Identification and characterization of melon circular RNAs involved in powdery mildew responses through comparative transcriptome analysis.

Circular RNAs (circRNAs) are a class of newly discovered non-coding RNAs that are typically derived from a genome's exonic, intronic, and intergenic regions. Recent studies of circRNAs in animals and plants have shown that circRNAs are vital in response to various abiotic and biotic stresses. Powdery mildew disease (PM) is a serious fungal disease threatening the melon industry.

Comparative transcriptome analysis uncovers regulatory roles of long non-coding RNAs involved in resistance to powdery mildew in melon.

Background: Long non-coding RNAs (lncRNAs) are a class of non-coding RNAs with more than 200 nucleotides in length, which play vital roles in a wide range of biological processes. Powdery mildew disease (PM) has become a major threat to the production of melon. To investigate the potential roles of lncRNAs in resisting to PM in melon, it is necessary to identify lncRNAs and uncover their molecular functions.

Genome-wide characterization, evolutionary analysis of WRKY genes in Cucurbitaceae species and assessment of its roles in resisting to powdery mildew disease.

The WRKY proteins constitute a large family of transcription factors that have been known to play a wide range of regulatory roles in multiple biological processes. Over the past few years, many reports have focused on analysis of evolution and biological function of WRKY genes at the whole genome level in different plant species. However, little information is known about WRKY genes in melon (Cucumis melo L.

Genome-wide analysis of basic/helix-loop-helix gene family in peanut and assessment of its roles in pod development.

The basic/helix-loop-helix (bHLH) proteins constitute a superfamily of transcription factors that are known to play a range of regulatory roles in eukaryotes. Over the past few decades, many bHLH family genes have been well-characterized in model plants, such as Arabidopsis, rice and tomato. However, the bHLH protein family in peanuts has not yet been systematically identified and characterized.

Assessment of the integrity of compounds stored in assay-ready plates using a kinase sentinel assay.

Sentinel assays are a convenient adjunct to LC-MS purity assessment to monitor the integrity of compounds in pharmaceutical screening collections over time. To assess the stability of compounds stored both at room temperature and at -20°C in assay-ready plates that were either vacuum pack-sealed using a convenient industrial vacuum sealing method or individually sealed using conventional foil seals, a diverse collection of ~ 5,000 compounds was assayed using a robust biochemical kinase assay at intervals over a one year period. Assay results at each time point were compared to those of initial assay using a series of correlations of compound Percent of Control (POC) values as well as IC50 values of a subset of compounds in 200 nL or 500 nL plates.

Molecular basis of sphingosine kinase 1 substrate recognition and catalysis.

Sphingosine kinase 1 (SphK1) is a lipid kinase that catalyzes the conversion of sphingosine to sphingosine-1-phosphate (S1P), which has been shown to play a role in lymphocyte trafficking, angiogenesis, and response to apoptotic stimuli. As a central enzyme in modulating the S1P levels in cells, SphK1 emerges as an important regulator for diverse cellular functions and a potential target for drug discovery. Here, we present the crystal structures of human SphK1 in the apo form and in complexes with a substrate sphingosine-like lipid, ADP, and an inhibitor at 2.

Rational design and binding mode duality of MDM2-p53 inhibitors.

Structural analysis of both the MDM2-p53 protein-protein interaction and several small molecules bound to MDM2 led to the design and synthesis of tetrasubstituted morpholinone 10, an MDM2 inhibitor with a biochemical IC50 of 1.0 μM. The cocrystal structure of 10 with MDM2 inspired two independent optimization strategies and resulted in the discovery of morpholinones 16 and 27 possessing distinct binding modes.

Inhibiting NF-κB-inducing kinase (NIK): discovery, structure-based design, synthesis, structure-activity relationship, and co-crystal structures.

The discovery, structure-based design, synthesis, and optimization of NIK inhibitors are described. Our work began with an HTS hit, imidazopyridinyl pyrimidinamine 1. We utilized homology modeling and conformational analysis to optimize the indole scaffold leading to the discovery of novel and potent conformationally constrained inhibitors such as compounds 25 and 28.

Synthesis and optimization of substituted furo[2,3-d]-pyrimidin-4-amines and 7H-pyrrolo[2,3-d]pyrimidin-4-amines as ACK1 inhibitors.

Two classes of ACK1 inhibitors, 4,5,6-trisubstituted furo[2,3-d]pyrimidin4-amines and 4,5,6-trisubstituted 7H-pyrrolo[2,3-d]pyrimidin-4-amines, were discovered and evaluated as ACK1 inhibitors. Further structural refinement led to the identification of potent and selective dithiolane inhibitor 37.

