The Rate Enhancement for Prolyl Cis-to-Trans Isomerization of Cyclic CPFC Peptide Is Caused by an Increase in the Vibrational Entropy of the Transition State

The conformational preferences and prolyl cis−trans isomerization of oxidized and reduced Ac-Cys-Pro-Phe-Cys-NH2 (CPFC peptides) have been carried out using the ab initio HF/6-31+G(d) and hybrid density functional B3LYP/6-311++G(d,p) levels of theory. The most preferred conformations of oxidized and reduced CPFC peptides with the trans prolyl peptide bond have a type-I β-turn for the Pro-Phe sequence in common. In particular, the transition states for both forms are stabilized by the intramolecular hydrogen bonds between the prolyl nitrogen and the N−H group of the Phe3 residue. The rotational barrier ΔGct⧧ to the cis-to-trans isomerization for the oxidized CPFC peptide is calculated to be 19.37 kcal/mol at the B3LYP/6-311++G(d,p)//HF/6-31+G(d) level of theory, which is lower by 0.88 kcal/mol than that of the reduced CPFC peptide. This may indicate that the rate constant kc→t of the prolyl cis-to-trans isomerization for the oxidized form is about 4 times larger than that of the reduced form, which is reasonably consistent with the value deduced from NMR experiments. In particular, the increase in vibrational entropy for the transition state of the oxidized form over that of the reduced form contributes to enhance the rate constant for the prolyl cis-to-trans isomerization of the oxidized form

Conformational Preferences and p<i>K</i>_a Value of Cysteine Residue

The conformational preferences of the Cys dipeptides with thiol and thiolate groups (Ac-Cys-NHMe and Ac-Cys−-NHMe, respectively) and the apparent (i.e., macroscopic) pKa value of the Cys dipeptide have been studied at the hybrid density functional B3LYP/6-311++G(d,p)//B3LYP/6-31+G(d) level with the conductor-like polarizable continuum model in the gas phase and in water. The hydrogen bonds and/or favorable interactions between the backbone and the thiol group of the side chain resulted in the different conformational preferences of the Cys and Cys− dipeptides from those of the Ala dipeptide in the gas phase and in water, although the preferred conformations of the Cys dipeptide are in part similar to those of the Ala dipeptide. In particular, the interactions between the thiolate group and the backbone amide groups appear to play a role in stabilizing the α- or 310-helical conformations for the Cys− dipeptide in the gas phase and in water. The pKa value of the Cys residue is estimated to be 8.58 at 25 °C using the statistically weighted free energies of all feasible conformations for the Cys and Cys− dipeptides in the gas phase and solvation free energies, which is consistent with the observed values of 8.3 and 8.22 ± 0.16

KY-05009 inhibits TGF-β1-induced activation of focal adhesion and non-Smad signaling pathways.

<p>Serum-deprived A549 cells were treated with TGF-β1 or its combination with KY-05009 for 48 h. Effects of KY-05009 on TGF-β1-induced activation of focal adhesion-related and ERK and JNK MAP kinase signaling molecules was evaluated by Western blot analysis. The ERK1 and 2 (p44 and p42, respectively) or JNK1 and 2 (p46 and p54, respectively) isoforms could be separated and differentially evaluated by gel electrophoresis, according to their molecular weight differences. The expression of p-FAK, p-Src, p-Paxillin, p-ERK1/2, and p-JNK2/1 was normalized by endogenous FAK, Src, Paxillin, ERK1/2, and JNK2/1, respectively. Actin was used as a loading control. Reported results are representatives of triplicate experiments.</p

A Novel Aminothiazole KY-05009 with Potential to Inhibit Traf2- and Nck-Interacting Kinase (TNIK) Attenuates TGF-β1-Mediated Epithelial-to-Mesenchymal Transition in Human Lung Adenocarcinoma A549 Cells

<div><p>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 (<i>K</i>_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.</p></div

KY-05009 inhibits TGF-β1-induced Wnt signaling.

<p>(A) Effects of KY-05009 on cell viability and TCF4 transcriptional activity. Serum-deprived A549 cells were treated with TGF-β1 or its combination with KY-05009 for 48 h, and then cell viability and TOPflash luciferase activity were measured. FOPflash-normalized TOPflash luciferase activity was represented to the relative TCF/LEF luciferase activity. The expression of TNIK and β-catenin in cytosolic and nuclear fractions (B), and the protein levels of TCF4-interacting proteins c were measured by Western blot analysis and immunoprecipitation assay, respectively. Actin, histone H3, and IgG were used as loading controls. The expression of cytosol and nucleus proteins was normalized by actin and histone H3, respectively. Reported results are representatives of triplicate experiments. ^###<i>p</i><0.001 (versus ‘the control’); ** <i>p</i><0.01, *** <i>p</i><0.001 (versus ‘the group treated with TGF-β1 only’).</p

KY-05009 inhibits TGF-β1-induced migration and invasion.

