Broad Chain-Length Specificity of the Alkane-Forming Enzymes NoCER1A and NoCER3A/B in Nymphaea odorata.

Many terrestrial plants produce large quantities of alkanes for use in epicuticular wax and the pollen coat. However, their carbon chains must be long to be useful as fuel or as a petrochemical feedstock. Here, we focus on Nymphaea odorata, which produces relatively short alkanes in its anthers. We identified orthologs of the Arabidopsis alkane biosynthesis genes AtCER1 and AtCER3 in N. odorata and designated them NoCER1A, NoCER3A and NoCER3B. Expression analysis of NoCER1A and NoCER3A/B in Arabidopsis cer mutants revealed that the N. odorata enzymes cooperated with the Arabidopsis enzymes and that the NoCER1A produced shorter alkanes than AtCER1, regardless of which CER3 protein it interacted with. These results indicate that AtCER1 frequently uses a C30 substrate, whereas NoCER1A, NoCER3A/B and AtCER3 react with a broad range of substrate chain lengths. The incorporation of shorter alkanes disturbed the formation of wax crystals required for water-repellent activity in stems, suggesting that chain-length specificity is important for surface cleaning. Moreover, cultured tobacco cells expressing NoCER1A and NoCER3A/B effectively produced C19-C23 alkanes, indicating that the introduction of the two enzymes is sufficient to produce alkanes. Taken together, our findings suggest that these N. odorata enzymes may be useful for the biological production of alkanes of specific lengths. 3D modeling revealed that CER1s and CER3s share a similar structure that consists of N- and C-terminal domains, in which their predicted active sites are respectively located. We predicted the complex structure of both enzymes and found a cavity that connects their active sites.

The DROL1 subunit of U5 snRNP in the spliceosome is specifically required to splice AT-AC-type introns in Arabidopsis.

An Arabidopsis mutant named defective repression of OLE3::LUC 1 (drol1) was originally isolated as a mutant with defects in the repression of OLEOSIN3 (OLE3) after seed germination. In this study, we show that DROL1 is an Arabidopsis homolog of yeast DIB1, a subunit of the U5 small nuclear ribonucleoprotein particle (snRNP) in the spliceosome. It is also part of a new subfamily that is specific to a certain class of eukaryotes. Comprehensive analysis of the intron splicing using RNA sequencing analysis of the drol1 mutants revealed that most of the minor introns with AT-AC dinucleotide termini had reduced levels of splicing. Only two nucleotide substitutions from AT-AC to GT-AG enabled AT-AC-type introns to be spliced in drol1 mutants. Forty-eight genes, including those having important roles in abiotic stress responses and cell proliferation, exhibited reduced splicing of AT-AC-type introns in the drol1 mutants. Additionally, drol1 mutant seedlings showed retarded growth, similar to that caused by the activation of abscisic acid signaling, possibly as a result of reduced AT-AC-type intron splicing in the endosomal Na+ /H+ antiporters and plant-specific histone deacetylases. These results indicate that DROL1 is specifically involved in the splicing of minor introns with AT-AC termini and that this plays an important role in plant growth and development.

Gateway binary vectors with organelle-targeted fluorescent proteins for highly sensitive reporter assay in gene expression analysis of plants.

