Biomedical subjects

Miki Akamatsu

Publications and source records attributed to Miki Akamatsu.

At least 19 recordsLinked to original sources

PubMed2006

Structural basis for androgen receptor agonists and antagonists: interaction of SPEED 98-listed chemicals and related compounds with the androgen receptor based on an in vitro reporter gene assay and 3D-QSAR.

The androgen receptor (AR) activity of listed chemicals, so called SPEED 98, by the Ministry of the Environment, Japan, and structurally related chemicals was characterized using MDA-kb2 human breast cancer cells stably expressing an androgen-responsive luciferase reporter gene, MMTV-luc. Since our results suggested that chemicals with diverse chemical structures were capable of disrupting the endocrine systems mediated by AR, a comparative molecular field analysis (CoMFA) model was developed to analyze the structural requirements necessary to disrupt AR function. A significant CoMFA model with r(2)=0.825 and q(2)=0.332 was developed for AR antagonist activity of 35 pure antagonists excluding procymidone. On the other hand, a good CoMFA model with r(2)=0.983 and q(2)=0.555 was obtained for antagonist activity of 13 chemicals with both agonist and antagonist activities. The steric and electrostatic properties were sufficient to describe the structural requirements for AR antagonist activity. In addition, the structural difference of AR agonists and antagonists was explained based on CoMFA results and the AR-LBD crystal structure. As several ERalpha agonists such as diethylstilbestrol (DES) acted as AR antagonists, the surface area of the AR ligand-binding domain (LBD) was compared with that of the ERalpha-LBD based on their reported crystal structures to analyze how those ligands interact with LBDs. The surface area of AR-LBD was shown to be smaller than that of ERalpha-LBD and therefore compounds with both estrogenic and antiandrogenic activities can fit well into the ERalpha-LBD but may protrude from the AR-LBD. It is likely that this subtle difference of the surface areas of the LBDs determines whether an ERalpha agonist acts as an AR antagonist or an agonist.

Androgen Antagonists↗

PubMed2006

Role in the selectivity of neonicotinoids of insect-specific basic residues in loop D of the nicotinic acetylcholine receptor agonist binding site.

The insecticide imidacloprid and structurally related neonicotinoids act selectively on insect nicotinic acetylcholine receptors (nAChRs). To investigate the mechanism of neonicotinoid selectivity, we have examined the effects of mutations to basic amino acid residues in loop D of the nAChR acetylcholine (ACh) binding site on the interactions with imidacloprid. The receptors investigated are the recombinant chicken alpha4beta2 nAChR and Drosophila melanogaster Dalpha2/chicken beta2 hybrid nAChR expressed in Xenopus laevis oocytes. Although mutations of Thr77 in loop D of the beta2 subunit resulted in a barely detectable effect on the imidacloprid concentration-response curve for the alpha4beta2 nAChR, T77R;E79V double mutations shifted the curve dramatically to higher affinity binding of imidacloprid. Likewise, T77K;E79R and T77N;E79R double mutations in the Dalpha2beta2 nAChR also resulted in a shift to a higher affinity for imidacloprid, which exceeded that observed for a single mutation of Thr77 to basic residues. By contrast, these double mutations scarcely influenced the ACh concentration-response curve, suggesting selective interactions with imidacloprid of the newly introduced basic residues. Computational, homology models of the agonist binding domain of the wild-type and mutant alpha4beta2 and Dalpha2beta2 nAChRs with imidacloprid bound were generated based on the crystal structures of acetylcholine binding proteins of Lymnaea stagnalis and Aplysia californica. The models indicate that the nitro group of imidacloprid interacts directly with the introduced basic residues at position 77, whereas those at position 79 either prevent or permit such interactions depending on their electrostatic properties, thereby explaining the observed functional changes resulting from site-directed mutagenesis.

Acetylcholine↗

PubMed2006

Estimation of the hydrophobicity of 2,4-diphenyl-1,3-oxazoline analogs and QSAR analysis of their ovicidal activity against Tetranychus [corrected] urticae.

Partition coefficients of six 2-phenyl-1,3-oxazoline congeners containing 2-I, 2-NO2, 2-CF3, 2,6-(CH3)2, 2,6-F2, and 2-F-6-Cl substitutions on the phenyl moiety were measured in a 1-octanol/water system using the flask-shaking method. The effect on the hydrophobicity (LogP) of substituents on the phenyl moiety of 2-phenyl-1,3-oxazolines linearly correlated with that of benzamide congeners. logP values of other 2-(substituted phenyl)-1,3-oxazoline analogs were empirically estimated from the corresponding substituted benzamides. The ovicidal activity of 2-(substituted phenyl)-4-phenyl-1,3-oxazoline analogs against the two-spotted spider mite Tetranychus [corrected] urticae was quantitatively analyzed using the classical QSAR (Hansch-Fujita) method. Results showed that ovicidal activity increases with hydrophobicity. The introduction of inductive electron-withdrawing groups at ortho-positions increased ovicidal activity, but addition of steric bulk was unfavorable. Substitution at either the meta- or para-position was detrimental to the acaricidal activity.