<p>The effect of KY-05009 on TGF-β1-induced migration and invasion of A549 cells was evaluated using (A) IncuCyte software and (B) Boyden chambers, respectively. The red and white dashed lines a represent the wounded area and the edge of migrated cells, respectively. Values (% RWD; Relative Wound Density) represent mean ± SD of triplicate samples, and reported images are representatives of triplicate experiments. Numbers of invaded cells were represented by an average number of cells per randomly selected three high-power field (HPF). Effects of KY-05009 on TGF-β1-induced expression and activation of MMP-2 and MMP-9 were measured by (C) quantitative RT-PCR and (D) gelatin zymography, respectively. (E) The effect of KY-05009 on NF-κB transcriptional activity was determined by reporter assay. ^##<i>p</i><0.01, ^###<i>p</i><0.001 (versus ‘the control’); * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001 (versus ‘the group treated with TGF-β1 only’).</p

KY-05009 inhibits TGF-β1-induced Smad signaling.

<p>(A) Effects of KY-05009 on TGF-β1-activated Smad signaling. Serum-deprived A549 cells were treated with TGF-β1 or its combination with KY-05009 for 48 h, and then the levels of p-Smad2 and endogenous Smad2/3 in cytosolic and nuclear fractions were evaluated by Western blot analysis. The nuclear expression levels of Snail and Twist were also evaluated. The expression of cytosol p-Smad2 was normalized by endogenous Smad2/3 and all nucleus proteins were normalized by histone H3. (B) Immunocytochemical confirmation of KY-05009 inhibition of TGF-β1-activated p-Smad2. Nuclei were counterstained with Hoechst 33342. All scale bars represent 50 µm. The expression of p-Smad2 was represented by the relative intensity of green fluorescence (n = 30), and reported images are representatives of triplicate experiments. ^##<i>p</i><0.01 (versus ‘the control’), ** <i>p</i><0.01 (versus ‘the group treated with TGF-β1 only’).</p

KY-05009 inhibits TGF-β1-mediated modulation of EMT markers.

<p>The effect of KY-05009 on TGF-β1-mediated modulation of EMT markers was evaluated by (A) Western blot analysis and (B) immunofluorescence microscopy. Serum-deprived A549 cells were treated with TGF-β1 or its combination with KY-05009 for 48 h. Actin was used as a loading control. Nuclei were counterstained with Hoechst 33342, and all scale bars represent 20 µm. The expressions of E-cadherin and vimentin were represented by the relative intensity of green fluorescence (n = 30), and reported images are representatives of triplicate experiments. ^##<i>p</i><0.01,* <i>p</i><0.05 (versus ‘the group treated with TGF-β1 only’).</p

Binding mode and <i>K</i>_i of KY-05009 for TNIK.

<p>(A) Chemical structure of KY-05009. (B) Binding mode of KY-05009 for TNIK. KY-05009 has two H-bond interactions with Cys108 (red dotted lines) in the hinge region, and CH/π interactions with Val31, Gly111, and Leu160. The yellow ball represents the CH/π interactions among Val31, ligand, and Gly111. (C) The binding constant, <i>K</i>_i, of KY-05009 for TNIK was determined using an ATP competition assay.</p

Praeruptorin A Inhibits <i>in Vitro</i> Migration of Preosteoclasts and <i>in Vivo</i> Bone Erosion, Possibly Due to Its Potential To Target Calmodulin

Excessive activity and/or increased number of osteoclasts lead to bone resorption-related disorders. Here, we investigated the potential of praeruptorin A to inhibit migration/fusion of preosteoclasts <i>in vitro</i> and bone erosion <i>in vivo</i>. Praeruptorin A inhibited the RANKL-induced migration/fusion of preosteoclasts accompanied by the nuclear translocation of NFATc1, a master regulator of osteoclast differentiation. Antimigration/fusion activity of praeruptorin A was also confirmed by evaluating the mRNA expression of fusion-mediating molecules. <i>In silico</i> binding studies and several biochemical assays further revealed the potential of praeruptorin A to bind with Ca²⁺/calmodulin and inhibit its downstream signaling pathways, including the Ca²⁺/calmodulin-CaMKIV-CREB and Ca²⁺/calmodulin-calcineurin signaling axis responsible for controlling NFATc1. <i>In vivo</i> application of praeruptorin A significantly reduced lipopolysaccharide-induced bone erosion, indicating its possible use to treat bone resorption-related disorders. In conclusion, praeruptorin A has the potential to inhibit migration/fusion of preosteoclasts <i>in vitro</i> and bone erosion <i>in vivo</i> by targeting calmodulin and inhibiting the Ca²⁺/calmodulin-CaMKIV-CREB-NFATc1 and/or Ca²⁺/calmodulin-calcineurin-NFATc1 signaling axis