Fluorescent proteins are valuable tools in the bioscience field especially in subcellular localization analysis of proteins and expression analysis of genes. Fusion with organelle-targeting signal accumulates fluorescent proteins in specific organelles, increases local brightness, and highlights the signal of fluorescent proteins even in tissues emitting a high background of autofluorescence. For these advantages, organelle-targeted fluorescent proteins are preferably used for promoter:reporter assay to define organ-, tissue-, or cell-specific expression pattern of genes in detail. In this study, we have developed a new series of Gateway cloning technology-compatible binary vectors, pGWBs (attR1-attR2 acceptor sites) and R4L1pGWB (attR4-attL1 acceptor sites), carrying organelle-targeted synthetic green fluorescent protein with S65T mutation (sGFP) (ER-, nucleus-, peroxisome-, and mitochondria-targeted sGFP) and organelle-targeted tag red fluorescent protein (TagRFP) (nucleus-, peroxisome-, and mitochondria-targeted TagRFP). These are available for preparation of promoter:reporter constructs by an LR reaction with a promoter entry clone attL1-promoter-attL2 (for pGWBs) or attL4-promoter-attR1 (for R4L1pGWBs), respectively. A transient expression experiment with particle bombardment using cauliflower mosaic virus 35S promoter-driven constructs has confirmed the correct localization of newly developed organelle-targeted TagRFPs by a co-localization analysis with the previously established organelle-targeted sGFPs. More intense and apparent fluorescence signals were detected by the nucleus- and peroxisome-targeted sGFPs than by the normal sGFPs in the promoter assay using transgenic Arabidopsis thaliana. The new pGWBs and R4L1pGWBs developed here are highly efficient and may serve as useful platforms for more accurate observation of GFP and RFP signals in gene expression analyses of plants.

Jasmonic acid facilitates flower opening and floral organ development through the upregulated expression of SlMYB21 transcription factor in tomato.

Plants coordinate the timing of flower opening with pollen and gynoecium maturation to achieve successful pollination. However, little is known about how the coordination is executed. We found that flower bud development was paused immediately before flower opening in a jasmonic acid (JA)-insensitive tomato mutant, jai1-1. Phytohormone measurement and RNA analysis in flower buds revealed that newly synthesised JA peaked at two days before flower opening and the expression of a transcription factor gene SlMYB21 delayed in jai1-1. Buds of transgenic tomato plants expressing an artificial repressor, AtMYB24-SRDX, which was expected to impede the function of SlMYB21, aborted flower opening and resembled those of jai1-1. Furthermore, the AtMYB24-SRDX plants produced abnormal pollen grains deficient in germination and pistils that did not support pollen tube elongation. We concluded that JA facilitates the expression of SlMYB21, which coordinates flower opening, pollen maturation, and gynoecium function in tomato.

EPIDERMAL PATTERNING FACTOR LIKE5 peptide represses stomatal development by inhibiting meristemoid maintenance in Arabidopsis thaliana.

Stomatal development in Arabidopsis epidermis is both positively and negatively regulated by a family of Cys-rich peptides, EPIDERMAL PATTERNING FACTOR LIKEs (EPFLs). We synthesized biologically active synthetic EPFL5 (sEPFL5) peptide, which reduced the number of stoma in leaves and cotyledons. The sEPFL5 possesses three disulfide bonds at positions identical to those of a positive development factor, stomagen. Application of sEPFL5 had little inhibitory effect on protodermal cells entering the stomatal lineage, but did inhibit the maintenance of meristemoid activity, resulting in the differentiation of arrested meristemoids into pavement cells. This phenotype was enhanced in the too many mouths (tmm) mutant background. RNA analysis revealed that sEPFL5 application halved SPEECHLESS expression and abolished MUTE expression in tmm mutants, explaining the phenotype observed. The action of sEPFL5 was mediated by ERECTA family receptors. We propose that EPFL5 functions to establish the differentiation of stomatal lineage cells to pavement cells.

Gateway binary vectors with the bialaphos resistance gene, bar, as a selection marker for plant transformation.

We constructed two series of Gateway binary vectors, pGWBs and R4pGWBs, possessing the bialaphos resistance gene (bar) as a selection marker for plant transformation. The reporters and tags employed in this system are sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, TagRFP, 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7 and TAP. Selection of Arabidopsis transformants with BASTA was successfully carried out using both plate-grown and soil-grown seedlings. Transformed rice calli and suspension-cultured tobacco cells were selected on plates containing BASTA or glufosinate-ammonium. These vectors are compatible with existing pGWB and R4pGWB vectors carrying kanamycin and hygromycin B resistance.