Animals↗

PubMed2005

Synthesis of 26,27-bisnorcastasterone analogs and analysis of conformation-activity relationship for brassinolide-like activity.

Three castasterone (CS) derivatives with varied side-chain moieties, 26,27-bisnorcastasterone (20S-bisnorCS), 20-epi-26,27-bisnorcastasterone (20R-bisnorCS), and 21,26,27-trisnorcastasterone (trisnorCS), were synthesized stereoselectively from either stigmasterol or dehydroisoandrosterone. The 50% effective doses (ED50, nmol/plant) in the concentration-response curve for brassinolide-like activity in the rice lamina inclination assay were determined to be 0.020 nmol (pED50 = 10.7) for 20S-bisnorCS, 3.2 nmol (pED50 = 8.5) for 20R-bisnorCS, and 2.0 nmol (pED50 = 8.7) for trisnorCS. An analog containing an ester linkage between the steroid and the side-chain moiety of 20S-bisnorCS was also synthesized and its activity was evaluated to be 3.2 nmol (pED50 = 8.5), being equipotent to 20R-bisnorCS and trisnorCS. The activity of 20S-bisnorCS was 1/40 that of CS. The conformation analysis was conducted using a systematic search, showing that the activity decreases with an increase in the degree of freedom of the side chain of the steroidal skeleton.

Brassinosteroids↗

PubMed2005

High-throughput screening of ecdysone agonists using a reporter gene assay followed by 3-D QSAR analysis of the molting hormonal activity.

In this study, 172 diacylhydrazine analogs were examined for their ability to activate an ecdysone (molting hormone)-dependent reporter gene in a silkworm (Bombyx mori) cell-based high-throughput screening assay. The measured EC(50) values (concentration required to cause an effect in 50% of the cells) were used to construct a 3-D QSAR model that describes the ecdysone agonist activities of the diacylhydrazine analogs. Of these compounds, 14 exhibited no activity and were excluded from the 3-D QSAR analysis. The resulting equation described approximately 74% of the activity for 158 compounds. The final equation consisted of 42% electrostatic and 58% steric effects (r(2) = 0.74 and q(2) = 0.45). Comparative molecular field analysis (CoMFA) was used to visualize the steric and electrostatic potential fields that were favorable and unfavorable for biological activity. Of particular interest was the observation that the hydrophobic parameter (logP) was not necessary for describing the observed activities, although previous studies have cited the importance of hydrophobic parameters in both classical and 3-D QSAR analyses of these compounds. Modeling studies of the B. mori ecdysone receptor supported the observed physicochemical parameters required for activity reported by the CoMFA models. Comparison of the present analysis with those performed using other lepidopteran assay systems evidenced a high degree of correlation (r(2) = 0.81 for a Sf-9 cell-based assay and r(2) = 0.89 for a Chilo suppressalis integument-based assay), indicating that it is valid to compare the results generated with the B. mori cell-based system to those generated with previous lepidopteran assays. This novel assay system is amendable to a high-throughput screening format and should greatly increase our ability to discover novel agonists of molting hormone (ecdysone) activity.

Amino Acid Sequence↗

PubMed2005

Relationships between structure and high-throughput screening permeability of diverse drugs with artificial membranes: application to prediction of Caco-2 cell permeability.

To evaluate the absorption of drugs with diverse structures across a membrane via the transcellular route, their permeability was measured using the parallel artificial membrane permeation assay (PAMPA). The permeability coefficients obtained by PAMPA were analyzed using a classical quantitative structure-activity relationship (QSAR) approach with simple physicochemical parameters and 3D-QSAR, VolSurf. We formulated correlation equations for diverse drugs similar to the equation obtained for peptide-related compounds in our previous study. The hydrogen-bonding ability of molecules, not only the hydrogen-accepting ability but also the hydrogen-donating ability, in addition to hydrophobicity at a particular pH, was significant in determining variations in PAMPA permeability coefficients. Based on this result, an in silico good prediction model for the passive transcellular permeability of diverse structural compounds was obtained. The artificial lipid-membrane permeability coefficients of the drugs, except salicylic acid, were well correlated with the Caco-2 permeability in a previous report suggesting the importance of absorption by the transcellular mechanism for these drugs.

