Englerin A-sensing charged residues for transient receptor potential canonical 5 channel activation

정승주,고주연,김민지,박기철,Eunice Yon June Park,김진성,백영주,위진홍,Art E Cho,전주홍,서인석 대한약리학회 2019 The Korean Journal of Physiology & Pharmacology Vol.23 No.3

The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EAinduced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or GTPγS. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.

Englerin A-sensing charged residues for transient receptor potential canonical 5 channel activation

SeungJoo Jeong,Juyeon Ko,Minji Kim,Ki Chul Park,Eunice Yon June Park,Jinsung Kim,Youngjoo Baik,Jinhong Wie,Art E,Cho,Ju-hong Jeon,Insuk So 대한약리학회 2019 The Korean Journal of Physiology & Pharmacology Vol.23 No.3

The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EA-induced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or GTPγS. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.

Englerin A-sensing charged residues for transient receptor potential canonical 5 channel activation

Jeong, SeungJoo,Ko, Juyeon,Kim, Minji,Park, Ki Chul,Park, Eunice Yon June,Kim, Jinsung,Baik, Youngjoo,Wie, Jinhong,Cho, Art E.,Jeon, Ju-hong,So, Insuk The Korean Society of Pharmacology 2019 The Korean Journal of Physiology & Pharmacology Vol.23 No.3

The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EA-induced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or $GTP{\gamma}S$. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.

Extension of QM/MM docking and its applications to metalloproteins

Cho, Art E.,Rinaldo, David Wiley Subscription Services, Inc., A Wiley Company 2009 Journal of computational chemistry Vol.30 No.16

<P>To overcome the limitation of conventional docking methods which assume fixed charge model from force field parameters, combined quantum mechanics/molecular mechanics (QM/MM) method has been applied to docking as a variable charge model and shown to exhibit improvement on the docking accuracy over fixed charge based methods. However, it has also been shown that there are a number of examples for which adoption of variable-charge model fails to reproduce the native binding modes. In particular, for metalloproteins, previously implemented method of QM/MM docking failed most often. This class of proteins has highly polarized binding sites at which high-coordinate-numbered metal ions reside. We extend the QM/MM docking method so that protein atoms surrounding the binding site along with metal ions are included as quantum region, as opposed to only ligand atoms. This extension facilitates the required scaling of partial charges on metal ions leading to prediction of correct binding modes in metalloproteins. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2009</P> <B>Graphic Abstract</B> <P> <img src='wiley_img/01928651-2009-30-16-JCC21270-gra001.gif' alt='wiley_img/01928651-2009-30-16-JCC21270-gra001'> </P>

Incorporating QM and solvation into docking for applications to GPCR targets

Kim, Minsup,Cho, Art E. Royal Society of Chemistry 2016 Physical Chemistry Chemical Physics Vol. No.

<P>A great number of GPCR crystal structures have been solved in recent years, enabling GPCR-targeted drug discovery using structure-based approaches such as docking. GPCRs generally have wide and open entrances to the binding sites, which render the binding sites readily accessible to solvent. GPCRs are also populated with hydrophilic residues in the extracellular regions. Thus, including solvent and polarization effects can be important for accurate GPCR docking. To test this hypothesis, a new docking protocol which incorporates quantum mechanical/molecular mechanical (QM/MM) calculations along with an implicit solvent model is developed. The new docking method treats the ligands and the protein residues in the binding sites as QM regions and performs QM/MM calculations with implicit solvent. The results of a test on all solved GPCR cocrystals show a significant improvement over the conventional docking method.</P>

Using reverse docking to identify potential targets for ginsenosides

Kichul Park,Art E. Cho 고려인삼학회 2017 Journal of Ginseng Research Vol.41 No.4

Background: Ginsenosides are the main ingredients of ginseng, which, in traditional Eastern medicine, has been claimed to have therapeutic values for many diseases. In order to verify the effects of ginseng that have been empirically observed, we utilized the reverse docking method to screen for target proteins that are linked to specific diseases. Methods: We constructed a target protein database including 1,078 proteins associated with various kinds of diseases, based on the Potential Drug Target Database, with an added list of kinase proteins. We screened 26 kinds of ginsenosides of this target protein database using docking. Results: We found four potential target proteins for ginsenosides, based on docking scores. Implications of these “hit” targets are discussed. From this screening, we also found four targets linked to possible side effects and toxicities, based on docking scores. Conclusion: Our method and results can be helpful for finding new targets and developing new drugs from natural products.

Using reverse docking to identify potential targets for ginsenosides

Park, Kichul,Cho, Art E. The Korean Society of Ginseng 2017 Journal of Ginseng Research Vol.41 No.4

Background: Ginsenosides are the main ingredients of ginseng, which, in traditional Eastern medicine, has been claimed to have therapeutic values for many diseases. In order to verify the effects of ginseng that have been empirically observed, we utilized the reverse docking method to screen for target proteins that are linked to specific diseases. Methods: We constructed a target protein database including 1,078 proteins associated with various kinds of diseases, based on the Potential Drug Target Database, with an added list of kinase proteins. We screened 26 kinds of ginsenosides of this target protein database using docking. Results: We found four potential target proteins for ginsenosides, based on docking scores. Implications of these "hit" targets are discussed. From this screening, we also found four targets linked to possible side effects and toxicities, based on docking scores. Conclusion: Our method and results can be helpful for finding new targets and developing new drugs from natural products.

