b,h,i that show potent agonist activity for the V(2) receptor.

Administration, Oral↗
PubMed2000

Isolation of cDNA clones corresponding to genes expressed during fruit ripening in Japanese pear (Pyrus pyrifolia Nakai): involvement of the ethylene signal transduction pathway in their expression.

Thirty cDNA clones of genes corresponding to mRNAs up-regulated during fruit ripening of Japanese pear (Pyrus pyrifolia Nakai cv. Kikusui) were obtained by differential screening of a ripe fruit cDNA library. All of these cDNAs were sequenced and gathered into 11 non-redundant groups after database searches. These clones represented genes associated with stress responses, protein catabolism or pathogenesis. The accumulation of transcripts of 3 out of 11 genes was inhibited by 1-methylcyclopropene (MCP), an inhibitor of ethylene action.

Blotting, Northern↗
PubMed2000

Retinoidal pyrimidinecarboxylic acids. Unexpected diaza-substituent effects in retinobenzoic acids.

Several pyridine- and pyrimidine-carboxylic acids were synthesized as ligand candidates for retinoid nuclear receptors, retinoic acid receptors (RARs) and retinoic X receptors (RXRs). Although the pyridine derivatives, 6-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl]pyri dine-3-carboxylic acid (2b) and 6-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]py ridine-3-carboxylic acid (5b) are more potent than the corresponding benzoic acid-type retinoids, Am80 (2a) and Am580 (5a), the replacement of the benzene ring of Am580 (5a), Am555 (6a), or Am55 (7a) with a pyrimidine ring caused loss of the retinoidal activity both in HL-60 cell differentiation assay and in RAR transactivation assay using COS-1 cells. On the other hand, pyrimidine analogs (PA series, 10 and 11) of potent RXR agonists (retinoid synergists) with a diphenylamine skeleton (DA series, 8 and 9) exhibited potent retinoid synergistic activity in HL-60 cell differentiation assay and activated RXRs. Among the synthesized compounds, 2-[N-n-propyl-N-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)a mino]pyrimidine-5-carboxylic acid (PA013, 10e) is most active retinoid synergist in HL-60 assay.

Animals↗
PubMed1999

Structure-activity study of estrogenic agonists bearing dicarba-closo-dodecaborane. Effect of geometry and separation distance of hydroxyl groups at the ends of molecules.

Dicarba-closo-dodecaboranes (carboranes), which have spherical geometry and hydrophobicity, are applicable as a hydrophobic pharmacophore of biologically active molecules. We have investigated structure-activity relations based on the structure of the potent estrogenic agonist, 1-hydroxymethyl-12-(4-hydroxyphenyl)-1,12-dicarba-closo-d odecaborane, which we have previously reported. The geometry and separation distance of the phenolic and alcoholic hydroxyl groups play a critical role in the appearance of biological activity.

Animals↗
PubMed1999

Protein kinase C modulators bearing dicarba-CLOSO-dodecaborane as a hydrophobic pharmacophore.

The size and position of a hydrophobic moiety on a benzolactam skeleton, which reproduces the active conformation and biological activity of teleocidins, play an important role in the appearance of the activity. We have designed and synthesized benzolactams bearing dicarba-closo-dodecaborane. These compounds showed potent binding affinity to protein kinase C, providing a further example of the application of carborane as the hydrophobic pharmacophore of biologically active molecules.

Boron Compounds↗
PubMed1999

Synthesis, computer modeling and biological evaluation of novel protein kinase C agonists based on a 7-membered lactam moiety.

4-Hydroxymethyl-5a-methyl-1,3,4,5,5a beta,6,7,8,9,9a alpha-decahydro-2H-benz[d]azepin-2-ones (4-12), which were designed to mimic the biologically active conformation of teleocidins and benzolactams, were synthesized and evaluated for the ability to compete with [3H]phorbol 12,13-dibutyrate in a PKC delta binding assay. Among the compounds, 10-12 showed potent binding affinity, with inhibition constants (Ki) of low nanomolar order. Computational docking simulation also indicates that the relative positions of the hydrogen-bonding sites and hydrophobic regions of the compounds are well matched to the PKC delta binding site.

Benzazepines↗
PubMed1999

Identification of 1-aminocyclopropane-1-carboxylic acid synthase genes controlling the ethylene level of ripening fruit in Japanese pear (Pyrus pyrifolia Nakai).

The shelf life of Japanese pear fruit is determined by its level of ethylene production. Relatively high levels of ethylene reduce storage potential and fruit quality. We have identified RFLP markers tightly linked to the locus that determines the rate of ethylene evolution in ripening fruit of the Japanese pear. The study was carried out using sequences of two types of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase genes (PPACS1 and pPPACS2) and a ACC oxidase gene (PPAOX1) as probes on 35 Japanese pear cultivars expressing different levels of ethylene (0.0 to approximately 300 microl/kg fresh weight/h) in ripening fruit. When total DNA was digested with HindIII and probed with pPPACS1, we identified a band of 2.8 kb which was specific to cultivars having very high ethylene levels (> or = 10 microl/kg f.w./h) during fruit ripening. The probe pPPACS2 identified a band of 0.8 kb specific to cultivars with moderate ethylene levels (0.5 microl/kg f.w./h-10 microl/kg f.w./h) during fruit ripening. The cultivars that produce high levels of ethylene possess at least one additional copy of pPPACS1 and those producing moderate levels of ethylene have at least one additional copy of pPPACS2. These results suggest that RFLP analysis with different ACC synthase genes could be useful for predicting the maximum ethylene level during fruit ripening in Japanese pear.