Synthesis and biological evaluation of novel phthalazinone derivatives as topically active phosphodiesterase 4 inhibitors.

Inhibitors of phosphodiesterase 4 (PDE4) are an important class of anti-inflammatory drug that act by inhibiting the production of proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha). We have synthesized and evaluated a series of 2-substituted phthalazinone derivatives as PDE4 inhibitors. Structure-activity relationship studies led to the identification of benzylamine-substituted phthalazinones as potent PDE4 inhibitors that also suppressed TNF-alpha production by whole rat blood cells. The most potent of these, when topically administered, were effective in a mouse model of dermatitis.

Discovery and structure-activity relationships of 4-aminoquinazoline derivatives, a novel class of opioid receptor like-1 (ORL1) antagonists.

Synthesis and structure-activity relationship studies of a series of 4-aminoquinazoline derivatives led to the identification of (1R,2S)-17, N-[(1R,2S)-2-({2-[(4-chlorophenyl)carbonyl]amino-6-methylquinazolin-4-yl}amino)cyclohexyl]guanidine dihydrochloride, as a highly potent ORL1 antagonist with up to 3000-fold selectivity over the mu, delta, and kappa opioid receptors. Molecular modeling clarified the structural factors contributing to the high affinity and selectivity of (1R,2S)-17.

Comparison of imatinib, dasatinib, nilotinib and INNO-406 in imatinib-resistant cell lines.

We compared the growth-inhibitory effects and inhibition profile of the SRC family kinases (SFKs) of imatinib, dasatinib, nilotinib and INNO-406. Dasatinib exhibited the strongest potency against BCR-ABL with little selectivity over SFKs. Nilotinib exhibited a weaker affinity than the other inhibitors, but was highly specific for ABL and may be useful for the treatment of P-glycoprotein overexpressing leukemic cells. INNO-406 had an intermediate affinity for BCR-ABL between that of dasatinib and nilotinib, and inhibited only SFKs LCK and LYN among SFKs. Both nilotinib and INNO-406 were potent inhibitors of the dasatinib-resistant T315A, F317L and F317V BCR-ABL mutations.

Structure-activity relationship study on the 6-membered heteroaromatic ring system of diphenylpyrazine-type prostacyclin receptor agonists.

A series of prostacyclin receptor agonists was prepared by modifying the central heteroaromatic ring of lead compound 2, and a docking study was performed to investigate their structure-activity relationships by using a homology-modeled structure of the prostacyclin receptor. Compound 2 and its derivatives could be docked to the prostacyclin receptor in two ways depending on the position of the nitrogen atom within the heteroaromatic ring. Furthermore, hydrogen bonding between the nitrogen atom in the heteroaromatic ring and the hydroxyl group of Ser20 or Tyr75 of the receptor appears to be important for the potent expression of biological activity.

Structural factors contributing to the Abl/Lyn dual inhibitory activity of 3-substituted benzamide derivatives.

To investigate why 3-substituted benzamide derivatives show dual inhibition of Abl and Lyn protein tyrosine kinases, we determined their inhibitory activities against Abl and Lyn, carried out molecular modeling, and conducted a structure-activity relationship study with the aid of a newly determined X-ray structure of the Abl/Lyn dual inhibitor INNO-406 (formerly known as NS-187) bound to human Abl. We found that this series of compounds interacted with both kinases in very similar ways, so that they can inhibit both kinases effectively.

NS-187 (INNO-406), a Bcr-Abl/Lyn dual tyrosine kinase inhibitor.