Development of a time-resolved fluorescence resonance energy transfer assay for cyclin-dependent kinase 4 and identification of its ATP-noncompetitive inhibitors.

Protein kinases are recognized as important drug targets due to the pivotal roles they play in human disease. Many kinase inhibitors are ATP competitive, leading to potential problems with poor selectivity and significant loss of potency in vivo due to cellular ATP concentrations being much higher than K(m). Consequently, there has been growing interest in the development of ATP-noncompetitive inhibitors to overcome these problems.

Novel pyrrolidine melanin-concentrating hormone receptor 1 antagonists with reduced hERG inhibition.

We discovered novel pyrrolidine MCHR1 antagonist 1 possessing moderate potency. Profiling of pyrrolidine 1 demonstrated that it was an inhibitor of the hERG channel. Investigation of the structure-activity relationship of this class of pyrrolidines allowed us to optimize the MCHR1 potency and decrease the hERG inhibition.

Identification of potent, noncovalent fatty acid amide hydrolase (FAAH) inhibitors.

Starting from a series of ureas that were determined to be mechanism-based inhibitors of FAAH, several spirocyclic ureas and lactams were designed and synthesized. These efforts identified a series of novel, noncovalent FAAH inhibitors with in vitro potency comparable to known covalent FAAH inhibitors. The mechanism of action for these compounds was determined through a combination of SAR and co-crystallography with rat FAAH.

Identification and optimization of N3,N6-diaryl-1H-pyrazolo[3,4-d]pyrimidine-3,6-diamines as a novel class of ACK1 inhibitors.

A new series of pyrazolo[3,4-d]pyrimidine-3,6-diamines was designed and synthesized as potent and selective inhibitors of the nonreceptor tyrosine kinase, ACK1. These compounds arose from efforts to rigidify an earlier series of N-aryl pyrimidine-5-carboxamides. The synthesis and structure-activity relationships of this new series of inhibitors are reported.

A homogeneous enzyme fragment complementation-based beta-arrestin translocation assay for high-throughput screening of G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) represent one of the largest gene families in the human genome and have long been regarded as valuable targets for small-molecule drugs. The authors describe a new functional assay that directly monitors GPCR activation. It is based on the interaction between beta-arrestin and ligand-activated GPCRs and uses enzyme fragment complementation technology.

High throughput screening for orphan and liganded GPCRs.

GPCRs had significant representation in the drug discovery portfolios of most major commercial drug discovery organizations for many years. This is due in part to the diverse biological roles mediated by GPCRs as a class, as well as the empirical discovery that they have proven relatively tractable to the development of small molecule therapeutics. Publication of the human genome sequence in 2001 confirmed GPCRs as the largest single gene superfamily with more than 700 members, furthering the already strong appeal of addressing this target class using efficient and highly parallelized platform approaches.

A highly sensitive high-throughput luminescence assay for malonyl-CoA decarboxylase.

Malonyl-CoA decarboxylase (MCD) catalyzes the conversion of malonyl-CoA to acetyl-CoA and thereby regulates malonyl-CoA levels in cells. Malonyl-CoA is a potent inhibitor of mitochondrial carnitine palmitoyltransferase-1, a key enzyme involved in the mitochondrial uptake of fatty acids for oxidation. Abnormally high rates of fatty acid oxidation contribute to ischemic damage.

An ultrasensitive high-throughput electrochemiluminescence immunoassay for the Cdc42-associated protein tyrosine kinase ACK1.

Several drugs inhibiting protein kinases have been launched successfully, demonstrating the attractiveness of protein kinases as therapeutic targets. Functional genomics research within both academia and industry has led to the identification of many more kinases as potential drug targets. Although a number of well-known formats are used for measuring protein kinase activity, some less well-characterized protein kinases identified through functional genomics present particular challenges for existing assay formats when there is limited knowledge of the endogenous substrates or activation mechanisms for these novel kinase targets.

High throughput screening for protein kinase inhibitors.

The pivotal role of kinases in signal transduction and cellular regulation has lent them considerable appeal as pharmacological targets across a broad spectrum of pathologies. Since the discovery that the v-Src oncogene encoded a protein kinase in 1978, kinases have remained a focus of research for pharmaceutical laboratories and academic groups alike. Many have sought to develop orally available low molecular weight synthetic kinase modulators (predominantly inhibitors) and thus capitalize on the links between aberrant regulation and disease.