Amino Acid Oxidoreductases↗
PubMed1998

A new method for predicting binding free energy between receptor and ligand.

A practical method to estimate binding free energy, deltaG(bind), of a given ligand structure to the target receptor has been developed. The method assumes that deltaG(bind) is given by the summation of intermolecular interaction energy, deltaG(inter), and partial desolvation energy, deltaG(desolv). DeltaG(desolv) is calculated from the buried surface area in the complex between the ligand and receptor, based on solvation energy, deltaG(solv), formulated by an equation which can be calibrated with observed values. Then, the method was applied to arabinose-binding protein (ABP) and dihydrofolate reductase (DHFR), after recalibrating the weights for deltaG(inter) and each term of deltaG(desolv) using observed deltaG(bind) data for 29 known ligands to avidin (AV). The usefulness of our method was confirmed by the fact that correlation coefficients between the calculated and observed deltaG(bind)'s in AV, ABP and DHFR were 0.92, 0.77, and 0.88, whereas the corresponding values obtained by simple force field calculation were 0.79, 0.30, and 0.79, respectively. Further investigations to improve the method and validate the parameters are in progress.

Animals↗
PubMed1998

Clarification of the binding mode of teleocidin and benzolactams to the Cys2 domain of protein kinase Cdelta by synthesis of hydrophobically modified, teleocidin-mimicking benzolactams and computational docking simulation.

Phorbol esters (12-O-tetradecanoylphorbol 13-acetate; TPA) and teleocidins are known to be potent tumor promoters and to activate protein kinase C (PKC) by binding competitively to the enzyme. The relationship between the chemical structures and the activities of these compounds has attracted much attention because of the marked structural dissimilarities. The benzolactam 5, with an eight-membered lactam ring and benzene ring instead of the nine-membered lactam ring and indole ring of teleocidins, reproduces the active ring conformation and biological activities of teleocidins. Herein we describe the synthesis of benzolactams with hydrophobic substituents at various positions. Structure-activity data indicate that the existence of a hydrophobic region between C-2 and C-9 and the steric factor at C-8 play critical roles in the appearance of biological activities. We also computationally simulated the docking of teleocidin and the modified benzolactam molecules to the Cys2 domain structure observed in the crystalline complex of PKCdelta with phorbol 13-acetate. Teleocidin and benzolactams fitted well into the same cavity as phorbol 13-acetate. Of the three functional groups hydrogen-bonding to the protein, two hydrogen-bonded with protein atoms in common with phorbol 13-acetate, but the third one hydrogen-bonded with a different protein atom from that in the case of phorbol 13-acetate. The model explains well the remarkable difference in activity between 5 and its analogue having a bulky substituent at C-8.

Binding Sites↗
PubMed1998

Proton transfer in benzyl alcohol dehydrogenase during catalysis: alternate proton-relay routes.

His51 in horse liver alcohol dehydrogenase (ADHE) has been proposed to act as a proton donor/acceptor in the NAD+/NADH-dependent oxidation/reduction of alcohol/aldehyde. The residue corresponding to His51 of ADHE is Val51 (Val45 in the protein sequence) in benzyl alcohol dehydrogenase (BADH) encoded by TOL plasmid pWW0. The 3-D structure of BADH modeled from the crystal structure of ADHE suggests that His47 (His41 in the protein sequence, corresponding to Arg47 in ADHE) of BADH would play the role of His51 in ADHE. To test this hypothesis, mutants of BADH, in which His47 was replaced by Gln(His47Gln) and/or Val51 was replaced by His (Val51His), were constructed. The kcat/K(m) value of the His47Gln mutant for benzyl alcohol was 125-fold lower than that of wild-type BADH, while the kcat/K(m) value of the His47Gln/Val51His double mutant was 12-fold higher than that of the His47Gln mutant. The kcat/K(m) value of the His47Gln mutant increased with increasing concentration of exogenous amines. These results suggest that His47 in wild-type BADH, exogenous amines in the His47Gln mutant, and His51 in the double mutant act as a general base catalyst during alcohol oxidation.

Alcohol Oxidoreductases↗
PubMed1998

Novel thiazolidinedione derivatives with retinoid synergistic activity.

Several arylmethylidene thiazolidinediones were synthesized and their retinoidal activities were examined. TZ181 (7a), having a benzanilide skeleton, exhibited differentiation-inducing activity in HL-60 cell assay, while TZ191 (7b), the N-methylated analog of TZ181 (7a), TZ245 (9) and TZ335 (10) acted as retinoid synergists like the RXR-selective ligand, LGD1069 (5).

Cell Differentiation↗