Protein kinases catalyze the transfer of the gamma-phosphoryl group of adenosine triphosphate (ATP) to the hydroxyl groups of protein side chains, and they play critical roles in regulating cellular signal transduction and other biochemical processes. They are attractive targets for today's drug discovery and development, and many pharmaceutical companies are intensively developing various kinds of protein kinase inhibitors. A good example is the recent success with the Bcr-Abl tyrosine kinase inhibitor imatinib mesylate (Gleevec) in the treatment of chronic myeloid leukemia. Though imatinib has dramatically improved the treatment of Bcr-Abl-positive chronic myeloid leukemia, resistance is often found in patients with advanced-stage disease. Several mechanisms have been proposed to explain this resistance, including point mutations within the Abl kinase domain, amplification of the bcr-abl gene, overexpression of the corresponding mRNA, increased drug efflux mediated by P-glycoprotein, and activation of the Src-family kinase (SFK) Lyn. We set out to develop a novel drug whose affinity for Abl is higher than that of imatinib and whose specificity in inhibiting Lyn is higher than that of SFK/Abl inhibitors such as dasatinib (Sprycel) or bosutinib (SKI-606). Our work has led to the development of NS-187 (INNO-406), a novel Abl/Lyn dual tyrosine kinase inhibitor with clinical prospects. To provide an overview of how a selective kinase inhibitor has been developed, this review presents chemical-modification studies carried out with the guidance of molecular modeling, the structural basis for the high potency and selectivity of NS-187 based on the X-ray structure of the NS-187/Abl complex, and the biological profiling of NS-187, including site-directed mutagenesis experiments.

Elucidation of characteristic structural features of ligand binding sites of protein kinases: a neural network approach.

Protein kinases play important roles in regulating cellular signal transduction and other biochemical processes, and they are attractive targets for drug discovery programs in many disease areas. Most kinase inhibitors under development as drugs act by directly competing with ATP at the ATP-binding site of the kinase. There are more than 500 protein kinases, and the ATP-binding site is highly conserved among them. Therefore selectivity is an essential requirement for clinically effective drugs, and understanding the structural characteristics of ATP-binding sites is of crucial importance. The objective of the present study was to elucidate the structural characteristics of the adenosine-binding site of four major kinase groups, AGC (PKA, PKG, and PKC families), CaMK (calcium/calmodulin-dependent protein kinases), CMGC (CDK, MAPK, GSK3, and CLK families), and TK (tyrosine kinases). To do this, we classified the kinases into groups by using feed-forward multilayer perceptron (MLP) neural networks and structural, electronic, and hydrophobic descriptors of the amino acids at the adenosine-binding site. A total of 275 kinases were classified in two ways: (1) kinases belonging to a certain group were distinguished from those not belonging to that group, and (2) all of the kinases were classified into four groups. More than 85% of the kinases were correctly classified by both methods. Trained neural networks clarified which amino acids and which properties characterize the adenosine-binding site of each group, and the results were visualized by molecular graphics. Comparison of the modeled neural networks and the distributions of amino acids provided more detailed information on the structural characteristics of each group. Application of the present results to drug development is also discussed.

Quantitative structure-activity relationship studies on inhibition of HERG potassium channels.

The human ether-a-go-go-related gene (HERG) protein forms the ion channel responsible for the rapidly acting delayed rectifier potassium current, I(Kr), and its blockade is a significant contributor to prolongation of the QT interval. Using descriptors which have clear physicochemical meanings and are familiar to medicinal chemists, we have carried out 2D-quantitative structure-activity relationship (2D-QSAR) studies on 104 HERG channel blockers with diverse structures collected from the literature, and we have formulated interpretable models to guide chemical-modification studies and virtual screening. Statistically significant descriptors were selected by a genetic algorithm, and the final model included the octanol/water partition coefficient, topological polar surface area, diameter, summed surface area of atoms with partial charges from -0.25 to -0.20, and an indicator variable representing the experimental conditions. The statistics were r = 0.839, r2 = 0.704, q2 = 0.671, s = 0.763, and F = 46.6. The correspondence of the molecular determinants derived from the 2D-QSAR models with the 3D structural characteristics of the putative binding site in a homology-modeled HERG channel is also discussed.

Design and synthesis of 3-substituted benzamide derivatives as Bcr-Abl kinase inhibitors.