Leukotriene B4 receptor 2 gene polymorphism (rs1950504, Asp196Gly) leads to enhanced cell motility under low-dose ligand stimulation

장재현,WEIJUNDONG,김민섭,김주영,Art E Cho,김재홍 생화학분자생물학회 2017 Experimental and molecular medicine Vol.49 No.-

Recently, single-nucleotide polymorphisms (SNPs) in G-protein-coupled receptors (GPCRs) have been suggested to contribute to physiopathology and therapeutic effects. Leukotriene B4 receptor 2 (BLT2), a member of the GPCR family, plays a critical role in the pathogenesis of several inflammatory diseases, including cancer and asthma. However, no studies on BLT2 SNP effects have been reported to date. In this study, we demonstrate that the BLT2 SNP (rs1950504, Asp196Gly), a Gly-196 variant of BLT2 (BLT2 D196G), causes enhanced cell motility under low-dose stimulation of its ligands. In addition, we demonstrated that Akt activation and subsequent production of reactive oxygen species (ROS), both of which act downstream of BLT2, are also increased by BLT2 D196G in response to low-dose ligand stimulation. Furthermore, we observed that the ligand binding affinity of BLT2 D196G was enhanced compared with that of BLT2. Through homology modeling analysis, it was predicted that BLT2 D196G loses ionic interaction with R197, potentially resulting in increased agonist-receptor interaction. To the best of our knowledge, this report is the first to describe a SNP study on BLT2 and shows that BLT2 D196G enhances ligand sensitivity, thereby increasing cell motility in response to low-dose ligand stimulation.

Hologram and Receptor-Guided 3D QSAR Analysis of Anilinobipyridine JNK3 Inhibitors

Chung, Jae-Yoon,Cho, Art-E,Hah, Jung-Mi Korean Chemical Society 2009 Bulletin of the Korean Chemical Society Vol.30 No.11

Hologram and three dimensional quantitative structure activity relationship (3D QSAR) studies for a series of anilinobipyridine JNK3 inhibitors were performed using various alignment-based comparative molecular field analysis (COMFA) and comparative molecular similarity indices analysis (CoMSIA). The in vitro JNK3 inhibitory activity exhibited a strong correlation with steric and electrostatic factors of the molecules. Using four different types of alignments, the best model was selected based on the statistical significance of CoMFA ($q_2\;=\;0.728,\;r_2\;=\;0.865$), CoMSIA ($q_2\;=\;0.706,\;r_2\;=\;0.960$) and Hologram QSAR (HQSAR: $q_2\;=\;0.838,\;r_2\;=\;0.935$). The graphical analysis of produced CoMFA and CoMSIA contour maps in the active site indicated that steric and electrostatic interactions with key residues are crucial for potency and selectivity of JNK3 inhibitors. The HQSAR analysis showed a similar qualitative conclusion. We believe these findings could be utilized for further development of more potent and selective JNK3 inhibitors.

Fates of water and salts in non-aqueous solvents for directional solvent extraction desalination: Effects of chemical structures of the solvents

Choi, Ohkyung,Kim, Minsup,Cho, Art E.,Choi, Young Chul,Kim, Gyu Dong,Kim, Dooil,Lee, Jae Woo Techno Press 2019 Membrane Water Treatment Vol.10 No.3

Non-aqueous solvents (NASs) are generally known to be barely miscible, and reactive with polar compounds, such as water. However, water can interact with some NASs, which can be used as a new means for water recovery from saline water. This study explored the fate of water and salt in NAS, when saline water is mixed with NAS. Three amine solvents were selected as NAS. They had the same molecular formula, but were differentiated by their molecular structures, as follows: 1) NAS 'A' having the hydrophilic group (<TEX>$NH_2$</TEX>) at the end of the straight carbon chain, 2) NAS 'B' with symmetrical structure and having the hydrophilic group (NH) at the middle of the straight carbon chain, 3) NAS 'C' having the hydrophilic group (<TEX>$NH_2$</TEX>) at the end of the straight carbon chain but possessing a hydrophobic ethyl branch in the middle of the structure. In batch experiments, 0.5 M NaCl water was blended with NASs, and then water and salt content in the NAS were individually measured. Water absorption efficiencies by NAS 'B' and 'C' were 3.8 and 10.7%, respectively. However, salt rejection efficiency was 98.9% and 58.2%, respectively. NAS 'A' exhibited a higher water absorption efficiency of 35.6%, despite a worse salt rejection efficiency of 24.7%. Molecular dynamic (MD) simulation showed the different interactions of water and salts with each NAS. NAS 'A' formed lattice structured clusters, with the hydrophilic group located outside, and captured a large numbers of water molecules, together with salt ions, inside the cluster pockets. NAS 'B' formed a planar-shaped cluster, where only some water molecules, but no salt ions, migrated to the NAS cluster. NAS 'C', with an ethyl group branch, formed a cluster shaped similarly to that of 'B'; however, the boundary surface of the cluster looked higher than that of 'C', due to the branch structure in solvent. The MD simulation was helpful for understanding the experimental results for water absorption and salt rejection, by demonstrating the various interactions between water molecules and the salts, with the different NAS types.