Caco-2 Cells↗

PubMed2005

Quantitative structure-activity relationship studies for antioxidant hydroxybenzalacetones by quantum chemical- and 3-D-QSAR(CoMFA) analyses.

Antioxidant activities for a series of hydroxybenzalacetones, OH-BZ, evaluated by their inhibitory potencies against lipid peroxidation induced by gamma-ray irradiation or t-BuOOH, were analyzed quantitatively using quantum-chemical parameters calculated by semi-empirical molecular orbital (MO) calculations. The energy of the highest occupied molecular orbital (E(HOMO)) and frontier electron densities (HOMO) on the phenolic oxygen atom (F(H,O)), together with the steric parameter (E(s)) for the substituent ortho to the phenolic oxygen, showed excellent correlations. We also performed 3D-QSAR studies by using the comparative molecular field analysis (CoMFA) model. The results were compared with the corresponding classical QSAR correlations.

Acetone↗

PubMed2005

Surface plasmon resonance-based immunoassay for 17beta-estradiol and its application to the measurement of estrogen receptor-binding activity.

A rapid and simple surface plasmon resonance (SPR)-based immunoassay for detection of 17beta-estradiol was developed. The assay was designed as an inhibitive format, in which 17beta-estradiol-BSA conjugates are immobilized on an SPR sensor chip and the binding of antibody to the chip is measured. The binding was inhibited by 17beta-estradiol in the concentration range 0.468 to 21.4 nmol L-1 with an IC50 value of 2.29+/-0.10 nmol L-1. Although not as sensitive as traditional radioimmunoassay (RIA) and enzyme-linked immunoassay (ELISA), this method requires no separation and washing after addition of the antibody, steps which are relatively time-consuming. Estrogen receptor (ER)-binding was then investigated using this SPR immunoassay for the determination of the amount of unbound 17beta-estradiol after competition with test compounds for the ER-binding. Inhibition of the binding of 17beta-estradiol to ER by diethylstilbestrol (DES) was successfully measured by injecting the reaction mixture into the SPR sensor after addition of the antibody. This binding assay requires no separation of unbound 17beta-estradiol from the mixture and no radioisotope- or fluorescence-labeling of 17beta-estradiol. These results show the potential usefulness of the SPR sensor both detecting 17beta-estradiol and evaluating the ER-binding activity of xenoestrogens such as DES in a single assay system.

Animals↗

PubMed2005

Neonicotinoids show selective and diverse actions on their nicotinic receptor targets: electrophysiology, molecular biology, and receptor modeling studies.

Neonicotinoid insecticides, which act selectively on insect nicotinic acetylcholine receptors (nAChRs), are used worldwide for insect pest management. Studies that span chemistry, biochemistry, molecular biology, and electrophysiology have contributed to our current understanding of the important physicochemical and structural properties essential for neonicotinoid actions as well as key receptor residues contributing to the high affinity of neonicotinoids for insect nAChRs. Research to date suggests that electrostatic interactions and possibly hydrogen bond formation between neonicotinoids and nAChRs contribute to the selectivity of these chemicals. A rich diversity of neonicotinoid-nAChR interactions has been demonstrated using voltage-clamp electrophysiology. Computational modeling of nAChR-imidacloprid interaction has assisted in the interpretation of these results.

Amino Acid Motifs↗

PubMed2004

Relationship between structure and permeability of dipeptide derivatives containing tryptophan and related compounds across human intestinal epithelial (Caco-2) cells.

The permeability of dipeptide derivatives containing tryptophans and indole derivatives through Caco-2 cells was used as an in vitro intestinal absorption model in order to clarify structural factors which influence their intestinal epithelial permeation and metabolism. Most peptide derivatives were hydrolysed not only by the cytosolic enzymes in Caco-2 cells during permeation but also by enzymes released to the apical solution before cell permeation. The N-terminal blocked dipeptides were more resistant to hydrolases expressed in the Caco-2 cells and indole derivatives were not entirely degraded. Based on compound concentration dependency and comparison of permeability coefficients in apical-to-basolateral and basolateral-to-apical directions, the main absorption mechanism of compounds were determined. Compounds were then classified into three groups; (1) passively transported compounds, (2) actively transported compounds and (3) compounds excreted by P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Relationships between structure and high-throughput screening permeability of peptide derivatives and related compounds with artificial membranes: application to prediction of Caco-2 cell permeability.