A series of 3-substituted benzamide derivatives structurally related to STI-571 (imatinib mesylate), a Bcr-Abl tyrosine kinase inhibitor used to treat chronic myeloid leukemia (CML), was prepared and evaluated for antiproliferative activity against the Bcr-Abl-positive leukemia cell line K562. About ten 3-halogenated and 3-trifluoromethylated benzamide derivatives were identified as highly potent Bcr-Abl kinase inhibitors. One of these, NS-187 (9b), is a promising new candidate Bcr-Abl inhibitor for the therapy of STI-571-resistant chronic myeloid leukemia.

NS-187, a potent and selective dual Bcr-Abl/Lyn tyrosine kinase inhibitor, is a novel agent for imatinib-resistant leukemia.

Although the Abelson (Abl) tyrosine kinase inhibitor imatinib mesylate has improved the treatment of breakpoint cluster region-Abl (Bcr-Abl)-positive leukemia, resistance is often reported in patients with advanced-stage disease. Although several Src inhibitors are more effective than imatinib and simultaneously inhibit Lyn, whose overexpression is associated with imatinib resistance, these inhibitors are less specific than imatinib. We have identified a specific dual Abl-Lyn inhibitor, NS-187 (elsewhere described as CNS-9), which is 25 to 55 times more potent than imatinib in vitro. NS-187 is also at least 10 times as effective as imatinib in suppressing the growth of Bcr-Abl-bearing tumors and markedly extends the survival of mice bearing such tumors. The inhibitory effect of NS-187 extends to 12 of 13 Bcr-Abl proteins with mutations in their kinase domain but not to T315I. NS-187 also inhibits Lyn without affecting the phosphorylation of Src, Blk, or Yes. These results suggest that NS-187 may be a potentially valuable novel agent to combat imatinib-resistant Philadelphia-positive (Ph+) leukemia.

Prediction of biological targets using probabilistic neural networks and atom-type descriptors.

Prediction of biological targets for molecules from their chemical structures is beneficial for generating focused libraries, selecting compounds for screening, and annotating biological activities for those compounds whose activities are unknown. We studied the ability of a probabilistic neural network (PNN), a variant of normalized radial basis function (RBF) neural networks, to predict biological activities for a set of 799 compounds having activities against seven biological targets. The compounds were taken from the MDDR database, and they were carefully selected to comprise distinct biological activities and diverse structures. The structural characteristics of compounds were represented by a set of 24 atom-type descriptors defined by 2D topological chemical structures. The modeling was done in two ways: (1). compounds having one certain activity were discriminated from those not having that activity and (2). all compounds were classified into seven biological classes. In both cases, around 90% of the compounds were correctly classified. Further validation of the modeled PNNs was done with 26 317 compounds having biological activities against various targets except for the seven targets used for modeling, and 67-98% compounds were correctly classified depending upon the targets. A PNN trains much more quickly than widely used neural networks such as a feed-forward neural network with error back-propagation. Calculation of atom-type descriptors is easy even for a large-size chemical library. Combination of PNN and atom-type descriptors thus provides a powerful way to predict biological activities from structural information.

Using general regression and probabilistic neural networks to predict human intestinal absorption with topological descriptors derived from two-dimensional chemical structures.

The objective of this study was to develop rapid and reliable methods to predict the percent human intestinal absorption (%HIA) of compounds based on their 2D descriptors. The analyzed data set included 86 drug and drug-like molecules and was the same as that studied by Wessel and co-workers. Instead of using three-dimensional descriptors such as polar surface area, which require lengthy computations, we employed only two-dimensional topological descriptors derived from information about the two-dimensional structure of molecules. The %HIA values were modeled using a general regression neural network (GRNN) and a probabilistic neural network (PNN), variants of normalized radial basis function networks. Both networks performed well to model the %HIA values. The root-mean square (rms) error was 22.8 %HIA unit for the external prediction set for a GRNN model, and 80% of the external prediction set was correctly classified for a PNN model, indicating the potential of our approach to estimate the %HIA values for a large set of compounds as virtual libraries.