Hu protein R (HuR) binds to the AU-rich element (ARE) in the 3'UTR to stabilize TNF-α mRNA. Here, we identified chemical inhibitors of the interaction between HuR and the ARE of TNF-α mRNA using RNA electrophoretic mobility gel shift assay (EMSA) and filter binding assay. Of 179 chemicals screened, we identified three with a half-maximal inhibitory concentration (IC 50 ) below 10 μM. The IC 50 of quercetin, b-40, and b-41 were 1.4, 0.38, and 6.21 μM, respectively, for binding of HuR protein to TNF-α mRNA. Quercetin and b-40 did not inhibit binding of tristetraprolin to the ARE of TNF-α mRNA. When LPS-treated RAW264.7 cells were treated with quercetin and b-40, we observed decreased stability of TNF-α mRNA and decreased levels of secreted TNF-α. From these results, we could find inhibitors for the TNF-α mRNA stability, which might be used advantageously for both the study for post-transcriptional regulation and the discovery of new anti-inflammation drugs.

Transforming growth factor (TGF)-β triggers the epithelial-to-mesenchymal transition (EMT) of cancer cells via well-orchestrated crosstalk between Smad and non-Smad signaling pathways, including Wnt/β-catenin. Since EMT-induced motility and invasion play a critical role in cancer metastasis, EMT-related molecules are emerging as novel targets of anti-cancer therapies. Traf2- and Nck-interacting kinase (TNIK) has recently been considered as a first-in-class anti-cancer target molecule to regulate Wnt signaling pathway, but pharmacologic inhibition of its EMT activity has not yet been studied. Here, using 5-(4-methylbenzamido)-2-(phenylamino)thiazole-4-carboxamide (KY-05009) with TNIK-inhibitory activity, its efficacy to inhibit EMT in cancer cells was validated. The molecular docking/binding study revealed the binding of KY-05009 in the hinge region of TNIK, and the inhibitory activity of KY-05009 against TNIK was confirmed by an ATP competition assay (K i, 100 nM). In A549 cells, KY-05009 significantly and strongly inhibited the TGF-β-activated EMT through the attenuation of Smad and non-Smad signaling pathways, including the Wnt, NF-κB, FAK-Src-paxillin-related focal adhesion, and MAP kinases (ERK and JNK) signaling pathways. Continuing efforts to identify and validate potential therapeutic targets associated with EMT, such as TNIK, provide new and improved therapies for treating and/or preventing EMT-based disorders, such as cancer metastasis and fibrosis.

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A Glu/Asp7.32 residue in the extracellular loop 3 of the mammalian GnRH receptor (GnRHR) is known to interact with Arg8 of mammalian GnRH (mGnRH), which may confer preferential ligand selectivity for mGnRH than for chicken GnRH-II (cGnRH-II). However, some nonmammalian GnRHRs also have the Glu/Asp residue at the same position, yet respond better to cGnRH-II than mGnRH. Amino acids flanking Glu/Asp7.32 are differentially arranged such that mammalian and nonmammalian GnRHRs have an S-E/D-P motif and P-X-S/Y motif, respectively. We presumed the position of Ser7.31 or Pro7.33 of rat GnRHR as a potential determinant for ligand selectivity. Either placing Pro before Glu7.32 or placing Ser after Glu7.32 significantly decreased the sensitivity and/or efficacy for mGnRH, but slightly increased that for cGnRH-II in several mutant receptors. Among them, those with a PEV, PES, or SES motif exhibited a marked decrease in sensitivity for mGnRH such that cGnRH-II had a higher potency than mGnRH, showing a reversed preferential ligand selectivity. Chimeric mGnRHs in which positions 5, 7, and/or 8 were replaced by those of cGnRH-II revealed a greater ability to activate these mutant receptors than mGnRH, whereas they were less potent to activate wild-type rat GnRHR than mGnRH. Interestingly, a mutant bullfrog type I receptor with the SEP motif exhibited an increased sensitivity for mGnRH but a decreased sensitivity for cGnRH-II. These results indicate that the position of Pro and Ser near Glu7.32 in the extracellular loop 3 is critical for the differential ligand selectivity between mammalian and nonmammalian GnRHRs.

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Conformational analyses of some thiamin-related compounds have been performed in order to find the relationship between their conformational and biochemical properties. Relaxed 2-D potential energy maps of free thiamin, its antagonists, and C(2) adducts were obtained using the molecular mechanics (MM) method. The antagonists include 4'-deaminothiamin, oxythiamin, pyrithiamin, thiamin thiazolone, thiamin thiothiazolone, 6,-methyl-4/f-thiamin and ó'-methylthiamin, and the C(2) adducts include 2-(<*-hydroxyethyl)thiamin (HET), 2-(a-lactyl)thiamin (LT), 6'-methyl-4/í-lactylthiamin and ó'-methyllactylthiamin. All of the local minima conformers were also identified for the active intermediates LT and HET. In numerous crystal structures, free thiamin assumes mostly the F form and less frequently the S form. The C(2) adducts assume only the S form. However, neither thiamin nor its active intermediates are found in the V form in crystal, but the cofactor assumes the V form in the active site of the protein.The MM map of free thiamin shows that the F form is truly the global minimum, while the S form occupies another minimum slightly higher in energy and lower in existence probability than the F form. The V form is also a local minimum but with very low existence probability. In addition, the availability of only one 4,-amino H atom for the intermolecular hydrogen bond makes it very unlikely that the thiamin molecule assumes the V form in solution and thereby in crystals. For various antagonists, either the V form is the global minimum or its existence probability is higher than that of thiamin. The V form instead of the crystalline S form is the global minimum conformer for both LT and HET. However, the V form of the C(2) adducts also would not be observed in the crystalline state due to the conformational characteristics of free thiamin; their V forms accordingly may be unique conformers available only inside the protein but not in solution. Based on the results of MM calculations and the crystal structures of holoenzymes, it is proposed that the active conformers of both intermediates are V forms with an intramolecular N-H-O(hydroxyl) hydrogen bond. A putative stereochemical model for thiamin catalysis is presented in which N-H-O hydrogen bonds contribute to the acceleration of the enzymic reaction by lowering the energies of the various species occurring along the reaction path. The principles of least motion and maximum orbital overlap which were originally applied to the decarboxylation reaction for the intermediates in the S form still hold for the V form.

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