To evaluate absorption of compounds across the membrane via a transcellular route, the permeability of peptide derivatives and related compounds was measured by the parallel artificial membrane permeation assay (PAMPA). The permeability coefficients by PAMPA were analyzed quantitatively using classical QSAR and Volsurf approaches with the physicochemical parameters. The results from both approaches showed that hydrogen bonding ability of molecules in addition to hydrophobicity at a particular pH were significant in determining variations in PAMPA permeability coefficients. The relationship between Caco-2 cell permeability and artificial lipid membrane permeability was then determined. The compounds were sorted according to their absorption pathway in the plot of the Caco-2 cell and PAMPA permeability coefficients.

Caco-2 Cells↗

PubMed2004

Two- and three-dimensional QSAR of carrier-mediated transport of beta-lactam antibiotics in Caco-2 cells.

In this study, we investigated whether such a topological descriptor-based approach is suitable for predicting the carrier-mediated transport of 20 beta-lactam antibiotics that are substrates of peptide transporters. To select the molecular descriptors that can effectively predict a targeted property in QSAR analysis, the genetic algorithm-combined partial least squares approach was used. The feasibility of the two-dimensional (2D)-QSAR approach was compared with that of comparative molecular field analysis (CoMFA). The logarithm of the uptake values of 20 beta-lactam antibiotics in Caco-2 cells obtained from the literature ranged from -1.15 to 1.09 (nmol/cm2/2 h). When preliminary leave-one-out cross-validated partial least squares analyses implemented in the SYBYL/CoMFA program were conducted, the r2pred was 0.759 and the standard error of prediction (s) was 0.373. However, the 2D-QSAR approach based on Molconn-Z descriptors gave a better predictability (r2pred = 0.923, s = 0.211), where 14 descriptors were selected and the optimal number of principal components was 4. Considering that the 2D-topological descriptors are less computationally intensive and practically completely automated, the simple 2D-QSAR model is also of great importance in drug discovery settings.

Anti-Bacterial Agents↗

PubMed2004

Enantioselective recognition of mono-demethylated methoxychlor metabolites by the estrogen receptor.

Metabolites of methoxychlor such as 2-(p-hydroxyphenyl)-2-(p-methoxyphenyl)-1,1,1-trichloroethane (mono-OH-MXC) and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (bis-OH-MXC), have estrogenic activity. Mono-OH-MXC is a chiral compound in which the carbon atom bridging two benzene rings is the chiral centre. In previous studies the estrogenic activity of racemic mono-OH-MXC has been measured, and the activity of each enantiomer of this compound has not yet been elucidated. In this study, we evaluated the estrogen receptor-binding activity of each enantiomer of mono-OH-MXC to clarify the enantioselective recognition by the estrogen receptor. (S)-mono-OH-MXC showed 3-fold higher binding activity than that of the (R) enantiomer. The activity of bis-OH-MXC was only 1.7-fold higher than that of (S)-mono-OH-MXC. This result suggests that the one hydroxy group and the orientation of the CCl3 group of mono- and bis-OH-MXCs are important for the interaction with the estrogen receptor. The result also points out the estrogenic activity of methoxychlor after metabolic activation in vivo, which predominantly produces the (S)-mono-OH-MXC, may be higher than estimated from the in vitro activity of racemic mixtures.

Estradiol↗

PubMed2003

Combinatorial mutations in loops D and F strongly influence responses of the alpha7 nicotinic acetylcholine receptor to imidacloprid.

The nitro group of a neonicotinoid, imidacloprid, plays a key role in its selective actions on insect nicotinic acetylcholine receptors (nicotinic AChRs) and is postulated to bind close to residues Q79 in loop D and G189 in loop F of the chicken alpha7 nicotinic AChR. To evaluate the relative contributions of these residues to interactions with imidacloprid, Q79 and G189 were replaced in tandem by first basic then acidic residues. Changes in the currents evoked by imidacloprid and acetylcholine (ACh) on the alpha7 wild type and mutant receptors expressed in Xenopus laevis oocytes were investigated using two-electrode voltage clamp electrophysiology. An increase in the efficacy of imidacloprid for the alpha7 receptor resulting from the Q79K and Q79R mutations was suppressed by a G189E mutation in loop F. However, the increases in efficacy resulting from such Q79 mutations were scarcely influenced by a G189D substitution. Three-dimensional modeling of the alpha7 nicotinic AChR, based on the acetylcholine-binding protein (AChBP) of Lymnaea stagnalis, suggests that the reduced efficacy of imidacloprid following the G189E mutation is likely to result from carboxylate interference with the electronic interactions between the nitro group of imidacloprid and the basic residues in loop D.

Animals↗

PubMed2003

Use of classical and 3-D QSAR to examine the hydration state of juvenile hormone esterase inhibitors.

Carboxylesterases are important enzymes in the metabolism of numerous pharmaceuticals and agrochemicals. They are of importance in many detoxification pathways, but their endogenous role remains unclear. The most potent esterase inhibitors found to date are trifluoromethylketone (TFK) containing compounds, which have been shown to inhibit both mammalian and insect esterases at the low nM level. The detailed mechanism by which these compounds inhibit the enzyme is still unclear. They are highly hydrated in aqueous solutions, but their mechanism of inhibition suggests that inhibition occurs through the ketone, not the hydrated gem-diol. Some studies have stated that the ketone is the inhibitor, while others have reported the gem-diol as the active form. Using juvenile hormone esterase (JHE) as a model system, we examined this question using both classical QSAR and 3-D QSAR with comparative molecular field analysis (CoMFA). Classical QSAR analyses demonstrated the high dependence of inhibitor potency upon log P as well as the limitations of sterically unfavorable substituents. The ketone form of the inhibitor consistently provided improved correlations over the gem-diol, with the final equations describing 72 and 69% of inhibitor activity, respectively, for 97 compounds. Initial CoMFA analyses for the ketone provided a significant equation for 108 compounds (q(2)=0.412, m=6); however all cross-validated values for the gem-diol form of the inhibitors were not statistically significant (q(2)<0.3). Inclusion of hydrophobicity descriptors in both CoMFA equations increased their significance; however the final ketone equation (q(2)=0.500, m=7) was still statistically improved over the gem-diol (q(2)=0.506, m=8). These results support those obtained for the classical QSAR analysis and further illustrate the importance of log P in the inhibition mechanism of these inhibitors. The CoMFA models also identified novel target areas for the synthesis of new JHE inhibitors. These results suggest that the ketone is the active form of TFK-containing inhibitors.

Carboxylic Ester Hydrolases↗

PubMed2003

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.

Circular Dichroism↗

PubMed2003

Correlations of the electrophysiological activity of neonicotinoids with their binding and insecticidal activities.

The electrophysiological actions of various neonicotinoids, including substituted benzyl derivatives, against recombinant Drosophila SAD/chicken beta2 hybrid nicotinic acetylcholine receptor (nAChR) were measured to analyze the relationships between the in vivo (insecticidal) and in vitro (binding and agonist) activities. Most of the neonicotinoids tested were capable of inducing inward currents by activating the hybrid nAChRs expressed in Xenopus laevis oocytes, whereas some compounds had no agonist activity and only blocked the acetylcholine-induced currents. Variations in the agonist activity were well correlated with those in the binding potency evaluated using [3H]imidacloprid as well as insecticidal activities.

Algorithms↗

PubMed2003

Diverse actions of neonicotinoids on chicken alpha7, alpha4beta2 and Drosophila-chicken SADbeta2 and ALSbeta2 hybrid nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes.

The 2-nitroimino-imidazolidine and related moieties are structural features of neonicotinoid insecticides acting on nicotinic acetylcholine receptors (nicotinic AChRs). To evaluate these moieties in neonicotinoid interactions with nicotinic AChR alpha subunits, the actions of imidacloprid and related compounds on the chicken alpha7, alpha4beta2 and Drosophila melanogaster-chicken hybrid (SADbeta2 and ALSbeta2) receptors expressed in Xenopus laevis oocytes were studied by voltage-clamp electrophysiology. Imidacloprid and nitenpyram were partial agonists and a nitromethylene analog of imidacloprid (CH-IMI) was a full agonist of the alpha7 receptor, whereas their agonist actions on the alpha4beta2 receptor were very weak, contrasting with full agonist actions of DN-IMI, a desnitro derivative of imidacloprid. The neonicotinoids and DN-IMI were either full or partial agonists of the SADbeta2 receptors. Nitenpyram and DN-IMI were partial agonists of the ALSbeta2 receptor, whereas imidacloprid and CH-IMI scarcely activated the ALSbeta2 receptor. Imidacloprid and CH-IMI in fact suppressed ACh-induced responses of the ALSbeta2 receptor, whereas imidacloprid potentiated and CH-IMI suppressed ACh-induced responses of the alpha4beta2 receptor. These results suggest that interactions with alpha subunits of the 2-nitroimino-imidazolidine moiety of imidacloprid play a role in determining not only agonist and antagonist actions on all four receptors, but also the potentiation of ACh-induced responses of the alpha4beta2 receptor.

Amino Acid Sequence↗